[Advances in Experimental Medicine and Biology] The Molecular Immunology of Complex Carbohydrates-3 Volume 705 || Aberrant Glycosphingolipid Expression and Membrane Organization in

643A.M. Wu (ed.), The Molecular Immunology of Complex Carbohydrates-3, Advances in Experimental Medicine and Biology 705, DOI 10.1007/978-1-4419-7877-6_34, © Springer Science+Business Media, LLC 2011

Keywords GM3 • Caveolin-1 • Tumor cell motility

Abbreviations

CHO Chinese hamster ovaryEGFR Epidermal growth factor receptorEM Electron microscopyGPI GlycosylphosphatidylinositolGSL Glycosphingolipid(s)

34.1 Introduction

More than 20 years ago [1], the pioneering work of Dr. Sen-itiroh Hakomori formed the basis for the concept that aberrant glycosylation is a general feature of human cancer. The term “aberrant glycosylation” describes the altered expression of oligosaccharide epitopes associated with both glycolipid and glycoprotein anti-gens in human cancer. This event is the consequence of at least two different meta-bolic mechanisms: (1) the impairment of specific glycosylation steps (“incomplete synthesis”) and (2) the transcriptional induction of genes encoding for glycosyl-transferases or carbohydrate transporters (“neosynthesis”) [2]. Both mechanisms contribute to the accumulation of antigen-carrying tumor-associated epitopes that

A. Prinetti (*) Department of Medical Chemistry, Biochemistry and Biotechnology, Center of Excellence on Neurodegenerative Diseases, University of Milan, Via Fratelli Cervi 93, 20090 Segrate, Milano, Italy e-mail: [email protected]

Chapter 34Aberrant Glycosphingolipid Expression and Membrane Organization in Tumor Cells: Consequences on Tumor–Host Interactions*

Alessandro Prinetti, Simona Prioni, Nicoletta Loberto, Massimo Aureli, Valentina Nocco, Giuditta Illuzzi, Laura Mauri, Manuela Valsecchi, Vanna Chigorno, and Sandro Sonnino

* Ganglioside and glycosphingolipid nomenclature is in accordance with Svennerholm L (1980) Ganglioside designation. Adv Exp Med Biol 125:11 and the IUPAC-IUBMB recommendations Nomenclature of glycolipids (1998) Carbohydr Res 312:167–175.

644 A. Prinetti et al.

were originally defined by their ability to raise the production of specific antibodies and subsequently characterized on the basis of their molecular structure. The dis-covery of oligosaccharide tumor-associated antigens provided useful diagnostic tools and opened the field of tumor glycobiology, which developed tremendously in the following decades.

It soon became clear that aberrant glycosphingolipid (GSL) expression is not simply an epiphenomenon accompanying neoplastic transformation (Fig. 34.1). The modification of GSL expression deeply affects several properties of tumor cells that are directly relevant to the growth and progression of the tumor and to metas-tasis formation: cell adhesion (to the extracellular matrix or to the endothelium of blood vessels), motility, and recognition and invasion of host tissues. In particular, GSLs might contribute to the modulation of integrin-dependent interactions of tumor cells (determining their adhesion, motility, and invasiveness) with the extra-cellular matrix as well as with host cells present in the stromal compartment of the tumor. The involvement of GSL in these events is of great interest as, after the initial tumorigenic events triggered by genetic mutations of oncogenes and tumor suppressor genes, tumor–host interactions represent a critical feature of each step leading to cancer disease progression.

Recently, it has been proposed that many aspects of tumor cell social life are mediated by cell surface signaling complexes regulated by GSL. For example, GSLs at the cell surface interact with plasma membrane receptors (including both adhesion receptors, such as integrin receptors, and classical tyrosine kinase growth factor receptors) forming signaling complexes that are able to influence the activity of signal transduction molecules oriented at the cytosolic surface of the plasma membrane (mainly the Src kinases pathway members). Highly hydrophobic adapter membrane proteins belonging to the family of tetraspanins are essential for the organization of these complexes. On the other hand, their function seems strictly dependent on their GSL composition and likely on specific sphingolