GM3 Signaling [1st ed.] 9789811556517, 9789811556524

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Cheorl-Ho Kim

GM3 Signaling

GM3 Signaling

Cheorl-Ho Kim

GM3 Signaling

Cheorl-Ho Kim Molecular and Cellular Glycobiology Lab Department of Biological Sciences College of Science, Sungkyunkwan University Suwon, Korea

ISBN 978-981-15-5651-7 ISBN 978-981-15-5652-4 https://doi.org/10.1007/978-981-15-5652-4

(eBook)

© Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Contents

1

History of Sialic Acids, Gangliosides, and GM3 . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2

2

Synthesis of GM3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

3

Molecular Localization of GM3 in Cells . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 8

4

Basic Function of GM3 as an Interacting Molecule . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9 12

5

GD3 Mimetics with a Neurite Forming Capacity . . . . . . . . . . . . . . 5.1 GD3 Mimetic of Deuterostome Echinoderms . . . . . . . . . . . . . 5.2 GM4-Type and Hematoside-Type Ganglioside Show Immune-Modulating Capacities . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

13 13

. .

14 14

6

GM3 as a Pathogenic Infection Receptor . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17 19

7

GM3 and Related Gangliosides Prevent Inflammation and Atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

21 28

8

GM3 Has an Anti-tumor Capacity . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

31 36

9

GM3 Suppresses Tumor Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37 38

10

Interaction Between EGFR and GM3 . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 45

v

vi

11

12

13

14

15

16

17

Contents

Membrane Ganglioside-Specific Neuraminidase 3 (NEU3) Regulates GM3 Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49 53

Regulation of GM3-Mediated EGFR Signaling by NEU3 Sialidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55 58

VEGFR–GM3 Interaction in Angiogenesis . . . . . . . . . . . . . . . . . . 13.1 Soluble sFLT1 Directly Recognize GM3 Embedded in the Lipid Rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.2 FLK1/KDR (VEGFR-2)–GM3 Interaction . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

61

. . .

63 64 74

GM3, Competing with GM1, Interaction with Urokinase Plasminogen Activator Receptor (uPAR) in Endothelial Caveolar-Lipid Rafts Inhibits Angiogenesis . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77 78

GM3 Interacts with TGFβRs in the Epithelial–Mesenchymal Transition (EMT) During Posterior Capsular Opacification (PCO) Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

79 84

Galectin-1 Promotes Tumor Growth and Escapes Immune Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 91

GM3-HGFR, FGFR, and PDGFR Cancer Cell Behavior and IGF-1R in Diabetic Wound Healing . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

93 97

18

GM3, Caveolin-1 and Insulin Receptor in Insulin Resistance . . . . . 99 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

19

GM3 Suppresses Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

20

GM3 Protects Cochlear Hair Cells and Hearing from Corti Degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

21

GM3 Increases Osteoclast Differentiation Via Direct Cooperation with RANKL and IGF-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Contents

22

23

GM3 Induces Terminal Differentiation of Leukemic Cells . . . . . . . 22.1 GM3 Differentiation Role in Human Leukemic Monocyte Model: THP-1 Cell . . . . . . . . . . . . . . . . . . . . . . . . 22.2 GM3 Roles in Both Monocytoid and Granulocytoid Differentiation of HL-60 Cells . . . . . . . . . . . . . . . . . . . . . . . . 22.3 GM3 Role in a Megakaryocytoid Differentiation Inducer of Human Leukemic K562 Cells . . . . . . . . . . . . . . . . 22.4 GM3 Is an Inducing Factor of Megakaryocytic Differentiation by CAPE in the Human CML K562 Cells . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . α2,3-Sialyllactose (3SL) or α2,6-Sialyllactose (6SL) of GM3 Glycan in Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.1 3SL/6SL and Gangliosides in Innate Immunity . . . . . . . . . . . . 23.2 3SL Interaction with Toll-Like Receptor (TLR)-4 on DCs in Intestinal Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . 23.3 3SL Stimulates CD40, CD80, and CD86 Expression and Driving CD11c+, Ly-6Chi, CD4+, Th-1, and Th-17T Cells with Secreted TNF-α, IL-6, IL-12, and CCL5 . . . . . . . . . . . . . 23.4 6SL Restores Sialylation of GNE-Related Myopathy . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

. 115 . 117 . 117 . 121 . 124 . 127 . 133 . 133 . 134

. 135 . 136 . 137

Chapter 1

History of Sialic Acids, Gangliosides, and GM3

Ganglioside was first named by the German scientist Ernst Klenk in 1942 since he for the first time identified it as lipids from ganglion cells of the brain [1]. By 2011, 188 gangliosides are classified in vertebrate alone. For 70 years since the first naming of ganglioside by Klenk, globular acknowledgments have been accumulated on the GM3. In 1951, the sialic acid as a form of sialic acid-containing substance was first found at cell surface membranes of dried ghosts from horse erythrocytes and the isolated acidic sugar compound named hematoside, nowadays GM3 [2, 3]. During the isolation, the dried membranes of packed horse erythrocytes were treated with diethyl ether-methanol (1:1) to extract substances and then further extracted with chloroform-methanol (1:1). The prepared substance was examined to react with reagent orcinol, which is used as the authentic neuraminic acid (sialic acid)-reactive reagent. He, for the first time, called this glycolipid isolated from horse erythrocytes as hematoside in order to distinguish this glycolipid hematoside from the ganglioside isolated by Klenk’s preceding report. However, in fact, the hematoside is GM3 [4]. The hematoside isolated by Yamakawa constituted of an acidic sugar without detailed structural basis but named hemataminic acid that showed a purple color upon orcinol reaction. Thus, his hemataminic acid was also a family of neuraminic acid, what Klenk had earlier discovered them from horse species [1]. In early 1935, Klenk had prepared and isolated an orcinol-reacting purple-colored substance from the brain tissues of human patients of specific lysosomal storage disease, Niemann– Pick diseases [5, 6]. In 1942, Klenk designated this substance, purple-colored substance, to ganglioside [1] because the isolated substances occupied in very high concentration level in neuronal tissue, ganglions. After treatment and incubation of the isolated substances of gangliosides with methanolic hydrochloric acid, crystalline substance with the orcinol-reacted purple color was precipitated, and they named neuraminic acid [7]. In earlier 1936, a scientist Blix isolated polyhydroxyamino acid-like substances from submaxillary mucins of bovine. Thereafter, in 1952, he named it sialic acid as different designation [8]. In another side, a biochemist, R. Kuhn isolated lactaminic acid from cow colostrum [9]. Now, the orcinol-responded substances with the four different names of lactaminic acid, © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_1

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1 History of Sialic Acids, Gangliosides, and GM3

hemataminic acid, sialic acid, and neuraminic acid meant the same sugar. The conclusive description of the name has been summarized by a biochemist, A. Gottschalk in Australia [10, 11]. Now, it is well-known that the simple GM3 ganglioside is abundantly occupied in mammalian cell plasma membrane (PM). In addition, the GM3 has been documented for the functional roles of GM3 in migration, proliferation, senescence, and apoptosis. Genetic mutant mice in ganglioside synthesis, including GM3, have been established with some preventive and therapeutic targets.

References 1. Klink E (1942) Uber die Ganglioside, eine neue Gruppe von zuckerhaltigen Gehirnlipoiden. Physiol Chem 273:76–86 2. Yamakawa T, Suzuki S (1951) The chemistry of the lipids of posthemolytic residue or stroma of erythrocytes. I. Concerning the ether-insoluble lipids of lyophilized horse blood stroma. J Biochem 38:199–212 3. Yamakawa T (2005) Studies on erythrocyte glycolipids. Proc Jpn Acad Ser B 81:52–63 4. Suzuki A, Yamakawa T (2009) Dawn of glycobiology. J Biochem 146(2):149–156 5. Klenk E (1935) Über die Natur der Phosphatide und anderer Lipoide des Gehirns und der Leber. Z Physiol Chem 235:24–36 6. Klenk E (1939) Beiträge zur Chemie der Lipoidosen – Niemann-Picksche Krankheite und amaurotische Idiotie. Z Physiol Chem 262:128–143 7. Klenk E (1941) Neuraminsäure, das Spaltprodukt eines neuen Gehirnlipoids. Z Physiol Chem 268:50–58 8. Blix G, Svennerholm L, Werner I (1952) The isolation of chondrosamine from gangliosides and from submaxillary mucin. Acta Chem Scand 6:358–362 9. Kuhn R, Brossmer R (1954) Über die prosthetische Gruppe der Mucoproteine des Kuh-Colostums. Chem Ber 87:123–127 10. Gottschalk A (1955) Structural relationship between sialic acid, neuraminic acid and 2-carboxypyrrole. Nature 176:881–882 11. Blix FG, Gottschalk A, Klenk E (1957) Proposed nomenclature in the field of neuraminic acid and sialic acids. Nature 179:1088

Chapter 2

Synthesis of GM3

Sialic acid (SA) is a 9-carbon sugar in eukaryotic cells. SA metabolism and catabolism are cooperatively linked in the cells. The SA transfer, modification, catabolic reactions, biosynthesis, and activation in extracellular region, plasma membrane, cytosol, endoplasmic reticulum, Golgi apparatus, and nucleus are simply described (Fig. 2.1). GM3 is a staring ganglioside during biosynthesis for serial gangliosides including a-, b- and c-series⁣ (Fig. 2.2). Ganglioside species are predominantly present in the extracellular and outer side of cellular membrane leaflets and recognize cellular plasma membrane (PM) proteins through basically non-covalent electrostatic and hydrophobic interactions. Consequently, they form microdomains or lipid rafts. GM3 is enzymatically biosynthesized by GM3 synthase enzyme, CMPN-acetylneuraminic acid/lactosyl-Cer α2,3N-acetylneuraminyltransferase (EC 2.4.99.9) or SAT-I and ST3Gal V (Fig. 2.3). Ganglioside GM3 and GD3 structures are shown in Fig. 2.4. GM3 is a second abundantly enriched type of glycosphingolipid (GSL) in caveolin-associated lipid raft microdomains, and it facilitates biologic responses in cells. For the initial sialic acid synthesis, a responsible enzyme, UDP-Nacetylglucosamine (GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE), catalyzes the two steps in enzymatic biosynthesis of Neu5Ac. The enzyme has two domains, which regulate activities of the epimerase and kinase, important for cellular sialylation and sialylation of specific glycoproteins. GNE regulates ST3Gal5 expression for GM3 synthesis. As the simplest form of gangliosides, GM3 is the precursor molecule for all other ganglioside biosynthesis, and blocking the GM3 synthase is associated with diseases. Ganglioside GM3 has advantages as a potential therapeutic agent. As ganglioside, studies of monosialylglycolipid GM3 on human function and cell membrane surface’s sialic acid have been initiated by Tamio Yamakawa, who found GM3. Because ganglioside is normally synthesized in a cell-type manner or tissue-type manner, they bind to various molecules in non-covalent bonding mode by forming dynamic functional complex to affect cellular activity. In content, distribution and composition of gangliosides in animals, level of biosynthesis, and content and total content of gangliosides vary by organism © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_2

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2 Synthesis of GM3

Sialic acid transfer and modification

catabolic reactions SIALIDASE

Plasma membrane

ManNAc + pyruvate

O-acetyltransferase O-Ac

8-O-methyltransferase SAM

OMe

ST

esterase

lyase

CoA-Ac

SIALIDASE

AT

esterase

CMP Hydroxylase

CT

CMP

lysosome Cytosol Golgi apparatus

CMP CS

+CTP

Glc +ATP

Neucleus

Fru-6-P +Gln GlcNH2-6-P +CoA-Ac GlcNac-6-P

endoplasmic reticulum

CS: CMP-NeuAc synthesis CT: CMP-Sia transporter ST: sialyltransferase

CoA-Ac: acetyl coenzyme A AT: acetyl coenzyme A transporter SAM: A-adenosylmethionine

Neu5Ac -P Neu5Ac-9-P

+PEP ManNAc-6-P

GlcNAc-1-P ManNAc +UTP -UDP UDP-GlcNAc

+ATP

Sialic acid biosynthesis and activation

Fig. 2.1 Sialic acid transfer, modification, catabolic reactions, biosynthesis, and activation in extracellular region, plasma membrane, cytosol, endoplasmic reticulum, Golgi apparatus, and nucleus. Many molecules including sialidases, esterases, O-acetyltransferase, ManNAc pyruvate lyase, CMP-SA hydroxylase, and 8-O-methyltransferase are cooperatively related for the SA metabolism. CS CMP-NeuAc synthesis, CT CMP-Sia transporter, ST sialyltransferase, CoA-Ac acetyl coenzyme A, AT acetyl coenzyme A transporter, SAM A-adenosylmethionine

species and tissues within the same species, with highly accumulation in the CNS of the brains, depending on transcriptional and posttranslational events. Composition of ganglioside has variability of neuraminic acid configuration, glycan chain size, and ceramide length, giving relevant cellular localization and functionality. In cell PMs, gangliosides are belonged to membrane-specific glycolipids, where GM3 is the simplest one. Thus, GM3 is named the precursor of all the ganglioside and located in the outer membrane layer of PM of the animal kingdom. GM3 as the primary ganglioside is expressed in the PM outer leaflet with activities of the recognition, interaction, binding, adhesion, migration, and motility of cells.

Fig. 2.2 Gangliosides structures

2 Synthesis of GM3 5

6

2 Synthesis of GM3

A)

B)

Serine + Palmitoyl-CoA

GM3 synthase (ST3Gal V)

3 Ketosphinganine Sphinganine + Fatty Acid CoA Serine palmitoyl transferase Dihydroceramide

Gal

Sphingosine-1-Phosphate S-1-P Phosphatase

Glc

Ceramide synthase

1,1

UDP-Gal

UDP

Ceramide

Ceramide UDP-Glc

Glucosyltransferase

Glc-Cer UDP Galactosyltransferase Gal 1,4Glc-Cer

CMP-NeuAc

NeuAc

1,4

Sphingosine

Dihydroceramide desaturase

2,3

GM3 synthase (ST3Gal-V/SAT-I

CMP Gal 1,4Glc-Cer 3 Neu5Ac 2

GM3

Fig. 2.3 Synthesis pathway (a) and structure (b) of GM3. Ganglioside GM3 involves in cellular communications via cell to cell interaction, attachment, adhesion, cell cycle arrest, differentiation, motility, proliferation, signal transduction, tumorigenesis, migration, and metastasis. How does GM3 modulate the cell growth in cancer cells?

Lactosyl-Ceramide Glucosyl-Ceramide Typical Ceramide

Ganglioside GM3 Ganglioside GD3 Fig. 2.4 Ganglioside GM3 and GD3 structures

Chapter 3

Molecular Localization of GM3 in Cells

The question why they are located on the outer sides of cell PM has long been raised in this molecular biology and glycobiology. The outer world or border side of any organization is endlessly recognizing and interacting with their meaning counterparts or meaningless counterparts. This process is specific in the multicellular tissues, organs, or individuals, as a naturally formed ways, depending on the environmental situation. This process is similar to the memory, learning, and education in the brain. Thus, they are regarded as highly differentiated biomolecule in higher organisms. Finally, they act for intermolecular interaction. Gangliosides act for cell-cell recognition, adhesion, and signal transduction, where sialic acids of ganglioside do the works of recognization of specific counterparts and then regulate the functions of the plasma membrane-based signaling and cell behavior [1, 2]. They are together with allogenic sphingolipids in raft microdomains. Ganglioside is also pivotal in central myelination of neuronal responses. Cell-type expression of gangliosides and diversity may solve the clues what kinds of gangliosides interact with their proteins and how do interact? Formation of cell membrane’s diverse biomolecular complex, named membrane microdomain lipid rafts, characterizes specificity of each individual cell type. Then, why gangliosides are accumulated in microdomain? The answer is that hydrogen donors, acceptor, and long acyl fatty chains in ceramide backbone are aggregated together. Since sialic acid (SA) residues of gangliosides are exposed in outside extracellular membrane and some cases are shed forms, they are easily interacted and accumulated in the microdomain. Consequently, the microdomain forms and potentiates the functions of supra-biomolecular complex between gangliosides and membrane functional proteins such as growth factor receptors for intracellular signaling or caveola-1 for endocytosis. However, to date the known functions are limited in their information, except for the GM1-based bacterial toxin interaction.

© Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_3

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References 1. Hakomori S (2000) T raveling for the glycosphingolipid path. Glycoconj J 17:627–647 2. Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3:97–130

Chapter 4

Basic Function of GM3 as an Interacting Molecule

Expression of cellular ganglioside regulates various extracellular stimuli. Gangliosides are plenary parts to the physical structure and biological function of PMs in eukaryotic cells. Distribution, composition, and clustering of PM gangliosides decide the functional direction of cells. For example, the apical villi surfaces of the colon and small intestinal brush border membrane influence and regulate commonly homeostatic functions of the cells [1]. The current knowledges on the cells include morphology, active transport, cell division, cell death, differentiation, endocytosis, signaling, bacterial attachment, and exotoxin recognition [2]. Uncontrolled degradation and catabolic loss of carbohydrate moieties attached to gangliosides are frequently observed in intestinal diseases with diseases including immune disorders of autoimmune self-attacks and enterocolitis caused by increased proinflammatory signaling. In fact, the inflammatory responses in intestinal mucosa reduce the amount of GM3 and GD3 in the mucosa region. GM3 production level is involved in expression level of proinflammatory mediators such as cytokines, chemokine networks, and arachidonic acid metabolites. Dietary supplementation of gangliosides can increase the contents in intestinal membrane mucosa [3]. Ganglioside distribution and species compositions are largely altered during certain disease development and manifests. As especially well studied, several lysosomal storage diseases such as Gaucher, Sandhoff, and Tay–Sachs diseases belonged to lysosomal enzymes-defected disorders, resulting in incomplete GSL metabolism. In the most cases, the related genes are mutated and deficient for lysosomal catabolic enzymes and consequently lead to accumulation of undegraded gangliosides. In tissues, gangliosides also play crucial roles in prevention of lipopolysaccharide (LPS)induced reduction of in cellular tight junction that is formed by junction protein occluding. Therefore, loss of GM3 levels in the intestinal villi membrane mucosa leads to lack of cellular tight junction proteins due to their degradation. Considering that the intestinal integrity based on protein occluding of the cellular tight junction, healthy maintenance and function of intestinal homeostasis require normal distribution and composition of gangliosides. The management of intestine-borne diseases including diarrhea, infection, allergy, and autoimmune responses should be © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_4

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Sialic acid Nucleuo

ER

Golgi

Glycoprotein

Cell–Cell Recognition Cell–Substrate interaction Cell Adhesion

Cell Growth DNA mRNA

Glycolipid

Differentiation Protein targeting

Sialyltransferase

Fig. 4.1 Biosynthesis and biological functions of glyco-glycoconjugates

considered for improvement of ganglioside integrity. Gangliosides inhibit proinflammatory responses, which are triggered by disruption of membrane integrity of tight junction-associated enterocytes. Apart from the intestinal importance, gangliosides are also involved in nervous system function and maintenance such as the stability of myelin and axons. Thus, their expression levels are related with neurodegenerative lesions including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington disease, and virus-associated dementia. As glycosylated derivatives of sphingolipids, GSLs are structurally integral components of cellular membranes in mammals. Their composition, distribution, and structures are characterized by heterogeneous diversity among tissues. The heterogeneity is seen even in the same cell, as a cell type or condition specificity, depending on cell differentiation and development. GSLs are localized on the membrane surfaces of mammalian cells. GSLs regulate the epithelial differentiation and maturation [4]. Quantitative and qualitative changes on the cell-surfaced GSLs profile such as distribution and composition are related to malignant transformation as the main trait, as they are surface-bound or shed to modulate cellular behaviors. Biological functions of synthesized glyco-glycoconjugates including glycoproteins and glycolipids have broad spectrums including cell-cell recognition, cell-substrate interaction, cell adhesion, cell growth, differentiation, and protein targeting (Fig. 4.1). The key sialyltransferases catalyze the biosynthesis of the SA-containing glycoconjugates in the cellular organelle of ER and Golgi apparatus. The synthesized glycoconjugates regulate various cell functions through neuronal cell adhesion molecules such as NCAM, proteoglycan-mediated growth and differentiation factor, cell-cell interaction by lectin and mucin, and tumor progression and viral propagation by glycan genes (Fig. 4.2). For example, transformed tumor cells acquire the malignant properties through the GSLs expression to promote tumor growth, adhesion, migration, metastasis, invasion, and angiogenesis [4]. Gangliosides, sialic acid-contained GSLs, regulate various biologic events such as proliferation, interactions, differentiation, maturation, and signal transduction of cells [5–8]. Also, gangliosides regulate various

4 Basic Function of GM3 as an Interacting Molecule

11

Fig. 4.2 Cellular functions of glycoconjugates

phenomena including inflammatory obesity for GD1a, GM1, and GM2, monocytoid differentiation for GD3 and GM3, and angiogenic signaling pathway for GM3 and GD1a [9–13]. GM3 expressed in human lymphocytes modulates lipid raft-mediated cell functions including signal transduction and adhesion [14, 15]. The level of GM3 defines the direction and stages of maturation and differentiation of myeloid lineage cells and differentiation precursors into matured monocytes or tissue-type macrophages by a mechanism of GM3 promotion of differentiation and maturation [16]. GM3 regulates various pathological events and suppresses the cellular internalization associated with insulin resistance, obesity, and tumor progression [10, 12]. On the cellular PM sides, GM3 ganglioside modulates the Tyr phosphorylation and stimulation of certain growth factor receptors (GFRs) including plateletderived growth factor receptor (PDGFR), FGF receptor (FGFR), and insulin receptor (IR) [17]. Also, ganglioside GM3 reduces the ligand binding of the extracellular domain and inhibition of dimerization and phosphorylation of Tyr residue in the vascular endothelial growth factor receptor (VEGFR)-2 in VEGF-induced endothelial cells and EGF receptor (EGFR) and EGF-induced mammary cells [12, 13]. Specifically, they are also identified as tumor-associated carbohydrate antigens (TACAs). There are also minor roles of GM3 in inflammation suppression in rheumatoid arthritis and in tumor development inhibition. Some gangliosides are the key molecules of stem cells.

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4 Basic Function of GM3 as an Interacting Molecule

References 1. Miklavcic JJ, Schnabl KL, Mazurak VC, Thomson AB, Clandinin MT (2012) Dietary ganglioside reduces proinflammatory signaling in the intestine. J Nutr Metab 2012:280–286 2. Park EJ, Suh M, Thomson B, Ma DW, Ramanujam K, Thomson AB, Clandinin MT (2007) Dietary ganglioside inhibits acute inflammatory signals in intestinal mucosa and blood induced by systemic inflammation of Escherichia coli lipopolysaccharide. Shock 28(1):112–117 3. Xu J, Anderson V, Schwarz SM (2013) Dietary GD3 ganglioside reduces the incidence and severity of necrotizing enterocolitis by sustaining regulatory immune responses. J Pediatr Gastroenterol Nutr 57(5):550–556 4. Tao RV, Kovathana N, Shen YW (1982) Isolation and partial characterization of fucose- and Nacetylglucosamine-containing neutral glycosphingolipids from human senile cataracts. Curr Eye Res 2:427–434 5. Birklé S, Zeng G, Gao L, Yu RK, Aubry J (2003) Role of tumor-associated gangliosides in cancer progression. Biochimie 85(3–4):455–463 6. Varki A (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 3:97–130 7. Hakomori S (2002) Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci U S A 99:10231–10233 8. Chung TW, Choi HJ, Lee YC, Kim CH (2005) Molecular mechanism for transcriptional activation of ganglioside GM3 synthase and its function in differentiation of HL-60 cells. Glycobiology 15:233–244 9. Kim OS, Park EJ, Joe EH, Jou I (2002) JAK-STAT signaling mediates gangliosides-induced inflammatory responses in brain microglial cells. J Biol Chem 277:40594–40601 10. Wentworth JM, Naselli G, Ngui K, Smyth GK, Liu R, O’Brien PE, Bruce C, Weir J, Cinel M, Meikle PJ, Harrison LC (2016) GM3 ganglioside and phosphatidylethanolamine-containing lipids are adipose tissue markers of insulin resistance in obese women. Int J Obes (Lond) 40 (4):706–713 11. Bennaceur K, Popa I, Chapman JA, Migdal C, Peguet-Navarro J, Touraine JL, Portoukalian J (2009) Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells. Glycobiology 19:576–582 12. Liu Y, McCarthy J, Ladisch S (2006) Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling. Cancer Res 66:10408–10414 13. Chung TW, Kim SJ, Choi HJ, Kim KJ, Kim MJ, Kim SJ, Lee HJ, Ko JH, Lee YC, Suzuki A, Kim CH (2009) Ganglioside GM3 inhibits VEGF/VEGFR-2-mediated angiogenesis: direct interaction of GM3 with VEGFR-2. Glycobiology 19:229–239 14. Zhu Y, Gumlaw N, Karman J, Zhao H, Zhang J, Jiang JL, Maniatis P, Edling A, Chuang WL, Siegel C, Shayman JA, Kaplan J, Jiang C, Cheng SH (2011) Lowering glycosphingolipid levels in CD4+ T cells attenuates T cell receptor signaling, cytokine production, and differentiation to the Th17 lineage. J Biol Chem 286(17):14787–14794 15. Garofalo T, Sorice M, Misasi R, Cinque B, Mattei V, Pontieri GM, Cifone MG, Pavan A (2002) Ganglioside GM3 activates ERKs in human lymphocytic cells. J Lipid Res 43(6):971–978 16. Nojiri H, Takaku F, Terui Y, Miura Y, Saito M (1986) Ganglioside GM3: an acidic membrane component that increases during macrophage-like cell differentiation can induce monocytic differentiation of human myeloid and monocytoid leukemic cell lines HL-60 and U937. Proc Natl Acad Sci U S A 83(3):782–786 17. Sabourdy F, Kedjouar B, Sorli SC, Colié S, Milhas D, Salma Y, Levade T (2008) Functions of sphingolipid metabolism in mammals—lessons from genetic defects. Biochim Biophys Acta 1781:145–183

Chapter 5

GD3 Mimetics with a Neurite Forming Capacity

5.1

GD3 Mimetic of Deuterostome Echinoderms

GM3-like forms, which are monomethylated or monosialylated gangliosides, are found from the lipid phase of the starfish Luidia maculata using the chloroform/ methanol extracts [1]. Ceramide moieties are quite different from the animal sources in their chemical structures, consisting of phytosphingosine, sphingosine, unsubstituted fatty acids, and 2-hydroxy fatty acids as constituents [1]. The L. maculata species contains disialyl ganglioside GD3-like component, termed LMG. The isolated GD3-like LMG exhibits the neuron-generating potentials of neuritogenic effects. The neurites have been formed when the pheochromocytoma PC12 cells were treated with the LMG, as similarly observed with the nerve growth factor (NGF) treatment [1]. In another case, the Comanthus japonica species known as feather starfish contains trisialyl-inositolphosphoceramide-like form, and this ganglioside CJP also forms the neurites when the PC12 cells were treated with the CJP, indicating the potential neuritogenic capacity like NGF capacity [2]. Also, LLG ganglioside prepared from the water and lipid extracts of A. pectinifera and Linckia laevigata as other species of starfishes showed the similar neuritogenesis potentials [3, 4]. The neuritogenic effects of the starfish preparations are much higher than those obtained from the NGF-treated controls. The effects of the starfish preparations are also stronger than that obtained from the brain GM1-treated PC12 cells. Chemically, glycosyl inositolphosphoceramide-like CSP ganglioside of the starfish Comanthina schlegeli is the monosialyl-type ganglioside with the structure of 9-O-methyl-N-acetylαD-neuraminosyl-2,3-inositol-phospho-Cer [5]. Gangliosides isolated have neuritogenic and axon plastic capacities in the same PC-12 cell types [6, 7]. Therefore, the prototype gangliosides can attract for the CNS and peripheral neuronal regeneration [2].

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GM4-Type and Hematoside-Type Ganglioside Show Immune-Modulating Capacities

A hematoside-like LLG1 ganglioside, O-N-glycolyl-α-D-neuraminosyl-2,3β-D-Gal1,4β-D-glucopyranosyl-Cer, has been isolated from starfish Linckia laevigata [3]. GM4-like ganglioside has been isolated from the fingerlike projection, pyloric caeca, of Protoreaster nodosus known as the Okinawan starfish. Its structures are O-8-O-methyl-N-acetyl-α-neuraminosyl-2,3βGal-R, 1-O-βGal1,3αGal1,4,8Omethyl-N-acetyl-α-neuraminosyl2,3βGal-R and 1-OβGal1,3αGal1,9Nacetyl-α-neuraminosyl-2,3-βGal-R [8]. The GM4 was discovered from the human brains in 1964 [9]. Interestingly, GM4 is enriched in the human myelins [10]. The GM4 has been confirmed to selectively recognize myelin-specific proteins, and it has an inhibitory activity of ganglioside-degrading neuraminidase [11]. Thus, GM4 has been recognized as human myelin-specific biomarker and has a capacity to maintain oligodendroglial perikarya function [12]. GM4 also inhibit the immune responses when tetanus toxoid induces CD8 cytotoxic T cells activation in myelin of the human brain [13, 14]. GM4 is expressed in many animal tissues of the intestines of red sea bream, rat kidney, shark liver, chicken thymus, and chicken embryonic liver [7, 15–18]. GM4 species are also expressed in animals of lower vertebrates like the liver of frogs and bony fishes [19], although GM4 species are largely expressed in invertebrates with unknown reason. Considering that gangliosides involve in cellular functions in the tumor-associated microenvironments, lower organism-expressed gangliosides are suggested to exert multiple functions in each organism. For example, the simple ganglioside GM3 regulates the basic FGFR (bFGFR), cell adhesion molecules (CAMs), insulin-like growth factor-1 receptor (IGF-1R), EGFR, PDGFR, and VEGFR as well as the integrins for their functions. GM3 suppresses VEGF-driven VEGFR function.

References 1. Kawatake S, Inagaki M, Isobe R, Miyamoto T, Higuchi R (2002) Isolation and structure of monomethylated GM3-type ganglioside molecular species from the starfish Luidia maculata. Chem Pharm Bull (Tokyo) 50(10):1386–1389 2. Arao K, Inagaki M, Higuchi R (2001) Constituents of crinoidea. 2. Isolation and structure of the novel type gangliosides from the feather star Comanthus japonica. Chem Pharm Bull (Tokyo) 49(6):695–698 3. Tamai H, Ando H, Tanaka HN, Hosoda-Yabe R, Yabe T, Ishida H, Kiso M (2011) The total synthesis of the neurogenic ganglioside LLG-3 isolated from the starfish Linckia laevigata. Angew Chem Int Ed Engl 50(10):2330–2333 4. Higuchi R, Inoue S, Inagaki K, Sakai M, Miyamoto T, Komori T, Inagaki M, Isobe R (2006) Biologically active glycosides from asteroidea, 42. Isolation and structure of a new biologically active ganglioside molecular species from the starfish Asterina pectinifera. Chem Pharm Bull (Tokyo) 54(3):287–291

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5. Inagaki M, Shiizaki M, Hiwatashi T, Miyamoto T, Higuchi R (2007) Constituents of Crinoidea. 5. Isolation and structure of a new glycosyl inositolphosphoceramide-type ganglioside from the feather star Comanthina schlegeli. Chem Pharm Bull (Tokyo) 55(11):1649–1651 6. Inagaki M (2008) Structure and biological activity of glycosphingolipids from starfish and feather stars. Yakugaku Zasshi 128(8):1187–1194. Review. Japanese. 7. Chisada S, Shimizu K, Kamada H, Matsunaga N, Okino N, Ito M (2013) Vibrios adhere to epithelial cells in the intestinal tract of red sea bream, Pagrus major, utilizing GM4 as an attachment site. FEMS Microbiol Lett 341(1):18–26 8. Pan K, Tanaka C, Inagaki M, Higuchi R, Miyamoto T (2012) Isolation and structure elucidation of GM4-type gangliosides from the Okinawan starfish Protoreaster nodosus. Mar Drugs 10 (11):2467–2480 9. Kuhn R, Wiegandt H (1964) Weitere Ganglioside Aus Menschenhirn. Z Naturforsch 19:256 10. Mullin BR, Decandis FX, Montanaro AJ, Reid JD (1981) Myelin basic protein interacts with the myelin-specific ganglioside GM4. Brain Res 222(1):218–221 11. Yohe HC, Jacobson RI, Yu RK (1983) Ganglioside-basic protein interaction: protection of gangliosides against neuraminidase action. J Neurosci Res 9:401–412 12. Arvanitis DN, Min W, Gong Y, Heng YM, Boggs JM (2005) Two types of detergent-insoluble, glycosphingolipid/cholesterol-rich membrane domains from isolated myelin. J Neurochem 94 (6):1696–1710 13. Kawashima I, Nakamura O, Tai T (1992) Immunosuppression by human gangliosides: I. Relationship of carbohydrate structure to the inhibition of T cell responses. Biochim Biophys Acta 1125:180–188 14. Yuki N, Takahashi Y, Ihara T, Ito S, Nakajima T, Funakoshi K, Furukawa K, Kobayashi K, Odaka M (2012) Lack of antibody response to Guillain-Barré syndrome-related gangliosides in mice and men after novel flu vaccination. J Neurol Neurosurg Psychiatry 83(1):116–117 15. Dasso JF, Obiakor H, Bach H, Anderson AO, Mage RG (2000) A morphological and immunohistological study of the human and rabbit appendix for comparison with the avian bursa. Dev Comp Immunol 24(8):797–814 16. Shiraishi T, Uda Y (1986) Characterization of neutral sphingolipids and gangliosides from chicken liver. J Biochem 100(3):553–561 17. Tsuboi N, Utsunomiya Y, Kawamura T, Kikuchi T, Hosoya T, Ohno T, Yamada H (2003) Shedding of growth-suppressive gangliosides from glomerular mesangial cells undergoing apoptosis. Kidney Int 63(3):936–946 18. Li YT, Sugiyama E, Ariga T, Nakayama J, Hayama M, Hama Y, Nakagawa H, Tai T, Maskos K, Li SC (2002) Association of GM4 ganglioside with the membrane surrounding lipid droplets in shark liver. J Lipid Res 43:1019–1025 19. Saito M, Kitamura H, Sugiyama K (2001) Liver gangliosides of various animals ranging from fish to mammalian species. Comp Biochem Physiol B 129:747–758

Chapter 6

GM3 as a Pathogenic Infection Receptor

The fact that the GM3 or related gangliosides are localized on cell surfaces allows the possibility that the extracellular interaction of pathogenic agents such as bacteria and viruses is mediated via those gangliosides. Glycans of gangliosides are recognized with high affinity and specificity by infectious protein factors. GSLs frequently act as recognition and interaction sites for pathogenic bacteria and parasites as well as infectious viruses. Therefore, the GSLs are the entry sites for invaders [1]. The gangliosides function as mediators in viral fusion. For example, the glycan moieties can be interacted with glycoproteins or other carbohydrates expressed on their own cellular PM in the same cells or in the neighbored surrounding cells. For example, with regard to the simian rotavirus receptors, infection of host cells with the simian rotavirus SA11 strain is inhibited by neuraminidase, and the result indicates the involvement of SA-linked receptors present in the cell surfaces of host [2]. Host infection with rotavirus strain SA11 was also blocked by treatments with sialylated glycoproteins [3] and by the GM1 ganglioside [4]. Rotavirus strain SA11 binds to the nonacid GSL gangliotetraosylceramid GA1 or another name of asialo-GM1, but not other gangliosides. GM3 in a form of either N-glycolylneuraminic acid (NeuGc) or N-acetylneuraminic acid (NeuAc) is recognized to be the cell surface receptors for attachment and infection of the porcine rotavirus strains [5, 6]. Another type of GM1a similarly mediates a certain rotaviral infection by the newly classified rotavirus strain, a neuraminidase-insensitive human rotavirus [7]. Representatively, GM3 functions as a receptor of bovine rotavirus [8], human immunodeficiency virus (HIV) [9], and simian rotavirus [8]. HIV-2 and HIV-1 glycoproteins are recognized by GM3, and GM3 is the abundantly expressed ganglioside, in the CD4(+) lymphocytes PM and macrophages PM. Adsorption and attachment of HIVs to the target cell surfaces require a recognition and binding of the envelope glycoprotein present on cell surface and certain cell types of GalCer, which is recognized by CD4( ) cells and GM3 by CD4(+) cells for their molecular interactions. The cellular binding sites for HIV antigens on CD4+ lymphocytes are GM3 [10]. GM3 represents HIV-1 gp120-binding sites on the CD4+ cell surfaces. Ganglioside-binding motif of the gp120 protein on the host cell surfaces is reported © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_6

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to be XXXGPGRAFXXX [11–13]. The gp120 motif homologous sites are also observed in synucleins, galectins, transmembrane receptors, and TNF-a receptor superfamily [14, 15]. As disialogangliosides, GD3 is also a receptor for HIV [10, 16], GD1b is for BK polyoma virus, JC is for polyoma virus, murine is for polyoma virus [17], and GD1a is for the simian rotavirus [8]. In a recent study [18], GD1a glycan is known to be an attachment receptor for adenovirus infection that causes for epidemic keratoconjunctivitis in human eye diseases. GD1a is glycosphingolipid with two sialic acids in the structure of Neu5Acα2,3βGal1,3βGalNAc1,4 [Neu5Acα2,3]β Gal1,4βGlc1,1Cer. Adenovirus type 37 is the leading cause of the epidemic keratoconjunctivitis. The type 37 adenovirus binds to α2,3-linked SA-containing surface molecules, but SA residues solely serve as a reality of attachment and binding receptor on the cell surfaces. Using a glycan-arrayed technology in the Ad37 knob binding to 260 different glycans, the disialylated glycan of GD1a was identified. Moreover, using another analytical technology of X-ray crystallography and functional analysis of the bound glycans, the binding complex between Ad37 knob and GD1a glycan confirmed two terminally linked SA residues. Ad37 virus, virion particles specifically bind the GD1a glycan-containing molecules attached to the cell hosts. The glycan structure linked to GD1a as the infectious receptor for Ad37 virus implicates in its application as a drug-design platform, if applicable, to new development of low molecular inhibitors as entry blockers to the host cells. Four sialic acid-containing ganglioside, GT1b is used as a receptor for BK polyoma virus. A different type of GSL, GalCer, is also reported to mediate endocytotic internalization of Semliki forest virus via membrane fusion event of host cells [19]. Similarly, neutral glycolipids, not acidic GSLs, serve as a functional receptor molecule to mediate the same endocytotic fusion upon myxovirus-induced membrane fluidity [20]. As the most well-studied case, GM1 is well characterized as a receptor for the cholera toxin [21]. Cholera toxin (CTx) that is extracellularly produced by marine source-derived, harmful bacteria Vibrio cholerae is a representative AB5 exotoxin with a single subunit A having enzymatic catalytic activity (CTA) and five identical B subunit polypeptides having the host cell-recognizing activity. The toxin binds to host cells to enter its target cytoplasm. The 5 B subunits recognize GM1 as receptor present in the host cell PM. The GM1 receptor-toxin 5B complex is delivered to the endoplasmic reticulum (ER) through endocytosis [22], calling the process to retrograde transport. The CTx retrograde transport to the ER is specifically progressed by GM1 recognition. Simian virus 40 (SV40) and bovine rotavirus also utilize GM1 as receptor [23]. During the SV40 viral infection, synergy between focal adhesion kinase and GM1 enhances the population-determined pattern of SV40 infection. As a non-sialic acid-containing glycosphingolipid, Gb3 is also known as HIV, Shiga toxin, and verotoxin receptor [9, 24, 25]. In addition, Gb4, Forssmann, SSEA-3, SSEA-4, and nLc3 are known as receptors for human and simian parvovirus [26, 27]. Gb4 as a major neutral GSL is recognized by B19 capsid, and the B19 capsid protein binds to complexed type of globo series glycans including SSEA-3 and SSEA-4 as well as paragloboside series such as neolactotetraglycosylceramide in B19 parvovirus-associated disease [27].

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In botulinum species, different serotypes of botulinum neurotoxin (BotN) are known with seven names of BotN-A, BotN-B, BotN-C, BotN-D, BotN-E, BotN-F, and BotN-G. They are separately produced and later assembled as a large body of protein complexes, which are composed of non-toxic hemagglutinin (HA) components and non-toxic non-HA components. The non-toxic protein part causes the oral toxicity during administration or infection. HA delivers toxins to the intestinal epithelium by carbohydrate-binding activity. Among the serotypes, HA of serotype C (HA/C) directly disrupt the epithelial cell barrier function, through an acting mechanism that HA/C binds sialic acid in GM3 and a-series gangliosides as the target molecule. The GM3 binding capacity of HA is abolished when ganglioside synthesis is blocked [28]. In cells, the mouse fibroblast cells resistant to HA/Cbinding lack GM3 synthase.

References 1. Groux-Degroote S, Cavdarli S, Uchimura K, Allain F, Delannoy P (2020) Glycosylation changes in inflammatory diseases. Adv Protein Chem Struct Biol 119:111–156 2. Kawagishi T, Nurdin JA, Onishi M, Nouda R, Kanai Y, Tajima T, Ushijima H, Kobayashi T (2020) Reverse genetics system for a human group A rotavirus. J Virol 94(2), pii: e00963-19. 3. Li Z, Gao C, Zhang Y, Palma AS, Childs RA, Silva LM, Liu Y, Jiang X, Liu Y, Chai W, Feizi T (2018) O-Glycome beam search arrays for carbohydrate ligand discovery. Mol Cell Proteomics 17(1):121–133 4. Superti F, Donelli G (1991) Gangliosides as binding sites in SA-11 rotavirus infection of LLC-MK2 cells. J Gen Virol Pt 10:2467–2474 5. Bergner DW, Kuhlenschmidt TB, Hanafin WP, Firkins LD, Kuhlenschmidt MS (2011) Inhibition of rotavirus infectivity by a neoglycolipid receptor mimetic. Nutrients 3(2):228–244 6. Martínez MA, López S, Arias CF, Isa P (2013) Gangliosides have a functional role during rotavirus cell entry. J Virol 87(2):1115–1122 7. Coulson BS (2015) Expanding diversity of glycan receptor usage by rotaviruses. Curr Opin Virol 15:90–96 8. Fleming FE, Böhm R, Dang VT, Holloway G, Haselhorst T, Madge PD, Deveryshetty J, Yu X, Blanchard H, von Itzstein M, Coulson BS (2014) Relative roles of GM1 ganglioside, N-acylneuraminic acids, and α2β1 integrin in mediating rotavirus infection. J Virol 88 (8):4558–4571 9. Seddiki N, Ben Younes-Chennoufi A, Benjouad A, Saffar L, Baumann N, Gluckman JC, Gattegno L (1996) Membrane glycolipids and human immunodeficiency virus infection of primary macrophages. AIDS Res Hum Retroviruses 12(8):695–703 10. Hammache D, Yahi N, Piéroni G, Ariasi F, Tamalet C, Fantini J (1998) Sequential interaction of CD4 and HIV-1 gp120 with a reconstituted membrane patch of ganglioside GM3: implications for the role of glycolipids as potential HIV-1 fusion cofactors. Biochem Biophys Res Commun 246(1):117–122 11. Hammache D, Yahi N, Maresca M, Piéroni G, Fantini J (1999) Human erythrocyte glycosphingolipids as alternative cofactors for human immunodeficiency virus type 1 (HIV-1) entry: evidence for CD4-induced interactions between HIV-1 gp120 and reconstituted membrane microdomains of glycosphingolipids (Gb3 and GM3). J Virol 73(6):5244–5248

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12. Richichi B, Pastori C, Gherardi S et al (2016) GM-3 lactone mimetic interacts with CD4 and HIV-1 Env proteins, hampering HIV-1 infection without inducing a histopathological alteration. ACS Infect Dis 2(8):564–571 13. Nehete PN, Vela EM, Hossain MM, Sarkar AK, Yahi N, Fantini J, Sastry KJ (2002) A postCD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains. Antiviral Res 56 (3):233–251 14. Delézay O, Hammache D, Fantini J, Yahi N (1996) SPC3, a V3 loop-derived synthetic peptide inhibitor of HIV-1 infection, binds to cell surface glycosphingolipids. Biochemistry 35 (49):15663–15671 15. Ideo H, Seko A, Ishizuka I, Yamashita K (2003) The N-terminal carbohydrate recognition domain of galectin-8 recognizes specific glycosphingolipids with high affinity. Glycobiology 13(10):713–723 16. Andersson LM, Fredman P, Lekman A, Rosengren L, Gisslén M (1998) Increased cerebrospinal fluid ganglioside GD3 concentrations as a marker of microglial activation in HIV type 1 infection. AIDS Res Hum Retroviruses 14(12):1065–1069 17. Neu U, Allen SA, Blaum BS et al (2013) A structure-guided mutation in the major capsid protein retargets BK polyomavirus. PLoS Pathog. 9(10):e1003688 18. Nilsson EC, Storm RJ, Bauer J, Johansson SM, Lookene A, Ångström J, Hedenström M, Eriksson TL, Frängsmyr L, Rinaldi S, Willison HJ, Pedrosa Domellöf F, Stehle T, Arnberg N (2011) The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis. Nat Med 17(1):105–109 19. Samsonov AV, Chatterjee PK, Razinkov VI, Eng CH, Kielian M, Cohen FS (2002) Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus. J Virol 76 (24):12691–12702 20. Huang RT (1982) Myxovirus-induced membrane fusion mediated by phospholipids and neutral glycolipids. Adv Exp Med Biol 152:393–400 21. Jobling MG, Yang Z, Kam WR, Lencer WI, Holmes RK (2012) A single native ganglioside GM1-binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway. MBio 3(6). pii e00401-12. 22. Ewers H, Helenius A (2011) Lipid-mediated endocytosis. Cold Spring Harb Perspect Biol. 3(8): a004721 23. Snijder B, Sacher R, Rämö P, Damm EM, Liberali P, Pelkmans L (2009) Population context determines cell-to-cell variability in endocytosis and virus infection. Nature 461 (7263):520–523 24. Chan YS, Ng TB (2016) Shiga toxins: from structure and mechanism to applications. Appl Microbiol Biotechnol 100(4):1597–1610 25. Iwamura K, Furukawa K, Uchikawa M et al (2003) The blood group P1 synthase gene is identical to the Gb3/CD77 synthase gene. A clue to the solution of the P1/P2/p puzzle. J Biol Chem 278(45):44429–44438 26. Sequeira J, Calado A, Dias M, Manita M (2017) Parvovirus B19 infection associated with hemolytic anemia and cranial polyneuropathy. J Neurovirol 23(5):786–788 27. Mao J, Zhang Q, Ye X, Liu K, Liu L (2014) Efficient induction of pluripotent stem cells from granulosa cells by Oct4 and Sox2. Stem Cells Dev 23(7):779–789 28. Sugawara Y, Iwamori M, Matsumura T, Yutani M, Amatsu S, Fujinaga Y (2015) Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3. FEBS J 282(17):3334–3347

Chapter 7

GM3 and Related Gangliosides Prevent Inflammation and Atherosclerosis

Gangliosides are implicated in inflammation. Ganglioside sustains and maintains intestinal cell architectures and functions, supporting outer membrane barrier integrities. Ganglioside content is decreased in the inflammatory intestines due to inflammation response. Gangliosides replace mucosal compounds composition of glycolipids and carbohydrates, decrease proinflammatory cytokines biosynthesis, and prevent autoimmune necrosis and cell injury. However, the level of ganglioside is increased in the healthy intestines and the mucosa. Certain disease states under inflammation responses and infection process lead to changes in a variety of modification and expression of hosts. Among the host responses, the remarkable changes are the host glycosylation. Regulation of host glycosylation events results in global changes in host protein and host GSLs towards adaptation of cell function. Changes in host protein, host glycolipids, as well as resulting cell function are accomplished through alterations in glycosylation events. Consequently, various cellular responses including inflammation initiation and progression, infection sensitivity, host cell transformation, and immune suppression occur. In certain microenvironments, microbes also regulate host glycosylation to make easier infection ability and promotion through host immune downregulation by alteration of host glycan biosynthesis. Totally, changes in host glycome expression yield differences of molecular and cellular functionalities in hosts. Therefore, the levels of infection, inflammation, and transformation are correlated with the glycome changes. Some gangliosides have antibacterial activities and the resistance against microbial pathogens. Gangliosides also prevent secondary infection and inhibit the related inflammatory signaling cascade. Gangliosides reduce proinflammatory signaling in the intestinal membrane lipid rafts of the villi mucosa [1]. Dietary supplementation of gangliosides and the resultant changes in ganglioside composition and content on the lipid raft microdomain gangliosides are important for cellular homeostasis. For example, a simple GSL GM3 mediates and involves in various cellular functions. In membrane lipid rafts, GM3 ganglioside is associated and co-localized with multiple membrane molecules to exert signaling pathways on cellular PM. The signaling molecules include adaptor molecules such as Rho, c-Src, and Fak in lipid rafts © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_7

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microdomains [2]. Thus, reduction in GM3 amounts largely alters signal transduction pathways activated by these signaling molecules. The elevated amounts of GD3 on PM lipid rafts activate immune cell responses and enhance immune functions of each working immune cell. For example, gut functions are known to protect during development stages because the expressed GD3 stimulates host T cells [3] and exhibits an anticarcinogenic activity [4]. The increased accumulation level of dietary gangliosides in lipid raft microdomains enriches GM3 level in lipid raft microdomains from the dietary sources. Thereafter, their accumulated GM3 activates phospholipase (PL) A2 activity and consequently increases in high level of arachidonic acid release from membrane phospholipids in human peripheral blood lymphocytes [5]. Thus, the decrease in GM3 levels reduces PLA2 activity leading to decreased PAF content where PAF recognizes a PAF receptor present in cellular PM to trigger inflammatory responses. For the beneficial side, therefore, the dietary or exogenous supplementation of ganglioside exerts some effects on anti-inflammation and autoimmune responses in the intestinal gut of animals and humans. The most outstanding effects of GM3 are found in protecting the neonatal intestine [6]. Apart from the ganglioside-related disease, the defective gangliosides in biosynthesis and degradation exert specific pathological developments. Ganglioside species compositions differ among several disease states. Gangliosides are associated with the pathophysiology of many diseases. In some lysosomal storage diseases such as Sandhoff, Gaucher, and Tay–Sachs diseases, GSL storages derived from defected enzymes by genetic variant of lysosomal sialidases or other enzymes are issued as the genetic diseases. For example, β-hexosaminidase enzyme activity is high in patient monocytes of peripheral bloods with autoimmune diseases when the peripheral blood monocytes are treated with LPS. In the β-hexosaminidase action, the enzyme produces GM3 from GM2; increased ganglioside degradation in catabolic process leads to pathogenic state like autoimmune diseases. As described earlier, membrane gangliosides integrally maintain the structural architecture and functionality of cellular PMs. Conversely, increased metabolic degradation of membrane gangliosides in local tissues such as intestinal mucosa may increase in enhanced disease states through proinflammatory signaling. In fact, GM3 content in intestinal brush border mucosa is gradually decreased with inflammation manifests. Thus, the decreased and low levels of membrane GM3 expression are associated with the increased level of inflammation progress with upregulated generation of proinflammatory mediators and cytokines. Recent representative case is an influenza “flu,” where sialic acid residues are the target of influenza A viruses in its invasion into host cells [7]. Another representative case is the Alzheimer’s neurodegenerative disease. Gangliosides affect in the causing aggregation of amyloid-β peptide [8]. Null mice of all ganglioside exhibit severe lethality [9]. In insulin resistance, GM3 has been suggested to be related in this disease. Thus, GM3 is a potential target for new drugs. GM3 is an anti-inflammation agent for RA and arteriosclerosis and inflammatory diseases treatment. Although not established, negative point is that GM3 induces the insulin resistance as the type 2 diabetes. GM3 activity inhibition means the type 2 diabetic prevention and treatment (please note that the author does not accept). GM3 is associated with the

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loss of eyesight in a rare infant-related genetic disease in the Old Amish population. GM3 has a significant potential for new therapies, but it is not the easy lipids to be studied. Its function in cell recognition and adhesion and structural biomembrane function will give design of future drugs. Tumor development and suppression are also associated with gangliosides. GM3 inhibit the development of the tumor cell, while GD1a proliferates tumor cell [10]. GM3 as the simplest form acts as an insulin resistance inducer and a tumor suppressor and plays in rheumatoid arthritis. In non-invasive bladder cancer, GM3 is highly expressed; however, the invasive cancer cells express it in a low level [11]. When the Golgi-disrupting agent, GSLd synthesis-inhibiting brefeldin A (BFA), a macrocyclic lactone structured compound of fungal strains, was treated to the cells, GSL production and invasive potentials of bladder cancers were together reduced. The results are obtained by the increased production of GM3 that is stimulated by BFA treatment. Bladder cancer cells express tetraspanin CD9. Interestingly, CD9 and GM3 act collaboratively as co-factors in tumor cell migration and motility. The decreased CD9 production is related to decreased invasive potentials of tumor cells and also improved survival rate of colon cancer patients. Accelerated GM3 synthesis, which is stimulated by BFA, decreases metastatic potentials in bladder cancer, via interaction between GM3 and integrin CD9. Thus, GM3 has a therapeutic benefit to bladder tumor with downregulating role of GM3 in bladder tumor metastasis [12]. When MBT-2 cells known as mouse bladder carcinoma are introduced with the cDNA encoding for the GM3 synthase gene cDNA for the gene transfection, GM3-expressing transfectants reduced growth, migration, motility, metastasis, and xenograft tumor proliferation transplanted in nude mice and increased in the apoptotic cell death. GM3 increased number of apoptotic cells, presenting a novel therapeutic modality. Inflammation is in general related with initial cancer development and linked with angiogenesis. Inflammation responses are associated with extensive expressions of a variety of related molecules to vascular endothelial proteins, secondary metabolites, lipid 2nd messengers, angiogenic inducers, and proinflammatory cytokines. The protein molecules as cytokines and growth factors include IL-6, IL-8, tumor necrosis factor-α (TNF-α), IL-1β, transforming growth factor-β (TGF-β) and VEGF [13]. These cytokines promote the tumor proliferation, blood vessel angiogenesis, tissue-destroying inflammation, and invasive metastasis. Inflammatory reaction in endothelial cell system involves in the synthesis of angiogenic and inflammatory IL-8/IL-6/IL-1β/TNF-α cytokines and growth factor TGF-β and VEGF [13]. These inflammation-related molecules are related to the tumor metastasis and angiogenesis by assistance of inter-CAM-I (ICAM-1) and vascular CAM-1 (VCAM-1) [14]. The other proteins such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), VEGF, IL-6, IL-1β, TNF-α, and TGF-β are transcriptionally upregulated by transcriptional factors of AP-1 and NF-κB [13]. Thus, VCAM-1 and ICAM-1 expressions are upregulated through phosphorylation-based NF-κB activation directed by the upstream of PI3K and Akt signaling [15]. ICAMs as an Ig superfamily are structurally related and ligands for the integrin molecules on the leukocytes. Out of the five ICAMs, ICAM-1 is representative for leukocyte recruitment to inflamed sites. ICAM-1 ligands are ITGAL/LFA-1,

24

7 GM3 and Related Gangliosides Prevent Inflammation and Atherosclerosis

fibrinogen, and Muc1, where these ligands recognize the ICAM-1 extracellular domain, having the ITGAL/LFA1 binding site, towards mediation of cell–matrix interactions and cell–cell adhesions. ICAM-1 is expressed in the processes of immune and inflammatory responses, where proinflammatory cytokines TNF-α and IFN-γ induce ICAM-1 expression in endothelial and lymphocytic cells. For example, the inflammatory milieu is ubiquitously reserving for the TNF-α and IFN-γ in the tumor microenvironment. In tumor cells, ICAM-1 is constitutively expressed to keep their extravasation. This is because inflammation in tumor cells promotes tumorigenesis and invasiveness. This phenotype is meaningful in certain cancer types including melanoma, pancreatic, and breast cancer. ICAM-1 expression is modulated through ICAM-1 gene transcriptional regulation and in minor posttranscriptional mechanism. The ICAM-1 gene promoter is complexed with several cis-elements for transcription factors binding sites, including AP-1 and NF-κB via PKC and MAPKs such as ERK, JNK, and p38. In normal conditions without inflammation, ICAM-1 is ubiquitously generated, but very lowly, on lymphocytes and endothelial cells. The proinflammatory cytokines, hormones, cellular stress, and virus infections induce the ICAM-1 expression. Ganglioside GM1 and GD1a activate the JAK–STAT-mediated inflammatory responses in brain microganglia activation [16]. Also, obesity, which is one of the mild chronic inflammatory disorders which occurred in adipose tissues, induces the expression level of adipose gangliosides of GM1, GM2, and GD1a, which are associated with the potential pathophysiology of obesity-associated diseases [17]. GM3 blocks the expression of CAMs related to inflammation in VEGFinduced endothelial cells. GM3 inhibits transcriptional regulation of CAMs promoters through the translocation of NF-κB. In addition, GM3 inhibits the monocyte adhesiveness to VEGF-treated endothelial cells. Therefore, it was concluded that GM3 downregulates the inflammatory cytokine-elicited CAM level and enzymatic production of the known inflammatory metabolites in experimental mice. With respect to receptor regulation, some gangliosides regulate the signaling of various receptors; ganglioside GT1b inhibits adhesion and migration of keratinocytes bound with the α5β1 receptor [18, 19]. Ganglioside GM3, which is the simplest ganglioside as a monosialic form, inhibits the angiogenic modulation in endothelium induced by neoplastic cells and the VEGF- and/or GD1a-induced angiogenesis in vivo and in vitro [20]. Although VEGF acts in angiogenic activation on endothelial cells, it also is a proinflammatory cytokine in terms of its induction capability of leukocyte–endothelial adhesion during inflammatory responses. However, GM3 blocks the VEGFstimulated VCAM-1 and ICAM-1 gene expressions through protein kinase B (AKT)-NF-κB signaling. Thus, this event resembles with leukocyte recruitment process. Moreover, GM3 remarkably reduce the monocytes or dendritic cells (DCs) adhesion to human umbilical vascular endothelial cells (HUVECs), although this inhibitory effect has been shown by the adhesion bioassay in vitro. Also, in in vivo VEGF injection mice established for the inflammation, GM3 strongly

7 GM3 and Related Gangliosides Prevent Inflammation and Atherosclerosis

25

suppress the levels of VCAM-1 and ICAM-1 expressions in vascular veins. The blocking and inhibition of monocyte-endothelial adhesion and proinflammatory cytokines production are mediated by GM3, but not other gangliosides, in HUVECs and mice. For determination of the glycolipid specificity, various monosialic and disialic gangliosides of lactosylceramide, GM3, GM2, GD2, GM1, GD3, GD1a, and GD1b have been studied. At the transcriptional and protein level, GM3 only suppress the VEGF-induced VCAM-1 and ICAM-1 expressions in vitro and in vivo. Additionally, GM3 completely block the monocyte–HUVECs adhesion related with the inhibition of CAMs of HUVECs during inflammation. For the leukocyte adhesion, the monocyte adhesion assay has been applied using a myeloid– monocyte cell line, THP-1, which CAMs in extracellular matrix (ECM) such as ICAM-1, VCAM-1, and fibronectin are bound on cell surfaces [21]. The human-type macrophage cells, THP-1 monocyte adhesion level to VEGF-stimulated endothelial cells, are increased, while the GM3 inhibits the monocyte-activated endothelial adhesion (Fig. 7.1). Additionally, the transcriptional level of GM3 synthase in THP-1 monocyte was higher than other leukocytes as well as leukemic cells of HL-60 and K-562. However, the ganglioside GM3 level in THP-1 may not inhibit the VEGF-mediated activation in HUVECs, because the concentration of the exogenous ganglioside GM3 is higher than endogenous level in THP-1 cells. GM3 ganglioside inhibits the VCAM-1/ICAM-1 expressions related with inflammation and leukocyte recruitment using HUVECs in vitro. In in vivo experiments using a low-pressured tail vein injections to the model animal, BALB/c mice, various pro-inflammatory mediators such as CAMs, MMP, COX2, iNOS, IL-1β, TNF-α, and TGF-β have been apparently downregulated by GM3 injection. The mice injected with GM3 decreased the expression levels of the CAMs as well as COX-2 and iNOS induced by VEGF or VEGF and TNF-α cotreatment. The serum levels of IL-1β, TNF-α, and TGF-β enhanced on the VEGF or TNF-α-injected mice were significantly decreased by GM3 (Fig. 7.2). Therefore, GM3 exhibits an antiinflammatory activity through blocking of the production of inflammation-mediating metanolite molecules and cytokines in the inflammatory response [22]. In the arteriosclerotic lesion, GM3-producing macrophages are known to perform phagocytosis of the inflamed and injured cells to remove them from the inflammatory sites. In experimental observation [23], GM3 level was increased in atherosclerotic rupture area of aortic vessels in contrast to normal tissues. Using GM3-specific and GM3 synthase-specific antibodies, GM3 ganglioside and GM3 synthase proteins were confirmed to be highly generated in the monocyte- and macrophage-derived cells that infiltrated intima in atherosclerotic and inflammatory sites. On interests, monocyte-derived macrophages and DCs in human highly express GM3 product and GM3 synthase protein [24]. Therefore, it is mentioned that differentiation of monocytes to macrophage types is associated with the increased GM3 production in the arterial wall and this should be the mechanism in vivo, explainable for the increased GM3 levels in vascular atherosclerosis and cardiovascular diseases [23]. On the other hand, GM3 amounts are increased in atherosclerotic lesion and enriched in ateriosclerotic microenvironments. Currently, there is no mechanistic explanation on the question of how the increased GM3 content or GM3-associated

VEGF

Control

100 m

100 m

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Monocyte Adhesion, Fold

+ +

*

+

: CAMs

VEGF+GM3

: Endothelium,

Inflammation Inducer (VEGF)

+ -

#

Fig. 7.1 GM3 is an inhibitor of macrophage THP-1 and endothelial HUVEC interaction. (a) HUVECs treated with GM3 prior to VEGF treatment were co-cultured with the leukocyte THP-1 cells. THP-1 and HUVECs adhesion was checked by phase-contrast microscopy. (b) The leukocyte adhesion. (c) Scheme of THP-1 adhesion to the HUVEC

A)

26 7 GM3 and Related Gangliosides Prevent Inflammation and Atherosclerosis

GM3

VEGFR2

Treatment of VEGF and GM3

Inactivation of VEGFR2

VEGF

-

Nucleus

I

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p50

p50

p65

I

-

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p50 Expression of

Leukocyte

VCAM-1 or ICAM-1

VCAM-1 or ICAM-1

Inflammation

Promoter

p65

p65

AKT

PI-3K

of VEGF

Treatment

Fig. 7.2 Schematic illustration of the inflammation inhibition by GM3 in vitro and in vivo

Cytosol

Leukocyte

Inhibition of VEGF binding and leukocyte adhesion

Anti-inflammation

7 GM3 and Related Gangliosides Prevent Inflammation and Atherosclerosis 27

28

7 GM3 and Related Gangliosides Prevent Inflammation and Atherosclerosis

microenvironment influences atherosclerotic development or progression. GM3 is experimentally incorporated to low-density lipoprotein (LDL) particles, and consequently GM3-enriched LDLs are formed. The constituted GM3–LDL particles are structurally stable with resistance to aggregation. Therefore, GM3 increases the LDL size and stability and consequently decreases LDL oxidation (oxLDL) and recognition by target cells such as HUVECs. GM3 decreases the monocytic adhesiveness to vascular endothelial cells and lipid accumulation in macrophagic foam cells. GM3 decreases lipid contents of blood and atherosclerotic levels of experimental atherosclerotic animal of the high-fat diet-administered apoE-/- mice [25]. GM3 inhibits atherogenesis through GM3-enriched atherosclerotic microenvironment, indicating an anti-atherosclerotic GM3 possibility.

References 1. Park EJ, Suh M, Thomson B, Thomson AB, Ramanujam KS, Clandinin MT (2005) Dietary ganglioside decreases cholesterol content, caveolin expression and inflammatory mediators in rat intestinal microdomains. Glycobiology 15(10):935–942 2. Zhang Y, Iwabuchi K, Nunomura S, Hakomori S (2000) Effect of synthetic sialyl 2-->1 sphingosine and other glycosylsphingosines on the structure and function of the “glycosphingolipid signaling domain (GSD)” in mouse melanoma B16 cells. Biochemistry 39(10):2459–2468 3. Villanueva-Cabello TM, Mollicone R, Cruz-Muñoz ME, López-Guerrero DV, MartínezDuncker I (2015) Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters. Glycobiology 25 (12):1454–1464 4. Parodi PW (1997) Cows’ milk fat components as potential anticarcinogenic agents. J Nutr 127 (6):1055–1060 5. Garofalo T, Sorice M, Misasi R, Cinque B, Mattei V, Pontieri GM, Cifone MG, Pavan A (2002) Ganglioside GM3 activates ERKs in human lymphocytic cells. J Lipid Res 43(6):971–978 6. Miklavcic JJ, Schnabl KL, Mazurak VC, Thomson AB, Clandinin MT (2012) Dietary ganglioside reduces proinflammatory signaling in the intestine. J Nutr Metab 2012:280–286 7. Wasik BR, Voorhees IEH, Barnard KN, Alford-Lawrence BK, Weichert WS, Hood G, Nogales A, Martínez-Sobrido L, Holmes EC, Parrish CR (2019) Influenza viruses in mice: deep sequencing analysis of serial passage and effects of sialic acid structural variation. J Virol 93(23). pii: e01039-19. 8. Matsuzaki K, Kato K, Yanagisawa K (2010) Aβ polymerization through interaction with membrane gangliosides. Biochim Biophys Acta 1801:868–877 9. Yamashita T, Wu YP, Sandhoff R, Werth N, Mizukami H, Ellis JM, Dupree JL, Geyer R, Sandhoff K, Proia RL (2005) Interruption of gangliosides synthesis produces central nervousl system degeneration and altered axon-glial interactions. Proc Natl Acad Sci U S A 102:2725–2730 10. Mukherjee P, Faber AC, Shelton LM, Baek RC, Chiles TC, Seyfried TN (2008) Ganglioside GM3 suppresses the pro-angiogenic effects of vascular endothelial growth factor and ganglioside GD1A. J Lipid Res 49:929–938 11. Satoh M, Ito A, Nojiri H, Handa K, Numahata K, Ohyama C, Saito S, Hoshi S, Hakomori SI (2001) Enhanced GM3 expression, associated with decreased invasiveness, is induced by brefeldin A in bladder cancer cells. Int J Oncol 19(4):723–731

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12. Watanabe R, Ohyama C, Aoki H, Takahashi T, Satoh M, Saito S, Hoshi S, Ishii A, Saito M, Arai Y (2002) Ganglioside G(M3) overexpression induces apoptosis and reduces malignant potential in murine bladder cancer. Cancer Res 62(13):3850–3854 13. Angelo LS, Kurzrock R (2007) Vascular endothelial growth factor and its relationship to inflammatory mediators. Clin Cancer Res 13:2825–2830 14. Rosenberg HF (2006) Interview with Dr. Andrew Issekutz regarding pivotal advance: endothelial growth factors VEGF and bFGF differentially modulate monocyte and neutrophil recruitment to inflammation. J Leukoc Biol 80(2):245–246 15. Dieterich LC, Huang H, Massena S, Golenhofen N, Phillipson M, Dimberg A (2013) αBcrystallin/HspB5 regulates endothelial-leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin. Angiogenesis 16 (4):975–983 16. Kim OS, Park EJ, Joe EH, Jou I (2002) JAK-STAT signaling mediates gangliosides-induced inflammatory responses in brain microglial cells. J Biol Chem 277:40594–40601 17. Tanabe A, Matsuda M, Fukuhara A, Miyata Y, Komuro R, Shimomura I, Tojo H (2009) Obesity causes a shift in metabolic flow of gangliosides in adipose tissues. Biochem Biophys Res Commun 379:547–552 18. Sung CC, O’Toole EA, Lannutti BJ, Hunt J, O’Gorman M, Woodley DT, Paller AS (1998) Integrin alpha 5 beta 1 expression is required for inhibition of keratinocyte migration by ganglioside GT1b. Exp Cell Res 239(2):311–319 19. Wang XQ, Sun P, Paller AS (2005) Gangliosides inhibit urokinase-type plasminogen activator (uPA)-dependent squamous carcinoma cell migration by preventing uPA receptor/alphabeta integrin/epidermal growth factor receptor interactions. J Invest Dermatol 124(4):839–848 20. Mukherjee P, Faber AC, Shelton LM, Baek RC, Chiles TC, Seyfried TN (2008) Thematic review series: sphingolipids. Ganglioside GM3 suppresses the pro-angiogenic effects of vascular endothelial growth factor and ganglioside GD1A. J Lipid Res 49(5):929–938 21. Ronald J, Nixon AB, Marin D, Gupta RT, Janas G, Chen W, Suhocki PV, Pabon-Ramos W, Sopko DR, Starr MD, Brady JC, Hurwitz HI, Kim CY (2017) Pilot evaluation of angiogenesis signaling factor response after transcatheter arterial embolization for hepatocellular carcinoma. Radiology 285(1):311–318 22. Kim SJ, Chung TW, Choi HJ, Jin UH, Ha KT, Lee YC, Kim CH (2014) Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol 46:32–38 23. Gracheva EV, Samovilova NN, Golovanova NK, Kashirina SV, Shevelev A, Rybalkin I, Gurskaya T, Vlasik TN, Andreeva ER, Prokazova NV (2009) Enhancing of GM3 synthase expression during differentiation of human blood monocytes into macrophages as in vitro model of GM3 accumulation in atherosclerotic lesion. Mol Cell Biochem 330(1–2):121–129 24. Bobryshev YV, Golovanova NK, Tran D, Samovilova NN, Gracheva EV, Efremov EE, Sobolev AY, Yurchenko YV, Lord RS, Cao W, Lu J, Saito M, Prokazova NV (2006) Expression of GM3 synthase in human atherosclerotic lesions. Atherosclerosis 184:63–71 25. Ao M, Wang K, Zhou X, Chen G, Zhou Y, Wei B, Shao W, Huang J, Liao H, Wang Z, Sun Y, Zeng S, Chen Y (2019) Exogenous GM3 ganglioside inhibits atherosclerosis via multiple steps: a potential atheroprotective drug. Pharmacol Res 148:104445

Chapter 8

GM3 Has an Anti-tumor Capacity

Gangliosides as SA or NeuAc-containing GSLs are composed of the cellular PMs of vertebrates and abundantly expressed in the CNS [1]. Among them, GM3 has a carbohydrate structure of Neu5Acα2,3Galβ1,4Glcα1,1Cer and is a basic component in most cells. GM3 is enzymatically generated in the starting step of GSL biosynthetic pathway. Other GSLs are thus generated from the starting substrate GM3 by various glycosyltransferases [2]. Exogenous addition of mammal cells with gangliosides allows cellular ganglioside incorporation to the cellular PM. The gangliosides in PM play various roles responsible for biological activities. The simple ganglioside GM3 involves in basic and various physiological events including recognition, differentiation, and growth [3]. GM3 potentially suppress tumor cells division, growth, cell motility, cytoskeletal organization, and invasion. Recently, the potential function of GM3 was recognized to suppress proliferation of tumor cells. The simple ganglioside GM3 inhibits growth of several tumor cells. Previously, the exogenously treated GM3 inhibited growth of tumor cells. The precise mechanism underlying the cell cycle inhibition has been known. A decade ago, Kim group [4] reported that an oncoprotein MDM2 negatively control tumorigenesis via a p53-independent or p53-dependent manner. Moreover, PTEN, a tumor suppressor of tensin homolog deleted on chromosome ten, completely suppress MDM2 translocation and MDM2 protein destabilization in colon cancer cells HCT116. Although the PTEN is positionally located on chromosome 10q23.3 locus, in malignant tumors, the PTEN genes are frequently mutated at a very high rate. The malignant tumors in human include breast cancer, glioblastoma, lung cancer, melanoma, and prostate cancer for the PTEN mutations [5]. The disruption of PTEN gene also develops tumors to malignant types. In function, PTEN phosphatase catalyzes dephosphorylation reaction of the phosphatidylinositol 3,4,5-triphosphate (PIP-3), which is generated by the PI-3K kinase enzyme. The overexpression of PTEN suppresses tumor formation. PTEN mutation activates protein kinase B (PKB) and AKT downstream signaling of the PI-3K signaling cascade. Another tumor suppressor p53 raises cell cycle arrest at G1 phase via p21WAF1 gene. p53-induced p21WAF1 activity inhibits cell cycle with several forms of cyclin © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_8

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GM3 Has an Anti-tumor Capacity

and cyclin-dependent kinase (CDK) complex. p21WAF1 recognizes the proliferating cell nuclear antigen (PCNA) and prevents DNA replication [6]. PTEN-p53 axis regulates the MDM2 levels in order to maintain the MDM2 nuclear localization and consequently ubiquitinates the p53 via the PI-3K/AKT downstream signaling. In protein level, MDM2 downregulates p53 function through binding and proteasomal degradation [7]. PTEN-elicited G1 phase arrest increases in the cyclin level and CDK2 complex inhibitor p27kip1. PTEN inhibits PI-3K and AKT signaling via the CDK inhibitor p27kip1. During the exogenous GM3 treatment, the PTEN expression is increased, depending on CDK inhibitor p21 (Fig. 8.1). Thus, GM3 is the p53 protein stabilizer in cells [8]. GM3 is also an inducer of p53-dependent upregulator of p21. Using the plasmids having p21WAF1 promoter-designated luciferase reporters, the GM3 function was further confirmed. GM3 enforces CKI p27kip1 expression by the PTEN-blocked PI-3K and AKT transduction. GM3-treated colon cancer cells showed dysfunction of the cyclin E and CDK2. GM3-induced PTEN inhibits cell cycle proteins and cell cycle arrest. Ganglioside GM3 inhibits cancer cell growth by downregulation of cellular signalings. GM3 regulates the expression of tumor suppressor PTEN [9]. PTEN gene promoter has an AP-2 binding site responsible for the PTEN gene expression. The molecular mechanism(s) for PTEN gene regulation is explained by the involvement of GM3 [10]. GM3 activates AP-2α-bound PTEN transcription. The PTEN expression is enhanced by GM3 in cancer cells. However, the expression is not associated with p53 function. In the analysis of transcriptional regulation by 50 -flanking promoter region present in the PTEN gene upstream, the transcriptional regulatory sequence between nucleotides at 1175 and at 1077 bears the specific binding cis-element for AP-2α. This promoter site acts as the GM3-regulating region of tumor cells. The AP-2α presence is critical for the PTEN-associated signaling in the GM3-induced tumor cells. The silenced expression experiment, using AP-2α siRNA, eliminated the AP-2α stimulation effect and PTEN effect in the GM3-treated tumor cells. AP-2α-involved PTEN expression was also coupled with blocking of autocrine-acted EGFR signaling (Fig. 8.2). Thus, GM3 is a negative controller of tumor cell growth for colorectal cancer therapy. On the other hand, the lipid parts of the gangliosides are exogenously incorporated into lipid bilayer of PM of the cells [11, 12]. However, exogenously treated GM3 is localized on the surface area of PM in another experiment [9]. Thus, GM3 has been suggested to be involved in various cell surface events including cell–cell recognition, binding, and interaction phenomena, eventually, in membrane-clustered signaling events [13, 14]. Therefore, if the cells are exogenously treated with GM3, the GM3 is fast incorporated into PM of the cells for biological and physiological activities [9]. For example, the EGF- and EGFR-induced growth of epidermal cells is reported to be suppressed by GM3 recognition to terminal GlcNAc residues in N-linked glycoproteins of EGFR [15]. Seemingly, GM3 is also reported to suppress the PDGFR- or bFGFR-induced growth potentials of fibroblastic cells and neuroblastoma cells with the blocking mechanism of receptor dimerization [16]. The GM3 suppression activity of those receptors on cell membrane of colon cancer cells was originated by regulation of transcriptional factor AP-2a and PTEN expression. GM3-mediated VEGFR inhibition inhibits a specific AP-2a and PTEN gene in tumorigenic colon cancers when VEGF was added for the interaction with

8 GM3 Has an Anti-tumor Capacity

A)

33

Growth Factor, Mitogens

p85

PIP3

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ub

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Stimulators

B)

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ub

Cytosol degradation

p53 Mdm2

Nucleus p53 p53

p53

p53

Survival Proliferation

p53 Mdm2

Mdm2

{™ˆ•šŠ™—›–•ˆ“GhŠ›ˆ›–• p21WAF1 Gene

Fig. 8.1 (a) Relationship of PTEN, Mdm2, and p53 expression. PI-3K/AKT activation/MDM2 axis is described. PTEN restricts MDM2 location to the cytoplasm. (b) GM3 regulation of p21WAF1 gene depending on p53 stability and PTEN expression. GM3-expressed PTEN regulates p21WAF1 gene expression depending on p53 stability. MDM2 is the E3 ubiquitin ligase, which the MDM2 gene encodes the MDM2 protein in humans. Mdm2 negatively regulate the p53 function. Mdm2 binds to the p%3 trans-activation domain in their N-terminal region and inhibits p53 transcriptional repression. GM3 suppresses the cancer cell growth through cell cycle inhibition and arrest by downstream signaling regulation. In brief, GM3 confers a tumor suppressor phosphatase PTEN function to suppress cancer cell growth. Oncoprotein MDM2 regulate oncogenesis and PTEN. PTEN inhibits MDM2 nuclear translocation with the MDM2 destabilization. GM3 stimulates CDK inhibitor expression by p53, which occurs by the PTEN-inhibited proliferating pathway in malignant tumor. GM3 increases the PTEN expression, and the enhanced PTEN activity additionally upregulates the p21WAF1 gene expression depending on p53 stability through MDM2. Mdm2 negatively modulates the tumor suppressor p53. MDM2 binds to p53 and inhibits p53 transcriptional repression

34

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GM3 Has an Anti-tumor Capacity

GFR

AP-2α

NF-kB

Apoptosis

B) α

α

α

α

α

α

Fig. 8.2 GM3 inhibit PTEN-mediated cell cycle arrest via p21WAF1, p27kip1, and PI-3K/AKT pathway and blocking AP-2α-PTEN transcriptional stream. (a) GM3 inhibition of PTEN signaling. Ganglioside GM3 recognizes the EGFR to regulate PTEN. (b) GM3 regulation of AP2α-PTEN transcription. GM3-EGFR interaction leads to AP-2α-PTEN-p53 axis. GM3 contributes to a specific transcription factor function of AP-2α. The resulted AP-2α upregulates expression of the PTEN gene in malignant cells such as colon cancer. The GM3 similarly induce the gene expression of PTEN in the same cells of both p53-positive and p53-null colon cancer cells. Therefore, the PTEN expression is independently enhanced from p53 function. Therefore, the GM3-induced PTEN gene expression is upregulated by the PTEN gene promoter region. Then, it has been found that PTEN promoter region consists of the AP-2α-recognition site and this site induces the GM3-mediated transcription factorbinding site in such malignant colon cancer cells and AP-2α is a key PTEN factor. Therefore, the endogenous or exogenous GM3 on the human cancers induces AP-2α/PTEN axis via EGFR inhibition. GSLs are linked with intracellular transcription factors such as AP-2α to the stimulated tumor suppressor genes such as PTEN. GM3 is indeed a modulator of colon cancer cell growth and an anti-colon cancer therapeutic candidate. Modified from Choi HJ et al. (2006) Glycobiology 16(7), 573–83 and Choi et al. (2008) Glycobiology 18(5), 395–407 [9, 10]

VEGFR. The GM3 role is to downregulate AP-2a/PTEN function linked with VEGFR-2 signaling. Brattain group in Ohio has clearly evidenced that autocrine TGF-β as an EGF family member induces activation of EGFR and growth in the colon cancer cells [17]. Several years ago, Kim group [3, 5] demonstrated that GM3 blocks the EGFR promotion and growth of colon tumor HCT116 cells. GM3 inhibits the EGFR in HCT116 cells. The exogenous AG1478 treatment, an EGFR tyrosine kinase inhibitor, activates AP-2α and PTEN expression. GM3 upregulates the AP-2α level and modulates AP-2α level via the activated EGFR signaling, where the AP-2α is a key element responsible for PTEN activation upon GM3 treatment (Fig. 8.3). The acting molecular mechanism underlyisng the question how does the GM3 transcriptionally regulate the PTEN gene induction? has clearly been explained. Mechanistic action of GM3 in anti-cancer cells has been summarized in Fig. 8.4.

8 GM3 Has an Anti-tumor Capacity

35

EGF

or

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P P

AG1478

AP-2

TGF-

EGFR Inhibition of EGFR phosphorylation

•Cell cycle arrest •Suppression of cell proliferation

Up-regulation and activation

PTEN gene

AP-2

Fig. 8.3 GM3–EGFR interaction in human colon cancer. GM3 regulates AP-2 α expression for PTEN expression via EGFR pathway in metastatic colon cancer cells. EGF or TNF-α induces the EGFR phosphorylation. Ganglioside GM3 recognizes the EGFR and inhibits the downstream signaling. AG1478 denotes a specific inhibitor of EGFR Tyr kinase. GM3-EGFR interaction contributes to a transcription factor AP-2α upregulation and activation to stimulate PTEN gene expression. Modified from Choi HJ et el. (2008) Glycobiology 18(5), 395-407 and Choi, H.J., Chung, T.W., et al. 2006. Glycobiology, 16, 573-583 [9, 10] GM3

Ganglioside GM3 PIP2

Plasma Membrane

PIP3

PTEN expression

PI-3K AKT inactivation MDM2 inactivation p53 stability

CDK2, cyclin E expression

p27kip1 expression

G1 cell cycle arrest

p21WAF1 expression

Fig. 8.4 Anti-cancer action of surfaced GM3 on colon cancer cells. PTEN-PI-3K-cell cycle arrest axis is illustrated. GM3 activates p53-dependent p21 and CKI p27 via the PTEN/PI-3K/AKT axis signals. Modified from the article of Choi HJ et al. 2006. Glycobiology 16(7), 573–83 [10]

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GM3 Has an Anti-tumor Capacity

References 1. Svennerholm L (1980) Ganglioside and synaptic transmission. Plenum, New York 2. Sandhoff K, van Echten G (1994) Ganglioside metabolism: enzymology, topology and regulation. Prog Brain Res 101:17–29 3. Chung TW, Choi HJ, Lee YC, Kim CH (2005) Molecular mechanism for transcriptional activation of ganglioside GM3 synthase and its function in differentiation of HL-60 cells. Glycobiology 15:233–244 4. Burdon D, Patel R, Challiss RA, Blank JL (2002) Growth inhibition by the muscarinic M (3) acetylcholine receptor: evidence for p21(Cip1/Waf1) involvement in G(1) arrest. Biochem J 367(Pt 2):549–559 5. El-Deiry WS (2016) p21(WAF1) mediates cell-cycle inhibition, relevant to cancer suppression and therapy. Cancer Res 76(18):5189–5191 6. Breton Y, Desrosiers V, Ouellet M, Deshiere A, Torresilla C, Cohen ÉA, Tremblay MJ (2019) Expression of MDM2 in macrophages promotes the early postentry steps of HIV-1 infection through inhibition of p53. J Virol 93(7), pii: e01871-18 7. Lu Y, Lin YZ, LaPushin R, Cuevas B, Fang X, Yu SX, Davies MA, Khan H, Furui T, Mao M, Zinner R, Hung MC, Steck P, Siminovitch K, Mills GB (1999) The PTEN/MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 18(50):7034–7045 8. Chung TW, Lee YC, Ko JH, Kim CH (2003) Hepatitis B Virus X protein modulates the expression of PTEN by inhibiting the function of p53, a transcriptional activator in liver cells. Cancer Res 63(13):3453–3458 9. Choi HJ, Chung TW et al (2006) Ganglioside GM3 modulates tumor suppressor PTENmediated cell cycle progression--transcriptional induction of p21(WAF1) and p27(kip1) by inhibition of PI-3K/AKT pathway. Glycobiology 16:573–583 10. Choi HJ, Chung TW, Kim SJ, Cho SY, Lee YS, Lee YC, Ko JH, Kim CH (2008) The AP-2alpha transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene. Glycobiology 18(5):395–407 11. Callies R, Schwarzmann G et al (1977) Characterization of the cellular binding of exogenous gangliosides. Eur J Biochem 80:425–432 12. Radsak K, Schwarzmann G et al (1982) Studies on the cell association of exogenously added sialo-glycolipids. Hoppe Seylers Z Physiol Chem 363:263–272 13. Fishman PH, Brady RO (1976) Biosynthesis and function of gangliosides. Science 194:906–915 14. Hakomori S (1981) Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. Annu Rev Biochem 50:733–764 15. Yoon SJ, Nakayama K, Takahashi N, Yagi H, Utkina N, Wang HY, Kato K, Sadilek M, Hakomori SI (2006) Interaction of N-linked glycans, having multivalent GlcNAc termini, with GM3 ganglioside. Glycoconj J 23(9):639–649 16. Rusnati M, Urbinati C, Tanghetti E, Dell’Era P, Lortat-Jacob H, Presta M (2002) Cell membrane GM1 ganglioside is a functional coreceptor for fibroblast growth factor 2. Proc Natl Acad Sci U S A 99(7):4367–4372 17. Carroll JS, Lynch DK, Swarbrick A, Renoir JM, Sarcevic B, Daly RJ, Musgrove EA, Sutherland RL (2003) p27(Kip1) induces quiescence and growth factor insensitivity in tamoxifentreated breast cancer cells. Cancer Res 63(15):4322–4326

Chapter 9

GM3 Suppresses Tumor Angiogenesis

Gangliosides, sialylated GSLs, are ubiquitously expressed components of mammal cells and affect cellular events including adhesion, differentiation, division, cell cycle, growth, signal transduction, invasion, metastasis, migration, angiogenesis, and tumorigenesis [1, 2]. Gangliosides with SA carbohydrate chains are implicated in basic cellular events [3]. For example, neuronal tissues are enriched with gangliosides, especially in neurons to modulate CNS function. Since gangliosides are present on the cellular PM and associate with membrane architecture, threedimensional organization, and structure, gangliosides modulate the GFRs of receptors for EGF, PDGF, FGF, and insulin [3, 4]. Among them, GM3 is particularly involved in cancer suppression in a way that inhibits intrinsic Tyr kinase activity coupled in cellular membrane receptors such as EGFR, VEGFR, and PDGFR. Ganglioside-targeting sialidases enhance the EGFR activity responsible for cell survival, migration, and spreading in some tumor cells including squamous and ovarian epidermal cells. This indicates the EGFR function is inhibited by GM3 [5– 7]. GM3 synthase transfection reduces invasiveness and tumorigenic potentials of bladder cancer cells. The enhanced GM3 synthesis increases cell death and apoptosis [8]. Cancer cells are characteristic for unregulated cell growth invading normal tissue. The tumor development and diverse tumor types are through different signaling pathways. Tumor cells recognize the host cells by releasing soluble factors to the tumor microenvironment (TME) [9]. Tumor cell ganglioside is one of the soluble factors for the formation of TME towards normal epithelial cells. Tumor cell gangliosides stimulate tumor development by shedding from the tumor cell membrane. Epidermal growth factors and vascular endothelial cells are controlled by the ganglioside, enhancing tumor angiogenesis in vitro. GM3 synthase and GM2 synthase gene double knockout mice [10] expressed lower tumor growth compared with the normal mice. Tumor growth was suppressed by ganglioside-specific antibody and ganglioside synthase inhibitors, suggesting that blocking the ganglioside synthase is a cancer therapeutic strategy. GM3 inhibit tumor growth while others enhance the tumor development. © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_9

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The tumors are communicated with tumor cells themselves, surrounded stromal cells, and the tumor-supporting stroma cells resident in the tumor-associated microenvironment (TAM) for their tumor progression. It involves cellular activation of endothelial cells for neovascularization, initiated VEGF secreted from cancer and normal cells by network of growth factors, and cytokines from fibroblasts, neutrophils, smooth muscle cells (SMCs), pericytes, endothelial cells, NK cells, lymphocytes, adipocytes, macrophages, and mast cells [11]. Gangliosides are normally present in shed forms in the surrounded microenvironment by transformed tumor cells and modulate the new vessel formation of angiogenic microvessels [12]. For example, GD3 expression is enhanced in many tumor cells and promotes VEGFmediated angiogenesis by certain tumor cells [13]. Moreover, gangliosides of GM1, GD3, and GT1b enhance newly formation of blood vessels, as confirmed in a model animal to target rabbit corneas [12, 14]. In addition, the angiogenic signalings including VEGFR-2 dimerization and phosphorylation were activated by GD1a treatment, and this process accelerates migration and growth of endothelial cells [15]. In contrast, depletion of GM3 products in GalNAc-transferase gene-engineered tumor cells of the mouse brain and the genetically reduced GM3 and GD3 ratio in the corneal cells accelerated the angiogenesis potentials [14, 16]. However, exogenous treatment of GM3 blocks the growth, migration, and motility of vascular endothelial cells [14]. Therefore, the correct molecular mechanism responsible for negative and positive tumor angiogenesis regulated by different gangliosides will be interested, although it has been partially understood.

References 1. Hakomori S (2002) Glycosylation defining cancer malignancy: new wine in an old bottle. Proc Natl Acad Sci USA 99:10231–10233 2. Birkle S, Zeng G, Gao L, Yu RK, Aubry J (2003) Role of tumor-associated gangliosides in cancer progression. Biochimie 85:455–463 3. Miljan EA, Meuillet EJ, Mania-Farnell B, George D, Yamamoto H, Simon HG, Bremer EG (2002) Interaction of the extracellular domain of the epidermal growth factor receptor with gangliosides. J Biol Chem 277:10108–10113 4. Mollinedo F, Gajate C (2015) Lipid rafts as major platforms for signaling regulation in cancer. Adv Biol Regul 57:130–146 5. Meuillet EJ, Mania-Farnell B, George D, Inokuchi JI, Bremer EG (2000) Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431. Exp Cell Res 256(1):74–82 6. Tang X, Powelka AM, Soriano NA, Czech MP, Guilherme A (2005) PTEN, but not SHIP2, suppresses insulin signaling through the phosphatidylinositol 3-kinase/Akt pathway in 3T3-L1 adipocytes. J Biol Chem 280(23):22523–22529 7. Wang XQ, Sun P, Paller AS (2003) Ganglioside GM3 inhibits matrix metalloproteinase-9 activation and disrupts its association with integrin. J Biol Chem 278(28):25591–25599

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8. Watanabe R, Ohyama C, Aoki H, Takahashi T, Satoh M, Saito S, Hoshi S, Ishii A, Saito M, Arai Y (2002) Ganglioside G(M3) overexpression induces apoptosis and reduces malignant potential in murine bladder cancer. Cancer Res 62:3850–3854 9. Kim Y, Stolarska MA, Othmer HG (2011) The role of the microenvironment in tumor growth and invasion. Prog Biophys Mol Biol 106:353–379 10. Liu Y, Wondimu A, Yan S, Bobb D, Ladisch S (2013) Tumor gangliosides accelerate murine tumor angiogenesis. Angiogenesis 17:563–571 11. Giordano FJ, Johnson RS (2001) Angiogenesis: the role of the microenvironment in flipping the switch. Curr Opin Genet Dev 11:35–40 12. Liu Y, Wondimu A, Yan S, Bobb D, Ladisch S (2014) Tumor gangliosides accelerate murine tumor angiogenesis. Angiogenesis 17(3):563–571 13. Zeng G, Gao L, Birkle S, Yu RK (2000) Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production. Cancer Res 60:6670–6676 14. Ziche M, Morbidelli L, Alessandri G, Gullino PM (1992) Angiogenesis can be stimulated or repressed in vivo by a change in GM3:GD3 ganglioside ratio. Lab Investig 67:711–715 15. Margheri F, Papucci L, Schiavone N, D’Agostino R, Trigari S, Serratì S, Laurenzana A, Biagioni A, Luciani C, Chillà A, Andreucci E, Del Rosso T, Margheri G, Del Rosso M, Fibbi G (2015) Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis. J Cell Mol Med 19(1):113–123 16. Seyfried TN, Mukherjee P (2010) Ganglioside GM3 is antiangiogenic in malignant brain cancer. J Oncol 2010:961243

Chapter 10

Interaction Between EGFR and GM3

GSLs are mainly located in the PMs with their carbohydrate parts protruding into the extracellular region. The gangliosides are distributed for recognition as roles of both ligands and receptors. Ganglioside recognition and binding results obtained to date suggested that certain GSLs, regardless ganglio series or non-ganglio series glycolipids, function as membrane receptors for specific invasive viral agents, bacterial toxins, and infectious microorganisms. Specific carbohydrates attached to both glycolipids and glycoproteins bind their target groups. Certain gangliosides interact with cellular integrins, indicating that ganglioside carbohydrates act as the integrin ligands. Upon recognition to their target molecules, gangliosides control the regulation factor-activated dimerization and Tyr residue phosphorylation, towards subsequent modulation of signaling events of receptor tyrosine kinases (RTKs). The well-known modulation of such receptors by GM3 includes the GM3-suppressed EGFR and bFGFR downstream signalings. The EGFR/ErbB1 is the mostly studied members of the RTKs family in human. The human EGFR as a general example of RTKs is ubiquitously involved in receptor-downstream signaling, growth, differentiation, migration, and motility. The EGFR resides in the cellular PM, in order to serve as a key signal receiver and thus to transduce the extracellular information and environment to intracellular information-treating center through adaptor molecule networks for the subsequent elicitation of a response [1]. Except for proteineous ligands, certain carbohydrates of lipid environment modulate the EGFR action. In the structural information, EGFR contains the four different domains of an extracellular ligand-binding site, which is resided as ectodomain of the receptor, a basic juxtamembrane site, a transmembrane helix site, and an intracellular protein kinase site [2]. The functional activity of EGFR is modulated by both regulators of GM3 and phosphatidylinositol-4,5-bisphosphate. Binding event of GM3 with EGFR is performed within the extracellular leaflet. The Neu5Ac-Gal part of GM3 is crucial for receptor protein interaction, as confirmed by GM3 lacking for the Neu5Ac residue or lactosylceramide (LacCer). GM3 interacts with the EGFR dimeric forms with a first minimum free Δ energy of 9 kJ/mol, which is implying for the state I. A © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_10

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second local minimum free Δ energy of 7 kJ/mol, which indicates the state-II, was also calculated [3]. N-terminally located helix dimers of EGFR binds to the carbohydrate part of GM3 headgroup. Lack of the Neu5Ac residue from the GM3 carbohydrate shows the reduced binding affinity to EGFR. Both the K618G mutant and Neu5Ac-lacking GM3 diminish recognition of the Lys side chain on EGFR to the carboxylate group on GM3. The decreased binding affinity is caused by the cationic Lys prosthetic group at position 618 and the anionic carbohydrate part of the GM3 headgroup during the GM3 and EGFR binding. In in vitro experimental assays, the neuraminidase-treated GM3, which cleaves off Neu5Ac residue from the substrate GM3 carbohydrate, eliminates receptor function, and thus K618 amino acid residue is a key interacting site for GM3 binding [4]. In view of carbohydrate– protein interaction, this result clearly implies that the polar part of the GM3 headgroup carbohydrates works as the recognition sites that enable EGFR to specifically bind to GM3. GM3 interaction with receptors is weak and transient [5], as the GM3 direct inhibition of EGFR function is observed only in lipid-disordered and lipid-ordered membranes, containing sphingomyelin and cholesterol, which is characteristically resembled to lipid rafts [0–2], but not in the condition of lipiddisordered phase bilayers. Stable EGFR conformation allows formation of the extracellular domain adoption, and typical glycosylation allows relevant glycation in glycan numbers. These protein and glycolipid organization form a better position in space close to the helices N-terminal region for the GM3-receptor recognition [6]. Thus, the glycans influence lateral lipid recognition to associate with the domain. GM3 inhibits the EGFR autophosphorylation in the phosphorylase domain. Glycolipids bind to integral membrane proteins and consequently control membrane protein function. Glycolipids and phospholipids embedded in membrane sites can interact with the EGFR membrane domain. The in vitro molecular interaction of ganglioside GM3 and EGFR has been explained in detail in the liposome system reconstituted with the purified receptors [1]. But the EGFR kinase inhibition by GM3 is abolished by removing the NeuAc of the GM3 headgroup. The sialylated Gal residue of GM3 interacts with terminal GlcNAc residues on EGFR N-glycan. For a molecular action mechanism to explain the GM3-driven functional suppression of the receptors, it is known that monosialyl GM3 modulates signaling of cellular receptors. In contrast, di- or polysialyl gangliosides rather activate the tyrosine kinase activities. These indicate that the neuraminic acid is a key regulator to bind the receptors. Gangliosides block the molecular dimerization of receptors and consequently lead to the inhibited receptor autophosphorylation activated by targeting ligands, even in the condition of the absence of ligands. RTKs are regulated by the glycan structure of gangliosides. Monosialyl gangliosides like GM3 or GM1 negatively regulate RTKs signaling. Direct binding of carbohydrates of gangliosides to RTKs in GM3 inhibition of EGFR via carbohydrate–carbohydrate interaction is another specific type of bindings. Changes in ganglioside distribution and composition frequently occurred in cancers. However, ganglioside-modified RTKs signaling change is a model for cancer therapeutic targets [7]. Apart from the ganglioside carbohydrates, phosphoinositide PIP2 and GM3 lipid part are also reported to partially regulate EGFR signaling through EGFR binding.

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Interaction Between EGFR and GM3

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Gangliosides regulate the function of GFRs, as GM3 inhibit human EGF receptor activity, regulating the composition, distribution, and functionality of carbohydrates in the surrounded membrane by EGFR. Mutation or deletion of key amino acids responsible for EGFR activation and signaling reduces its recognition capacity of the lipids and GSLs. The candidate amino acid residues to molecularly recognize the GSLs are demonstrated both in experiment and computer simulation [3]. During binding of gangliosides to cellular membrane receptors, GM3 directly recognizes extracellular domain of EGFR, as a work of Miljan et al. [8]. Interestingly, Hakomori group [9] showed a different mode of inhibition that EGFR recognizes GM3 by an unusual pattern of recognition between carbohydrate and carbohydrate resided both on receptor and ligand. GM3 has many intercellular interactions, and EGFR autophosphorylation is blocked by the exogenously treated GM3. Consequently, GM3 controls the cell growth of tumor cells [10]. Except for EGFR, GM3 has been reported to associate with other membrane proteins including EGF-R, α3β1-integrin, and tetraspanins [11, 12]. During GM3-driven suppression of proliferation and survival, which is conducted with the human bladder cancer cells [13], GM3 decreases cell–cell adhesion and EGFR phosphorylation during EGF treatment. The prevention of cellular adhesion and inhibition of cell division, which are observed by GM3 treatment, are molecularly explained by the inhibited EGFR and VEGFR activities. Laminin-induced cell spreading out was also disappeared in GM3-treated cells, suggesting that GM3 blocks integrin-mediated cell adhesion. GM3-CD9 binding negatively controls integrin-mediated cellular function and integrin-dependent downstream signaling [14]. However, GM3 induces CD9 expression and inhibits the tumor cell motility through downregulation of CD9 gene and protein expression. As a metastasis inhibitor, CD82-KAI-1 is a group of tetraspanin superfamilies and interacts with GSLs for suppression of cell migrative potentials induced by EGFR and HGFR/cMet. CD82 gene-transfected Hepa1-6 cells were reported to decrease migration potentials induced by EGF and HGF [15], through inhibition of EGF-activated EGFR phosphorylation at the position of amino acid Tyr1173. In the CD82-trasnfected Hepa1-6 cells, HGF-activated Tyr phosphorylation of cMet at the positions of amino acid Tyr1313 and Tyr1365 was also inhibited. The double condition of CD82-GM3 or CD82-GM2/GM3 showed the enhanced inhibitory effects. GM3 also enforce the GM2 binding to tetraspanin CD82, hence, blocking the motility of cells, since the molecular complex of GM2 and CD82 recognizes the Met receptor to bind [16]. Thus, the functional recognition of α3 or β1 integrin protein to Met receptor is specifically inhibited, and Tyr phosphorylation of the HGF-mediated Met receptor is remarkably inhibited. Cell adhesion events mediated by integrins cooperate with EGFR to regulate cell growth [14]. GM3 regulates such integrins and growth factor receptors like EGFR. GM3 binds to EGFR to suppress Tyr kinase activity of the EGF-activated receptor, which is strongly involved in tumor malignancy [8]. GM3 inhibits EGFR signaling [12] via binding to the terminal GlcNAc residues linked to N-glycans of the EGFR, exerting the role in blocking of EGF-activated EGFR Tyr kinase in epidermoid tumor A431 cells [10]. From the

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descriptions, the conclusion that GM3 is an anti-tumor growth effector is generalized. EGF receptor’s autophosphorylation was inhibited by GM3 and GM3 directly bound to EGF receptor [8]. In proteoliposome-embedded GM3, GM3 regulates human EGFR activity in the coding whole gene of human EGFR. Using a mixture of sphingomyelin (18:0), ternary lipid mixture (1,2-dioleoyl-sn-glycero-3phosphocholin), and cholesterol, two different proteoliposome phases (liquiddisordered phase, liquid-disordered/liquid-ordered separate phase) were established, and lipid-based GM3 has been examined to see effect on EGF receptor function [1]. The preventive activity of GM3 treatment is completely diminished by either removement of the NeuAc residue present in the GM3 headgroup or defected function-derived K642G mutation of Lys residue, which is located at a membrane proximal region of EGFR [1]. How gangliosides affect the EGFR function? To answer this question, the following results are explained. Changes in cellular and membrane GM3 distribution reverse activity of the Tyr kinase activity, which is linked to the EGFR in many different cells [17–19]. The GM3-mediated inhibition effect is obtained from the direct interaction of GM3 with the EGFR ectodomain [20]. Artificial depletion of cholesterol content from cell membranes activates the EGFR [21, 22]. Membrane caveolin, a cholesterol-binding protein, also regulates the recognition, binding, and interactions. Gangliosides modulate EGFR activity directly, as confirmed by reconstitution of human EGFR into proteoliposomes binds GM3 and receptor autophosphorylation. For understanding the GM3 roles on EGFR signaling and downstream activity, whole human EGFR has been reconstituted in forms of proteoliposome. The above lipids (1,2-dioleoyl-snglycero-3-phosphocholin), sphingomyelin (SM) (18:0) and cholesterol, two different proteoliposome phases of liquid-disordered phase and liquid-disordered/liquidordered separate phase, exhibit the coincided effect of EGFR activity obtained from the direct GM3 interaction [1]. GM3 inhibits EGFR autophosphorylation [23] by GM3 which directly binds to EGFR in the established design of proteoliposome [1]. Structurally, the sialic acid residue is a key molecule in the direct binding capacity of GM3, as confirmed by the neuraminidase-treated GM3. The precursor form, lactosylceramide does not bind to the EGFR, and neuraminic acid is a specific binding molecule to inhibit the EGFR activity. Therefore, EGFR Tyr kinase activity is regulated by GM3. More reversely, ganglioside regulates function of growth factor receptor. If GM3 was treated with neuraminidase, the inhibitory activity is diminished, suggesting that GM3’s precursor form, lactosylceramide does not exhibit the receptor activity, and neuraminic acid specifically inhibits the EGF receptor tyrosine kinase activity. The key point is that GM3–EGF receptor interaction or EGF binding is not affected by lipid environment and receptor–membrane glycolipid interaction regulates EGF receptor [1]. In addition, a complex between GM3 and tetraspanin CD82 interferes with HGFR function, motility, and proliferation of the cells [24]. The mechanism responsible for regulatory function of the GM2/GM3/CD82 complex axis in tumor cell inhibition for growth has not been precisely explained due to its limited information explored in research. However, its action mechanism resembles with the

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PKCα-involved inhibitory function of EGFR-activated growth phenotype, where GM3 and CD82 cooperatively potentiate the PKCα activation such as translocation and phosphorylation, where the cooperation phosphorylates the Thr 654 amino acid of EGFR, triggering EGFR internalization [25]. HGFR activation is suppressed by GM3 expressed on cell surfaces, in terms of GM3 that blocks CD82-involved recruitment of intracellular signaling adaptors to activate HGFR and integrin functions. The inhibition event is mediated with defected functions of PI3K/Akt and GRB2 downstream signaling. Moreover, through association between CD9 and gangliosides, the CD9-ganglioside complex regulates its function. A non-invasive but proliferating bladder cancer highly expresses the GM3/CD9 complex and exhibits additional association of GM3/CD9 complex with α3β1 integrin; consequently their phenotypes show relatively lowered cell motility. This reversed phenotype changes are generally characteristic for a highly invasive cells of bladder cancers. This is because the enhanced GM3 expression level yields a GM3/CD9/α3 integrin chain complex and thereafter CsK translocates to TEMs [26]. The event leads to the SRC suppression, Rac functional impairment, and PI3K/Akt activation [11]. Interestingly to a similar case, the chicken or mouse fibroblast cells, which v-jun gene is transformed, interfere with the gene expression of GM3 synthase, consequently reducing the complex formation of the GM3/CD9/integrin, enhanced motility, and colony formation. The enforced GM3 synthesis on the cells reorganizes the GM3/CD9/integrin complex form. However, the transformed cells lose tumorigenic potentials and motility as well as increase adhesiveness [25]. There are many different additives of GM3 because the GM3 has a core sugar structure of Sia-GalGlu-ceramide. Among them, GM1, glycolylNeuGM3, GD1a, GD3, and GT1b are well reported to inhibit the EGFR signaling [1, 27–31]. Interestingly, globo series Gb4 globoside interacts with EGFR [32].

References 1. Coskun U, Grzybek M, Drechsel D, Simons K (2011) Regulation of human EGF receptor by lipids. Proc Natl Acad Sci USA 108(22):9044–9048 2. Mitchell RA, Luwor RB, Burgess AW (2018) Epidermal growth factor receptor: structurefunction informing the design of anticancer therapeutics. Exp Cell Res 371(1):1–19 3. Hedger G, Shorthouse D, Koldsø H, Sansom MS (2016) Free energy landscape of lipid interactions with regulatory binding sites on the transmembrane domain of the EGF receptor. J Phys Chem B 120(33):8154–8163 4. Guéguinou M, Gambade A, Félix R, Chantôme A, Fourbon Y, Bougnoux P, Weber G, PotierCartereau M, Vandier C (2015) Lipid rafts, KCa/ClCa/Ca2+ channel complexes and EGFR signaling: novel targets to reduce tumor development by lipids? Biochim Biophys Acta 1848 (10 Pt B):2603–2620 5. Raghunathan K, Kenworthy AK (2018) Dynamic pattern generation in cell membranes: current insights into membrane organization. Biochim Biophys Acta Biomembr 1860(10):2018–2031

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6. Kaszuba K, Grzybek M, Orłowski A, Danne R, Róg T, Simons K, Coskun Ü, Vattulainen I (2015) N-glycosylation as determinant of epidermal growth factor receptor conformation in membranes. Proc Natl Acad Sci USA 112:4334–4339 7. Julien S, Bobowski M, Steenackers A, Le Bourhis X, Delannoy P (2013) How do gangliosides regulate RTKs signaling? Cell 2(4):751–767 8. Takebayashi M, Hayashi T, Su TP (2004) Sigma-1 receptors potentiate epidermal growth factor signaling towards neuritogenesis in PC12 cells: potential relation to lipid raft reconstitution. Synapse 53(2):90–103 9. Guan F, Handa K, Hakomori SI (2011) Regulation of epidermal growth factor receptor through interaction of ganglioside GM3 with GlcNAc of N-linked glycan of the receptor: demonstration in ldlD cells. Neurochem Res 36(9):1645–1653 10. Mirkin BL, Clark SH, Zhang C (2002) Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B. Cell Prolif 35 (2):105–115 11. Mitsuzuka K, Handa K, Satoh M, Arai Y, Hakomori S (2005) A specific microdomain (“glycosynapse 3”) controls phenotypic conversion and reversion of bladder cancer cells through GM3-mediated interaction of alpha3beta1 integrin with CD9. J Biol Chem 280:35545–35553 12. Park HJ, Chae SK, Kim JW, Yang SG, Jung JM, Kim MJ, Wee G, Lee DS, Kim SU, Koo DB (2017) Ganglioside GM3 induces cumulus cell apoptosis through inhibition of epidermal growth factor receptor-mediated PI3K/AKT signaling pathways during in vitro maturation of pig oocytes. Mol Reprod Dev 84(8):702–711 13. Wang H, Isaji T, Satoh M, Li D, Arai Y, Gu J (2013) Antitumor effects of exogenous ganglioside GM3 on bladder cancer in an orthotopic cancer model. Urology 81(1):210. e11–210.e15 14. Park SY, Yoon SJ, Freire-de-Lima L, Kim JH, Hakomori SI (2009) Control of cell motility by interaction of gangliosides, tetraspanins, and epidermal growth factor receptor in A431 versus KB epidermoid tumor cells. Carbohydr Res 344(12):1479–1486 15. Li Y, Huang X, Zhang J, Li Y, Ma K (2013) Synergistic inhibition of cell migration by tetraspanin CD82 and gangliosides occurs via the EGFR or cMet-activated Pl3K/Akt signalling pathway. Int J Biochem Cell Biol 45(11):2349–2358 16. Todeschini AR, Dos Santos JN, Handa K, Hakomori SI (2008) Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMetmediated pathway. Proc Natl Acad Sci USA 105(6):1925–1930 17. Kawashima N, Yoon SJ, Itoh K, Nakayama K (2009) Tyrosine kinase activity of epidermal growth factor receptor is regulated by GM3 binding through carbohydrate to carbohydrate interactions. J Biol Chem 284(10):6147–6155 18. Song WX, Vacca MF, Welti R, Rintoul DA (1991) Effects of gangliosides GM3 and De-Nacetyl GM3 on epidermal growth factor receptor kinase activity and cell growth. J Biol Chem 266(16):10174–10181 19. Wada T, Hata K, Yamaguchi K et al (2007) A crucial role of plasma membrane-associated sialidase in the survival of human cancer cells. Oncogene 26(17):2483–2490 20. Kawashima N, Nishimiya Y, Takahata S, Nakayama KI (2016) Induction of glycosphingolipid GM3 expression by valproic acid suppresses cancer cell growth. J Biol Chem 291 (41):21424–21433 21. Westover EJ, Covey DF, Brockman HL, Brown RE, Pike LJ (2003) Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers. J Biol Chem 278 (51):51125–51133 22. Saffarian S, Li Y, Elson EL, Pike LJ (2007) Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis. Biophys J 93:1021–1031 23. Kannagi R, Cai BH, Huang HC, Chao CC, Sakuma K (2018) Gangliosides and tumors. Methods Mol Biol 1804:143–171

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24. Kapitonov D, Bieberich E, Yu RK (1999) Combinatorial PCR approach to homology-based cloning: cloning and expression of mouse and human GM3-synthase. Glycoconj J 16 (7):337–350 25. Wang XQ, Yan Q, Sun P, Liu JW, Go L, McDaniel SM, Paller AS (2007) Suppression of epidermal growth factor receptor signaling by protein kinase C-alpha activation requires CD82, caveolin-1, and ganglioside. Cancer Res 67:9986–9995 26. Zhang X, Shi G, Gao F, Liu P, Wang H, Tan X (2019) TSPAN1 upregulates MMP2 to promote pancreatic cancer cell migration and invasion via PLCγ. Oncol Rep 41(4):2117–2125 27. Bergante S, Torretta E, Creo P et al (2014) Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells. J Lipid Res 55 (3):549–560 28. Palomo AG, Santana RB, Pérez XE, Santana DB, Gabri MR, Monzon KL, Pérez AC (2016) Frequent co-expression of EGFR and NeuGcGM3 ganglioside in cancer: it’s potential therapeutic implications. Clin Exp Metastasis 33(7):717–725 29. Palomo AG, Medinilla AL, Segatori V et al (2018) Synergistic potentiation of the antimetastatic effect of anti EGFR mAb by its combination with immunotherapies targeting the ganglioside NGcGM3. Oncotarget 9(35):24069–24080 30. Wang J, Yu RK (2013) Interaction of ganglioside GD3 with an EGF receptor sustains the selfrenewal ability of mouse neural stem cells in vitro. Proc Natl Acad Sci USA 110 (47):19137–19142 31. Yankelevich M, Kondadasula SV, Thakur A, Buck S, Cheung NK, Lum LG (2012) AntiCD3  anti-GD2 bispecific antibody redirects T-cell cytolytic activity to neuroblastoma targets. Pediatr Blood Cancer 59(7):1198–1205 32. Park SY, Kwak CY, Shayman JA, Kim JH (2012) Globoside promotes activation of ERK by interaction with the epidermal growth factor receptor. Biochim Biophys Acta 1820 (7):1141–1148

Chapter 11

Membrane Ganglioside-Specific Neuraminidase 3 (NEU3) Regulates GM3 Signaling

Sialidases or neuraminidase (N-acylneuraminosyl glycohydrolase, EC 3.2.1.18) belongs to a group of exotype glycosidase family. The enzymes remove the terminal α-linkage of SA residues at the non-reducing end, which are ketositically attached to mono-SA, di-SA, or oligosaccharide SA chains of glycoproteins and glycolipids. The mammalian enzymes are important in lysosomal catabolism, modulation of developmental modeling of myelin, T cell activation, cell differentiation, insulin resistance, and cancer behaviors [1]. Mammalian sialidases were initially divided through their localization properties. Their subcellular localization on cells determines the terminology of three enzyme types. They include lysosomal type, cytosolic type, and plasma membrane-type sialidases, later being abbreviated to the corresponding neuraminidase (NEU)1, NEU2, and NEU3, respectively [2]. The mammalian neuraminidases are characteristic for the different properties, which each type has a unique function due to their subcellular location and substrate specificities for enzymatic catalysis. A decade ago, only three mammalian enzyme types of the lysosomal neuraminidase (known as NEU1), cytosolic form (known as NEU2), and membrane-type sialidase (known as NEU3) have been cloned [1]. Recently, the three types have been renumbered to four types of sialidases in humans with NEU1, NEU2, NEU3, and NEU4. These enzymes are characteristics of subcellular localizations and substrate specificity [3, 4]. From the different NEU forms, membrane-type enzyme, NEU3, is essential for hydrolysis reaction of ganglioside-specific SAs. NEU3 has been detected in various tissues, although the expression was low except in testis, skeletal muscle, the thymus, and the adrenal gland. The NEU3 expression was observed in erythroleukemia K562, colon cancer, and neuroblastoma cells such as SK-N-MC and NB-1 [1, 5]. The enzyme with 48 kDa of a molecular weight efficiently hydrolyzes GM3, GD1a, GD1b, and GD3, whereas GM1, GM2, and non-ceramidic sialyllactose are poor substrates. Moreover, the enzyme is not active for sialoglycoproteins including fetuin, transferrin, orosomucoid, fetuin, etc. This concludes that NEU3 is specific for SA residues linked to gangliosides. Therefore, cellular membrane-type neuraminidase catalyzes the ganglioside-specific © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_11

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sialo-hydrolysis. This implies for its participation in modulation of cell-surfaced function via degradation of gangliosides such as GM3. For example, NEU3 expression effectively inhibit gene expression of MMP-9 in human aortic vascular smooth muscle cells (VSMC). This indicates that a NEU3 potentially prevent VSMC proliferative disorders. NEU3 involves in the biosynthesis, catabolism, and transfer of SA residues in the arterial walls to reduce the arterial SA content [2]. The ganglioside-specific NEU3 is therefore participated in proliferation, migration, and differentiation of the cells. NEU3 localized in plasma membranes acts on gangliosides within own membrane and neighboring cells [4, 6]. NEU3 gene is frequently expressed in cancer cells of melanoma, colon, prostate, and ovary cancers [5, 7–10] and adjacent non-tumor tissues in cancers. NEU3 involves particularly in modulating transmembrane signaling by reducing the gangliosides contents [6], and the aberrant NEU3 expression elaborates the tumor pathogenesis [11]. In the recent study of the head and neck squamous carcinoma cells, which migrate, invade, and metastasize into lymph node region, NEU3 showed a crucial participation in the downregulation of gene expressions of matrix-degrading enzymes of MMP2 and MMP9 by blocking of EGFR signaling and contributing to reduced invasiveness and migration of the cells [12]. NEU3 activity was upregulated in tumor cell tissues, and its mRNA transcriptional expression was increased during metastasis to lymph node region. NEU3-induced malignancy of squamous carcinoma cells of HSC-2 and SAS promotes its motility and invasiveness with the increased MMP-9 levels, while NEU3 silencing was negative for those phenotypes. NEU3 phosphorylated EGFR, but EGFR Tyr kinase inhibitor AG1478 abrogated the NEU3-mediated MMP9 upregulation [12], as MMP-9 is a prognosis marker of human cancers [13]. NEU3 activates the EGFR phosphorylation and consequent ERK stimulation and MMP expression. Interestingly, the genes of NEU3, EGFR, and MMP2/MMP9 are all regulated by the same transcription factors of Sp1 and Sp3, which are known as players in growth control and tumorigenesis through Ras/ERK signals [14]. NEU3 is a crucial enzyme activating the EGFR signaling and MMP expression regulated in the downstream EGFR through GM3 SA digestion, which is related to the lymphatic node metastasis of HNSCC. In the Wnt signaling regulation, NEU3 deficiency in HCT116 and HT-29 colon cancer cells decreased the cancer stem cell-like phenotypes including clonogenicity and tumorigenicity during tumor growth with reduced renewal-like stemness and expression of Wnt-coupled genes [15]. NEU3 phosphorylates the Wnt LRP6 receptor and activates β-catenin function through formation of LRP6 complex assembled with Axin and GSK3β. In addition, NEU3 phosphorylates ERK and Akt through regulation of the EGF receptor and Ras cascades, triggering tumor progression. NEU3 enhances tumorigenesis levels through maintenance of stemness-like and renewal properties through Wnt signaling in colon cancer cells. Also, NEU3 elevates the levels of tumor proliferation and progression through Ras and MAPK signaling. Therefore, it is emphasized that NEU3 is a target in drug creation of tumor therapeutics. On the other hand, NEU3 induces growth, migration, survival, attachment and EGF-activated EGFR Tyr phosphorylation in the cancer cells [16]. Because GM3 is a downregulator of EGFR and VEGFR, NEU3 activity may negatively regulate the

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Membrane Ganglioside-Specific Neuraminidase 3 (NEU3) Regulates GM3 Signaling

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receptor activities of EGFR and VEGFR. For example, NEU3 activity is directly related with EGFR kinase signaling pathway [17]. NEU3 previously known to recognize EGFR is co-immunoprecipitated with EGFR [18]. In colon cancer cells, cell growth modulation by NEU3 is acquired through induced Tyr phosphorylation on integrin β4 by Grb-2 and Shc recruitments. This process consequently activates ERK1/2 and focal adhesion kinase (FAK) phosphorylation [16]. NEU3 overexpression accumulates lactosylceramide in colon cancer tissue specimens [19]. NEU3 can reduce the GM3 level and consequently prevent the inhibition effect derived by the ganglioside on the growth factor receptor function. NEU3 influences EGFR activation, and NEU3 overexpression negatively regulates EGFR activity without alteration of EGFR gene transcription and protein expression. Due to NEU3–EGFR interaction, EGFR activation was observed in cancer cell lines. It is suggested that modification of sialylation on the cell-surfaced molecules is a regulator of malignant differentiation. EGFR activation is associated with NEU3 removal of SAs in the receptor activity. NEU3 directly regulate EGFR signaling, and its enhanced expression activates EGFR function and cell growth. Enhanced expression of wild-type NEU3 also enforces the level of EGFR phosphorylation, giving a conclusion of receptor function alteration. NEU3 expression strongly activate EGFR with its downstream pathways. Sialidase activity is essential for EGFR activation because it regulates EGFR sialylation level. Sialidase activity is involved in EGFR desialylation through gangliosides sialylation pattern alteration. EGFR desialylation catalyzed by the membrane-type sialidase reversely activates its receptor activity. In mass spectrometry analysis, glycosylation of N528 [3] on EGFR was a mannose-type biantennary sialylated glycan, and the negative charge of sialylated carbohydrates on the EGFR extracellular domain was suggested to be influenced in recognition of EGF ligand. In parallel with GM3-engaged EGFR activation, NEU3 is another type of regulators to activate the EGFR through its desialylation of GM3. In the leukemic differentiation, NEU3 inhibits differentiation potentials of chronic myeloid leukemia (CML) cells of K562 leukemia cells. NEU3 modulates megakaryocytoid differentiation of the leukemic cells when phorbol 12-myristate 13-acetate (PMA) was treated, responsible for differentiation. PMA-induced GM3 expression potentiates the differentiation into megakaryocytic cell phenotypes [20, 21]. GM3 motivates to differentiate human myeloid leukemic cell line HL60 and monocytoid leukemic cell line U937 into the monocytic phenotypes [22]. In addition, human erythroleukemic K562 cells are differentiated into megakaryocytoid types [23]. The ganglioside-type sialidase NEU3 thus alters the cell surface events via alteration of gangliosides sialylation level. NEU3 importantly regulates cell growth [24], tumor transformation [25], and reduction of cell densitydependent growth suppression. This enzyme is also regarded as a caveolinassociated modulating molecule [26]. The human GM3-synthesizing enzyme, hST3Gal V, generates ganglioside GM3 and NEU3 reversely eliminates it. NEU3 expression blocks the PMA-activated phosphorylation of kinases ERK1/2 and p38MAPK of the K562 cells. Reduced expression of CD41b molecule as a surface antigen on myeloid cells is coupled with the level of NEU3 gene expression. Thus, NEU3 inhibitory analog agent, Neu5Ac2en, contributed to morphological changes,

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Fig. 11.1 NEU3 degrades GM3 and inhibits surface CD41b expression during K562 cells differentiation via PKC/ERKs signal transduction pathway

indicating onset of megakaryocytic cell differentiation in K562 cells, with CD41b expression, while PDMP, a selective inhibitor of glucosylceramide (GlcCer) synthase, blocks the megakaryocytic differentiation in K562 cells. GM3 is an inducing modulator of megakaryocytoid cell differentiation, as confirmed by human erythroleukemic K562 cell line [27]. The expressed GM3 ganglioside is located on the PM in the PMA-added cells. But, NEU3 enzyme removes the surfaced GM3 content in the NEU3 gene-enforced K562 cells upon treatment with PMA. NEU3 expression blocks the PMA-activated phosphorylation of kinases ERK1/2 and p38MAPK in the PMA-added K562 cells. The cell state promotes gene expression of CD41b antigen via PKC/ERKs/p38 MAPK axis pathway. The signaling event is additionally demonstrated in PDMP and Neu5Ac2en-treated cells. Cleaves membrane SAs to block the PKC/ERKs/p38MAPK signaling, reducing the CD41b expression and blocking megakaryocytic differentiation in K562 cells. NEU3 also degrades the membrane-assembled GM3, and consequently, K562 differentiation is stopped (Fig. 11.1). Similarly, silenced NEU3 gene abolishes apoptotic resistance and leads to megakaryocyte differentiation of CML K562 cells via the enhanced production of GM3 ganglioside amount [23]. In K562 cells, lowly expressed GM3 content caused by NEU3 enzyme blocks the cellular differentiation. In the silenced NEU3 cells, the levels of GM3, GM1, and sialosyl-norhexaosylceramide were increased, while cell growth and DNA synthesis were diminished. In the intracellular phenotype, the mRNA, Myc, and cyclin D2 expression levels are significantly reduced, while the apoptotic Bax and Bad protein levels are upregulated with apoptotic changes in the Bcl-XL and Bcl-2 proteins known as anti-apoptosis molecules. Finally, the synthesized levels of the cell surfaced CD10, CD41, CD44, and CD61, known as

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megakaryocytic biomarkers, are gradually enhanced to the same levels of PMA-stimulated cells. The differentiation-related signaling molecules such as ERK1/2, JNK, PKC, PLC-β2, RAF, and RSK90 are induced. Like the GM3-producing patterns in differentiating K562 cells, the pattern was equal level of the NEU3-silenced cells. During NEU3 silencing, K562 cells undergo apoptosis and megakaryocytic differentiation.

References 1. Ha KT, Lee YC, Cho SH, Kim JK, Kim CH (2004) Molecular characterization of membrane type and ganglioside-specific sialidase (Neu3) expressed in E. coli. Mol Cells 17(2):267–273 2. Moon SK, Cho SH, Kim KW, Jeon JH, Ko JH, Kim BY, Kim CH (2007) Overexpression of membrane sialic acid-specific sialidase Neu3 inhibits matrix metalloproteinase-9 expression in vascular smooth muscle cells. Biochem Biophys Res Commun 356(3):542–547 3. Mozzi A, Forcella M, Riva A, Difrancesco C, Molinari F, Martin V, Papini N, Bernasconi B, Nonnis S, Tedeschi G, Mazzucchelli L, Monti E, Fusi P, Frattini M (2015) NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels. Glycobiology 25(8):855–868 4. Monti E, Miyagi T (2012) Structure and function of mammalian sialidases. Topics Curr Chem 366:183–208 5. Shiozaki K, Yamaguchi K, Sato I, Miyagi T (2009) Plasma membrane-associated sialidase (NEU3) promotes formation of colonic aberrant crypt foci in azoxymethane-treated transgenic mice. Cancer Sci 100:588–594 6. Miyagi T, Wada T, Yamaguchi K, Hata K, Shiozaki K (2008) Plasma membrane-associated sialidase as a crucial regulator of transmembrane signalling. J Biochem 144:279–285 7. Mozzi A, Forcella M, Riva A et al (2015) NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels. Glycobiology 25(8):855–868 8. Miyagi T, Wada T, Yamaguchi K (2008) Roles of plasma membrane-associated sialidase NEU3 in human cancers. Biochim Biophys Acta 1780(3):532–537 9. Kawamura S, Sato I, Wada T, Yamaguchi K, Li Y, Li D, Zhao X, Ueno S, Aoki H, Tochigi T, Kuwahara M, Kitamura T, Takahashi K, Moriya S, Miyagi T (2012) Plasma membraneassociated sialidase (NEU3) regulates progression of prostate cancer to androgen-independent growth through modulation of androgen receptor signaling. Cell Death Differ 19:170–179 10. Forcella M, Oldani M, Epistolio S et al (2017) Non-small cell lung cancer (NSCLC), EGFR downstream pathway activation and TKI targeted therapies sensitivity: effect of the plasma membrane-associated NEU3. PLoS One 12(10):e0187289 11. Miyagi T, Takahashi K, Hata K, Shiozaki K, Yamaguchi K (2012) Sialidase significance for cancer progression. Glycoconj J 29:567–577 12. Shiga K, Takahashi K, Sato I, Kato K, Saijo S, Moriya S, Hosono M, Miyagi T (2015) Upregulation of sialidase. NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis. Cancer Sci 106(11):1544–1553 13. Chung TW, Lee YC, Kim CH (2004) Hepatitis B viral HBx induces matrix metalloproteinase-9 gene expression through activation of ERK and PI-3K/AKT pathways: involvement of invasive potential. FASEB J 18(10):1123–1125 14. Yamaguchi K, Koseki K, Shiozaki M, Shimada Y, Wada T, Miyagi T (2010) Regulation of plasma membrane-associated sialidase NEU3 gene by Sp1/Sp3 transcription factors. Biochem J 430:107–117

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15. Takahashi K, Hosono M, Sato I, Hata K, Wada T, Yamaguchi K, Nitta K, Shima H, Miyagi T (2015) Sialidase NEU3 contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling. Int J Cancer 137(7):1560–1573 16. Kato K, Shiga K, Yamaguchi K, Hata K, Kobayashi T, Miyazaki K, Saijo S, Miyagi T (2006) Plasma-membrane-associated sialidase (NEU3) differentially regulates integrin-mediated cell proliferation through laminin- and fibronectin-derived signalling. Biochem J 394:647–656 17. Tringali C, Lupo B, Silvestri I, Papini N, Anastasia L, Tettamanti G, Venerando B (2012) The plasma membrane sialidase NEU3 regulates the malignancy of renal carcinoma cells by controlling beta1 integrin internalization and recycling. J Biol Chem 287:42835–42845 18. Orizio F, Triggiani L, Colosini A et al (2019) Overexpression of sialidase NEU3 increases the cellular radioresistance potential of U87MG glioblastoma cells. Biochem Biophys Res Commun 508(1):31–36 19. Okada Y, Kimura T, Nakagawa T et al (2017) EGFR downregulation after anti-EGFR therapy predicts the antitumor effect in colorectal cancer. Mol Cancer Res 15(10):1445–1454 20. Kang SK, Kim YS, Kong YJ, Song KH, Chang YC, Park YG, Ko JH, Lee YC, Kim CH (2008) Disialoganglioside GD3 synthase expression recruits membrane transglutaminase 2 during erythroid differentiation of the human chronic myelogenous leukemia K562 cells. Proteomics 8(16):3317–3328 21. Jin UH, Ha KT, Kim KW, Chang YC, Lee YC, Ko JH, Kim CH (2008) Membrane type sialidase inhibits the megakaryocytic differentiation of human leukemia K562 cells. Biochim Biophys Acta 1780(5):757–763 22. Nojiri H, Takaku F, Terui Y, Miura Y, Saito M (1986) Ganglioside GM3: an acidic membrane component that increases during macrophage-like cell differentiation can induce monocytic differentiation of human myeloid and monocytoid leukemic cell lines HL-60 and U937. Proc Natl Acad Sci USA 83:782–786 23. Tringali C, Lupo B, Cirillo F, Papini N, Anastasia L, Lamorte G, Colombi P, Bresciani R, Monti E, Tettamanti G, Venerando B (2009) Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3. Cell Death Differ 16 (1):164–174 24. Valaperta R, Chigorno V, Basso L et al (2006) Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts. FASEB J 20(8):1227–1229 25. Ledeen RW, Wu G, André S et al (2012) Beyond glycoproteins as galectin counterreceptors: tumor-effector T cell growth control via ganglioside. Ann NY Acad Sci 1253:206–221 26. Bonardi D, Papini N, Pasini M et al (2014) Sialidase NEU3 dynamically associates to different membrane domains specifically modifying their ganglioside pattern and triggering Akt phosphorylation. PLoS One 9(6):e99405 27. Choi HJ, Chung TW, Kang NY, Kim KS, Lee YC, Kim CH (2004) Involvement of CREB in the transcriptional regulation of the human GM3 synthase (hST3Gal V) gene during megakaryocytoid differentiation of human leukemia K562 cells. Biochem Biophys Res Commun 313:142–147

Chapter 12

Regulation of GM3-Mediated EGFR Signaling by NEU3 Sialidase

Sialylation patterns and distribution at the cellular surfaces or the extracellularly secreted milieu are a hallmark of parameters to evaluate the level of malignant differentiation [1, 2]. However, the mechanisms between sialylation and cancer are not well explained. For protein regulation, NEU3 controls cell growth and division by modulation of Tyr phosphorylation on membrane proteins and downstream target molecules including FAK, ERK1/2, and MAP38 kinase [3]. For ganglioside regulation, NEU3 accumulates lactosylceramide in colon cancers [4] and regulates GM3 function on EGFR activation [5–7], decreasing GM3 content and consequently eliminating the GM3 inhibitory effect on the receptor activation [8, 9]. For sialic acid role, it was suggested that the sialylated carbohydrates negatively charged on the EGFR extracellular domain influence both EGF ligand binding of an anionic small protein and its EGFR dimerization [10]. Because the importance of sialylation in glycolipid and glycoprotein become uncovered during the last two decades, the relation between the sialylated glycolipids and its degrading sialidase has been the hot topic in the progression of cancer and tumorigenic transformation. NEU3 is reported to be upregulated in many different neoplastic tumors. For example, the NEU3 gene expression is found in many cancer cells including colon, melanoma, ovarian, renal, and prostate cancer cells of human [11]. In transcriptional mRNA levels of NEU3 gene, the maximum increase in colon cancer tissues of human was in contrast to adjacently present non-cancer tissues [12]. NEU induced colorectal tumorigenesis in transgenic model of mouse [13]. The plasma membrane-type NEU3 is reported to be deregulated in colon cancer, and the NEU3 is also co-immunoprecipitated with EGFR in uterine cancer [14]. Recently, it was evident that NEU3 activates the EGFR signaling [15, 16]. For the role of NEU3 in EGFR function in colon cancer, NEU3 activated EGFR signaling through a direct SA degradation of the receptor glycans in EGFR-positive colon cancer cells, without alteration in the EGFR protein and mRNA expression levels [10]. In the regulation effect of NEU3 enforced expression on EGFR expression, downstream signaling, activation and cell viability, where EGFR mRNA and protein levels were not changed, NEU3 enhanced EGFR phosphorylation, © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_12

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Neu3 activated EGFR downstream signaling of the ERK1/2 MAP kinase and AKT-PI3K–mTOR pathways. Cell viability was significantly increased upon NEU3 expression. Thus, NEU3 overexpression activates EGFR and its downstream pathways, and sialidase activity is important for promotion of EGFR and regulates the sialylation level of EGFR. In a recent report [17], ST6Gal-I, β-Galα2,6ST, was shown to regulate EGFR function, and the reduced ST6Gal-I activity activated EGFR tyrosine kinase. NEU3 expression does not affect ST6Gal-I mRNA expression; however NEU3 overexpression decreased EGFR function by decreased sialic acid level. While indirectly acting NEU3 promotes tumorigenesis process, direct EGFR desialylation by NEU3 is involved. EGF induces activation of phospholipase D-1 known as PLD-1 to release phosphatidic acid, and the released phosphatidic acid translocates NEU3 to the cell surface [18]. EGFR stimulation uses NEU3 to remove sialic acid residues, and this should be a positive feedback for the EGFR activity. This information gives a conclusive remark that the NEU3 can be the molecular target of the cancer therapy in a method of monoclonal antibody-based or enzyme inhibitory compound-based drugs against human cancers. To meet the criteria of the NEU3 inhibitory drugs against human cancers, it was recently reported that naringin, a flavonoid, blocks tumor cell division and proliferation. Because GM3 role in cancer behavior has in detail been defined in human tumor cells including ovarian HeLa cells and A549 cells [19], the naringin-exposed cells showed the drastically increased level of GM3 by a mechanism of that naringin suppresses NEU3 sialidase activity as a GM3 SA-degrading sialidase. NEU3 inhibitory natural products are also identified from other flavanones including hesperidin and neohesperidin dihydrochalcone. However, the aglycones of the two candidates showed less inhibitory activities than the original naringin. In the cancer cells treated with naringin, EGFR and ERK phosphorylation levels were remarkably reduced, indicating that naringin suppressive effect of tumor growth and cell division is based on the alteration in sialylation level of glycolipid GM3. Mammal cells are coated with excess amounts of glycoconjugates including proteoglycans, GSLs and glycoproteins on the PM of the cells, and regulate using the glycoconjugates to transmembrane receptor signaling. The cell-surfaced glycoconjugates are associated with cellular phenotypes, largely in tumor cells. When the membrane glycolipid fractions were prepared and analyzed from human ovarian cancer cells such as HeLa cells and A549 cells, glycolipids stained by oricinol-H2SO4 reagent are mostly GM3 as sialyl GSLs, which bear the carbohydrate of Neu5Acα2-3Galβ1-4Glc1-1-Cer structure. This structure is increased in tumor cells of A549 cells and HeLa cells after naringin exposures. The contents of GM3 were approximately threefold more increased in A549 and HeLa cells, while other glycolipids were unchanged. When the glycoprotein profiles were examined by lectins of MAM, which is specific for α2-3 SA-structure on sialoglycoprotein, and SSA, which is specific for α2-6 SA structure on sialoglycoprotein, lectin affinity alterations were not seen in both MAM and SSA lectin analysis. Only GM3 was an altered parameter of naringin-leaded suppression of cell proliferation. For the mechanism responsible for GM3 enhancement in naringin-exposed cell, sialidase NEU3 was inhibited without effects on st3galV and NEU3 mRNA levels. NEU3 expression

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is upregulated in tumors or many tumor patients, and NEU3 increases positively tumor malignancies including division, motility, growth, and anti-apoptosis of cells. NEU3 sialidase is a ganglioside-specific membrane-type neuraminidase [20], and for the substrate, MU-NANA is used as for NEU1 and NEU4. As approximately calculated, 1–2 units of NEU3 enzyme approximately equally corresponded to total sialidase activity observed in 1  106 HeLa cells. Naringin dose-dependently inhibits NEU3 activity, and IC50 concentration of naringin to NEU3 was 1188 μM. Other glycosidic flavanones showed the similar NEU3 inhibitory activities. For example, hesperidin moderately inhibits NEU3 activity, which is lower than that of naringin. Aglycones of such glycosidic flavanones in the cases of hesperetin and naringenin exhibited the lower inhibitions, when compared to their glycosidic compounds. Dihydrochalcone neohesperidin holds NEU3 inhibition activity, and thus disaccharide chains constituted in hesperidin and naringin seem to be essential for NEU3 inhibition. Naringin increases GM3 levels in human tumor HeLa and A549 cells. GM3 suppresses tumor cell division, growth, and signaling alteration. EGFR signaling pathway was specially inhibited. EGFR phosphorylation is dose-dependently suppressed by naringin, and approximately half level of EGFR phosphorylation is blocked by 400 μM concentration of naringin. Thus, naringinmediated GM3 deposition is significant in tumor cells. In the ERK phosphorylation of the EGFR downstream signaling, the ERK phosphorylation was also abrogated in the treatment condition with 400 μM naringin in tumor cells, accompanying with the decreased level of EGFR phosphorylation. Another neuraminidase, lysosomal sialidase NEU1, also negatively modulates EGFR downstream signaling through desialylation of N-glycosylated EGFR glycans [21]. In addition, MMP-9 downregulation in naringin-treated VSMCs rather suppresses the EGFR signaling. However, NEU1 and MMP-9 expression levels were not altered in the naringin-treated A549 cells. Naringin-driven inhibition of EGFR downstream and ERK phosphorylation seems to be independently associated with NEU1 or MMP-9 in such tumor cells. Thus, naringin-mediated mechanism in suppression of tumor proliferation against tumor cells is based on GM3 accumulation in membrane. Naringin-inhibited NEU3 activity accumulates GM3 ganglioside, and this led the suppression of EGFR downstream functions with the reduced cell proliferation. GM3 recognizes EGFR through a Lys618 side chain and inhibits EGFR dimerization and EGFR downstream signaling [22]. GM3 accumulation by naringin decreases EGFR activation and growth inhibition of cancer cells. GM3 synthesis in cells is performed by the enzyme GM3 synthase known as ST3Gal V, and NEU3 controls the total GM3 contents. Thus, GM3 accumulation can be occurred by NEU3 activity inhibition, not by the GM3-associated proteins. NEU3 expression is upregulated in many human cancer types such as colon, ovary, prostate, head, neck, and renal carcinomas [23, 24]. Furthermore, NEU3-null mice show the suppression of azoxymethane-caused colon tumor formation and accelerate shift to malignant phenotypes through alteration of transmembrane signaling including Wnt/β-cathenin, integrins, and phospholipids [18]. EGFR is the molecule directly influenced by NEU3 sialidase. GM3 degradation activates EGFR function-associated downstream signaling including AKT, ERK, Raf, and Ras

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molecules in tumor cells. Human airway epithelial cells express enzyme-catalytic NEU1, a lysosome-locating sialidase. This NEU1 negatively modulates EGFRassociated phosphorylation of the ERK by NEU1-calatyzed cleavage of SA residues in the EGFR sialoglycans in tumor cells [21]. Thus, it is suggested that GM3-mediated EGFR signaling can be controlled by NEU3 to inhibit cancer growth. Neu3 is also useful to target in cancer therapy by monoclonal antibodybased or low molecular weights compound-based drugs against cancer.

References 1. Schauer R, Kamerling JP (2018) Exploration of the sialic acid world. Adv Carbohydr Chem Biochem 75:1–213 2. Schauer R (2009) Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol 19:507–514 3. Kato K, Shiga K, Yamaguchi K, Hata K, Kobayashi T, Miyazaki K, Saijo S, Miyagi T (2006) Plasma-membrane-associated sialidase (NEU3) differentially regulates integrin-mediated cell proliferation through laminin- and fibronectin-derived signalling. Biochem J 394:647–656 4. Pietrantonio F, Petrelli F, Coinu A et al (2015) Predictive role of BRAF mutations in patients with advanced colorectal cancer receiving cetuximab and panitumumab: a meta-analysis. Eur J Cancer 51(5):587–594 5. Yang HJ, Jung KY, Kwak DH, Lee SH, Ryu JS, Kim JS, Chang KT, Lee JW, Choo YK (2011) Inhibition of ganglioside GD1a synthesis suppresses the differentiation of human mesenchymal stem cells into osteoblasts. Dev Growth Differ 53:323–332 6. Huang X, Li Y, Zhang J, Xu Y, Tian Y, Ma K (2013) Ganglioside GM3 inhibits hepatoma cell motility via down-regulating activity of EGFR and PI3K/AKT signaling pathway. J Cell Biochem 114:1616–1624 7. Hakomori SI, Handa K (2015) GM3 and cancer. Glycoconj J 32:1–8 8. Cirillo F, Ghiroldi A, Fania C et al (2016) NEU3 sialidase protein interactors in the plasma membrane and in the endosomes. J Biol Chem 291(20):10615–10624 9. Scaringi R, Piccoli M, Papini N, Cirillo F, Conforti E, Bergante S, Tringali C, Garatti A, Gelfi C, Venerando B (2013) NEU3 sialidase is activated under hypoxia and protects skeletal muscle cells from apoptosis through the activation of the epidermal growth factor receptor signaling pathway and the hypoxia-inducible factor (HIF)-1alpha. J Biol Chem 288:3153–3162 10. Mozzi A, Forcella M, Riva A, Difrancesco C, Molinari F, Martin V, Papini N, Bernasconi B, Nonnis S, Tedeschi G, Mazzucchelli L, Monti E, Fusi P, Frattini M (2015) NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels. Glycobiology 25(8):855–868 11. Monti E, Miyagi T (2015) Structure and function of mammalian sialidases. Topics Curr Chem 366:183–208 12. Takahashi K, Hosono M, Sato I et al (2015) Sialidase NEU3 contributes neoplastic potential on colon cancer cells as a key modulator of gangliosides by regulating Wnt signaling. Int J Cancer 137(7):1560–1573 13. Shiozaki K, Yamaguchi K, Sato I, Miyagi T (2009) Plasma membrane-associated sialidase (NEU3) promotes formation of colonic aberrant crypt foci in azoxymethane-treated transgenic mice. Cancer Sci 100:588–594

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14. Shiga K, Takahashi K, Sato I et al (2015) Upregulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis. Cancer Sci 106 (11):1544–1553 15. Huang C, Hays FA, Tomasek JJ, Benyajati S, Zhang XA (2019) Tetraspanin CD82 interaction with cholesterol promotes extracellular vesicle-mediated release of ezrin to inhibit tumour cell movement. J Extracell Vesicles 9(1):1692417 16. Tringali C, Lupo B, Silvestri I, Papini N, Anastasia L, Tettamanti G, Venerando B (2012) The plasma membrane sialidase NEU3 regulates the malignancy of renal carcinoma cells by controlling beta1 integrin internalization and recycling. J Biol Chem 287:42835–42845 17. Park JJ, Yi JY, Jin YB, Lee YJ, Lee JS, Lee YS, Ko YG, Lee M (2012) Sialylation of epidermal growth factor receptor regulates receptor activity and chemosensitivity to gefitinib in colon cancer cells. Biochem Pharmacol 83:849–857 18. Shiozaki K, Takahashi K, Hosono M, Yamaguchi K, Hata K, Shiozaki M, Bassi R, Prinetti A, Sonnino S, Nitta K et al (2015) Phosphatidic acid-mediated activation and translocation to the cell surface of sialidase NEU3, promoting signaling for cell migration. FASEB J 29:2099–2111 19. Yoshinaga A, Kajiya N, Oishi K, Kamada Y, Ikeda A, Chigwechokha PK, Kibe T, Kishida M, Kishida S, Komatsu M, Shiozaki K (2016) NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation. Eur J Pharmacol 782:21–29 20. Heneghan CJ, Onakpoya I, Jones MA et al (2016) Neuraminidase inhibitors for influenza: a systematic review and meta-analysis of regulatory and mortality data. Health Technol Assess 20 (42):1–242 21. Feng C, Li J, Snyder G, Huang W, Goldblum SE, Chen WH, Wang LX, McClane BA, Cross AS (2017) Antibody against microbial neuraminidases recognizes human sialidase 3 (NEU3): the neuraminidase/sialidase superfamily revisited. mBio 8(3):e00078-17 22. Lillehoj EP, Hyun SW, Feng C, Zhang L, Liu A, Guang W, Nguyen C, Luzina IG, Atamas SP, Passaniti A, Twaddell WS, Puché AC, Wang LX, Cross AS, Goldblum SE (2012) NEU1 sialidase expressed in human airway epithelia regulates epidermal growth factor receptor (EGFR) and MUC1 protein signaling. J Biol Chem 287:8214–8231 23. Kawamura S, Sato I, Wada T, Yamaguchi K, Li Y, Li D, Zhao X, Ueno S, Aoki H, Tochigi T, Kuwahara M, Kitamura T, Takahashi K, Moriya S, Miyagi T (2012) Plasma membraneassociated sialidase (NEU3) regulates progression of prostate cancer to androgen-independent growth through modulation of androgen receptor signaling. Cell Death Differ 19:170–179 24. Shiga K, Takahashi K, Sato I, Kato K, Saijo S, Moriya S, Hosono M, Miyagi T (2015) Upregulation of sialidase NEU3 in head and neck squamous cell carcinoma associated with lymph node metastasis. Cancer Sci 106:1544–1553

Chapter 13

VEGFR–GM3 Interaction in Angiogenesis

Tumor-associated vessels display glycosylation change to delineate sensitivity against anti-VEGF trials. Cancer cells are specific for the VEGF–VEGFR signaling in the angiogenetic vascular tube formation and new vessel formation. VEGF is a protruding regulator responsible for development and pathologic conditions such as angiogenesis. Three VEGFR-1, VEGFR- 2, and VEGFR-3 subtypes are currently reported. Also, the two different forms of membrane-bound and soluble VEGFRs are generated in the cells from the variant transcripts through alternative splicing events. VEGF is a leading factor of angiogenesis, and VEGFR Tyr kinases include KDR/FLK1 known as VEGFR-2 and FLT1 known as VEGFR1. VEGF receptors such as FLT1 and FLK1/KDR as well as their ligand VEGF are essential for angiogenic regulation. VEGF and their VEGFRs of FLK1/KDR and FLT1 are crucial for the angiogenic events in vascular vessel formation. Although the function of FLK1/KDR is well studied to date, the FLT1 is still unknown. FLT1 is produced as two different isoforms of soluble sFLT1 and full-length proteins. Regulation of cellular events including migration, growth, and differentiation of endothelial cells is generally influenced by FLK1/KDR. While FLT1’s role is not known yet, FLT1 gene’s main different splice variants encode the transmembrane receptor of full length, soluble, secreted, and truncated forms. VEGFs are integral in angiogenesis process through VEGFR RTKs. Blocking of VEGF-A signaling with anti-VEGF Mab or low molecular weights RTK inhibitors has much improved survival rate against several cancers. The efficacy of anti-VEGF MAbs or inhibitors is lowered by the compensatory angiogenesis pathways. Thus, compensatory pathways in antiVEGF refractory or sensitive tumor are not well understood during the above antiVEGF treatment. VEGF is a major angiogenesis factor, and its two receptors with tyrosine kinase activity are (1) FLT1 (VEGFR1) and (2) FLK1/KDR (VEGFR-2). In the ST6Gal-KO mouse, sialic acid-recognizing SNA level was decreased. When ST6Gal-1 KO mouse injected with anti-VEGF-sensitive tumor was treated with antiVEGF antibody, the tumor size was not decreased compared with WT. Thus, glycome pattern will affect to the Gal-1-specific ligand, and thus it will determine the refractoriness against anti-VEGF. © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_13

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Fig. 13.1 Structure and function of VEGFR1 (FLT1) gene. (a) Two alternatively spliced variants of FLT1 gene. (b) Interaction of sFLT1 and GM3 in lipid raft microdomain. (c) sFLT1 function in podocytes. sFLT1 synthesized in the glomerular microvasculature of pericytes recognizes the GM3 in membrane lipid raft microdomain on the podocyte surfaces (b). If sFLT1 is deprived in podocyte, cytoskeleton rearrangement raises the protein urine. If sFLT1 is expressed, the protein urea is not released from blood to urine (b). sFLT1 acts as autocrine effector to manage cytoskeletal dynamics in specialized pericytes and perivascular cells. sFLT1 forms the microdomain lipid rafts enriched with GSLs. sFLT1 specifically interact with the GM3 ganglioside embedded in lipid rafts

VEGF family activates cellular functions through binding to receptor Tyr kinase receptors (RTK) of VEGFRs present on the cell membrane, dimerizing and transphosphorylating the VEGFRs. The VEGFRs consist of extracellular domains with an intracellular Tyr kinase domain, a TM domain, and seven immunoglobulin (Ig)like domains (Fig. 13.1). VEGF-A equally recognizes the Flt-1 form known as VEGFR-1 and KDR/Klk-1 form known as VEGFR-2. The VEGFR-2 has a broad spectrum to transduce various cellular responses, but VEGFR-1 is limited to its role. Phosphorylation of VEGFR-2 and VEGFR-2 ligand downstream signaling includes multiple responses such as PI3K/Akt, p38 MAPK, and ERK in endothelial cells induced by VEGF. Alternative variant form, named sFlt1, of VEGFR-1 as a spliced form is a soluble protein and secreted protein which acts as a decoy. VEGFR-3 is not recognized by VEGF-A, but VEGFR-3 leads to lymphangiogenesis upon VEGF-C or VEGF-D binding.

13.1

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Soluble sFLT1 Directly Recognize GM3 Embedded in the Lipid Rafts

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Soluble sFLT1 Directly Recognize GM3 Embedded in the Lipid Rafts

Soluble VEGFR1, sVEGFR1, or sFLT1 is an inhibitor of angiogenesis archived by competition with angiogenic VEGF and angiogenic placental growth factor (PlGF). Unlike the FLK1 or KDR, the FLT1 role is remained covered. FLT1 is formed as a soluble form (sFLT1) and also as a full-length isoform. Even though FLT1 function is not studied before, the two different splicing variants of FLT1 gene are transcribed. The two variants encode a transmembrane receptor having a full length, a soluble receptor, and truncated receptor as a secreted form, respectively. The sFLT1’s mRNA encodes a ligand recognition domain, losing the C-terminal Tyr kinase domain and TM domain. It is assumed that the mRNA for soluble FLT1 is translated to the protein lacked for both the transmembrane domain and Tyr kinase domain in the C-terminal region, while having its ligand-binding domain. Soluble FLT1 is found in several cells and tissues of endothelial cells, peripheral blood mononuclear cells (PBMCs), and placenta [1]. sFLT1 mediates the apoptosis induction of endothelial cells and consequently prevents thickening development of vascular intimal vessels in the labyrinthine layer in murine model. In addition, exogenous injection of sFLT1 reduces growth and vascular extravasation of tumor cells as well as tube formation and ascites formation in mice implanted with xenografts of ovarian cancer [2]. sFLT1 has a tumor suppression effect in a mice ovarian cancer model, indicating importance of sFLT1 as a tumor therapeutic [1]. sFLT1 is summarized to be kinase-defective mutant having the GM3 binding capacity to attach and rearrange to lipid raft of cell surface of the podocytes. In the pericyte, the sFLT1 function is important with regard to glomerulus podocyte [3]. sFLT1 stimulates cell adhesion and cytoskeleton reorganization of podocytes by binding to the cell surfaces. sFLT1 recognizes directly the lipid raft microdomain embedded with GM3. Then, sFLT1 allows pericyte adhesion with perivascular cells, through sFLT1 binding and activation of intracellular signaling. As a VEGF isoform, sFLT1 is considered to be a competitive inhibitor of pro-angiogenic function when it binds to target receptors. For its apparent activity in renal function, it was recently reported that differentiated cells of the two different pericytes and perivascular cells express sFLT1. Of course, vascular endothelial cells basically express sFLT1. As examined using the cholera toxin B subunit, the sFLT1 directly interact with the GM3 in the lipid raft microdomain on the podocyte surfaces followed by rapid endocytosis binding of sFLT1 with its counterpart such as syndecans activates the intracellular signaling. Pericytes produce sFLT1 in the glomerular microvasculature (Fig. 13.1a). The binding of sFLT1 and GM3 promotes actin organization and adhesion. sFLT1 also regulates pericyte cells in vessels [3], as sFLT1 produced by pericytes functions as autocrines for cytoskeletal plasticity and dynamics in pericytes and perivascular cells. Initially, sFLT1 is dispersed on the surface of podocytes and then internalized to microdomain-conjugated endocytic vesicle. sFLT1 is reported to be co-bounded with adaptor proteins. If sFLT1 is deprived in podocyte, cytoskeleton rearrangement is occurred and consequently

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raising the protein urine. But in normal state, the sFLT1 is expressed and keeping the protein urea in blood (Fig. 13.1b). Conventional FLT1 KO mice exhibit lethal vascular phenotype with hyperproliferation of endothelial cells [2, 3]. For the ligand binding, sFLT1 binds VEGF to suppress pro-angiogenic function. In mice, FLT1 KO mouse shows the increased VEGF activity and consequent hyperproliferation of endothelial cells with vascular lethality. Thus, FLT1’s signal is not operating; instead FLT1 captures the VEGF typical signaling, which interacts with VEGFR-2 and FLK1/KDR. Thus, the role of sFLT1 should be the decoy receptor. Glomerular podocyte is specialized pericyte to keep the skeletal system. The podocyte and perivascular cells express sFLT1 [4]. Unlike FLK1/KDR, the FLT1 roles are not well understood. The synthesis of FLT1 occurs as a sFLT1 (100 kDa), which is the soluble isoform, or as a Flt1 (170 kDa), which is a full-length isoform. Pericytes produce the sFLT1 form without understanding of the precise roles. In the glomerular pericytes known as podocytes in the glomerular microvasculature, Flt1 deficit or deletion causes the cytoskeleton reorganization, leading to massive proteinuria caused by kidney failure, which is representative for nephrotic impaired features. The Tyr kinase lost allele of Flt1 reverses this cellular phenotype, and this confirms dispensability of the isoform with full-length Flt1. sFLT1 protein binds to the ganglioside GM3 expressed on the membrane lipid rafts of the glomerular podocyte cells. The binding of sFLT1 and GM3 induces cellular adhesion level and reorganization of actin filaments in cells [4].

13.2

FLK1/KDR (VEGFR-2)–GM3 Interaction

GM3 plays a provisional role in modulating angiogenesis in vitro. Gangliosides, SAs-containing GSLs, are resided on the most out leaflet of the cellular PM of mammalian cells [5]. The gangliosides are functionally associated with various cellular events of cell-to-cell communication, recognition, binding, adhesion, binding, interaction, differentiation, and cell division. Changes of ganglioside composition on the cell membrane have been described in many cell types, indicating that gangliosides modulate various GFR functions [6]. In addition, most ganglioside forms shed on extracellular environment of cancer cells modulate cellular functions [7]. It was hypothesized that gangliosides expressed on the PM in many cell types are extracellularly excreted to the tumor microenvironment in certain physiological states or in certain cancer stages and alter VEGF signaling to adapt their environments. Expression of GM3 in normal tissues is higher than what is observed in cancer tissues. Interestingly, GM3 around blood vessels of cancer tissues was markedly lower in comparison with normal tissues. GM3 is directly involved in the suppressed angiogenesis through GM3-interacting microenvironment with molecular mediators secreted from or on the stroma cellular PM and tumor cells. The structure of gangliosides consists of ceramide as a common precursor of glycosphingolipids and oligosaccharides. GM3 (Neu5Acα2,3Galβ1,4Glc1,1Ceramide) is generated by the

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FLK1/KDR (VEGFR-2)–GM3 Interaction

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sequential addition of Glc, Gal, and SA onto ceramide through the action of glucosyl, galactosyl, and sialyltransferases. New blood capillaries are formed by angiogenesis event with activation of vascular endothelial cells as tumor metastasis [8]. A key angiogenic factor is a VEGF [9], and VEGF is secreted in surrounded stroma and various tumor cells in the tumor-associated microenvironment. VEGF is a powerful factor to influence the proliferation, migration, permeation, and transmigration of endothelial cells [10]. VEGF recognizes two RTKs such as VEGFR-2/Flk-1/KDR and VEGFR-1/Flt-1, produced by vascular endothelial cells. VEGFR-2 is important for tumor angiogenesis, proliferation, and metastasis [11]. Then, many scientists have developed agents targeting the VEGF–VEGFR-2 signaling inhibitors for cancer treatment of human solid tumors. VEGFR-2 is composed of 18 putative N-glycosylation sites, and it functions as a general receptor of VEGF, regulating endothelial cell migration, proliferation, and differentiation. VEGFR-2 is structured with the seven extracellular immunoglobulin (Ig)-like domains. Among them, domain Ig-2 and Ig-3 involve in VEGF-A binding, while domains Ig-4 to Ig-6 lead to receptor dimerization, and domain Ig-7 maintains stabilized dimer formation (Fig. 13.2a). It was previously known that GM3 levels are lower in several cancerous tissues in comparison with normal tissues and that GM3 is also detected around blood vessels of normal tissues, but not in cancer tissues. GM3 inhibits tumor neovascularization by modulating the biological function of vascular endothelial cells. It was demonstrated that GM3 is the sole case to directly bind to VEGFR-2, which blocks VEGF– VEGFR-2-leading cellular events driven by vascular endothelial cells and resulting angiogenic event and the tumor growth in mice with Lewis lung carcinoma. Intracellularly, cell motility and migration-related intracellular signaling are regulated in the membrane receptor level. Kim group in 2009 showed GM3 inhibits VEGFR-2 function, as VEGFR-2 is stimulated by VEGF for tumor development [11]. GM3 inhibits the VEGFR-2 activation by eliminating its dimerization and diminishing VEGFR-2 interaction with VEGF by direct recognition with VEGFR-2 in vitro. The blocking of neovascularization and inhibition of microvessel permeability induced by VEGF was blocked by GM3 in vivo. Tumor suppression in mice model with primary tumor growth is by GM3, concluding that GM3 suppresses the function of VEGFR-2 mediated pathway, which would be a target candidate in order to exert tumor therapeutic effect and angiogenic suppression. GM3-mediated anti-angiogenesis has been reported from the therapeutic effect on brain cancer in 2010 [12] and was reported. In GM3 on different brain cancer models, higher GM3 expression resulted in lower tumor development. GM3 expression leads to higher cell–cell adhesion while slows down cell growth rate. Ratio of GM3 to other ganglioside complex might affect the angiogenesis. In CT-2A astrocytoma, GM3 inhibited the angiogenesis, concluding GM3 as a therapeutic candidate for brain tumor by increasing the GM3 ratio. The anticancer activity of exogenously treated GM3 [13] was visible for bladder cancer cells. “This, bladder tumor has been suggested to be a prominent candidate for GM3-driven treatment, because ganglioside GM3 transported at an efficient concentration to target area but with minimal trafficking to non-desired sites.” It receives more attention in studying GM3 suppressing tumor development. GM3 inhibits hepatoma cell motility.

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Fig. 13.2 GM3–VEGFR-2 suppression in VEGF-acted endothelial cells. VEGFR function (a) and VEGFR-2–GM3 interaction (b). Biological functions of VEGFR-2 (KDR/Flk-1) in cell proliferation, migration, survival, adhesion, tube formation, and angiogenesis are described. GM3 directly recognize the endothelial cell-expressed VEGFR-2, but not VEGF. GM3 suppresses dimerization of VEGFR-2 and VEGF recognition to VEGFR-2. VEGFR-2–GM3 binding blocks VEGFR-2 dimerization and downstream signaling

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The proliferation rate of human solid tumors depends on neovascularization, and new generated capillary tubes at tumor sites are essential for tumor progression, cell division, invasion, and metastasis [5, 11]. The angiogenesis alters the microenvironments. In hypoxic conditions, the avascular tumor or stromal cells trigger neovascularization by releasing VEGF as an endothelial targeting mitogen and also by a specific factor of hypoxia-inducible factor (HIF)-1. VEGF recruits endothelial cells and induces the proliferative potentials of endothelial cells in a way that VEGF recognizes the VEGFR-2 expressed at the endothelial cell surfaces [14]. The VEGF binding to VEGFR-2 contributes to the sequential cascade reactions including VEGFR dimerization and kinase activation for autophosphorylation of certain Tyr residues [15]. VEGFR-2 activation stimulates intercellular signaling event via Ca2+ mobilization, AKT, ERK, Grb, eNOS, FAK, PLC, Src family Tyr kinases, VE-cadherin, Ras GTPase-activator, PI-3K, PKC, p38MAPK, Shc, STAT, SHP-1, and SHP-2 in endothelial cells. VEGF and its receptor VEGFR-2 essentially activate endothelial cell for blood vessels formation and the progression of solid tumors. In tissue microenvironments, GM3 modulates a local balancing state among the angiogenic and anti-angiogenic agents. This balancing negatively activates VEGF– VEGFR-2 driving forces of endothelial cells for the delayed tumor progression. In endothelial cells, GM3 blocks the VEGF-induced stimulation of ERK, PI-3K/AKT, FAK/paxillin, and VE-cadherin signalings and inhibits migration, proliferation, and tube formation. GM3 blocks VEGF-activated VEGFR-2 Tyr phosphorylation in endothelial cells, indicating the attenuation of VEGFR-2 signaling. The question is raised whether GM3 synthase gene-transfected HUVECs and exogenously administered GM3 suppress the phosphorylation event in the VEGF–VEGFR-2 interaction. VEGF-activated VEGFR-2 response and VEGFR-2 downstream signaling molecule are inhibited in the GM3 synthase gene-transfected HUVECs. Therefore, endogenously synthesized GM3 and exogenously treated GM3 block VEGFinduced VEGFR-2 function, indicating the anti-angiogenic GM3 role on the HUVECs PM, and shed GM3 roles from tumor or tumor-surrounded microenvironments. For the molecular mechanism(s) underlying the GM3-inhibited VEGFR-2 function, vascular endothelial cells were treated with VEGF. The effect based on the direct GM3–VEGFR-2 recognition was observed on the treated endothelial cell surface, not direct VEGF effect. GM3 also suppresses dimerization of VEGFR-2 and consequently inhibits the VEGF–VEGFR-2 recognition (Fig. 13.2b). GM3 regulates the physiological events through blocking of VEGFR-2 function in vitro, as confirmed in vascular endothelial cells. The ganglioside GM3 blocks VEGFstimulated neovascularization process in animals. In a molecular mechanism, GM3 blocks its dimerization event of VFGFR and VEGF–VEGFR-2 recognition in a PCI manner of GM3 ganglioside–GFR protein recognition. The precise protein part is the extracellular domain of VEGFR-2, suppressing angiogenic capacity, tumor cell proliferation, VEGF-stimulated microvessel permeability, and angiogenesis for anti-angiogenic therapy. GM3 inhibits phosphorylation event of VEGFR-2 and Akt downstream signaling in endothelial cells such as HUVECs (Fig. 13.2b).

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VEGFR–GM3 Interaction in Angiogenesis

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Fig. 13.3 The construction of VEGFR-2 ExD and direct visualization of the GM3 binding to VEGFR-2 ExD. (a) Construction of VEGFR-2 ExD on the myc-6xHis expression vector. (b) Direct recognition of ganglioside GM3 to VEGFR-2 of endothelial cells via GM3 binding to the VEGFR-2 ExD. Experimental illustration to demonstrate the direct recognition of GM3 and VEGFR-2 ExD. (c, d) GM3–VEGFR-2 ExD complex is immunologically precipitated by VEGFR-2-recognizing antibodies. The comprehensive method is illustrated by slight modification from the cited reference [11]

However, other ganglioside GD1a rather promoted the induction of VEGFR-2 signaling not blocking [16]. Thus, GD1a is pro-angiogenic, and the GD1a action is reversed by treatment with GM3 [17]. VEGFR-2 is composed of a transmembrane domain, a Tyr kinase region, and an extracellular domain. The extracellular domain (ExD) holds the VEGF-binding region and the dimerization site. Thus, GM3 directly recognize the VEGFR-2 ExD region for the VEGF–VEGFR-2-mediated behavior to exert anti-angiogenic effect. The carbohydrate and protein interaction is not easy to visualize from the experimental tools such as electrophoretic and blotting assay. For the exceptional cases which show the protein–carbohydrate interactions, two different analytic methods of the lectin blotting analysis and immunoblotting analysis using carbohydrate-specific antibodies are known to date. However, there has no direct assay to detect the glycolipid and proteins. In 2009, our group developed the direct assay to visualize the specific recognition of glycolipid GM3 with the ExD region of VEGFR-2. The comprehensive approach is illustrated in Fig. 13.3 from the cited reference [11]. The

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ExD of VEGFR-2 has been constructed using the expression vector with the myc-6x-His motif gene (Fig. 13.3a). GM3 is known to block VEGFR-2 function and activation by inhibiting VEGF–VEGFR binding and VEGFR dimerization. GM3 directly recognize the extracellular domain of the VEGFR [11]. Direct interaction of ganglioside GM3 with VEGFR-2 expressed on human umbilical vascular endothelial cells (HUVECs) has clearly demonstrated in immunoprecipitation and Western blot analysis. GM3 specifically recognizes the VEGFR-2 ExD which is featured with Ig-like domains. The in vitro recognition of ganglioside GM3 to VEGFR-2 protein demonstrates the carbohydrate–protein interaction. GM3 recognizes the VEGFR-2 ExD. The remaining third Ig-like domain is known as a VEGF recognition site. The IgG-like domain is located at the amino acids 219–330 positions in VEGFR-2 of human. The third IgG-like domain present on human VEGFR-2 carries β-sheets with eight strands and two short regions of α-helices [18]. GM3 seems to bind strongly to the specific amino acid of Asp-257 with the negative charge and the two Asn-259 and Ser-290 polar amino acids in VEGFR-2 via the hydrogen bonds and ionic bonds which are located on the concave surface. The hydroxyl group attached to the C-2 of Glc residue and SA residue is suggested to be necessary for inhibition of VEGFR-2 function. From the Biacore analysis, 3SL has clearly been known to recognize the second and third IgG-like domains present on VEGFR-2 ExD, and the second/third domains are the VEGF recognizing sites. Moreover, it has been demonstrated that the carbohydrate moiety of GM3 and sialyllactose (SL) recognizes the VEGFR-2 ExD at the molecular levels [18]. Therefore, SL can block the activation of VEGF-transduced VEGFR-2 by preferential interaction with its VEGF recognition site located on second/third IgG-like domains. SL blocks proliferation of VEGF-induced endothelial cells. Growth of the endothelial cells treated with VEGF is arrested with SL treatment with suppression of tube formation, migration, actin filament reorganization, neovascularization, and angiogenesis on tumor tissues allotransplanted with Lewis-type lung cancer, colon cancer, and melanoma cells. Using the VEGFR-2 ExD coding region and VEGFR-2 ExD protein, GM3 has been demonstrated to suppress direct binding to the VEGFR-2 ExD. The direct GM3 recognition with VEGFR-2 ExD is very interesting in its relationship between the structure and evolution. The VEGFR-2 ExD to recognize GM3 was IgG-like domain as verified using serially deleted mutants of VEGFR-2 ExD-encoding gene. In contrast, ceramide and lactosylceramide have no effect on VEGF responses to VEGFR-2 without the recognizing activity to VEGFR-2. Thus, carbohydrate moiety (Neu5Acα2,3Galβ1,4Glc) of GM3 blocks VEGFR-2 function stimulated by VEGF and interacts with VEGFR-2. Neu5Acα2,3Galβ1,4Glc treatment suppresses the level of VEGF-driven VEGFR-2 activation and its signaling event through binding to VEGFR-2, indicating that Neu5Acα2,3Galβ1,4Glc structure of GM3 is critical for interaction with VEGFR-2 ExD. GM3 recognizes VEGFR-2 ExD and Neu5Acα2,3Galβ1,4Glc oligosaccharide directly bind to VEGFR-2 ExD. It is reported that the EGFR and VEGFR structures and signaling are distinct [19]. However, the alignment analysis of two EGFR and VEGFR-2 sequences, which derived from the BLAST of National Center for Biotechnology Information, indicates

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existence of the common GM3 recognition sites both in EGFR and VEGFR-2. Homology of ExDs in the two receptor proteins has been identified in intracellular Tyr kinase motifs present both in EGFR and VEGFR-2. The three-dimensional crystal structure of the VEGFR-2 ExD–GM3 complexed form may give a solution. The in vivo anti-angiogenic GM3 function is also confirmed by the Matrigel plug tube formation and chicken chorioallantoic membrane experiments for VEGFmediated neovascularization. GM3 inhibits the new blood vessel formation in experimental animals of C57BL/6 mouse and VEGF-induced neovascularization in the chick chorioallantoic experiment. The GM3 anti-angiogenic activity is linked to the anti-tumor activity. GM3 abrogates the tumor growth in experimental model animals using the Lewis lung carcinoma-implanted C57BL/6 mice, where GM3 regresses primary Lewis lung tumor. Angiogenesis and proliferation of the Lewis lung tumor by GM3 indicate the negative angiogenic factor for tumor regression. Because the progression of solid tumors depends on angiogenesis through the activation of endothelial cells, VEGF secreted from tumor cells stimulates vascular endothelial cells. Anti-angiogenic inhibitor, GM3, blocks tumor angiogenesis through VEGF signaling blocking and direct VEGFR-2 binding. Gangliosides shed by tumor cells in the microenvironment can block the angiogenic microvessels formation triggered by angiogenic factors. VEGFR-based angiogenic behavior of vascular endothelium is operated by angiogenic factors such as VEGF released by tumors. Functions of many growth factor receptor (GFR)s are possibly regulated when they are present in the endothelial cell surfaces. If antiangiogenic molecules are expressed in the tumor microenvironmental tissue, tumor growth and progression are suppressed. As the candidate of antiangiogenic molecules, GM3 are produced by matured and differentiated macrophages. The GM3-induced differentiation of the promyelocyte lines of macrophage cells is also described in another chapter in this book. GM3 diminishes the endothelium growth triggered by neoplastic cells. GM3 blocks the proliferation when the endothelial cells such as HUVEC cells are preincubated with GM3. In addition, GM3 can block the fibronectin and collagen type (1 and 2) binding capacities of the endothelial cells. Matured and differentiated macrophages in tissue microenvironment secrete GM3 which blocks vascular endothelium. The nature of GM3 in the endothelial microenvironment is obviously demonstrated on the tumor-activated angiogenesis. The GM3 blocking of tumor-induced angiogenesis is summarized in Fig. 13.4. TAMs induce the angiogenesis, growth, immune suppression, and tumor metastasis events [20]. The function of TAMs is modulated by multiple factors produced by lymphocytes, stroma cells, and tumor cells resided in the tumor microenvironments. Biosynthesis of complex gangliosides enhances the expression levels of VEGF in tumor cells. Tumor-shed gangliosides directly induce endothelial angiogenesis. Among the stroma cell types present in the tumor microenvironment, macrophages are abundantly resided as tumor effector cells for tumor progression [21]. Angiogenic progression of tumor cells is stimulated by the interactive cooperation of endothelial cells, tumor cells, and infiltrating immune cells, therefore, consequently forming new types of blood vessel. From various immune cells, macrophages are frequently differentiated from circulatory monocytic cells and also recruited at cancerous

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Fig. 13.4 GM3 blocking of tumor-induced angiogenesis in the tumor microenvironmental tissue. Antiangiogenic GM3 is produced by healthy promyelo lines of macrophage cells. GM3 blocks the endothelium growth in neoplastic cells in human tumors. TAMs in the cancer growth, metastasis, and angiogenesis. The TAMs are modulated by lymphocytic, stromal, and cancer cells, in the TAMs. Angiogenesis of tumor cells is carried out by interaction of the endothelial and tumor cells and also by infiltrated immune cells. GM3 exerts anti-angiogenic or angiostatic effects

regions by attractive cytokines and chemotactic factors [22]. Tumor cells, overexpressing GalNAc transferase, produce complex GM1, GM2, and GD1a, which are necessary for angiogenic potentials of tumor cells. Such shed gangliosides of tumor cells activate the angiogenic induction, while a simple ganglioside GM3 inhibits the angiogenesis or angiostasis event [23]. For example, the level of tumor angiogenesis is reduced in mice i.v. administered with ganglioside-deficient or ganglioside-depleted cancer cells, which are constructed through double-negative KO cells of the ST-9 and Gal-T1 genes [24]. As the complex gangliosides generated induce the expression level of VEGF in mouse model bearing GalNAc transferase-expressed tumor cells, the same tumor cells exhibit the enhanced angiogenic potentials. Tumor-shed gangliosides also directly stimulate endothelial cells for angiogenesis. Tumor-infiltrating macrophages express several types of gangliosides such as o-series of GM1b, asialo-GM1, and GD1α. This o-series ganglioside phenotype is compared to those produced by peripheral macrophages [25]. Notably, nevertheless of the positive activity of blocking the GM3–VEGFR interacted downstream signaling to inhibit the tumor angiogenic metastasis, several evidences suggest specific and negative viewpoint that GM3 is associated with the potential pathogenetic role in diabetes mellitus with regard to insulin signaling

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suppression. Diabetic condition produces harmful glycans named advanced glycation products (AGE), increasing in contents of a-series gangliosides being the most GM3. GM3 is controversial for the renal and mesangial cell regeneration if it inactivates VEGFR and the VEGFR–Akt signalings [26]. GM3 is a pathological mediator of the diabetic nephropathy. GM3 blocks regenerative renal cells; however, the relation between GM3 levels and diabetic levels is still controversial [12, 27]. The GM3 role in the diabetic nephropathy is originated on the concept of lipid rafts. For insulin signaling, caveolae-linked lipid raft formation is explained, where sphingolipids and cholesterol form clusters [25]. Lipid raft-rich caveolae are 50–100 nm size in cell mosaic membranes formed by membranes such as caveolin [28] and then serve as receptor [29]. In the glycosynapse, GSLs associate with clusters [30]. AGEs are the featured phenotype and biochemically characteristic of diabetic patients [23]. In renal mesangial cells, AGE levels are correlated with the levels of GM1, GM3, and GM2, which are types of a-series gangliosides [26]. GM3 present in the renal tissues forms lipid rafts with GM1 [31], and GM3-associated lipid rafts are located on the microvilli sides in the apical renal cells, whereas GM1 lipid rafts are resided on the protrusion valleys on the membranes [32]. GM3 compromises the renal vascular endothelial regeneration, where GM3 has been linked with VEGF and Akt signaling towards renal dysfunction, as GM3 lipid rafts on human podocytes bind soluble VEGFR FLT1, promoting adhesion and rapid actin reorganization [33]. Of course, the cell-type and tissue-type specificity of GM3 should be further characterized in coming future. In addition, generally, it is broadly suggested that GM3 downregulates the several receptor functions implicated in vascular angiogenesis, where the target receptors include the EGFR, IGF-1R, PDGFR, VEGFR, b-FGFR, and integrin CAM molecules [12]. Furthermore, certain complex gangliosides of monosialyl to trisialyl gangliosides including GD3, GD1b, GD1a, GT1b, GM2, and GM1 induce the angiogenesis activities, while GM2 has the opposing activity [34]. These results implicate that GM3 has a valuable potential as future therapeutic target against tumor cell progression and vascular angiogenesis. For the molecular mechanistic elucidation of PCI, carbohydrate portion which directly interacts with receptor has been suggested in the recent study. Sialyllactose (SL) moiety in GM3 can be obtained from GM3 by enzymatic cleavage using ceramide glycanase (Fig. 13.5). The cleaved and liberated SL suppresses the phosphorylation of VEGF-induced VEGFR-2 via direct interaction with its ligand, VEGF-recognizing site. The precise position was the second and third domains like IgG domain of VEGFR-2. The biding blocks downstream signal activation and VEGF-stimulated proliferation of endothelial cells. SL blocked tube formation, migration, actin filament arrangement, VEGFinduced neovascularization, tumor growth, and angiogenesis in vivo. As an angiogenic regulator, VEGF also has an inflammatory cytokine-like function, which potentiates adhesion of leukocytes to vascular endothelial cells during inflammatory response. Ganglioside GM3 also suppresses the VEGF-activated production of VCAM-1 and ICAM-1 in HUVECs [35]. The VCAM-1/ICAM-1 gene expressions are upregulated by NF-κB through protein kinase B (PKB/AKT) downstream signaling, and they recruit myeloid leukocytes to endothelial cells in the

13.2

FLK1/KDR (VEGFR-2)–GM3 Interaction

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Fig. 13.5 Sialyllactose (SL) moiety is liberated from GM3 by ceramide glycanase

inflamed tissue sites. In fact, GM3 remarkably block the monocyte adhesion to HUVECs in the in vitro monolayer adhesion assay. In in vivo animal model, VEGF i.v.-administered mice exhibit the inflammation phenotype, but GM3 ganglioside injection dramatically reduces the levels of VCAM-1/ICAM-1 expressions in the mice blood vein tissues (Fig. 13.6). The VCAM-1 and ICAM-1 gene and protein expressions are inhibited by treatment of exogenous GM3 only but not by other types of gangliosides including neutral GSLs, GD1b, GD2, GD1a, GM1, and GM2 in the VEGF-treated HUVECs. GD1a enhances proliferation and migration potentials in the VEGF-treated HUVECs [36]. In tumor cells, ganglioside-deficient cells, which generated via the double knockout of the GM3 synthase gene, Siat9 gene, GM2 synthase gene, and Galgt1 gene, exhibit the retarded growth rates, compared with the normal tumor cells [37]. Ganglioside-poor double-deleted tumors enhance angiogenesis and tumor growth but no difference in production of VEGF or other angiogenic molecules [24]. Thus, certain ganglioside forms are responsible for angiogenesis and VEGF-triggered signaling of vascular endothelial cells. For ganglioside structure and angiogenic relationship, GM3 but not GD3, GM1 and GD1a present in normal tumor cells, inhibits interaction between growth factors and normal stromal cells, to respond [38, 39]. Therefore, ganglioside GM3 exhibits an anti-inflammatory function through blocked production of pro-inflammatory molecules in in vitro and in vivo inflammation.

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Fig. 13.6 GM3 inhibits adhesion of leukocytes to vascular endothelial cells during inflammatory response. Ganglioside GM3 suppresses the VEGF-activated VCAM-1/VCAM-1 biosynthesis in HUVECs. The ICAM-1/VCAM-1 is expressed through NF-κB signaling by PKB(AKT) signaling and recruits leukocyte to endothelial cells. GM3 blocks the monocyte adhesion to HUVECs

References 1. Miyake T, Kumasawa K, Sato N, Takiuchi T, Nakamura H, Kimura T (2016) Soluble VEGF receptor 1 (sFLT1) induces non-apoptotic death in ovarian and colorectal cancer cells. Sci Rep 6:4853 2. Sallinen H et al (2009) Antiangiogenic gene therapy with soluble VEGFR-1, VEGFR-2, and VEGFR-3 reduces the growth of solid human ovarian carcinoma in mice. Mol Ther 17:278–284 3. Shibuya M (2013) Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases. J Biochem 153(1):13–19 4. Jin J, Sison K, Li C, Tian R, Wnuk M, Sung HK, Jeansson M, Zhang C, Tucholska M, Jones N, Kerjaschki D, Shibuya M, Fantus IG, Nagy A, Gerber HP, Ferrara N, Pawson T, Quaggin SE (2012) Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function. Cell 151(2):384–399 5. Svennerholm L (1980) Ganglioside and synaptic transmission. Plenum, New York 6. Kaucic K, Liu Y, Ladisch S (2006) Modulation of growth factor signaling by gangliosides: positive or negative? Methods Enzymol 417:168–185 7. Liu Y, McCarthy J, Ladisch S (2006) Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling. Cancer Res 66:10408–10414 8. Li T, Kang G, Wang T, Huang H (2018) Tumor angiogenesis and anti-angiogenic gene therapy for cancer. Oncol Lett 16(1):687–702 9. Karaman S, Leppänen VM, Alitalo K (2018) Vascular endothelial growth factor signaling in development and disease. Development 145(14):dev151019

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10. Hicklin DJ, Ellis LM (2005) Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 23(5):1011–1027 11. Chung TW, Kim SJ, Choi HJ, Kim KJ, Kim MJ, Kim SH, Lee HJ, Ko JH, Lee YC, Suzuki A, Kim CH (2009) Ganglioside GM3 inhibits VEGF/VEGFR-2-mediated angiogenesis: direct interaction of GM3 with VEGFR-2. Glycobiology 19:229–239 12. Seyfried TN, Mukherjee P (2010) Ganglioside GM3 is antiangiogenic in malignant brain cancer. J Oncol 2010:961243 13. Wang H, Isaji T, Satoh M, Li D, Arai Y, Gu J (2012) Antitumor effects of exogenous ganglioside GM3 on bladder cancer in an orthotopic cancer model. Urology 81:210. e.11–210.e.15 14. Choi KS, Bae MK, Jeong JW, Moon HE, Kim KW (2003) Hypoxia-induced angiogenesis during carcinogenesis. J Biochem Mol Biol 36:120–127 15. Álvarez-Aznar A, Muhl L, Gaengel K (2017) VEGF receptor tyrosine kinases: key regulators of vascular function. Curr Top Dev Biol 123:433–482 16. Coskun Ü, Grzybek M, Drechsel D, Simons K (2011) Regulation of human EGF receptor by lipids. Proc Natl Acad Sci USA 108(22):9044–9048 17. Mukherjee P, Faber AC, Shelton LM, Baek RC, Chiles TC, Seyfried TN (2008) Thematic review series: sphingolipids. ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a. J Lipid Res 49(5):929–938 18. Chung TW, Kim EY, Kim SJ, Choi HJ, Jang SB, Kim KJ, Ha SH, Abekura F, Kwak CH, Kim CH, Ha KT (2017) Sialyllactose suppresses angiogenesis by inhibiting VEGFR-2 activation, and tumor progression. Oncotarget 8(35):58152–58162 19. Hiller NJ, Silva NAAE, Faria RX et al (2018) Synthesis and evaluation of the anticancer and trypanocidal activities of boronic tyrphostins. ChemMedChem 13(14):1395–1404 20. Shapouri-Moghaddam A, Mohammadian S, Vazini H et al (2018) Macrophage plasticity, polarization, and function in health and disease. J Cell Physiol 233(9):6425–6440 21. Ruffell B, Affara NI, Coussens LM (2012) Differential macrophage programming in the tumor microenvironment. Trends Immunol 33:119–126 22. Mantovani A, Locati M (2013) Tumor-associated macrophages as a paradigm of macrophage plasticity, diversity, and polarization: lessons and open questions. Arterioscler Thromb Vasc Biol 33(7):1478–1483 23. Alessandri G, Cornaglia-Ferraris P, Gullino PM (1997) Angiogenic and angiostatic microenvironment in tumors – role of gangliosides. Acta Oncol 36(4):383–387 24. Liu Y, Wondimu A, Yan S, Bobb D, Ladisch S (2014) Tumor gangliosides accelerate murine tumor angiogenesis. Angiogenesis 17:563–571 25. Nakagawa R, Serizawa I, Motoki K et al (2000) Antitumor activity of alphagalactosylceramide, KRN7000, in mice with the melanoma B16 hepatic metastasis and immunohistological study of tumor infiltrating cells. Oncol Res 12(2):51–58 26. Vukovic I, Bozic J, Markotic A, Ljubicic S, Ticinovic KT (2015) The missing link – likely pathogenetic role of GM3 and other gangliosides in the development of diabetic nephropathy. Kidney Blood Press Res 40(3):306–314 27. Kwak DH, Rho YI, Kwon OD et al (2003) Decreases of ganglioside GM3 in streptozotocininduced diabetic glomeruli of rats. Life Sci 72(17):1997–2006 28. Liu L, Pilch PF (2008) A critical role of cavin (polymerase I and transcript release factor) in caveolae formation and organization. J Biol Chem 283:4314–4322 29. Sowa G (2012) Caveolae, caveolins, cavins, and endothelial cell function: new insights. Front Physiol 2:120 30. Hakomori SI (2008) Structure and function of glycosphingolipids and sphingolipids: recollections and future trends. Biochim Biophys Acta 780:325–346 31. Aguilar RP, Genta S, Sánchez S (2008) Renal gangliosides are involved in lead intoxication. J Appl Toxicol 28(2):122–131 32. Chen Y, Qin J, Chen ZW (2008) Fluorescence-topographic NSOM directly visualizes peakvalley polarities of GM1/GM3 rafts in cell membrane fluctuations. J Lipid Res 49:2268–2275

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33. Jin J, Sison K, Li C, Tian R, Wnuk M, Sung HK et al (2012) Soluble FLT1 binds lipid microdomains in podocytes to control cell morphology and glomerular barrier function. Cell 151:384–399 34. Kucerova L, Zmajkovic J, Toro L, Skolekova S, Demkova L, Matuskova M (2015) Tumordriven molecular changes in human mesenchymal stromal cells. Cancer Microenviron 8 (1):1–14 35. Kim SJ, Chung TW, Choi HJ, Jin UH, Ha KT, Lee YC, Kim CH (2014) Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Biol 46:32–38 36. Wei Q, Zhang F, Richardson MM et al (2014) CD82 restrains pathological angiogenesis by altering lipid raft clustering and CD44 trafficking in endothelial cells. Circulation 130 (17):1493–1504 37. Liu Y, Yan S, Wondimu A, Bob D, Weiss M, Sliwinski K, Villar J, Notario V, Sutherland M, Colberg-Poley AM, Ladisch S (2010) Ganglioside synthase knockout in oncogenetransformed fibroblasts depletes gangliosides and impairs tumor growth. Oncogene 29:3297–3306 38. Uentes D, Avellanet J, Garcia A et al (2010) Combined therapeutic effect of a monoclonal antiidiotype tumor vaccine against NeuGc-containing gangliosides with chemotherapy in a breast carcinoma model. Breast Cancer Res Treat 120(2):379–389 39. Wang H, Isaji T, Satoh M, Li D, Arai Y, Gu J (2013) Antitumor effects of exogenous ganglioside gm3 on bladder cancer in an orthotopic cancer model. Urology 81:210 e211–210 e215

Chapter 14

GM3, Competing with GM1, Interaction with Urokinase Plasminogen Activator Receptor (uPAR) in Endothelial Caveolar-Lipid Rafts Inhibits Angiogenesis

The tumorigenic potential is related to the gangliosides [1, 2]. Among them, it was reported that many tumors express GM1 [3] and GM1 exert its pro-angiogenic activity. GM1 recruits tumor-produced soluble urokinase plasminogen activator receptor (uPAR) on HUVEC lipid rafts to trigger angiogenesis [4]. For valance of tumor cells and normal cells, GM1-dependent angiogenesis seems to be regulated by GM3 anti-angiogenesis [5, 6]. From the above chapters, GM3 is suggested as a non-aggressive tumor behavior, and this anti-tumor behavior can be explained from the facts of the anti-angiogenesis properties and blocking capacity of a caveolar-lipid rafts formation of uPAR. For tumor progressing capacity, it was known that GM1 recognizes uPAR more strongly than GM3. Such gangliosides and uPAR participate in microdomain lipid rafts formation. uPAR in caveolar-lipid rafts is essential for angiogenic reaction in endothelial cells [7, 8], as this process is similar to vascular angiogenesis in tumors [9]. uPAR in caveolar-lipid rafts has been shown to be recruited to angiogenesis-related system of endothelial cells [7] and caveolar-lipid rafts of HUVEC [4] and interacted with GM1 and GM3. Then, uPAR functions as an angiogenic initiation factor upon recruited to caveolar-lipid rafts. Among GM1 and GM3, caveolar-lipid rafts enriched with GM1 is pro-angiogenic, and GM3 is antiangiogenic, as demonstrated in the recent paper [10]. The uPAR interaction with lipid rafts with GM1 or GM3, on the artificial bilayers with lipid raft on solid polar surfaces, was analyzed by a method of surface plasmon resonance (SPR). This shows the recruited uPAR levels because uPAR can be clustered with GM1 or GM3 in caveolar rafts. However, GM3–uPAR complexes are readily detached in presence of GM1, and consequently GM1 promotes angiogenesis [5, 6]. However, GM3 exhibits an anti-angiogenesis activity in the same system [5]. From the molecular interaction between uPAR–GM1 and uPAR–GM3, it was shown that uPAR binds preferentially to GM1-enriched artificial matrix and this uPAR-GM1 interaction exerts pro-angiogenic capacity, as examined by Matrigel invasion and capillary morphogenesis. However, uPAR–GM3 interaction showed the antiangiogenesis activity. Moreover, exogenous GM1 treatment with the caveolar rafts depleted the GM3 by preference of GM1 towards uPAR, consequently initiating © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_14

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angiogenesis. Under resting vascular microenvironments, uPAR is localized in GM3 non-caveolar-lipid rafts. When GM1 is enriched, the uPAR–GM1 is complexed by detaching uPAR from the uPAR–GM3 non-caveolar-lipid rafts. Therefore, GM1 treatment stimulates the formation of uPAR–GM1 caveolar-lipid rafts to promote endothelial invasion and capillary angiogenesis through angiogenic MAPK phosphorylation. GM1-dependent uPAR localization in caveolar-lipid rafts indicates GM1-dependent tropism to angiogenesis.

References 1. Dam DHM, Paller AS (2018) Gangliosides in diabetic wound healing. Prog Mol Biol Transl Sci 156:229–239 2. Prinetti A, Aureli M, Illuzzi G et al (2010) GM3 synthase overexpression results in reduced cell motility and in caveolin-1 upregulation in human ovarian carcinoma cells. Glycobiology 20:62–77 3. Zheng C, Terreni M, Sollogoub M, Zhang Y (2019) Ganglioside GM3 and its role in cancer. Curr Med Chem 26(16):2933–2947 4. Rao JS, Gujrati M, Chetty C (2013) Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis. Oncogenesis 2:e53 5. Koochekpour S, Merzak A, Pilkington GJ (1996) Vascular endothelial growth factor production is stimulated by gangliosides and tgf-beta isoforms in human glioma cells in vitro. Cancer Lett 102:209–215 6. Ziche M, Morbidelli L, Alessandri G et al (1992) Angiogenesis can be stimulated or repressed in vivo by a change in GM3:GD3 ganglioside ratio. Lab Investig 67:711–715 7. Margheri F, Chillà A, Laurenzana A et al (2011) Endothelial progenitor cell-dependent angiogenesis requires localization of the full-length form of uPAR in caveolae. Blood 118:3743–3755 8. Hunt NJ, Lockwood GP, Warren A et al (2019) Manipulating fenestrations in young and old liver sinusoidal endothelial cells. Am J Physiol Gastrointest Liver Physiol 316(1):G144–G154 9. Li Calzi S, Neu MB, Shaw LC et al (2010) EPCs and pathological angiogenesis: when good cells go bad. Microvasc Res 79:207–216 10. Margheri F, Papucci L, Schiavone N, D’Agostino R, Trigari S, Serratì S, Laurenzana A, Biagioni A, Luciani C, Chillà A, Andreucci E, Del Rosso T, Margheri G, Del Rosso M, Fibbi G (2015) Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis. J Cell Mol Med 19(1):113–123

Chapter 15

GM3 Interacts with TGFβRs in the Epithelial–Mesenchymal Transition (EMT) During Posterior Capsular Opacification (PCO) Formation

Epithelial–mesenchymal transition (EMT) is a scheduled programing and differentiating transition from epithelial phenotype to mesenchymal phenotype. The EMT event is, therefore, the programmed cellular process for differentiation transition from epithelial to mesenchymal type cells. EMT involves in multiple cellular events including development, cataract, wound healing, mobility, tissue fibrosis, and tumor invasion [1, 2]. In lens development, EMT induces the proliferation and transdifferentiation in lens epithelial cells, causing cataract. TGF-β induces EMT. The EMT involves development, cataract, mobility, invasion, wound healing, and tissue fibrosis [1]. Among them, several clinical outcomes including cataract surgery complication, posterior capsular opacification (PCO), and anterior subcapsular cataract (ASC) are accompanied by the known EMT process initiated by lens epithelial cells [2]. The lens epithelial cells are transdifferentiated to mesenchymal-like cells such as myofibroblasts with the phenotypes such as membrane presence of fibronectin, type 1 collagen, and α-smooth muscle actin (SMA) [2, 3]. PCO is frequently caused by the EMT process in lens epithelial cells through mesenchymal-like transdifferentiation of myofibroblasts and expression of the SMA, extracellular type 1 collagen, and fibronectin in ocular tissues. Lens epithelial cells-mediated PCO event is promoted by certain growth factors including EGF, FGF, HGF, and TGF-β [4–7]. The TGF-β signaling involves several biochemical processes such as the TGFβ/Smads signaling pathway. As a factor of scleroderma and tissue fibrosis, TGF-β induces EMT event and extracellular matrix (ECM) production with expression of type 1 collagen and fibronectin in ocular tissues [1, 7]. Upon TGF-β treatment, TGF-β internalizes the target cells through phosphorylation of Ser/Thr residues present on TGF-β receptor I (TGFβRI) after pairing with TGFβRI and TGFβRII. The phosphorylated TGFβRI activates receptor-activated Smad2/Smad3 (R-Smad) [8]. The activated Smad2/Smad3 binds to the central mediator named Smad4 and translocates to the nucleus region. Smad complex induces EMT-related multifunctional gene [9]. TGF-β-related Smad also involves EMT-induced human

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lens epithelial (HLE) [10]. During the posterior capsular and anterior polar cataracts, TGF-β essentially act for EMT event, changes in cellular phenotype and production of ECM, which contains the type 1 collagen and fibronectin via new synthesis in ocular tissues [1, 2]. Ceramide, glycan-absent sphingolipid, induces apoptosis in bovine lens epithelial cells [11]. GSLs and gangliosides are deposited in cataract lens tissues of human [12, 13]. To elucidate the relation of sphingolipid and cataract progression is important for roles of TGF-β and GM3 in EMT-induced HLE cells. HLE cells express increased levels of ST3Gal-5 with concomitant accumulation of a-series GSL GM3 in TGF-β1-induced EMT-like progression. In parallel, silencing ST3Gal5 in the lens epithelial cells actively inactivate the functions of Smad2/Smad3. These findings suggest that TGF-β1R stimulation and downstream signaling need GM3 ganglioside [14]. TGF-β-induced EMT stimulates the proliferation and migrative activity of the HLE cells caused by the PCO after cataract surgery. The interaction of GM3 and TGFβR has also reported [14], because TGF-β-elicited EMT of the HLE cells is a great interest of GM3 binding with TGFβR. It has been clearly shown that GM3 participates in TGF-β1-induced EMT towards HLE. GM3 level is clearly upregulated in TGF-β1-stimulated HLE B-3 cells, as certified in thin layer chromatography and immunofluorescence. From the ganglioside GM3-depleted experiment using d-PDMP and shGM3S, it is known that the GM3 production regulates the TGF-β-promoted migration and EMT-associated signaling in HLE cells. In the exogenously treated cell with GM3, the activation of TGF-βR and Smad2/Smad3 was recovered from depletion of GM3 during TGF-β1-elicited EMT in the HLE B-3 cells. GM3 binds to TGFβR in TGF-β1-activated HLE B-3 cells. Cellular GM3 stimulated by TGF-β1 participates in the EMT by binding to TGFβR species during PCO formation (Fig. 15.1). Function of GM3 is interested in TGF-β-mediated EMT in lens epithelial cells of human. GM3 upregulates the migration potentials and EMT-driven signaling in TGF-β-promoted lens epithelial cells. The inhibition of the activated TGFβR and Smad2/Smad3 is reversed when GM3 was exogenously treated to the HLE B-3 cells depleted with GM3 during TGF-β1-mediated EMT process. GM3 recognizes TGF-β1-activated TGFβR in HLE B-3 cells. Ganglioside GM3 binds to TGFβR present in EMT-driven HLE cells. Therefore, it is concluded that GM3 involves in EMT-driven lens epithelial cells through binding to TGFβR species. For example, in mammary epithelial NM18 cells, GM3 is accumulated after prolonged exposure to TGF-β1. When GM3 is exposed to TGF-β1 in the Zeb1- or ST3Gal-5-silenced cells, Smad2 and Smad3 functions are remarkably stimulated, and additional Smad2 phosphorylation is reduced [15]. The phosphorylated TGFβRI stimulates the downstream receptor-stimulated Smads known as R-Smads. More specifically, Smad2 and Smad3 are targeted [8, 16–19]. The targeted Smad2/Smad3 binds to the downstream signaling molecules such as specificity protein 1 (Sp1), p300, and Co-Smad, and the Smad complex translocates to the nuclear region to express EMT-regulated target genes of E-cadherin and vimentin [10, 20, 21]. In contrast, the two forms of R-Smads and

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A

TGF- 1 GM3 Ab

+

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+ IgM

IP : Anti-GM3 Ab IB : Anti-TGF R I Ab

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TGF R II

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TGF1

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IP : Anti-TGF RII Ab Gangliosides I-TLC : Anti-GM3 Ab purification I-TLC : Anti-GM3 Ab

Fig. 15.1 GM3 bind with TGF-β1-induced TGFβR in EMT in human epithelial cells of lens

Smad4 are located in the nuclear region, and they recruit co-activators/repressors. The complexed forms recognize the cis-sites of promoters of target genes [18]. The Smad3/Smad4 complex recognizes a specific promoter DNA sequence with the Smad-binding element (SBE) of the nucleotide sequence 50 -GTCT-30 for SmadsDNA affinity binding [22, 23]. GSLs regulate the EMT process. GSL, GM3, and TGF-β are associated with EMT in HLE cells. It has been known that GM3 enhances the EMT of the TGF-β1regulted HLE cells [14]. GM3 also induce the TGF-β1-promtoed growth level of glomerular mesangial cells [24]. In TGF-β1-drived HLE cells, the gene expression of GM3 synthase is activated by the transcriptional upregulation activated by Sp1 transcription factor. GM3 binds to TGFβR species as a phenotype of TGF-β1-driven EMT event and a migration phenotype of HLE cells. If the GM3 production is inhibited, TGF-β1-drived EMT event and migration phenotype of HLE B-3 cells are downregulated because the membrane GM3 interacts with TGFβRs. During EMT process, the GM1, GM3, and GD1a are all found in human cataractous lenses. In human normal bladder cells and other normal MMGCs, gangliotetraosylceramide (Gg4) and GM2 levels but not GM1 and GM3 levels are reduced during TGF-β1driven EMT event [25]. Presumably, GM2 and Gg4 inhibit the TGFβ-induced EMT event, while GM3 and GM1 can activate TGFβ-induced EMT. In contrast, another GSL, Gg4 level, is decreased in the TGF-β-elicited EMT process using the normal mammary gland cells (MGCs) of mouse with the decreased

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transcriptional expression of the β1,3Gal-T4 gene, which encodes UDP-Gal:β1,3Gal-transferase-4 [26]. The β1,3Gal-T4 enzyme synthesizes Gg4 from Gg3. The Gg4 level was reduced, but other GM1, GD1a, and FucGM1 are not changed, although GM1 is also synthesized by the β3GalT4 gene [27]. Different acceptor GSLs present in Golgi can point out the difference between Gg4 and GM1 generation [28]. The decreased β1,3Gal-T4 expression in the TGF-β-elicited EMT process of human breast cancer cells and normal MGCs is linked with the progression of breast tumor cells. The SBE site of 50 -GTCTAGAC-30 nucleotide sequence at the 788 and 795, corresponding to the transcription start point of the β1,3Gal-T4 gene, is also implicated for Smad binding. Smads are essential for the EMT event, and normal MGCs largely express Smad3 or Smad4. The Smad3/Smad4-positive cells are susceptible for TGF-β sensing. Smad-4 and R-Smads docking shuttles between the nuclear region and cytoplasmic region. Activated Smads complex is lowly present in normal cells without phosphorylation [29]. Thus, the enhanced expression of Smads such as Smad3/Smad4 in normal MGCs contributes to expression of the biomarker proteins in mesenchymal cells, including vimentin and N-cadherin. The decreased level of the expressed epithelial biomarkers of E-cadherin and β-catenin acts as the intracellular signal transducers, and the signaling is also caused by the increased expression of the Smads of Smad3/Smad4. Therefore, expression of EMT markers is modulated by the transcription factor of Smad species [30]. During TGF-β-elicited EMT event observed in normal MMGCs, the β1,3Gal-T4 gene expression and Gg4 synthesis levels are suppressed because Gg4 form functions as an EMT suppressor through Gg4-binding to E-cadherin and β-catenin [31]. Activation of the function of Smad3/Smad4 complex contributes to the inhibited expression of β1,3Gal-T4 in TGF-β-treated cells. The levels of β-catenin, Gg4, and E-cadherin expression are all decreased in the TGF-β-induced target cells. Exogenous TGF-β induces the Smad complex formation to activate the nuclear translocation of the Smad complex to target gene expression for β1,3Gal-T4 during the TGF-β/Smads-mediated downstream signalings. Activation of the formed Smad3/Smad4 complex leads to the inhibited β1,3Gal-T4 gene expression. These results imply that Gg4 importantly regulates the TGFβ/Smads signaling [32]. Other GSLs and Gg4 involve in the TGF-β-driven EMT events. Thus, Gg4 can modulate the EMT event through its binding to E-cadherin and β-catenin of the normal MGCs, as the Gg4 level is reduced in the EMT cell surfaces when EMT is induced by TGF-β in the cell surfaces. The transcriptional expression level of β1,3Gal-T4 is also reduced in human breast cancer patients. The promoter sequence of the β1,3GalT4 gene consists of a putative region of the typical SBE for the Smad4 transcription factor during TGF-β responses, forming a complex with Smad3. The formed Smad3/ Smad4 complex inhibits the gene expression of β1,3Gal-T4 during EMT in normal MGCs. The formed Smad3/Smad4 complex recognizes directly the SBE site on β1,3Gal-T4 gene promoter region. TGF-β decreases the Gg4 level and β3GalT4 mRNA level in Smad3- and Smad4-expressing cells with the decreased expression

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levels of the other epithelial marker proteins of β-catenin and E-cadherin. Thus, the activation of the complex of Smad3/Smad4 blocks the expression levels of the Gg4 synthase gene and β1,3Gal-T4 gene expression via direct interaction with the promoter region of β1,3Gal-T4 gene. GM3 levels are regulated in the TGF-β1-elicited EMT process of epithelial lens cells in human. The level of GM3 product is elevated with the concomitant increase in the migrative potentials of cells and EMT-driven signal transduction in TGF-β1driven epithelial lens cells. The transcriptional upregulation of GM3 synthase gene promoter is also mediated by Sp1 in the HLE B-3 cells upon TGF-β1 induction. GM3 interacts with the TGF-β1-elicited TGFβR and is associated with TGF-β1driven EMT event with TGFβRs. GM1 and GM3 gangliosides are known to be highly expressed in human cataract tissues [33, 34]. The GM3 level is upregulated in TGF-β1-driven HLE B-3 cells through the 50 -flanking region in promoter sequence of the GM3 synthase transcription, which is located within TGF-β1-inducible promoter site. The 50 -flanking region located between nucleotide 432 and 178 acts as the essential transcriptional regulation site of the GM3 synthase gene promoter in TGF-β1-driven cells. The element CREB, termed p300/CBP, is responsible for TGF-β downstream signaling via Smad2 modification [35]. Sp1 as a TGF-β1-specific element is a zinc-finger transcription factors and acts as a Smad co-factor to regulate the TGF-β1-driven EMT event and consequent cellular migration [23, 36]. Sp1 also activates the transcription of GM3 synthase gene in the TGF-β1-driven EMT process in the epithelial HLE B-3 cells. Caveolin-1 molecule in the lens epithelial cells of human also involves in the EMT process [37]. TGF receptors including TGFβRI and TGFβRII present in lipid rafts microdomain activate MAPK activity in epithelial cells when TGF-β1 induces the EMT event [38, 39]. In TGF-β1-elicited EMT process of epithelial cells, the TGFβRI and TGFβRII phosphorylation upregulates downstream signaling of Smad2/Smad3 to produce the ECM components. After TGFβR downstream activation, TGF-β-driven epithelial cells are transdifferentiated [40, 41]. In the Ser phosphorylation of TGFβRI, GM3 depletion blocks TGFβRII signaling and Smad2/ Smad3 function in TGF-β-elicited HLE B-3 cells. GM3 biosynthesis is positively regulated by TGFβR signaling during TGF-β1-driven EMT process. Therefore, it is concrete that GM3 is stringently involved in EMT event progression in the TGF-β1elicited states in HLE B-3 cells. In TGF-β-treated cells, the Ser/Thr residue-induced kinase domain on TGFβRI binds to TGFβRII, and the stimulated TGFβRs internalize epithelial cells to lead to EMT event [42]. Ser-phosphorylation of TGFβRs occurs in the TGF-β1-elicited HLE-B-3 cells, but GM3 downregulates the phosphorylation of Ser residue in Smad2/Smad3, TGFβRI, and TGFβRII in the TGF-β1driven HLE B-3 cells. Additionally, GM3 enhances the binding to TGFβR in the TGF-β1-driven HLE B-3 cells (Fig. 15.2). Therefore, the results indicate the functional variability of GSL distribution and expression in each cell system, despite a common expression of ST3Gal V gene during TGF-β1-mediated cellular function.

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GM3 Interacts with TGFβRs in the Epithelial–Mesenchymal Transition. . .

TGF- 1

GM3

P

TGF RII

TGF RI

Smad2/3

P

GM3

Sp-1

Nucleus

GM3 synthase gene

Promoter -432 -178

Fig. 15.2 Interaction between GM3 and TGFβRI and TGFβRII in EMT in human epithelial cells of lens. Schematic illustration of the EMT-involved GM3 expression in lens epithelial cells is shown. GM3 synthesized by GM3 synthase through TGF-β1-activated Smad2/Smad3-dependent Sp1 binds to TGFβRs in human epithelial cells of lens. GM3 involved in TGF-β1-driven EMT event is transcriptionally synthesized by GM3 synthase gene through the Smad2/Smad3 signaling in the TGF-β1-driven HLE B-3 cells. GM3 confers the cellular migration potentials and EMT-mediated signaling event during TGF-β1-elicited EMT process. GM3 interacts with TGF-β1-triggered TGFβRs. GM3 is a therapeutic target in EMT-induced cataracts

References 1. Thiery JP (2003) Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol 15:740–746 2. Lee EH, Joo CK (1999) Role of transforming growth factor-beta in transdifferentiation and fibrosis of lens epithelial cells. Invest Ophthalmol Vis Sci 40:2025–2032 3. Lovicu FJ, Schulz MW, Hales AM, Vincent LN, Overbeek PA, Chamberlain CG, McAvoy JW (2002) TGFbeta induces morphological and molecular changes similar to human anterior subcapsular cataract. Br J Ophthalmol 86:220–226 4. Saint-Geniez M, Kurihara T, D’Amore PA (2009) Role of cell and matrix-bound VEGF isoforms in lens development. Invest Ophthalmol Vis Sci 50(1):311–321 5. VanSlyke JK, Boswell BA, Musil LS (2018) Fibronectin regulates growth factor signaling and cell differentiation in primary lens cells. J Cell Sci 131(22):jcs217240 6. Choi J, Park SY, Joo CK (2004) Hepatocyte growth factor induces proliferation of lens epithelial cells through activation of ERK1/2 and JNK/SAPK. Invest Ophthalmol Vis Sci 45:2696–2704 7. Nanu RV, Ungureanu E, Instrate SL et al (2018) An overview of the influence and design of biomaterial of the intraocular implant of the posterior capsule opacification. Rom J Ophthalmol 62(3):188–193

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8. Heldin CH, Moustakas A (2016) Signaling receptors for TGF-β family members. Cold Spring Harb Perspect Biol 8(8):a022053 9. Zhou XL, Xu P, Chen HH et al (2017) Thalidomide inhibits TGF-β1-induced epithelial to mesenchymal transition in alveolar epithelial cells via smad-dependent and smad-independent signaling pathways. Sci Rep 7(1):14727 10. Wormstone IM, Tamiya S, Eldred JA et al (2004) Characterisation of TGF-beta2 signalling and function in a human lens cell line. Exp Eye Res 78(3):705–714 11. Lim JY, Kumar P, Lee YI (2008) Investigation on C2-ceramide complexes with transition metal ions using electrospray ionization tandem mass spectrometry. Eur J Mass Spectrom (Chichester) 14(2):87–97 12. Ogiso M (1998) Implication of glycolipids in lens fiber development. Acta Biochim Pol 45 (2):501–507 13. Saito M, Sugiyama K (2000) Gangliosides of rat eye lens: a severe reduction in the content of C-series gangliosides following streptozotocin treatment. Life Sci 67(15):1891–1899 14. Kim SJ, Chung TW, Choi HJ, Kwak CH, Song KH, Suh SJ, Kwon KM, Chang YC, Park YG, Chang HW, Kim KS, Kim CH, Lee YC (2013) Ganglioside GM3 participates in the TGF-β1induced epithelial-mesenchymal transition of human lens epithelial cells. Biochem J 449 (1):241–251 15. Mathow D, Chessa F, Rabionet M, Kaden S, Jennemann R, Sandhoff R, Gröne HJ, Feuerborn A (2015) Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism. EMBO Rep 16(3):321–331 16. Li W, Li W, Zou L et al (2017) Membrane targeting of inhibitory Smads through palmitoylation controls TGF-β/BMP signaling. Proc Natl Acad Sci USA 114(50):13206–13211 17. Meng F, Li J, Yang X, Yuan X, Tang X (2018) Role of Smad3 signaling in the epithelialmesenchymal transition of the lens epithelium following injury. Int J Mol Med 42(2):851–860 18. Chen B, Huang S, Su Y et al (2019) Macrophage Smad3 protects the infarcted heart, stimulating phagocytosis and regulating inflammation. Circ Res 125(1):55–70 19. Sanders YY, Cui Z, Le Saux CJ et al (2015) SMAD-independent down-regulation of caveolin-1 by TGF-β: effects on proliferation and survival of myofibroblasts. PLoS One 10(2):e0116995 20. ChoiJ PSY, Joo CK (2007) Transforming growth factor-β1 represses E-cadherin production via slug expression in lens epithelial cells. Invest Ophthalmol Vis Sci 48:2708–2718 21. Jungert K, Buck A, von Wichert G, Adler G, König A, Buchholz M, Gress TM, Ellenrieder V (2007) Sp1 is required for transforming growth factor-β-induced mesenchymal transition and migration in pancreatic cancer cells. Cancer Res 67:1563–1570 22. Xu F, Liu C, Zhou D, Zhang L (2016) TGF-β/SMAD pathway and its regulation in hepatic fibrosis. J Histochem Cytochem 64(3):157–167 23. Shi Y, Wang YF, Jayaraman L, Yang H, Massagué J, Pavletich NP (1998) Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell 94:585–594 24. Guan F, Handa K, Hakomori SI (2009) Specific glycosphingolipids mediate epithelial-tomesenchymal transition of human and mouse epithelial cell lines. Proc Natl Acad Sci USA 106(18):7461–7466 25. Jacob F, Alam S, Konantz M et al (2018) Transition of mesenchymal and epithelial cancer cells depends on α1-4 galactosyltransferase-mediated glycosphingolipids. Cancer Res 78 (11):2952–2965 26. Guan F, Schaffer L, Handa K, Hakomori SI (2010) Functional role of gangliotetraosylceramide in epithelial-to-mesenchymal transition process induced by hypoxia and by TGF-(beta). FASEB J 24:4889–4903 27. Nishio M, Tajima O, Furukawa K, Urano T, Furukawa K (2005) Over-expression of GM1 enhances cell proliferation with epidermal growth factor without affecting the receptor localization in the microdomain in PC12 cells. Int J Oncol 26(1):191–199

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28. Giraudo CG, Daniotti JL, Maccioni HJ (2001) Physical and functional association of glycolipid N-acetyl-galactosaminyl and galactosyl transferases in the Golgi apparatus. Proc Natl Acad Sci USA 98:1625–1630 29. Inman GJ, Nicolas FJ, Hill CS (2002) Nucleocytoplasmic shuttling of Smads 2, 3, and 4 permits sensing of TGF-beta receptor activity. Mol Cell 10:283–294 30. Ma M, Fu Y, Zhou X, Guan F, Wang Y, Li X (2019) Functional roles of fucosylated and O-glycosylated cadherins during carcinogenesis and metastasis. Cell Signal 63:109365 31. Sarkar TR, Battula VL, Werden SJ et al (2015) GD3 synthase regulates epithelial-mesenchymal transition and metastasis in breast cancer. Oncogene 34(23):2958–2967 32. Guo J, Song B, Li X, Hε C, Yang G, Yang X, Guan F (2015) Downregulation of gangliotetraosylceramide and β1,3-galactosyltransferase-4 gene expression by Smads during transforming growth factor β-induced epithelial-mesenchymal transition. Mol Med Rep 11 (3):2241–2247 33. Ogiso M, Irie A, Kubo H, Hoshi M, Komoto M (1992) Senile cataract-related accumulation of Lewis(x) glycolipid in human lens. J Biol Chem 267(10):6467–6470 34. Panz T, Lepiarczyk M, Zuber A (2011) Comparing the content of lipids derived from the eye lenses of various species. Folia Histochem Cytobiol 49(3):425–430 35. Nagarajan RP, Chen F, Li W et al (2000) Repression of transforming-growth-factor-betamediated transcription by nuclear factor kappaB. Biochem J 348(Pt 3):591–596 36. Datta PK, Blake MC, Moses HL (2000) Regulation of plasminogen activator inhibitor-1 expression by transforming growth factor-β-induced physical and functional interactions between smads and Sp1. J Biol Chem 275:40014–40019 37. Magyar M, Zsiros VL, Kiss A, Nagy ZZ, Szepessy Z (2019) Caveolák szerepe a szürke hályog képződésében: humán szemlencse epithelsejtjeinek vizsgálata [The role of caveolae in cataractogenesis: examination of human lens epithelial cells]. Orv Hetil 160(8):300–308 38. Sciacovelli M, Frezza C (2017) Metabolic reprogramming and epithelial-to-mesenchymal transition in cancer. FEBS J 284(19):3132–3144 39. Hedrick E, Safe S (2017) Transforming growth factor β/NR4A1-inducible breast cancer cell migration and epithelial-to-mesenchymal transition is p38α (mitogen-activated protein kinase 14) dependent. Mol Cell Biol 37(18):e00306–e00317 40. Dawes LJ, Eldred JA, Anderson IK et al (2008) TGF beta-induced contraction is not promoted by fibronectin-fibronectin receptor interaction, or alpha SMA expression. Invest Ophthalmol Vis Sci 49(2):650–661 41. Walker EJ, Heydet D, Veldre T, Ghildyal R (2019) Transcriptomic changes during TGF-β-mediated differentiation of airway fibroblasts to myofibroblasts. Sci Rep 9(1):20377 42. Tripathi V, Sixt KM, Gao S et al (2016) Direct regulation of alternative splicing by SMAD3 through PCBP1 is essential to the tumor-promoting role of TGF-β [published correction appears in Mol Cell. 2016 Dec 1;64(5):1010]. Mol Cell 64(3):549–564

Chapter 16

Galectin-1 Promotes Tumor Growth and Escapes Immune Surveillance

Galectin-1 (Gal-1) recognizes βGal residue on N-glycan of growth factor receptors and O-glycans of mucins and promotes tumor growth and escape immune surveillance. In immune recognition of self or non-self-antigens, biological roles of galectins have been “Recognition of exogenous ligands in innate immunology”. For example, galectins recognize carbohydrates on the surfaces of viruses, bacteria, fungi, and parasites. For more specifically, the following ligands have been interacted by galectins: (1) complex type N-linked glycans from the HIV gp120 envelope protein, (2) meningococcal (Neisseria meningitidis) lipopolysaccharide (LPS), (3) gonococcal (N. gonorrhoeae) lipooligosaccharide (LOS), (4) Haemophilus influenzae LPS, (5) Helicobacter pylori LPS O-antigen side chain, (6) Streptococcus pneumoniae polysaccharide type XIV, (7) Leishmania major lipophosphoglycan (LPG), (8) Trichomonas vaginalis LPG, (9) Schistosoma mansoni LacdiNAc (LDN), and (10) Candida albicans oligomannan [0]. In the innate immunology, galectins act as pattern recognition receptors (PRRs) for selfrecognition vs. non-self-recognition. The Janeway and Medzhitov model shows that PRRs recognize pathogens through MAMPs, because MAMPs are absent from the host. The paradox of galectins, which bind similar “self” and “non-self” patterns, is raised due to limited knowledge on galectin compartmentalization and oligomerization as well as ligand presentation. Galectin as an animal lectin family has a high affinity for β-galactoside and shares similar amino acid sequences with family. Galectins are conserved in carbohydrate recognition domains (CDRs) with 130 a.a. There are 15 mammalian galectins. Galectin should be “Trojan horse” because galectins play key roles in host development and immune regulation, and some pathogens and parasites evolved them to mimic their hosts [1]. In tumor biology and recognition of self-antigens, roles of galectins in tumors have been suggested: (1) the modulation of tumorigenesis and growth, including apoptosis, transformation, and cell division/cycle; (2) the metastasis, including adhesion, angiogenesis, and migration; and (3) the inflammation response by the tumors and immune escape and avoidance by tumor cells. Glycans of cell surfaces promote cell detachment from ECM and metastasis, migration, tumor cell masking © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_16

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to escape from an immune surveillance, and tumor cell protection from apoptotic signals. Galectins during transformation and survival of tumors enhance transformation where galectin-1 and galectin-3 expressions are necessary. They can interact with oncogenic Ras. Galectins regulate apoptosis. When galectins are added or cells are transfected with a galectin cDNA, antiapoptotic activity is observed through galectin-3 Ser-6 phosphorylation. Other effects of galectins on tumorigenesis are in tumor cell regulation to inhibit growth with galectins during apoptosis. In fact, galectin-1 functions as an autocrine suppressor of cell growth and regulates tumorigenesis via the cell cycle control, where cyclin A and cyclin E are declined and suppressors p21, p27, and cyclin D1 are increased. Galectin-3 upregulates cyclin D1 through β-catenin binding. In these circumstances, the glycans are recognized by various lectins. To date many different lectins have been isolated and characterized. Well-studied lectins are as follows: L-PHA (L-phytohemagglutinin) has a carbohydrate-binding capacity for a complex carbohydrate containing Gal, GlcNAc, and Man. LEL (Lycopersicon esculentum lectin) has an affinity for GlcNAc oligomers, binds to endothelial cells, and is used to visualize the microvasculature. As a SA-binding lectin, SNA (Sambucus nigra agglutinin) binds to α2,6-linked SA, while MAL-II (Maackia amurensis agglutinin) recognizes α2,3-SA linkages, For Gal, PNA (peanut agglutinin) binds the carbohydrate sequence Galβ1,3-GalNAc and HPA (Helix pomatia agglutinin) has affinity for terminal N-acetyl-α-D-galactosaminyl residues [2]. On the other hand, Gal-binding lectins are evolutionarily diverse in their galactose ligand recognition capacity. Among them, Gal-1 is relatively well conserved in their sequences from animal lectins and activates tumor progression through tumor immune escape and metastasis. Galectin-1 expression is increased in hypoxia condition and modulates EC signaling, VEGFR trafficking, and tumor angiogenesis. Galectin-1 binds to multiple Gal-β1-4-GlcNAc (LacNAc) units. Galectin-1 activates the immune responses toward a phenotype which dampens Th-1 cells and Th-17 cells via IL-10 and IL-27 expressions [3]. Prostate epithelial cancer, LNCaP cells are generally resistant to apoptosis, an interesting feature of neoplastic transformed cells. Galectin-1 induces apoptosis to LNCaP cells rather than other prostate cancer cells. Then, galectin-1 was examined in the prostate tumor cells, and the cells were analyzed for the death susceptibility to galectin-1. O-glycoproteins are necessary for prostate tumor LNCaP cell susceptibility to apoptotic cell death through galectin-1. The resulting galectin-1-resistant PSA-LNCaP cells show the reduced level of core 2 GnT carbohydrates. Blocking Oglycan elongation using benzyl-alpha-GalNAc, which inhibits adding of initial GalNAc and consequently blocks O-glycan elongation in low sialylated O-glycans, protects the galectin-induced prostate epithelial LNCaP cell death [3]. For the mechanism to protect themselves from their own Gal-1-induced apoptosis, galectin-1-resistant tumor cells of prostate origins rather synthesize galectin-1, contributing to apoptosis of adherent T cells. O-glycans expressed on prostate epithelial cancer LNCaP cells are used as ligands of galectin-1, and O-glycans– galectin-1 binding triggers galectin-1-elicited apoptotic cell death. To avoid this Gal-1-mediated apoptosis, the prostate epithelial cancer LNCaP cells should mask

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Galectin-1 Promotes Tumor Growth and Escapes Immune Surveillance

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Fig. 16.1 VEGF-like signaling by Gal-1 lectin. High levels of β1,6-GlcNAc-braches by GlcNAcTV and decreased terminal α2,6sialyl residues by ST6Gal-1 increases in the Gal residues usable for Gal-1 ligand

the O-glycans by SA through overexpression of the ST3Gal-1 or ST6Gal-1, sialytransferases, which enable the cell resistance to galectin-1 (Fig. 16.1). Tumor cells are specifically well grown on the oxygen-deficient hypoxia environment. Hypoxia decorates carbohydrate remodeling and recognition of the lectin galectin-1 to vascular resident cells. For example, L-PHA or LEL-recognizing sugars are increased, while SNA-recognizing sugars are decreased. Lectin SNA recognizes the ST6Gal-1-synthesized sialic acid residue, and thus ST6Gal-1-synthesized sugar inhibits the L-PHA or LEL-recognizing sugar synthesis. L-PHA or LEL-recognizing sugar structure and SNA-recognizing sugars are reversely functioned. Lectin Gal-1 recognizes galactose-β1-4-N-acetylglucosamine (LacNAc) unit, and the lectin level is increased in the hypoxia status to bind the endothelial cells in the hypoxia environment. When Gal-1 is treated, an angiogenic parameter, tube formation level, is increased. After siRNA for the MGAT5 enzyme for N-glycan branching or C2GNT1 enzyme for O-glycan branching is treated to the cells, if tube formation is examined, in case of siMGAT5, Gal-1-mediated tube formation was decreased (Fig. 16.1). However, siC2GNT1 treatment does not affect the tube formation. This indicates that Gal-1-binding glycan is largely dependent on the Nglycan branching in the endothelial cells. Thus, together with VEGF-A, lectin Gal-1 recognizing sugar structures regulate the angiogenic tube formation, although only VEGF-A has been recognized importantly to date in the field of tumor invasion or angiogenesis [4].

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16 Galectin-1 Promotes Tumor Growth and Escapes Immune Surveillance

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Cell surface’s glycan structures are remodeled O-/N-glycan-synthesizing enzymes of glycosyltransferase or glycan-degrading glycosidase. This glycosylation event consequently controls the pathophysiological status of the cells. Thus, cell surface glycosylation indicates an essential modulator of immune cells and/or cancer cells to endothelial cells interaction. As representative case, interaction between lectin and growth factor receptor, which is differently glycosylated depending on pathological situation, is found. For example, galectin-1 specifically interacts with N-glycan of VEGFR-2, and this interaction mimics VEGF-A activity. If interaction of Gal-1 with N-glycans is blocked by binding site alteration, immune response is changed, and tumor growth is also affected. As Gal-1 directly interacts with VEGFR-2, Gal-1-dependent increase in the tube formation was decreased when the VEGFR-2-specific siRNA or VEGFR-2-blocking antibody was treated with the cells. Thus, Gal-1 binds endothelial VEGFR-2 by glycosylation-dependent manner and eventually mimics the VEGF signal transduction pathway. Then, it was speculated that changes in the extracellular glycosylation of the cells may affect the tumor growth. In this case, changes in glycan structures open the specific binding of the lectin with its ligand in specific receptors and consequently alter the receptor physiology. Thus, this indicates that differences in the glycosylation pattern determine the sensitivity against the receptor activity. If it is the case of VEGFR-2, effect of anti-VEGF treatment will not be expected. To facilitate this regulation of glycosyl structures of the asparagine-linked N-glycan and serine/threonine-linked O-glycans, glycosyltransferases and degrading enzymes are related. Among them, Nacetylglucosaminyltransferase-5 (GlcNAcT-V or MGAT5) adds β1-6-GlcNAc branch to the N-glycans, and α2,6sialyltransferase (ST6Gal-1) adds α-2,6 SA to Gal residues. Galectin-1 inhibits enzyme activity of ST6Gal-1 because of prior Gal-1 binding of β1,4-Gal residue to Siaα2.6-residue. In O-glycans, core 2 branching structure-synthesizing enzyme, core 2 β1,6GlcNAc-transferase 1(C2GNT1), influences the VEGFR-2-regulating glycan structures to be interacted with tumorproliferating lectins (Fig. 16.2). From the results, MGAT5 synthesizes the Gal-1binding substrates and ST6Gal-1 produces the glycan structure what Gal-1 cannot bind. In the MGAT5 KO mouse, L-PHA binding capacity specific for Gal-1recognizing glycan is decreased. MGAT5 KO mouse showed that tumor size was significantly decreased than WT. When endothelial cells were treated with Gal-1

References

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lectin, it exhibited the similar action to the VEGF treatment, indicating that Gal-1 induces the VEGFR function likely to the VEGF treatment. For signaling, Gal-1 treatment showed the increased phosphorylation of VEGFR-2 with the increased Akt and ERK 1/2 phosphorylation of the downstream pathway, likely to the VEGFA treatment. Previously, in order to target the VEGF in diseases such as cancer metastasis, angiogenesis, and intimal thickening atherosclerosis, although VEGF- or VEGFR-specific monoclonal antibody or VEGFR’s RTK inhibitors are regarded as future therapeutic strategies, those developments, so-called anti-VEGF therapy, seem to be limited. In addition, anti-VEGF-therapy-resistant tumors may be created by unique themselves signaling pathways. This circumstance in functional diversity caused from VEGFR-2 glycan structure requires the fundamental understanding for tumors glycan signaling. Apart from the direct interaction of Gal-1 with growth receptor glycan structure in tumor cells, another remarkable property of Gal-1 in immune escape should be explained. Gal-1 suppresses the immune cell functions. When Gal-1-inhibitory monoclonal F8.G7 MAb was treated, T cell growth was activated, and inflammatory cytokines of IFN-γ and IL-17 were increased, while immunosuppressive cytokine, IL-10, was decreased. Additionally, number of tumor immunity-related effector CD8 T cells was increased. Thus, Gal-1 inhibition induces the immune response against tumor cells. If cells have many Gal-1-recognizing sugars, anti-VEGF antibody cannot be bound; instead, Gal-1 can bind to the glycans on the VEGFR-2. Galectin-1 transduces and delivers angiogenic signals through glycosylationdependent mode in endothelial cells. Glycosylation-based recognition of Gal-1 with endothelial cells mimics VEGF function. Differential glycosylation events of tumor cells delineate acting sensitivity to VEGF-suppressing therapeutic treatment. Targeting galectin-1 in tumor microenvironments increases its efficiency of anti-VEGF therapeutic treatment. Targeting galectin-1 overcomes to anti-VEGF therapeutic treatment and improves vascular remodeling. Disruption of galectin-1N-glycan interaction controls immune compartments.

References 1. Vasta GR (2012) Galectins as pattern recognition receptors: structure, function, and evolution. Adv Exp Med Biol 946:21–36 2. Sundblad V, Morosi LG, Geffner JR, Rabinovich GA (2017) Galectin-1: a Jack-of-all-trades in the resolution of acute and chronic inflammation. J Immunol 199(11):3721–3730 3. Suzuki O, Abe M, Hashimoto Y (2015) Sialylation and glycosylation modulate cell adhesion and invasion to extracellular matrix in human malignant lymphoma: dependency on integrin and the rho GTPase family. Int J Oncol 47(6):2091–2099 4. Croci DO, Cerliani JP, Dalotto-Moreno T, Méndez-Huergo SP, Mascanfroni ID, Dergan-DylonS, Toscano MA, Caramelo JJ, García-Vallejo JJ, Ouyang J, Mesri EA, Junttila MR, Bais C, Shipp MA, Salatino M, Rabinovich GA (2014) Glycosylation-dependent lectin-receptor interactions preserve angiogenesis in anti-VEGF refractory tumors. Cell 156(4):744–758

Chapter 17

GM3-HGFR, FGFR, and PDGFR Cancer Cell Behavior and IGF-1R in Diabetic Wound Healing

Tumor cells express highly carbohydrates on the cell surfaces for immune escape or better tumor environments of tumor cells. Some tumor-associated carbohydrate antigens (TACC) are mainly detected in mucins. Mucins are heavily O-glycosylated proteins resided on the epithelial cell surfaces or tumors. An excess levels of tumor-associated antigens (TAA) on the carcinoma cell surfaces correlate with a poor prognosis of the patients. Carbohydrates normally belonged to T cellindependent antigens, due to incapability of a strong immune response. Therefore, carbohydrate mimetics, capable of eliciting anti-carbohydrate immune response, is future strategy of overcoming aberrant tumors in therapeutic regards. Peptide mimics of the GD2 or GD3 have been designed by phage display against anti-ganglioside antibodies. GM3 as the simplest ganglioside and vertebrate’s major ganglioside regulates Tyr kinase of GFRs. GM3 is a precursor substrate for biosynthesis of the gangliosides of complex types including a, b, and c series. The precise mechanism (s) responsible for GM3 engagement is not clear in the malignant transformation, where GM3 exerts anti-tumor actions or promoting actions on tumor behavior. The mechanism of GM3 inhibition of tumor cell function is still under investigation. From many studies, it is certain that GM3 functions as a negative regulator of EGFR, FGFR, PDGFR, nerve GFR (NGFR), hepatocyte GFR (HGFR, cMet), insulin-like GFR-I (IGFR-I) and VGFR by controlling tyrosine kinase of GFR. In early 1970S, Hakomori and his colleagues found their pioneer results that GM3 expression as an abundant membrane component is reduced in malignant tumor cells when transformed by oncogenes such as “v-Jun”. Enforced transfection of GM3 synthase gene potentiated the transformed cells to normal cell-like revertant phenotype. This is based on the facts that GM3 deactivates many GFR such as EGFR. In contrast, De-N-acetyl GM3 enhances functional activation of EGFR signaling. The SA residue role of GM3 was confirmed using the membrane-type neuraminidase NEU3. GM3 is enriched in the membrane microdomain termed “glycosynapse”, with signaling molecules of Src kinases; tetraspanins of CD9, CD81, and CD8; integrins; and different GFRs such as FGFR and HGFR/cMet [1]. GM3 exhibits its biological function in a cancer cell-type manner, differently its © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_17

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GM3-HGFR, FGFR, and PDGFR Cancer Cell Behavior and IGF-1R in Diabetic. . .

anti-tumor activity, although it suppresses the proliferation and invasion. However, an inverse relation is reported between the GM3 contents and cell proliferation and metastasis in certain type tumors. GM3 also activates non-tyrosine kinase receptors. Thus, the GM3 functions are complicated. In certain tumors, the molecules regulated by GM3 differ, and GM3 differently acts. This implies that GM3 exerts distinct or opposing actions in tumor-specific manner of different tumor types and the mechanism of tumor-type specificity remained unanswered. For the GM3 function on the HGFR (cMet) signaling, GM3 further enhances the HGF-induced HGFR autophosphorylation. HGF/scatter factor (SF) acts as a mitogen having dissociation and motility-inducing activities in several epithelial cells. In addition, HGF also exerts as a cytokine function during tumor cell adhesion, migration, invasion, and metastasis [2]. HGF controls aggressive and metastatic potentials of cancer cells. HGFR is also called cMet. HGF binds cMet and promotes multimerization and phosphorylation in the PI3K-Akt signaling, PLCγ signaling and ERK signaling pathway. GM3 phosphorylates the amino acid sites of Tyr-1313 and Tyr-1365 of cMet to regulate PI3K/Akt signaling pathway and promotes the HGF-induced cMet functions [3]. GM3 activates PI3K-Akt signaling, influencing in vitro mobility in mouse hepatoma cell. Thus, GM3-mediated cMet activation and PI3K-Akt signaling increase the mobility and migrative potentials of invasive tumor cells. Although GM3 generally regulates proliferation and metastasis, GM3 is also known as an negative modulator of certain GFRs in the specific cancer cells. Using a specific inhibitor of GlcCer synthetic enzyme, UDP-Glc ceramide glucosyltransferase, biological functions of cellular synthesis of GM3 have been explained with some extents. GM3 has been suggested to have opposite effects on the HGF- and EGF-stimulated Hepa1-6 cells in the parameter such as migration and motility in vitro [4]. HGF activity is initially forwarded to activate the cMet Tyr kinase function. The recognition, binding, and interaction of HGF/SF with the cMet extracellular domain induce receptor multimerization and Tyr residue phosphorylation in the cytoplasmic region. Thereafter, the phosphorylated receptor form influences downstream signalings including the PI3K/Akt, PLCγ, and ERK to exhibit cMet-controlled cellular phenotypes such as migration, motility, adhesion, and invasion of cells. For the GM3 effects on the HGFR/cMet signaling, the action mechanism(s) which regulates cMet signaling and HGF-induced cell phenotype changes has been explained in mouse hepatic cancer cells including Hepa1-6, Hca/16A3 and Hca/A2 cells [3]. Both two different conditions such as cellular loss of GM3 contents and a specific inhibitor P4-treated GlcCer deficiency inhibited the HGF-induced cMet phosphorylation and PI3K/Akt signaling force. However, the increased GM3 production and exogenous GM3 addition to the cells enhanced the HGF-induced cMet phosphorylation and PI3K/Akt downstream signaling. GM3 ganglioside blocks HGF-dependent cell migration and motility in vitro. The effects are reduced when GM3 amount is decreased but enhanced by increased GM3 level. The increased GM3 product induces HGF-dependent motility of murine hepatoma cells by cMet phosphorylation at specific Tyr amino acid residues and a PI3K/Akt-depended manner. Silenced GM3 synthase gene blocks the cMet signaling with blocked invasion and migration

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of the cells even in metastatic type of breast cancer 4T1 cells [5]. However, the complex between GM3, GM2, and CD82 blocks cMet signaling [6, 7], but the GM3 functions in cMet-mediated signaling is not answered. In the recent report [8], exogenously added GM3 induced the HGF-dependent cMet phosphorylation at Tyr-1313 and Tyr-1365 amino acid residues. GM3 induces the HGF-dependent HGFR phosphorylation at amino acid residues of Tyr-1313 and Tyr-1365. Therefore, GM3 upregulates the HGF-driven cMet phosphorylation and activation, clearly answering the question why GM3 exhibits opposite effects on the motility and migration of the different growth factors, in HGF-treated Hepa1-6 cells, compared to EGF effect [8]. With regard to GM3 effects on the signalings in Hepa16 cells for HGF-driven motility and migration in vitro, cellular signaling pathway is crucial for the cancer cell invasion and migration. Three major signaling pathways controlled by growth factor receptors include the PLCγ/PI3K/Akt/MAPK pathways. For GM3, the MAPK signaling seems not to be important in the HGF-induced Hepa1-6 cells. However, ganglioside GM3 roles in the PLCγ1 downstream signaling pathway includes the PLCγ phosphorylation and cellular PM translocation from the cytosols, where PLCγ1 phosphorylation form is not observed in the HGF-treated Hepa1-6 cells. In the cellular distribution of PLCγ1, membrane-bound PLCγ1 form is not found in the HGF-tread Hepa1-6 cells. However, GM3 inhibits the PLCγ1 expression in the HGF-induced cells. Therefore, GM3 does not regulate the PLCγ1 signaling. HGF-induced cells lack GM3 product obtained by silencing of GM3 synthase gene expression through shRNA plasmid loss the Akt phosphorylation at amino acid residue of Ser 473. The exogenous addition of GM3 induced the Akt phosphorylation at Ser 473 amino acid residue. Thus, GM3 enhances the HGF-driven PI3K/Akt downstream-related migrative potential of the cells. The HGF-driven migration enhanced by the GM3 treatment is also prevented by inhibition of the PI3K/Akt pathway. For FGFR, human embryonal WI38 fibroblasts proliferate in an FGF-dependent growth manner with its receptor FGFR. The ganglioside-enriched microdomain of the cells is stably composed of GM3 ganglioside and CD-9-/CD81-containing TSP components [9]. The cellular adhesion and motility potentials are enforced by laminin-5-associated microdomain in lipid rafts. Cross talks between membraneembedded integrins and FGFR are further enhanced by GM3 engagement. GM3 depletion induces FGFR Tyr phosphorylation and its downstream Akt signaling followed by activation of MAPK. GM3 depletion also contributes to the increased level of FGFR co-immunoprecipitation with α3α5β1 integrin and also enhances the level of coprecipitated integrins with CD9/CD81. Consequently, GM3 depletion activates the cell growth. Therefore, GM3 activates integrin-FGFR cross interaction within the ganglioside-enriched microdomain [6]. GM3 inhibits basic FGFR [10]. GM3 inhibits the PDGFR [10, 11]. In neurite genesis in SH-SY5Y human neuroblastoma, gangliosides of GM1, GM3, and GT1b are related with the action of PDGF, NGF, and IGF-I. PDGF induces new neurite outgrowth in SH-SY5Y neuroblastoma cell, while GM1 inhibits the neurite formation [12]. The similar inhibitory effects on neurite outgrowth formation were also observed in other gangliosides of GM3 and GT1b. Other types of growth factors including insulin

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and IGF-I are also known to elicit such neuritogenic responses, although less potent than that of PDGF. The gangliosides GM1 and GT1b are known to inhibit temporary and induction factor-driven outgrowth of neurites observed in SH-SY5Y cells. However, GM3 does not inhibit the neurite outgrowth. The GM1 and GT1binhibited neurite outgrowth is driven through induction factors as their receptors. In fact, GM3 and GT1b inhibit [3H]thymidine incorporation during neurite outgrowth. Tyr residue phosphorylation of a 170 kDa protein upon 20 ng/ml PDGF stimulation was blocked by treatments with GT1b, GD1a, GM2, and GM2, but not with GM3. Inhibition of the target receptor dimerization is also observed by GM1 treatment. In the SH-SY5Y cells, the binding receptors for PDGF-BB are expressed to exert their functional signaling, and, thus, the signaling is specifically controlled by treatment with gangliosides [13]. Interestingly, the autophosphorylation of PDGF-BB-dependent receptor and enzymatic activation of PLCγ1 are not directly influenced by GM3 treatment. However, the genesis of PDGF-BB-dependent InsP3 and liberation of [Ca2+]i are suppressed by GM3 treatment with the inhibited growth of cells. When gangliosides such as GM1 and GM2 were incubated with 125I-PDGFBB on VSMC, GM1 and GM2 effectively block the PDGF-BB binding to the VSMC cells. However, GM3 does not exhibit any blocking of the binding activity of 125I-PDGF-BB to the VSMC cells. Therefore, it is confirmed that GM1/GM2 potentially binds to the PDGF-BB or the PDGF-BB receptor and, consequently, prevents the PDGF-BB binding to the cellular receptors. In the case of GM3, the ganglioside can inhibit the InsP3 liberation in a PDGF-BB-dependent manner and blocks the [Ca2+]i downstream caused by autophosphorylation of the PDGF-BBdependent receptor as well as PLCγ1 activity [14]. In fact, the enforced GM3 expression cells exhibit the anchorage-independent proliferation phenotype and the reduced mRNA expression level of PDGFα receptor gene. Change in anchoragedependent growth is not detected in these cells, and tumorigenic signals were controlled selectively in both positive and negative directions [15]. Thus, the spatiotemporal gangliosides by gene expression of individual gangliosides are accumulated in the cell membrane lipid raft microdomain. Because GM3 involves in insulin resistance event of adipocytes, the GM3 functions are interested in physiological responses of diabetic wound healing process. The increased glycolipid level is characteristic in diabetic wound healing. In addition, a specific disease state like chronic cutaneous ulcerations shows the relatively high frequency with type 2 diabetic phenotype, with approximately over 15% of individuals with type 2 diabetes [16]. The poor wound healing levels in diabetic patients are correlated with the impaired levels of insulin receptor (IR) and IGF-1R signaling with the abnormal accumulation and deposition of advanced glycosylation end products and resulting vasculopathy diseases [17]. Wound healing response essentially involves the activation, proliferation, and migration of dermal keratinocytes to potentiate reepithelialization of the wound tissues. Generally, skin tissues do not belong to typical tissue types susceptible for the insulin targeting. Therefore, both stimulants of insulin and IGF-1 are associated with the other distinct cellular processes of normal cell migration, division, and proliferation via IR and IGF-1R responses [18]. In insulin resistance, GM3 involves in adipocyte insulin

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resistance. Because the insulin resistance is also related to wound healing event of diabetic cells as an integral morbidity factor, the GM3 roles are of interests in wound healing of diabetic mellitus. The question is raised whether GM3 loss or deficiency enhances diabetic wound healing event and potentiates insulin response of keratinocytes because GM3 synthase gene expression is upregulated in several tissues and cells such as obese diabetic mouse and human diabetic skins, ob/ob mice, and mouse glucose-treated keratinocytes [19]. GM3 synthase gene KO in obese mice was diabetic wound healing. In the GM3 synthase KO( / ) keratinocytes, growth and migration of keratinocyte and IGF-1R activation were increased by glucose treatment. Insulin signaling molecules such as IR, IGF-1R, and IR substrate-1 (IRS-1) are normally clustered with Akt phosphorylation upon receptor activation. For interesting effect of GM3, a recent report suggested that nanoparticle, spherical nucleic acid (SNA)-based silencing GM3 synthase increases in growth and migration of keratinocytes as well as insulin and IGF-1R activation [20]. The GM3 synthase silencing in wounds of diet obese diabetic mice heals wounds clinically and histologically. Several analytical evidences were all positive, as granulation tissue area, vascularity, and IGF-1R phosphorylation are improved and increased in the silenced GM3 synthase-administrated wounds tissue. The experiments raised two possibilities of in vivo or clinical application using GM3 synthase knockdown and silencing technology: (1) the natural penetration of specific target silencing genes on the local skin such as keratinocytes and (2) validation of local GM3 depletion via targeting of GM3 synthase gene for promising therapy as a pivotal regulator of delayed type of wound healing events in type 2 diabetes. The theoretical basis of the IGF-1R-based GM3 is derived from the fact that GM3 is suggested as a target of diabetic animals and insulin resistance and impeding wound healing event.

References 1. Zheng C, Terreni M, Sollogoub M, Zhang Y (2019) Ganglioside GM3 and its role in cancer. Curr Med Chem 26(16):2933–2947 2. Yi S, Tsao MS (2000) Activation of hepatocyte growth factor-met autocrine loop enhances tumorigenicity in a human lung adenocarcinoma cell line. Neoplasia 2(3):226–234 3. Li Y, Huang X, Zhong W, Zhang J, Ma K (2013 Oct) Ganglioside GM3 promotes HGF-stimulated motility of murine hepatoma cell through enhanced phosphorylation of cMet at specific tyrosine sites and PI3K/Akt-mediated migration signaling. Mol Cell Biochem 382 (1–2):83–92 4. Li Y, Huang X, Wang C, Li Y, Luan M, Ma K (2015 Apr) Ganglioside GM3 exerts opposite effects on motility via epidermal growth factor receptor and hepatocyte growth factor receptormediated migration signaling. Mol Med Rep 11(4):2959–2966 5. Bhuiyan RH, Kondo Y, Yamaguchi T et al (2016) Expression analysis of 0-series gangliosides in human cancer cell lines with monoclonal antibodies generated using knockout mice of ganglioside synthase genes. Glycobiology 26(9):984–998

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6. Meuillet E, Cremel G, Dreyfus H, Hicks D (1996) Differential modulation of basic fibroblast and epidermal growth factor receptor activation by ganglioside GM3 in cultured retinal Müller glia. Glia 17(3):206–216 7. Todeschini AR, Dos Santos JN, Handa K, Hakomori SI (2008) Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMetmediated pathway. Proc Natl Acad Sci U S A 105(6):925–930 8. Li Y, Huang X, Wang C, Li Y, Luan M, Ma K (2015 Apr) Ganglioside GM3 exerts opposite effects on motility via epidermal growth factor receptor and hepatocyte growth factor receptormediated migration signaling. Mol Med Rep 11(4):2959–2966 9. Toledo MS, Suzuki E, Handa K, Hakomori S (2005 Apr 22) Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor. J Biol Chem 280(16):16227–16234 10. Ernst AM, Brügger B (2014) Sphingolipids as modulators of membrane proteins. Biochim Biophys Acta 1841(5):665–670 11. Bergante S, Creo P, Piccoli M et al (2018) GM1 ganglioside promotes osteogenic differentiation of human tendon stem cells. Stem Cells Int 2018:4706943 12. Hung JT, Yeh CH, Yang SA et al (2016) Design, synthesis, and biological evaluation of ganglioside Hp-s1 analogues varying at glucosyl moiety. ACS Chem Neurosci 7(8):1107–1111 13. Yamagishi M, Hosoda-Yabe R, Tamai H et al (2015) Structure-activity relationship study of the neuritogenic potential of the glycan of starfish ganglioside LLG-3 ({). Mar Drugs 13 (12):7250–7274 14. Sachinidis A, Kraus R, Seul C, Meyer ZU, Brickwedde MK, Schulte K, Ko Y, Hoppe J, Vetter H (1996 Sep) Gangliosides GM1, GM2 and GM3 inhibit the platelet-derived growth factorinduced signalling transduction pathway in vascular smooth muscle cells by different mechanisms. Eur J Cell Biol 71(1):79–88 15. Russo D, Della Ragione F, Rizzo R et al (2018) Glycosphingolipid metabolic reprogramming drives neural differentiation. EMBO J 37(7):e97674 16. Lim JZ, Ng NS, Thomas C (2017) Prevention and treatment of diabetic foot ulcers. J R Soc Med 110(3):104–109 17. Christman AL, Selvin E, Margolis DJ, Lazarus GS, Garza LA (2011 Oct) Hemoglobin A1c predicts healing rate in diabetic wounds. J Invest Dermatol 131(10):2121–2127 18. Sakagami N, Umeki H, Nishino O et al (2012) Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows. J Reprod Dev 58(1):140–146 19. Wang XQ, Lee S, Wilson H, Seeger M, Iordanov H, Gatla N, Whittington A, Bach D, Lu JY, Paller AS (2014 May) Ganglioside GM3 depletion reverses impaired wound healing in diabetic mice by activating IGF-1 and insulin receptors. J Invest Dermatol 134(5):1446–1455 20. Randeria PS, Seeger MA, Wang XQ, Wilson H, Shipp D, Mirkin CA, Paller AS (2015 May 5) siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown. Proc Natl Acad Sci U S A 112(18):5573–5578

Chapter 18

GM3, Caveolin-1 and Insulin Receptor in Insulin Resistance

GM3 ganglioside involves various cellular events of receptor-associated signalings. GM3 regulates signaling pathway involved in the insulin resistance and diabetes process. The GM3 contents generated by the ST enzyme ST3Gal V/GM3 synthase/ SAT-I are upregulated in the adipose, kidney, liver, and murine muscles of animal diabetes [1]. The aberrant expression of GM3 in adipose tissue accelerates diabetic development and progression, especially in the type 2 diabetic mellitus (T2DM) and insulin resistance known for metabolic disorders. Increased GM3 level is found in the plasma fluids of T2DM, vasculopathies and microvascular lesions [2]. The GM3 synthase gene is upregulated in the T2DM patients with kidney pathies and nephropathies [3]. TNF-α is known to cause insulin resistance and increase GM3 synthase gene expression as well as GM3 product in insulin-interacting receiver cells such as hepatocytes, adipocytes, and myocytes. The obesity and diabetic role of GM3 has been attributed to impaired insulin action [4]. In human studies, aberrantly increased serum level of GM3 is detected in hyperglycemic, hyperlipidemic, and T2DM patients [5, 6]. Glucocerebrosidase deficient Gaucher disease shows insulin resistance with serum GM3 level [7]. Therefore, aberrant GM3 synthesis and its metabolic reutilization may contribute to defection of glucose and lipid metabolic homeostasis. Therefore, better understanding of the fundamental mechanism will open the way to potential therapies using targeting GM3. The level of blood sugar is increased in the insulin defective condition such as insulin-dependent diabetes (IDDM) and also insulin resistance condition of severe non-insulin-dependent diabetes (NIDDM). In this meaning, diabetes is classified into two major types of type 1 diabetes as IDDM and type 2 diabetes as NIDDM. Type 1 diabetes is caused by the destroyed pancreatic β cells by attacking of autoimmune T cells. As a minor and rare condition, type 3 diabetes referred for pregnancy women is offered. Among them, type 2 diabetes is so-called dysfunctional cell system for insulin and glucose metabolism. Type 2 diabetes is the disease that target cell does not react due to the IR dysfunction. Caveolae forms microdomain for insulin signaling in adipocyte. Caveolae-microdomain allows the phosphorylation of IRS-1 in clinical insulin resistance. Caveolin-binding peptide © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_18

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sequence motif is the specialized motif of fXXXXfXXf in the IR β subunit. Mutation in the motif of caveolin inhibits insulin signaling, as IR β subunit mutation is found from the type 2 diabetes patients. Insulin-driven cellular signaling is initiated from the IR kinase (IRK), and, consequently, family molecules of downstream signaling including IRS and PI3K are additionally activated [8]. The activated PI3K-driven genesis of PIP3 contributes to PKB/Akt molecular translocation to the cellular PMs, and amino acid residues at Thr308 and Ser473 are phosphorylated to active PKB/Akt [9]. Active PKB/Akt kinase triggers glycogen synthesis and glucose uptake stimulated with GLUT transporter through AS160 protein, Rab GTPaseactivator protein, and glycogen synthase kinase-3 (GSK-3) [10]. Impairing insulin action dysregulates glucose homeostasis, as obesity factor TNF-α mediates the insulin resistance [11]. Therefore, insulin resistance process is classified to a type of membrane microdomain-embedded lipid rafts disorders. Most diabetes patients are the type 2 caused by membrane microdomain disorder. Insulin induced the uptake of glucose, fatty acids, and amino acid to the target cells and glycogen biosynthesis in liver. GM3 has been suggested to link with the insulin resistance onset from the findings that TNF-α-driven insulin desensitization is accompanied with aberrant GM3 production in 3T3-L1 adipocytes [1]. GM3 directly blocks IR and IRS-1 Tyr phosphorylation in adipocytic cells and, consequently, reduces the level of glucose uptake [1]. Knocking out GM3 synthase gene in mice [12] and glucosylceramide synthase inhibitors for GM3 depletion improves insulin sensitivity [13] and inhibits the progress and initiation of diabetic renal hypertrophy development [14]. Specifically, exogenous GM3 treatment to the 3T3-L1 adipocytic cells inhibits the phosphorylation levels of insulin-promoted Tyr residues in the IR and IRS-1, and this event consequently leads to the decreased level of insulin-mediated glucose uptake [1]. Exogenous TNF-α with 3T3-L1 adipocytes accumulated GM3 [15]. The enhanced GM3 level was correlated with the reduced level of IR. GM3 disrupts the binding of IR to caveolin-1 and consequently increases IR mobility, leading to dissociation between caveolin and microdomain [16]. GM3 directly interacts with the lysine residue (Lys-944) in IRβ subunit and provokes insulin resistance through disturbing the caveolin-1–IR interaction. TNF-α-induced dissociation between IR and caveolin-1-based microdomain is prevented by D-PDMP with the restoration of insulin signaling [17]. GM3 is suggested to suppress insulin signaling via inhibition of IRS-1. It was suggested that GM3 induces insulin resistance, by mechanism (s) where insulin binds to IR and induces IR Tyr kinase. The activated kinase phosphorylates IRS and p-IRS mediates the GLUT-4 translocation. The IR–IRS– GLUT-4 signaling pathway is glucose metabolic action of cells. It was reported that TNF-α increases the level of insulin resistance in 3T3-L1 adipocytic cells and also increases in GM3 level [18]. GM3 synthase activity is also increased in insulin-resistant adipocytes [1]. However, GM3 synthase KO mice enhanced the insulin signaling [19]. In caveolin-1-null mice (a subunit of caveolae is a membrane microdomain-rich membrane protein) [20], the insulin signaling was inhibited because caveolin stabilized the IR protein. Interaction between caveolin and IR is inhibited by the GM3 [21]. GM3 weakens the IR–caveolin interaction. IR

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interacts with caveolin and GM3, but GM3 cannot interact with caveolin. IR interacts with caveolin and GM3 [22]. Because acidic GSLs in the outer leaflet are sulfatides and gangliosides, the protein recognition to lipid membrane is considered to mediate by electrostatic binding and interactions between acidic GSLs and basic domains in proteins. GM3 is located on outer leaflet of the negatively charged PM. From the situation, serum GM3 levels have been considered as a diagnostic biomarker of metabolic diseases. However, it is still controversial for the real possibility. The major form of ganglioside GM3 in serum is also clustered in an associated form with serum lipoproteins such as lipoproteins like high-density lipoprotein (HDL), low DL (LDL), very low LDL (VLDL), intermediate IDL, etc. Serum GM3 level is high in hyperglycemic patients of type 2 diabetes and hyperlipidemic patients. Thus, serum GM3 level is greatly influenced by glucose, lipid metabolites, abnormal metabolites, and visceral obesity. Further possibility in the therapeutic intervention of metabolic diseases has been considered by inhibition of complex ganglioside formation in its synthetic pathway. For example, TNF-α induces GM3 expression and TNF-α-mediated insulin resistance. This insulin sensitivity event is blocked by D-PDMP (glucosylceramide synthase inhibitor), as suppressed in GM3 synthase-deficient mice. GM3 induces the dissociation of insulin receptor in caveolae, because lysine residue (Lys-944) of insulin receptor interacts with sialic acid of GM3. Thus, IR’s basic amino acid lysine (IR944) interacts with the negative charge of sialic acid in GM3. Lysine of IR interacts with GM3. The reduced level of GM3 recovers insulin sensitivity in the type 2 diabetic patients, as GM3 synthase-deficient mice are insulin-sensitive and not glucosetolerant [23]. The event has been enhanced by insulin-driven Tyr phosphorylation of the IR and IRS-1. Therefore, GM3 synthase-inhibiting drugs are suggested to maintain glucose homeostatic responses and insulin sensitivity. For similar action to PDMP, GlcCer synthase inhibitor Genz-123346 resensitized and improved glucose tolerance response in a diabetic model animal of Zucker fatty rats with improved insulin sensitivity level [13]. Another drug, AMP-DNM also induced the levels of insulin sensitivity and glucose tolerance of leptin-deficient obese ob/ob mouse [24, 25]. Nevertheless, GM3-related modulation of insulin action is limited to generalization of the pharmacological application of GM3-related drugs without accurate understanding on the GM3-related biology (not levels of tissue and/or cell type) [26]. Exogenous or endogenous expression of GM3 in adipocytes suppressed insulin action. Therefore, GM3 may promote the responses involved in insulin resistance in specific types of adipose tissues. The levels of glucose uptake and insulin sensitivity are significantly enhanced in skeletal muscle tissues of animal model mice with GM3 synthase gene KO null mutation [26]. Thus, GM3 in insulinrelated diabetic and obesity conditions should be carefully approached. Thus, the conclusion is that GM3 induces insulin resistance response in type 2 diabetic patients. Although potential therapeutic application for prevention of metabolic syndrome has been suggested in order to block ganglioside synthesis pathway, however, more theoretical mechanistic explanation on the GM3-mediated insulin resistance should be cleared. The present author does not still fully accept the theory.

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For example, how does GM3 induce insulin resistance? If possible, likely to GM3– VEGFR.

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17. Sekimoto J, Kabayama K, Gohara K, Inokuchi J (2012) Dissociation of the insulin receptor from caveolae during TNFalpha-induced insulin resistance and its recovery by D-PDMP. FEBS Lett 586:191–195 18. Liu LS, Spelleken M, Röhrig K, Hauner H, Eckel J (1998) Tumor necrosis factor-alpha acutely inhibits insulin signaling in human adipocytes: implication of the p80 tumor necrosis factor receptor. Diabetes 47(4):515–522 19. Yamashita T, Hashiramoto A, Haluzik M, Mizukami H, Beck S, Norton A, Kono M, Tsuji S, Daniotti JL, Werth N, Sandhoff R, Sandhoff K, Proia RL (2003) Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci U S A 100:3445–3449 20. James DJ, Cairns F, Salt IP et al (2001) Skeletal muscle of stroke-prone spontaneously hypertensive rats exhibits reduced insulin-stimulated glucose transport and elevated levels of caveolin and flotillin. Diabetes 50(9):2148–2156 21. Inokuchi J (2007) Insulin resistance as a membrane microdomain disorder. Biol Pharm Bull 29 (8):1532–1537 22. Kabayama K, Sato T, Saito K, Loberto N, Prinetti A, Sonnino S, Kinjo M, Igarashi Y, Inokuchi J (2007) Dissociation of the insulin receptor and caveolin-1 complex by ganglioside GM3 in the state of insulin resistance. Proc Natl Acad Sci U S A 104:13678–13683 23. Inokuchi JI, Inamori KI, Kabayama K et al (2018) Biology of GM3 ganglioside. Prog Mol Biol Transl Sci 156:151–195 24. Chavez JA, Siddique MM, Wang ST, Ching J, Shayman JA, Summers SA (2014) Ceramides and glucosylceramides are independent antagonists of insulin signaling. J Biol Chem 289 (2):723–734 25. van Eijk M (2009) Reducing glycosphingolipid content in adipose tissue of obese mice restores insulin sensitivity, adipogenesis and reduces inflammation. PLoS One 4:e4723 26. Ersek A, Xu K, Antonopoulos A et al (2015) Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease. J Clin Invest 125(6):2279–2292

Chapter 19

GM3 Suppresses Arthritis

Homeostatic maintenance of bone articular cartilage is performed basically by resident chondrocytic cells. The chondrocytes synthesize and degrade the extracellular matrix (ECM). If the chondrocytes excessively degrade the components, osteoarthritis (OA) issue is raised for a progressive degradation of articular cartilages in the tissue. Rheumatoid arthritis (RA) is another type of OA but a type of autoimmune disease with chronic inflammation response in synovial tissues [1]. Therapies for blocking and prevention of the OA and RA development need to identify molecular targets involved in the component digestion and degradation as well as inflammatory responses. Among CD4+ T cells, Th-17, a subunit of CD4+ T cell, is a key in the inflammatory response [2], because synthesis of IL-6, IL-8, and TNF-α cytokines is activated by TH-17-produced IL-17 [3]. Th-17 is the primary inducer for RA, and the IL-17 is a main cytokine in destructive and chronic arthritis progression. GM3 product level is reduced in the synovial fluids of RA patients. The cross talk of GM3 and RA was claimed in a mice type 2 collagen-induced arthritis (CIA) model. GM3 deficiency is accompanied with arthritis in the RA mice. Defect in GM3 content activated T cell behaviors in vivo with the RA-inducing cytokines. GM3 contents of the synovial gangliosides of RA patients were reduced in the RA, when compared with OA patients [4]. In the CIA mice, the decreased levels of GM3 were coincided with the development and progress levels of CIA. In the homozygous GM3 synthase KO mice model (GM3S / ), the cell number of infiltrating Th-17 cells is increased, suggesting the lack of GM3, and recruits and accumulates Th-17. The Th-17 cytokine level was also increased in GM3S / . Decreased GM3 in RA and CIA is correlated with the increased Th-17 cells. GM3 suppresses the inflammation associated with endothelial cells [5]. CAMs such as VCAM-1 and ICAM-1 are synthesized on leukocytes. Therefore, GM3 is a potential target for antiarthritis inflammation therapy. On the other hand, cellularly synthesized GSLs are suggested to assist osteoclastogenic development in vitro and PDMP, an inhibitor of GlcCer synthase which inhibits osteoclastogenic development [6]. GM3 is abundantly present in © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_19

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primary myeloma tumor CD138+ cells as well as the human MM cell lines. LacCer over GM2 and GM3 is also abundantly produced in the bone marrow (BM) of healthy individuals. In malignant CD138+ plasma cells, GM3 is an abundant polar GSL, and, thus, GM3 is a predominant GSL constituent of MM cells and myeloma cells present in patients [7]. GM3 synergizes and corresponds with IGF-1 and RANKL to elicit osteoclastogenic maturation. Exogenously treated GM3 easily influences and consequently modifies the BM environments and leads to activation of paracrine osteoclastogenesis. The GM3 treatment with RANKL and M-CSF dosedependently enhances the osteoclastogenesis [8]; however, GM3 cocultures with M-CSF without RANKL molecule do not induce osteoclastogenesis. The pro-osteoclastogenic GM3 activity is also confirmed in human osteoclastogenesis experiments that PBMCs are differentiated to osteoclasts with RANKL and M-CSF. Therefore, a direct GM3 action on osteoclast cells is confirmed in the osteogenic response. Pro-osteoclastogenic GM3 directly acts to osteoclastogenic precursors in the presence of IGF-I and RANKL in osteoclast maturation process. Therefore, GM3 cooperates with both IGF-I and RANKL to activate osteoclast maturation process. In OA progression, the level of whole gangliosides produced in OA cartilage is reduced by approximately 40% [9, 10]. Gangliosides suppress the secretion of IL-17 cytokine, consequently preventing the CIA development and progression in mice [11] with critical roles in OA pathogenesis. In mice strain, lacking for GM3 synthase (GM3S), the GM3 functions in OA pathogenesis and thus the mice can be used for validation of target molecules in the treatment of OA [12]. GSLs are reported to inhibit arthritis in a mouse model of arthritis [13]. GM3 and its synthetic mimetics modulate arthritis diseases [9–11]. Using OA models such as age-related, instabilityactivated, and IL-1α-driven models in GM3S KO mice, the GM3 deficiency enhanced OA development in three models [12]. In aged GM3S / KO mice, level of age-associated arthritis is severe, indicating GM3 depletion linked age-associated progression on chondrocyte activities. GM3 is suggested to be associated with inflammation. Because RA model with GM3S / showed the accelerated inflammation [11], OA development may be accelerated in GM3S / mice. The IL-17 elevation in synovium is not found in OA patients but observed in RA patients [14], giving an insight into the difference between RA and OA. GM3S deficiency enhanced the IL-1α-driven chondrocyte apoptosis with the related gene expression. Therefore, GM3 would suppress OA pathogenesis. The GM3 depletion produces severe clinical OA signs [10], suggesting that GM3 is functional dominant species in chondrocytes. For the real action of OA development in GM3-null animals, where GM3 may protect chondrocytes by suppressing apoptosis, further studies are required. Despite the current restrictions, the role for GM3 in OA development emphasizes the involvement of GM3. GM3S-deficient KO mice activate OA development [15], and, therefore, GM3 is a candidate target for therapies of OA. The GM3S expression in vitro suppresses the MMP-13 and ADAMTS-5 levels, suggesting that GM3 suppresses OA formation and is a therapeutic candidate for targeting of the OA.

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References 1. Tas SW, Maracle CX, Balogh E, Szekanecz Z (2016) Targeting of proangiogenic signalling pathways in chronic inflammation. Nat Rev Rheumatol 12(2):111–122 2. Yoon BR, Chung YH, Yoo SJ, Kawara K, Kim J, Yoo IS, Park CG, Kang SW, Lee WW (2016) Referential induction of the T cell auxiliary signaling molecule B7-H3 on synovial monocytes in rheumatoid arthritis. J Biol Chem 291(8):4048–4057 3. Friday SC, Fox DA (2016) Phospholipase D enzymes facilitate IL-17- and TNFα-induced expression of proinflammatory genes in rheumatoid arthritis synovial fibroblasts (RASF). Immunol Lett 174:9–18 4. Tsukuda Y, Iwasaki N, Seito N, Kanayama M, Fujitani N, Shinohara Y, Kasahara Y, Onodera T, Suzuki K, Asano T, Minami A, Yamashita T (2012a) Ganglioside GM3 has an essential role in the pathogenesis and progression of rheumatoid arthritis. PLoS One 7:e40136 5. Kim SJ, Chung TW, Choi HJ, Jin UH, Ha KT, Lee YC, Kim CH (2013) Monosialic ganglioside GM3 specifically suppresses the monocyte adhesion to endothelial cells for inflammation. Int J Biochem Cell Bio 46:32–38 6. Bahtiar A, Matsumoto T, Nakamura T et al (2009) Identification of a novel L-serine analog that suppresses osteoclastogenesis in vitro and bone turnover in vivo. J Biol Chem 284 (49):34157–34166 7. Kiesewetter B, Simonitsch-Klupp I, Kornauth C et al (2018) Immunohistochemical expression of cereblon and MUM1 as potential predictive markers of response to lenalidomide in extranodal marginal zone B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT lymphoma). Hematol Oncol 36(1):62–67 8. Ersek A, Xu K, Antonopoulos A, Butters TD, Santo AE, Vattakuzhi Y, Williams LM, Goudevenou K, Danks L, Freidin A, Spanoudakis E, Parry S, Papaioannou M, Hatjiharissi E, Chaidos A, Alonzi DS, Twigg G, Hu M, Dwek RA, Haslam SM, Roberts I, Dell A, Rahemtulla A, Horwood NJ, Karadimitris A (2015) Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease. J Clin Invest 125(6):2279–2292 9. David MJ, Hellio MP, Portoukalian J, Richard M, Caton J, Vignon E (1995) Gangliosides from normal and osteoarthritic joints. J Rheumatol Suppl 43:133–135 10. Choi HS, Im S, Park JW, Suh HJ (2016) Protective effect of deer bone oil on cartilage destruction in rats with monosodium iodoacetate (MIA)-induced osteoarthritis. Biol Pharm Bull 39(12):2042–2051 11. Tsukuda Y, Iwasaki N, Seito N, Kanayama M, Fujitani N, Shinohara Y, Kasahara Y, Onodera T, Suzuki K, Asano T, Minami A, Yamashita T (2012b) Ganglioside GM3 has an essential role in the pathogenesis and progression of rheumatoid arthritis. PLoS One 7:e40136 12. Sasazawa F, Onodera T, Yamashita T, Seito N, Tsukuda Y, Fujitani N, Shinohara Y, Iwasaki N (2014) Depletion of gangliosides enhances cartilage degradation in mice. Osteoarthr Cartil 22 (2):313–322 13. Seito N, Yamashita T, Tsukuda Y, Matsui Y, Urita A, Onodera T, Mizutani T, Haga H, Fujitani N, Shinohara Y, Minami A, Iwasaki N (2012) Interruption of glycosphingolipid synthesis enhances osteoarthritis development in mice. Arthritis Rheum 64:2579–2588 14. Cho ML, Ju JH, Kim KW et al (2007) Cyclosporine A inhibits IL-15-induced IL-17 production in CD4+ T cells via down-regulation of PI3K/Akt and NF-kappaB. Immunol Lett 108(1):88–96 15. Shui G, Stebbins JW, Lam BD et al (2011) Comparative plasma lipidome between human and cynomolgus monkey: are plasma polar lipids good biomarkers for diabetic monkeys? PLoS One 6(5):e19731

Chapter 20

GM3 Protects Cochlear Hair Cells and Hearing from Corti Degeneration

Deficiency of GM3 synthase leads to deficits in newborn hearing and auditory in humans. For example, deficiency of GM3 synthase causes severe neuronal diseases, showing deafness and neurological disability. The auditory function is characterized by stereocilia of outer hair cells. GM3-associated membrane microdomain of lipid raft formation is crucial for the relevant organization stereocilia in auditory hair cells [1]. GM3 is the dominant cochlear GSL. In postnatal periods, GM3 level is increased with GlcCer, sulatides of SM3 and SM4, GM1, GD1a, GD3, GD1b, and GT1b. GM3 is required for both the neural sound and cochlear editing in vertebrates, especially mammals. In the GM3 synthase, SAT-I KO mice, the hearing attitude was almost completely impaired with spatial stereocilia damage and degeneration of hair cells resided in the Corti organ [2]. The close relation between hearing ability and local ganglioside synthesis is suggested, but the ganglioside’s action and function are still unknown in their roles. The GM3 defection in mice, which are null in GM3 synthase SAT-I gene, caused hearing ability, impairing the development of hearing, and hearing was almost disappeared by 17 days after birth without hair cell formity in the Corti organ [3]. Causing factor of the defected hearing capacity of SAT-I-KO mice was attributed to the absence of GM3 product. GM3 is important during the cochlea development and maturation for the hearing acquisition and maintenance. Ganglioside roles for formation of lipid raft microdomains in auditory systems are developmentally important issue. Cochlea is an auditory organ, receiving the sound in inner ear. Many different gangliosides were expressed in the region of Corti, tectorial membrane, stria vascularis, Reissner’s membrane, and spiral ganglion of the cochlea. Especially, polysialic acid and GM3 were highly expressed in spiral ganglion region. Ganglioside GM3 recovers from hearing dysfunctional region due to selective regeneration of the Corti organ. The gangliosides of a/b/c series are modified to o series such as GD1 and GM1b. However, GM3 synthase KO mice are phenotypically characteristic for hearing defect or loss because of selectively targeted degenerative event in the Corti organ. When normal mouse produces different species of gangliosides including GM1, GM3, GT1b, GD1b, and GD1a, the GM3 KO mice produce only GM1b and GD1α. Thus, GM3 © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_20

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loss is a causing factor for the primary degeneration of the Corti organ. Thus, other factors such as metabolic changes in the endolymph, where Cochlea maturation requires the ganglioside for the hearing ability, are not considered. Then how GM3 works for the Cochlea maturation? Spatially produced GMs are important with temporal production for the formation and organization of the normal function and structure of auditory hair cells. From the results obtained in the GM3 synthase KO mice, it is mentioned that the auditory hearing loss is clearly caused by GM3 deficiency due to its lack of the synthetic enzyme, but the GM3-related conclusion does not always fully explain the complete mechanism. Interestingly, GM3-deficient mice showed some dysfunctional phenotypes including the interference with cerebral neuron myelination and electrical plasticity. Then, another possibility of the GM3-associated lipid raft microdomain has been raised to explain the mechanistic action of GM3. The role of lipid raft microdomain embedded with GM3 is linked in the CNS development and its functional maintenance. As hearing attitude levels are reduced at human birth due to mutations of GM3 synthase gene, in the experimental animal, the St3gal5 / mice showed the similar mode to the animal phenotype. However, the hair cell remodeling and functional degeneration of the St3gal5 / mice can be regenerated and reformed by ganglioside repletion therapy. In the direct protective effect of GM3 treatment on gentamicin ototoxicity to Corti organ explants during young stage from p3 to p5 rats, a drug (gentamicin)-caused hair cell loss was decreased by GM1 and GM3. Aminoglycoside antibiotic, gentamicin, is known to cause loss in inner ear hair cell and sensorineural hearing functions through apoptosis, and the injury is a key event. However, GM3 protects hair cells from the gentamicin-caused cell death, while ceramide rather increases hair cell death from gentamicin toxic apoptosis. Other S1P and GM1 with GM3 acted as cochlear protectants [4]. The roles of gangliosides in embryonic development of CNS and neuronal tissue are limited but are crucial for postnatal function and growth of brain.

References 1. Yoshikawa M, Go S, Suzuki S, Suzuki A, Katori Y, Morlet T, Gottlieb SM, Fujiwara M, Iwasaki K, Strauss KA, Inokuchi J (2015) Ganglioside GM3 is essential for the structural integrity and function of cochlear hair cells. Hum Mol Genet 24(10):2796–2807 2. Inokuchi J (2011) Physiopathological function of hematoside (GM3 ganglioside). Proc Jpn Acad Ser B Phys Biol Sci 87(4):179–198 3. Yoshikawa M, Go S, Takasaki K, Kakazu Y, Ohashi M, Nagafuku M, Kabayama K, Sekimoto J, Suzuki S, Takaiwa K, Kimitsuki T, Matsumoto N, Komune S, Kamei D, Saito M, Fujiwara M, Iwasaki K, Inokuchi J (2009) Mice lacking ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti. Proc Natl Acad Sci U S A 106 (23):9483–9488 4. Nishimura B, Tabuchi K, Nakamagoe M, Hara A (2010) The influences of sphingolipid metabolites on gentamicin-induced hair cell loss of the rat cochlea. Neurosci Lett 485(1):1–5

Chapter 21

GM3 Increases Osteoclast Differentiation Via Direct Cooperation with RANKL and IGF-1

GSLs play integral roles in the tissue and organ development of mammals and their maintenances. For example, in osteoclastogenesis, which BM cells are differentiated into tartrate-resistant acid phosphatase (TRAP)-expressing multinuclear cells or mononuclear cells, ganglioside GM3 and GM1 are synthesized. More in detail, when the BM cells are utilized as osteoclast-like cells and induced with M-CSF and RANKL, GM3 and GM1 gangliosides are predominantly detected [1]. However, the two GM3 and GM1 are not produced if the cells are not treated with M-CSF and RANKL. The GSLs in cultured osteoclasts are comprised of GlcCer, LacCer, GM3, and GM1 on the microdomain lipid rafts of TRAP positive cells. However, RANKLdriven GM3 and GM1 production in BM cells are blocked by treatment with a low molecular inhibitor of GlcCer synthase of D-PDMP. When D-PDMP is treated to the BM cells, the treated cells showed the lowered osteoclastogenesis even in the induction condition by M-CSF and RANKL [2]. Treatment with D-PDMP blocks almost every appearance of GSLs on the BM cells, and this suggests that GSLs synthesis is essential for osteoclastogenesis of BM cells [1]. When the BM cells were treated with RANKL, GM3 and GM1 levels were also increased. In addition, RANK recruited TNF-associated factor (TRAF)-2 and TRAF-6 to the cytoplasmic tail domain of RANKL with activated IkB kinase and IkB phosphorylation. For the lipid raft formation, their machinery molecules of RANK, TRAF2, TRAF6, and LacCer were colocalized to associate with the lipid rafts, suggesting that glycosphingolipids are required for the RANKL-induced osteoclastogenesis [1]. GM3 as the main GSL component of myeloid plasma cells membrane is a factor of osteoclastogenesis in vitro as well as in vivo, as the polar GM2 and GM3 components are the dominant forms in myeloma cells. GSLs profiles including the distribution and composition in human MM cell lines and primary myeloma cells indicate that GM3 is an abundantly expressed. For example, GSLs in primary myeloma CD138+ tumor and human MM cells include GM3 as a major GSL. GM3 does not reside in CD138– nonmalignant BM cells, which are derived from myeloma patients. Thus, GM3 is the representative GSL component of MM cells and patient-derived myeloma cells [3]. Exogenous treatment with GM3 can © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_21

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reorganize the BM environments through membrane modification and activate OC functions. GM3 treatment accelerates the differentiation to form matured cell types in mouse and human OC, supporting the pro-osteoclastogenic GM3 effects where PBMCs are differentiated into osteoclastic cells, likely in the treatment with M-CSF and relevant doses of RANKL. Thus, the effects are derived from the direct regulation of GM3 in OC. The pro-osteoclastogenic GM3 effects appear when it directly interacts with OC precursors under physiological conditions including the treatments with IGF-I and RANKL to activate the maturation of OC. GM3 promotes development and activation of OC in vivo. For the mechanism underlying the exogenous GM3 enhancement to the M-CSF and RANKL effects in vitro, the following explanation is made: GSLs are easily incorporated into the microdomain lipid rafts of cellular PM to associate with them or interact with EGF and VEGF receptors [4]. Exogenous GM3 incorporated into the cell PM of developing OC enhances the M-CSFR and RANK responses to the M-CSF and RANKL ligands, respectively. Stromal cell-derived GM3 is incorporated to the cellular PM lipid rafts in the myeloid progenitors, and GM3 recognition to GM-CSF receptor activates cellular growth responses upon GM-CSF treatment [5]. Direct role of GM3 in pro-osteoclastogenesis is suggested [6]. Apart from the GM3 lipid raft-associated direct effects, alternatively GM3 can indirectly promote production and liberation of pro-osteoclastogenic molecules from their BM cells including T cells, macrophages, monocytes, and osteoblasts. For conclusion, GM3 effectively promotes OC development and functionality. Exogenously added GM3 is associated with cellular PM microdomain of lipid rafts constructed in myeloid lineage cells to upregulate the related intracellular signaling of the cells [5].

References 1. Khavandgar Z, Murshed M (2015) Sphingolipid metabolism and its role in the skeletal tissues. Cell Mol Life Sci 72(5):959–969 2. Bahtiar A, Matsumoto T, Nakamura T et al (2009) Identification of a novel L-serine analog that suppresses osteoclastogenesis in vitro and bone turnover in vivo. J Biol Chem 284 (49):34157–34166 3. Jarvis RM, Chamba A, Holder MJ et al (2007) Dynamic interplay between the neutral glycosphingolipid CD77/Gb3 and the therapeutic antibody target CD20 within the lipid bilayer of model B lymphoma cells. Biochem Biophys Res Commun 355(4):944–949 4. Nagafuku M, Kabayama K, Oka D et al (2003) Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins but does not impair signal transduction via the T cell receptor. J Biol Chem 278 (51):51920–51927 5. Martino S, Cavalieri C, Emiliani C et al (2002) Restoration of the GM2 ganglioside metabolism in bone marrow-derived stromal cells from Tay-Sachs disease animal model. Neurochem Res 27 (7–8):793–800 6. Ersek A, Xu K, Antonopoulos A, Butters TD, Santo AE, Vattakuzhi Y, Williams LM, Goudevenou K, Danks L, Freidin A, Spanoudakis E, Parry S, Papaioannou M, Hatjiharissi E,

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Chaidos A, Alonzi DS, Twigg G, Hu M, Dwek RA, Haslam SM, Roberts I, Dell A, Rahemtulla A, Horwood NJ, Karadimitris A (2015) Glycosphingolipid synthesis inhibition limits osteoclast activation and myeloma bone disease. J Clin Invest 125(6):2279–2292

Chapter 22

GM3 Induces Terminal Differentiation of Leukemic Cells

Immune cells, which are derived from bone marrow (BM), are differentiated into each of finally differentiated terminal cell to act as functional self-defense system. This course of cell differentiation is phenotypically characteristic of appearance or disappearance of specific clusters of differentiation (CD) as specific biomarkers. This is distinguished from the cell proliferation or growth, although the transformed cells also express their markers on cell surfaces named tumor maker or cancerassociated marker. In transformed cells, the tumor or cancer cells show the resembled cell markers. For example, the innate immune cells like macrophages and their precursors such as monocytes function in inflammatory regulation to activate the response of adaptive immune responses through antigen ingestion, digestion, processing, and presentation. The precursor cells like monocytic cells are differentiated into macrophage lineages or DCs with phenotypic changes in cell surface antigens of CD markers and the expression of pro- or anti-inflammatory molecules. Apart from the changes in CDs biomarkers, the monocytoid cell differentiation into the terminal DCs or macrophages induces changes in glycosylation status of cell surface glycans [1, 2]. Especially, the glycosylation in glycosphingolipids is large subject of the changes in cell phenotype. The glycosyltransferases responsible for synthesis of N-acetyllactosamine (LacNAc) sequences, and related sialylcarbohydrates are induced and activated during the DC differentiation [2]. Many glycosyltransferases engaged in monocytes differentiation into macrophages synthesize glycolipids, as parameter of functional and structure changes in glycolipids. Such cell surface glycosylation appears during the differentiation and maturation of certain immune cell types [3]. Glycolipids basically confer structural integrity to the cell PM. However, functionally glycolipids are a member of membrane microdomains or lipid rafts to convey the functions in recognition, binding, adhesion, and interaction of the cells as well as intracellular signal transduction events. Signaling process mediated by glycolipids or GSLs is therefore pivotal in immune cells, including myeloid cells and lymphocytes [4]. For example, GM3 regulates membrane lipid raft-driven signaling after cellular recognition and adhesion in lymphocytes [5, 6]. Therefore, the increased GM3 level indicates the levels of © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_22

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differentiation, activation, and maturation of myeloid precursor cells into matured DCs, macrophages, and monocytes, and GM3 is a factor of the differentiation and maturation [7]. Further information is required about changes in glycosylation that are frequently observed in the events of activation, maturation, and differentiation of monocytoid cell types into macrophage lineage cells. Leukemic monocytic cell lines of human origins, including HL-60 cells, THP-1 cells, and U937 cells as well as megakaryocytoid cell lines of K562, have long been studied as model cells of macrophage-like or megakaryotic cells. Human THP-1 cells of AML and human HL-60 cells of CML are the models for studies on regulation of macrophages and monocytes in inflammation, allergy, asthma, immune response, cancer, and vascular diseases [8, 9]. When the cells are treated with some differentiating factors such as phorbol myristate acetate (PMA) or RA, they are easily differentiated into each of specific cell type. For example, THP-1 or HL-60 cells differentiate into typical macrophage-type lineages, which express general phenotypical antigens and are characteristically featured of native monocyte-derived macrophages [8, 9]. The differentiated cells exhibit phagocytic and adhesion capacity with surface marker molecules such as CD11 [10]. In contrast, PMA-induced differentiation of the human leukemic monocytic cells disregulate in destruction of the dynamic sialylation [11]. Recently, structure analysis has elucidated the exact property and nature of the glycosylation changes. Changes in glycosylation direction and level during differentiation of monocytic cells or precursor cells into macrophages are crucial to elucidate cell fate. Carbohydrate chains attached to GSLs are used as binding ligands specific for pathogenic bacterial lectins as well as galectins, selectins, or Siglecs [12, 13]. The specific carbohydrate-binding lectin-carbohydrate interactions modulate cellular adhesion in cell to cell, cell to ECM, and cell differentiation, apoptotic cell death, and signalings associated with in proinflammatory production of cytokines and phagocytosis of pathogenic invaders. In mass spectrometry analysis, the increased glycosylation pattern of GSLs showed numerous gangliosides including GM2, GM3, GM1, GD1, and GD3 in THP-1 differentiation. The enhanced overexpression of ST3GAL5, GM3 synthase, was prominent, while B4GALNT1 (GM2/GD2 synthase) was not [1]. PMA-driven differentiation of human leukemic cells including U937 and HL-60 to macrophagic cell types involves in the PKC/ERK signaling activation and continuously activates GM3 synthase gene expression and also enhances the GM3 products [14]. The increased gene expression of GM3 synthase is not limited only to promyelocytic cells, because such similar actions following human blood monocyte differentiation into macrophage lines were observed. GM3 synthase is a metabolic keeper for monocyte-to-macrophage differentiation.

22.2

22.1

GM3 Roles in Both Monocytoid and Granulocytoid Differentiation of HL-60 Cells

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GM3 Differentiation Role in Human Leukemic Monocyte Model: THP-1 Cell

An established cell line named THP-1 is a human acute leukemic monocyte, used as the monocyte/macrophage differentiation model. This cell line is terminally differentiated to monocyte and macrophage type. There are some similar aspects and dissimilar aspects between the two different cells of human PBMC-derived monocytes/macrophages and THP-1 cell line [14]. Adult myelogenic leukemia (AML) is the most general adult acute type of leukemia. In AML, cells of normal BM are replaced b leukemic cells. For the causing factors, chromosomal abnormalities were demonstrated, although the fundamental causing factor is unknown. For possible suspected factors, dysfunction of cell surface carbohydrates required for myelopoiesis was raised, because carbohydrate antigens involve in the facilitated vascular adhesion to endothelium, maturation, activation, and phagocytosis of granulocytes and monocytes. Moreover, ganglioside synthesis is accompanied with neutrophilic and monocytic differentiations [15, 16]. As studied for a long time in HL-60 cells, monocytic and granulocytic differentiation events are molecularly associated with specific changes in ganglioside synthesis and their surface expression on the precursor cells [17]. GSLs have been recognized as biomarkers of differentiation in leukemic cells [18, 19]. In fact, AML patients and M1 subtype of AML are highly expressed for the lactotriaosylceramide (LacCer3), GM3, and neolactotetraosylceramide (nLacCer4) more than the normal donors [20]. LacCer3 and nLacCer4 were relatively highly expressed in AML patients rather than in normal donors. Trace amounts of isoglobotriosylceramide (iGb3) were also observed in AML patients [20]. LacCer3Lc3, the LacCer3 synthase, β1,3-Nacetylglucosaminyltransferase5 (β3GnT-5), was 16-fold highly expressed in the BM tissues of AML patients than those of normal person. Thus, GSLs of LacCers, nLacCer4, and GM3 are associated with differentiation and activation of AML cells. Among them, GM3 is a major form in AML cells. As a starting molecule form of the downstream biosynthesis of complex gangliosides, GM3 involves in the cell proliferation, apoptosis, migration and metastasis. Therefore, GM3 identification as a major form in the BM tissues of AML patients is meaningful.

22.2

GM3 Roles in Both Monocytoid and Granulocytoid Differentiation of HL-60 Cells

The AML cells obtained from human leukemia, promyelocytic leukemic HL-60 cells, expresses high level of LacCer3, whereas little LacCer3 is detected in the acute lymphoblastic leukemia (ALL) cell line, Jurkat cells [21]. AML cell and clinically isolated cells highly express LacCer3 on cell surface. However, the expression levels of LacCer vary in specific cell types of AML subpopulation, indicating that the LacCer3 or nLacCer3 is closely associated with its differentiation. The LacCer3

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Fig. 22.1 Changes in morphology and surface attachment during GM3-induced HL-60 cell monocytic differentiation. (a) Before differentiation. (b) Morphological changes and surface attachment after monocytic differentiation

expression in the M1 subtypes as an AML subtype without maturation is increased rather than normal controls. Hence, LacCer3 is pivotal to differentiation due to the fact GM3 sialylation, which acts as a key inducer of human bone marrow [22]. In chemical induction of differentiation, exposure to several chemical agents elicits the HL-60 cell differentiation into the monocytes or macrophagic lineage or granulocytic direction [23–25]. Representatively, several inducers like dimethyl sulfoxide (DMSO) and RA accelerate the HL-60 cell differentiation induction into the granulocyte lineage direction [23, 24], whereas PMA directs to differentiate HL-60 cells into the monocyte/macrophage lineages [25]. As a carbohydrate inducer, GM3 induces differentiation to monocytic direction of both the HL-60 cells and U937 cells, which are myeloid lineage and monocytic leukemia cell types of human (Fig. 22.1) [26]. Among them, the HL-60 cell is used as an experimental model frequently in studies on monocyte differentiation through phenotype changes because HL-60 cell as a promyelocytic lineage can be differentiated into monocyte/macrophage lineage during treatment of a chemical inducer, 12-O-tetradecanoylphorbol-13-acetate (TPA) that is a PKC activator [27, 28]. The production level of GM3 was increased in parallel with GM3 synthase (ST3Gal V) enzyme activity during monocytic differentiation by the monocyte/macrophage differentiation inducers such as TPA [29–32]. However, the upregulation of GM3 product and activity of ST3Gal V enzyme were not correlated to the extent of differentiation level induced by the TPA [30]. Therefore, it is concluded that PKC preferentially activates ST3Gal V gene in order to upregulate the GM3 level in the TPA-induced HL-60 cell differentiation. In addition, PTA-driven PKC transcriptionally activates the ST3Gal V gene expression. Hence, the ST3Gal V gene expression is a prerequisite for the GM3 biosynthesis of the HL-60 cells. Because PKC is suggested to be linked as an endogenous signaling mediator, the ST3Gal V gene promoter was previously isolated in hepatoma HepG2 cells of human as well as SK-N-MC of human neuroblastoma cells [33]. As expected, the gene expression of ST3Gal V gene of human during the HL-60 monocytoid differentiation explains that the transcriptional factor, CREB, binds to its cis-element on the 50 -flanking region for activation of the ST3Gal V transcription. The TPA-stimulated activation of the

22.2

GM3 Roles in Both Monocytoid and Granulocytoid Differentiation of HL-60 Cells

Monocyte/ Macrophage

PMA

HL-60

GM3 Increased

Differentiation RA

Granulocyte

DMSO

119

GM3 Decreased

GM3

HL-60

Differentiation

Monocyte/ Macrophage

PMA : Phorbol 12-myristate 13-acetate RA : Retinoic acid DMSO : Dimethyl sulfoxide

Fig. 22.2 Differentiation direction system of the monocytoid leukemic HL-60 cells

ST3Gal V gene of human is transcriptionally controlled at the 50 -flanking 177 to 83 responsible for a CREB binding site located at 143 position. This cis-element region in the functional sequence is the TPA-responding promoter in HL-60 cells. Multiple transcription factor-binding sites are known for CREB, EGR3, MZF1, NFY, and SP1 [34]. Among them, CREB cis-element is involved in TPA-driven activation of hST3Gal V expression. Therefore, CREB element is crucial for a transcriptional activation in the TPA-driving HL-60 cell differentiation to monocytic phenotypes. The key CREB binding sequence is located at the nucleotide 143 position during the TPA-activated induction of the ST3Gal V gene expression in HL-60 cells, [35]. For the working mechanism underlying the GM3-associated HL-60 cell differentiation, the GM3 production through transcriptional activation has been established in order to phenotypically differentiate the PMA-elicited HL-60 cells. The detailed mechanistic approaches are previously dedicated mostly by Kim’s group [36], which clarified that the GM3 synthase activity and GM3 ganglioside level are essentially enhanced for the HL-60 cell differentiation. On the other hand, HL-60 is another type of model to be differentiated into monocyte/macrophage lineages. This HL-60 is regulated by another type of inducer PMA for the increase in the enzyme GM3 synthase activity and ganglioside GM3, too [37–39]. Additionally, the exogenous addition of GM3 accelerates the HL-60 cell differentiation into monocyte/macrophage types [40, 41]. PMA-driven differentiation of leukemia cells like HL-60 cells is well described (Fig. 22.2). The efficacy of PMA treatment is also derived from the PKCβ activation, followed by fast and sustained ERK1/2 activation in the HL-60 cell differentiation. In addition, PMA-driven terminal differentiation of leukemic cells is involved in the cytochrome C, stress-activated protein kinases, caspase activation, p21WAF and p27kip1 induction, bcl-2 down-regulation, and eventually apoptotic cell death [36]. The leukocyte integrin (α) subunit CD11b known as a macrophage-1 antigen is present on the monocyte/macrophage and granulocyte surfaces, which is coupled

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with CD18(β) subunit to form a heterodimeric α1/β1 complex. The heterodimeric α1/β1 complex confers leukocyte adherence capacity in the myeloid lineage cells [42]. CD11b subunit expression is dependently derived by the differentiation stage with macrophage maturation, through the maximum expression levels of CD11b and its mRNA. In addition, CD11b expression is reached at maximum level during the PMA-driven HL-60 cell differentiation [43]. Therefore, GM3 is indeed a maturating factor for HL-60 cell differentiation into monocytic/macrophage lineages [24, 25]. Recently, CD11b expression event is well documented in the steps of PMA-mediated GM3 production and exogenous treatment with GM3 in HL-60 cells. When several chemicals such as DMSO and RA trigger to induce the HL-60 cell differentiation to granulocytes and PMA promotes the macrophagic differentiation, the level of CD11b expressions is highly increased, and the CD11b/CD18 form is localized on these cells’ surface [44, 45]. Likely, the CD11b expression, a differentiation marker, is correlated with the GM3 production during PMA-activated HL-60 cell differentiation. The pathway for PKC/ERK-directed CREB stimulation during the HL-60 cell differentiation is the acting mechanism. The promoter region containing 50 -flanking sequence of GM3 synthase gene of human has been characterized using the sequence obtained from human fetal brain, HepG2 hepatoma cells, and SK-N-MC neuroblastoma cells [46]. As the GM3 synthase enzyme activity and GM3 amounts are upregulated in the HL-60 cell differentiation into the monocyte/ macrophagic phenotype cells upon PMA treatment, GM3 is the differentiation factor, causing for CD11b expression. The GM3 motivates PKC/ERKs/CREB pathway to express surface antigen CD11b and increase adherence potentials of HL-60 cells. Using PDMP, GlcCer synthase inhibitor, to inhibit the GM3 formation, the CD11b expression and cellular adherence were demonstrated to decrease. Exogenous GM3-induced CD11b expression and GM3 synthase gene expression through the activation pathway of PMA/PKC/ERKs/CREB axis are accompanied with the cellular differentiation levels. The GM3 expression is induced by CREB motif regulation in the transcriptional sequence of the human GM3 synthase promoter region. PMA-induced GM3 biosynthesis and GM3 treatment upregulate the CD11b transcriptional activity via PKC/ERK/CREB/GM3S gene activation and, consequently, enhance the differentiation capacity of the HL-60 cells through CD11b upregulation (Fig. 22.3). Currently, inducing differentiation is regarded as the most promising therapy for AML, if it is possible for the M3 type, an acute promyelocytic leukemia (APL). Enforced therapeutic strategy for cellular differentiation includes all-trans retinoic acid (ATRA) as an effective trial, and it has been recognized as an APL therapy [46–48]. Human leukemia cells of HL-60 and NB4 are the models differentiated into the neutrophile phenotypes by ATRA or into the monocyte phenotypes by PMA treatment. The phenotype features are changes in morphology, function, and drug sensitivity with the lowered proliferation.

GM3 Role in a Megakaryocytoid Differentiation Inducer of Human Leukemic. . .

22.3

121

Phorbol Esters GM3 Enhanced CD11b by GM3

p85 p110

Cytosol X

Wortmannin

PKC

X SAPK/JNK

ERK1/2

P38 MAPK

GÖ6976

SP600125 SB203580 c-jun

U0126

Nucleus

c-jun CREB

X

ATF-2

Trancriptional activation (GM3 synthase)

CREB Binding site

Fig. 22.3 Mechanism responsible for ganglioside GM3 and CD11b expression via GM3/PKC/ ERK/CREB axis in the HL-60 cell differentiation

22.3

GM3 Role in a Megakaryocytoid Differentiation Inducer of Human Leukemic K562 Cells

Hematopoietic stem cells in the BM produce various descendant cells including macrophages, eosinophils, megakaryocytes, platelets, erythrocytes, basophils, neutrophils, DCs, B/T cells, NK cells, and other unnamed subpopulations [49]. The leukemia cells, which are transformed from the BM-differentiated leukocytic cells, are classified as early hematopoietic stem cells that could not appropriately be responsive to environmental stimuli in the BM microenvironments, and they are eventually differentiated into cancerous cells of blood cells [50]. Accordingly, leukemic cells are characterized of immortalization based on abnormal proliferation in the bone marrow and peripherally dispersed into systemic blood. The marrow stem cell lineage (BMSC) is a model to study the megakaryocyte or platelet differentiation. The BMSC is multipotent hematopoietic cells differentiating into erythrocyte, monocyte, granulocyte, and megakaryocyte. BMSC differentiation can be monitored by morphology changes and adhesion ability, cell cycle arrest, endomitosis, megakaryocyte, and intrinsically platelet biomarkers. Megakaryocytes (MKs) are mature hematopoietic cells, keeping the homeostasis by a cellular controller. It was discovered that expression of GM3 synthase, ST3Gal V, is directly related with megakaryocytic differentiation of BMSC. Kim’s group reported that GM3 is crucially important for direction decision of the

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differentiation and megakaryocyte-type differentiation [51]. Megakaryocytic lineage cells normally express a lot of GM3 ganglioside. BMSC accumulation of GM3 is the induced activator for differentiation into megakaryocytic cells. The present status on the development of new activator drugs for megakaryocytic differentiation is currently limited. GM3 is indeed the agent of megakaryocytic development, but not a cell growth inhibitor [52]. Changes in morphology include low nucleus-to-cytosolic ratio, low adhesion, smaller vacuoles, a large size of cells, and low gene regulation. These parameters include the upregulation of megakaryocytic markers of CD10 and CD41 antigens. By recent advances in studying the megakaryocytic differentiation of BMSC, it is known that megakaryocytic differentiation of BMSC is directed by the MEK/MAPK signaling pathway. For example, ERK1/2 is involved in PMA-driven megakaryotic differentiation of BMSC. There are remarkable changes in the adhesive capacity of the BMSC with the activation of the integrin GpIIb/IIIa expression, named CD41 and CD61, on cell surfaces. On the one hand, MEK and ERK regulate cell cycle arrest during differentiation. PMA treatment of BMSC increases the expression level of p53-independent cell cycle inhibitor p21WAF1/CIP1. Additionally, PMA-induced differentiation of BMSC involves fast and sustained ERK2 phosphorylation event, leading to proliferation or differentiation. Cell surface gangliosides regulate or inhibit the cell division via binding to GFR kinases. If the GM3 roles are defined, GM3 has a specific binding protein in hematopoietic cells. Because polyploidy inducer is considered to a promising strategy to give a polyploidization during megakaryocytic differentiation, the author proposes that cell surfaced lipid raft (microdomain) containing GM3 as a SA-based mimic can be a potential polyploidy former for higher level of the megakaryocytic differentiation. GM3 or sialic acidbased mimic can inhibit cell cycle, but not influencing on its cell viability. The high ploidy cells can be accumulated with the cell population. The polyploidization is coupled with the expression of CD41, a complex of platelet glycoprotein IIb/IIIa and GPIIb/IIIa. When megakaryotic differentiation is successful, platelet-like fragments are released. Platelet-like fragments produced from GM3-treated cells carry GPIIb/ IIIa receptor protein, which has capacity for platelet adhesion. The chronic myeloid leukemia K562 cells as human immortalized CML cells were originally isolated from the pleural fluid of CML patients’ blast crisis [52]. The K5632 cells are being utilized as experimental model cells to examine the growth and differentiation of hematopoietic cells, because they are differentiated into an erythroid lineage cells upon treatments with hemin, sodium butyrate, and nicotinic acid [53]. The number of cells with ruffled surfaces and surface expression of certain megakaryocyte and platelet antigens are essential parameters as its hallmarks of megakaryocytic differentiation and platelet production. The events such as cytoskeleton backbone modeling morphologically appear in differentiated megakaryocytes and blebbed platelets. Platelet parts split off from bigger megakaryocytes store the platelet surface markers like CD61 and CD49 antigens. Differentiation is easily validated by examination of benzidine staining or hemoglobin expression [54, 55]. K562 cells are also differentiated into megakaryotic lineages upon treatment with PMA [51]. K562 cells show the low level of expression of Lc3 without

22.3

GM3 Role in a Megakaryocytoid Differentiation Inducer of Human Leukemic. . .

123

Fig. 22.4 Morphological changes in megakaryocytically differentiated K562 cells induced by PMA. (a) Before differentiation. (b) Morphological changes after megakaryocytic differentiation

GM3 expression [56]. But during differentiation to megakaryocytic phenotype, GM3 content is increased together with the increasing degree of differentiation. The blocking of differentiation is due to low levels of GM3 if GM3 is degraded by GM-degrading enzyme sialidase NEU3 activation [57]. Kim’s group has shown that cell surfaced GM3 acts as a true agonist of platelets towards the megakaryocytic lineage. GM3 is an inducer in megakaryocyte/platelet physiology to terminal differentiation. Leukemic K562 cells can produce in vitro platelet-derived particles. It was found that the GM3 amount increases correlatedly with hST3Gal V activity during megakaryocytic differentiation with an inducer, but not with an erythrocytic inducers such as hemin. The hST3Gal V activity is increased time-dependently during megakaryocytic differentiation. Thus, exogenously treated GM3 induces megakaryocytoid differentiation of BMSC or K562 cells with CREB-mediated GM3 accumulation. Thus, GM3 is a trigger of differentiation of BMSC or K562 cells. However, GM3 is a glycolipid and alternative GM3-replaceable mimetics will be synthesized and developed for platelet differentiation. Thus, GM3 functions as a megakaryocytoid differentiation factor of erythroleukemia K562 cells (Fig. 22.4) [55]. Therefore, megakaryotic K562 cells also undergo the similar morphologic changes during megakaryocytoid differentiation of PMA-induced K562 cells. GM3 synthase functions as a megakaryocytoid differentiation factor of K562 cells, a human erythroleukemia cells. Upon PMA treatment, the K562 cells can differentiate into megakaryocytes [58, 59]. The GM3 contents are increased with the increased activities of GM3 synthase and ST3Gal V in the megakaryocytoid differentiation of K562 cells upon PMA induction known as a megakaryocytoid differentiation factor. However, the megakaryocytoid differentiation of K562 cells is not stimulated by erythrocytic differentiation factor, hemin [60]. PKC activates human ST3Gal V gene expression with a concomitant increase in GM3 content at the transcriptional, protein, and product levels.

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GM3 Is an Inducing Factor of Megakaryocytic Differentiation by CAPE in the Human CML K562 Cells

The leukemia cells are not easily progressed to apoptotic cell death, inducing the leukemia expression such as hematopoietic cell differentiation and apoptosis [61]. Many different leukemic cells have been established in order to understand the detailed differentiation events of unipotent and multipotent hematopoietic cells. Human CML K562 cells are one of the cases. K562 cells are used for better understanding of leukemia differentiation. Seemingly, differentiation model system of the chronic myelogenous leukemia (CML) K562 cells of human has also been illustrated for erythroid and megakaryocyte (platelets). Some chemical agents including Ara-C, butyric acid, hemin, and their mimics are used as erythrocytoid differentiation factors for K562 cells (Fig. 22.5) [62]. Also, the K562 cells are used as experimental cells to study on the megakaryocytic cells by PMA. PMA induces megakaryocytic appearance, development, growth suppression, phenotype change, and gene controls (Fig. 22.6). Genetic regulations include megakaryocytic antigens like CD10 and CD41 [63]. PMA-mediated K562 cells response is based on the MEK/MAPK pathway [64]. ERK1/2 activation is also linked, while ERK1/2 inhibition and p38 MAPK activation are coupled in erythrocytic differentiation [63]. The adhesion change of the K562 cells is caused by the integrin phenotype changes and cytoskeleton distribution [65, 66]. In addition, the level of GpIIb and GpIIIa (CD41 and CD61, respectively) integrin complex is enhanced, functioning as an induction biomarker of cellular differentiation [67]. PMA treatment increases the p21WAF1/CIP1 expression in a p53-independent manner [68], and the MEK activity induces phosphorylation of ERK2 [64] in the K562 cells, indicating its multipotent hematopoietic potency, capable of differentiating into multiple cells such as erythrocytic, monocytic, granulocytic, and megakaryocytic lineages [63].

Ara-C RA

Erythroid

Hemin NaBut

GM3

Platelet peroxidasepositive

PMA

GM3 content

Hemin

GM3 content

K562 Megakaryocyte

PMA NaBut RA : Retinoic acid NaBut : sodium butyrate Ara-C : 1- -D-arabinofuranosyl cytosine

Fig. 22.5 Differentiation model system of the human CML K562

platelet

22.4

GM3 Is an Inducing Factor of Megakaryocytic Differentiation by CAPE in the. . .

125

* Change in cell morphology * Adhesive properties

K562 cell

* Cell growth arrest

PMA stimulates

* Expression of markers associated with the megakaryocytes Myeloid lineage marker : CD15 Megakaryocyte markers : Erythroid lineage markers :

CD41a, CD61, -naphthyl acetate esterase, IL-6 CD71, Glycophorin A, -globin

Fig. 22.6 PMA as a megakaryocytic developer and proliferation inhibitor with phenotypic changes

Gangliosides of leukemia cells are biochemical markers for human hematopoietic cells in their steps of differentiation and direction [69–71]. Exogenous GM3 is an inducing factor of monocytoid differentiation. The ganglioside synthetic level of neolacto-series is indeed the granulocytoid differentiation factor of HL-60 cells [72, 73]. In fact, GM3, GM2, and GD1a gangliosides are the major ganglioside constituents produced by K562 cells [74]. After TPA or hemin-dried differentiation, however, GD1a and GM2 contents are increased. GM3 content is increased in TPA-driven megakaryocytic differentiation. GM3 content is decreased in heminderived erythrocytic direction. The megakaryocytic lineage is enriched with GM3, compared to erythrocytic lineage. Differentiation is also involved by systemic increase in megakaryocytic markers, where GM3 product and hST3Gal V activity are increased by its inducers, but not by an erythrocyte inducer, hemin [69]. hST3Gal V activity is elevated during megakaryocytic differentiation [75]. Changes in GSLs synthesis are well documented in human K562 cells [69]. Thus, GM3is a trigger in differentiation of K562 cells. A distinct differentiation inducer, caffeic acid phenethyl ester (CAPE) has been known to have antagonistic activities against oxidation, inflammation, and tumor growth. CAPE is also a NF-κB inhibitor, as this is linked to tumor growth and invasion [76]. Regarding its mechanism on the biological activities on growth factorassociated phenotypes, CAPE inhibits VEGF-associated events such as growth, tube formation, migration, actin fiber genesis, and VE-cadherin loss in endothelial cells. Therefore, it inhibits VEGF-VEGFR-2 and downstream signals, as CAPE terminates VEGF-linked neovascularization and vascular blood vessel formation [77]. Furthermore, the CAPE-exerted apoptotic cell death of myelocytoid leukemic U937 cells of human was dose- and time-dependently shown. Oligonucleosomal and DNA fragments are detected in CAPE-treated U937 cells. The nuclear DAPI staining and nuclear condensation were also found. Apoptotic CAPE displays for typical phenotypes including cytochrome C, Bcl-2 and Bax, caspase-3, and PARPs, but not for Fas protein or phospho-eIF2 alpha and CHOP, an ER-mediated apoptotic marker. Thus, CAPE is an effective inducer of the mitochondrial apoptosis but not ER apoptosis or death receptor in U937 cells [78].

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Fig. 22.7 K562 cells treated with CAPE or PMA. GM3 or CD41 is detected by fluorescence microscope using anti-41, anti-GM3, or anti-mouse FITC/IgM. The upper is CAPE

CAPE also induces ATRA-mediated apoptotic cell death and granulocytic differentiation of HL-60 cells known as human promyelocytic cells [79] and also induces megakaryocytic differentiation [80]. GM3 is linked to the differentiation induced by CAPE. CAPE treatment differentiates to the megakaryocytic lineage from K562 cells with characteristic physiological presence of vacuoles and demarcation membranes. CAPE induces the cell cycle arrest, having similar changes in PMA treatment. CAPE stimulates the expression of specific megakaryocytic differentiation markers and cell cycle inhibitors of p21WAF1/CIP1 and p27kip1. However, expression of the cyclins and CDKs associated with the cell cycle progression is rather reduced by CAPE treatment. Moreover, CAPE stimulates the ERK1/2 but inhibits p38. Therefore, CAPE as stimulus leading to direction for megakaryocytic differentiation provokes the same symptoms in signal transduction induced by PMA. CAPE effect was selectively mediated by ganglioside GM3. The transcription of ST3Gal V gene and GM3 synthesis levels is increased by CAPE treatment in the experiment used by hST3Gal V promoter and D-PDMP. Morphological modifications were also involved in K562 cells. The siRNA for hST3Gal V also blocks hST3Gal V and megakaryocytic marker gene expressions induced by CAPE. Thus, GM3 stimulates the differentiation to megakaryocytic phenotype in K562 cells upon treatment with CAPE, and this supports the GM3-driven differentiation. Endogenous hST3Gal V expression and CREB DNA binding are coupled [75]. The promoter of hST3Gal V mRNA transcription involves the CREB factor regulated by CAPE with ERK1/2 activation and p38 MAPK inhibition. The ST3Gal V siRNA blocks the CAPE-stimulated expression of the phenotypical marker molecules in K562 cells. CREB binding site located at the TGACGTCA sequence at 143 to 136 in human ST3Gal V promoter is a key region for upregulation by PMA [75] in K562 cells. CAPE is a PMA-like CREB inducer for GM3 synthesis [81]. Therefore, GM3-binding molecules might act in hematopoietic cells. GM3 produced by CAPE induces K562 megakaryocytic differentiation, and CAPE’s role in K562 megakaryocytic differentiation is mediated through CREB-GM3 axis (Fig. 22.7).

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Chapter 23

α2,3-Sialyllactose (3SL) or α2,6-Sialyllactose (6SL) of GM3 Glycan in Innate Immunity

Sialooligosaccharides benefit the body because sialooligosaccharides as probiotics serve as carbohydrate substrates for beneficial bacteria. Sialooligosaccharides prevent pathogen attachment to intestinal epithelial cells because they function as glycan receptor decoys and as antiadhesive and antimicrobial agent [1]. Sialooligosaccharides directly influence intestinal epithelial and enterocytic cells and also modulate the cell responses. For example, sialooligosaccharides regulate selectin-driven cell–cell recognition and interaction of immune cells, decreasing leukocyte homing and rolling on activated vascular endothelial cells and, consequently, and decrease mucosal leukocyte infiltration. Sialooligosaccharides modulate cytokine production in innate immune cells and consequent appropriate Th-1/Th-2 response. Sialooligosaccharides are also a supplied SA source required in neuron and cognition.

23.1

3SL/6SL and Gangliosides in Innate Immunity

Milk oligosaccharides have been reported to contain shorter chain trisaccharides such as sialyllactose or fucosyllactose or complex higher molecular weight glycans [2]. The functions of milk oligosaccharides are not well-known, although some beneficial milk oligosaccharides are mentioned in literature. At present, the scientific mechanisms regarding the functions in glycan receptor regulation, antiadhesive and antimicrobial “sensing” or “signaling” of the sialyllactose, fucosyllactose, or complex glycans is still in dawn. The function of sialylα2,3 or α2,6 lactose (3SL or 6SL) is interested in intestinal immunity and disease (Fig. 23.1). Certain milk GSLs and oligosaccharides include 3SL or 6SL, and they elicit intestinal immune responses through direct binding to DCs in order to activate the cells. 3SL directly recognized by DCs reduces an inflammatory response, and this indicates dietary 3SL or 6SL modulation of mucosal immunity [3, 4]. More specifically, milk sialooligosaccharides such as 3SL and 6SL have been reported to reduce © Springer Nature Singapore Pte Ltd. 2020 C.-H. Kim, GM3 Signaling, https://doi.org/10.1007/978-981-15-5652-4_23

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α2,3-Sialyllactose (3SL) or α2,6-Sialyllactose (6SL) of GM3 Glycan in Innate. . .

Fig. 23.1 Sialyllactose structures

α2,3

ȼ1,4

α2,6

aGuŒœhŠ

aGnˆ“ˆŠ›–šŒ

aGn“ŠuhŠ

zˆ“ “OȻYSZP “ˆŠ›–šŒGOZzsPG zˆ“ “OȻYS]P “ˆŠ›–šŒGO]zsPG

autoimmune enterocolitis in mice via specific colonization of intestinal bacteria [5], indicating the mechanisms mediating the “sensing or signaling” [6]. GM3- and 3SL-containing gangliosides are major plasma membrane constituents, and SA-linked gangliosides function as binding receptors of host cells for pathogenic bacterial toxins [7] and some viruses [8–10]. Sialic acid residues in gangliosides expressed in the viral coats also function as capture ligands of pattern recognition receptors of mDC [4]. Because virus captures the cell surfaced sialyl residues on gangliosides, 3SL or 6SL is a pattern recognition molecule.

23.2

3SL Interaction with Toll-Like Receptor (TLR)-4 on DCs in Intestinal Innate Immunity

Sialooligosaccharides of breast milk promote growth of beneficial bacteria, prevent attachment of pathogens in the gastrointestinal tract as soluble receptors, reduce adhesion of leukocytes, and affect anti-inflammation and maturation of intestinal mucosal immunity as prebiotics [11–13]. Therefore, it is suggested that sialooligosaccharides exert protective effects. DCs are indeed dominant players of intestinal inflammation, and intestinal DCs exert either proinflammatory or tolerogenic responses. Sialooligosaccharides stimulate intestinal innate immune responses by interaction with DCs. DC maturation is induced by MyD88-dependent TLR signalings and produces inflammatory cytokines via the NF-κB pathway [14]. 3SL was reported to exacerbate acute colitis in mice model of spontaneous chronic intestinal inflammation [15]. Thus, the acting mechanism of the sialoglycan signaling in the intestine is interesting, because identification of molecular targeted cells by sialooligosaccharides is related in its downstream signaling. The boundary interface of the immune system in intestinal immunity involves diverse epithelial enterocytic cells and cell-specific pattern recognition and carbohydrate-binding lectin receptors on cell surface of intestinal-resident leukocytes. These receptor molecules of the interfaced cells recognize and bind foreign antigenic antigens resided in the intestinal lumen. The interaction and contact of the mucosal immune cells with glycolipid or glycoprotein sialoligosaccharides input from exogenous dietary sources and detached from the surfaces of host cells potentiate immunemodulating roles [16]. The molecular mechanism such as sialoligosaccharide

23.3

3SL Stimulates CD40, CD80, and CD86 Expression and Driving

135

sensing and signaling by DCs is initiated from the interaction with many carbohydrate-binding receptors including TLRs, DC-SIGN as C-type lectin, and Siglecs [16]. 3SL activates DCs through direct recognition of TLR-4 and MyD88 signaling [17], while the structural mimic 6SL failed to stimulate DC, concluding a certain sensing of 3SL or 6SL in a TLR-4 sensing and signaling. If 3SL only promotes inflammation, the molecular recognition is interesting. Because SA appears to α2,3linked to Gal residue, this conformation is the same as 3SL [18]. The α2,3sialylglycans can be used as a signaling transducer for activation of innate myeloid cells present in the intestinal mucosal area. In fact, 3SL was reported to prime such signals in early infancy during lactation [17]. As 3SL binds to TLR-4 on DCs, the gram-negative bacterial lipooligosaccharides (LPS) or sialylated bacterial glycans target the TLR-4 of DCs. A well-known case is the α2,3-sialyl-LPS of C. jejuni, and the sialyl LPS targets the TLR-4 [19]. Therefore, it is suggested that TLR-4dependent signaling senses 3SL. CD80 and CD86 expressions were increased, whereas the CD40 level is decreased, while the synthesis is not abolished in 3SL-activated DCs. Absence of TLR-4, but not TLR-2, strongly decreased activation of 3SL stimulation. Thus, it was concluded that TLR-4 on DCs directly binds to 3SL for sensing and activates the intestinal immune responses. Therefore, it is considered that 3SL provides structural basis to educate and learn the early innate immunity in order to prepare the infant for a possible future emergency such as invasion or encounter with α2,3-sialyl mimicking pathogens. For example, 3SL first colonize certain α2,3-sialylated pathogens and induces colitis, although 3SL directly affects the immune system.

23.3

3SL Stimulates CD40, CD80, and CD86 Expression and Driving CD11c+, Ly-6Chi, CD4+, Th-1, and Th-17T Cells with Secreted TNF-α, IL-6, IL-12, and CCL5

The α2,3-sialyltransferase-4 (ST3GAL-4) yields the α2,3sialyllactose, 3SL, in lactating mammary gland, and 3SL regulates intestinal mucosal immunity [17] after 3SL induces bacterial colonization. 3SL induces its sensitivity to dextran sulfate sodium (DSS)-provoked acute enterocolitis [6]. However, 6SL does not affect the susceptibility. In mouse acute colitis model 3SL induced bacterial colonization [15], although some differences in intestinal bacterial colonization were observed compared to the 3SL effect on DSS-induced acute enterocolitis. Direct stimulation of 3SL on DCs exerts intestinal homeostasis, although direct interaction of milk oligosaccharides on mucosal immune responses is complicated. For example, administration of newborn rats with human milk oligosaccharides, which are composed of di-sialyl-Lac-N-tetraose or SL, blocked necrosis and enterocolitis [20]. Direct administration by lacto-N-neotetraose i.v. injection in mice increased

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α2,3-Sialyllactose (3SL) or α2,6-Sialyllactose (6SL) of GM3 Glycan in Innate. . .

the level of accumulation of suppressive immune cells with IL-10 production [20]. The above beneficial results were attributed to the 3SL and 6SL for the antiinflammatory and functional roles in intestinal homeostasis [15]. The 3SL bound to DCs stimulates a proinflammatory response, and α2,3-SA leads to the early development of enterocolitis, as DCs are pivotal cells in intestinal immune responses. Sialyl oligosaccharides modulate mucosal immunity, as 3SL in intestine and disease was reported in colitis development mice [1]. Milk 3SL directly stimulates DCs to produce cytokines driving Th-1- and Th-17-dependent inflammation. Monocytederived DCs (mo-DCs) present in the colon are modulated mainly by 3SL sensing; however, the 3SL sensing effect is not observed in TLR-4-deficient CD11c(+) cells, indicating the 3SL induces TLR-4-signaling in inflammation. Thus, 3SL directly managed to regulate intestinal mucosal immune responses, which elicit susceptibility to enterocolitis. ST3Gal-4 deficiency decreases leukocyte infiltration. Development of intestinal inflammatory responses is inhibited in the ST3GAL-4-deficient mice (ST3Gal4 / ), where ST3Gal-4 synthesizes 3SL. Supplementation of newborn ST3Gal-4( / ) mice with 3SL increased the level of colitis. 3SL directly stimulated lymph node CD11c-positive DCs and elicited cytokine expression for Th-1 and Th-17 T cells. CD11c+ DCs transduce and sense 3SL-mediated signaling in order to elicit proinflammatory responses, secreting proinflammatory IL-6, IL-12, and TNF-α cytokines. CD45+ cell infiltration into colon tissues is increased in the 3SL-administrated mice, and CD11c+ cells largely infiltrate colons of ST3Gal-4(+/ +) mice, but not ST3Gal-4( / ) mice. This indicates an engagement of DCs in 3SL-involved enterocolitis. DCs stimulated with 3SL also enhance the expression levels of CD80, CD86, and CD40 up to the level induced by LPS stimulation. The inflammatory IL-6/IL-12/TNF-α cytokines and C–C motif ligand 5 (CCL5) as an inflammatory chemokine are also increased in 3SL-treated DCs, not in 6SL-treated DCs. The levels of CD4+ cells, CD11c+ cells, and Ly-6Chi cells are increased, and this correlated with aggravated intestinal inflammation. Finally, 3SL increased TGF-β1 production, which induces T-helper 17 differentiation.

23.4

6SL Restores Sialylation of GNE-Related Myopathy

GNE-caused myopathy syndrome is classified to a distal myopathy, which displays a specific phenotype characteristic of rimmed vasculopathy or inherited inclusion body myopathy. These diseases are pathologically specific for amyloid accumulation with related proteins in the intracellular fraction. GNE myopathy is raised by the GNE-coding gene mutations, where the GNE gene encodes a bifunctionally active enzyme for SA synthesis. In human, alternatively spliced six variants of different GNE transcripts are known. The representative alternative variant transcript with the GenBank No. NM_005476 encodes 722 amino acids. However, the longest variant transcript with the GenBank No. NM_001128227 encodes 753 amino acids [21]. The GNE enzyme is a bifunctional UDP-GlcNAc2-epimerase and N-acetylmannosamine (ManNAc) kinase. The GNE enzyme produces SA via two

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steps. GNE myopathy can recover and is a therapeutically treatable disorder. Sialylation can recover the defected syndromes, as well established in skeletal muscle dysfunction. Human patients of GNE mutation myopathy exhibit a progressive dysfunctional symptoms raised by defection in SA generating pathway. GNE myopathy can be cured by SA replacement and supplementation on GNE myopathy symptom model mice. Experimental therapy of a SA-linked carbohydrates of 3SL, 6SL, or free SA (NeuAc type) is applicable by oral administration to GNE myopathy mice. In treated mice, sialic acid content is measurable in the skeletal muscle. Temporary and spontaneous locomotion capacity is recovered in 3SL- or 6SL-receiving mice, while NeuAc-received mice have been progressed to the disease. Administration of 6SL, but not SA, restores sialylation in muscle, because 6SL is metabolically degraded to free SA during metabolism. 6SL ameliorated muscle atrophic symptoms and improved symptomatic GNE myopathy in mice. Thus, 6SL is superior to free sialic acid, providing clinical use in patients [22].

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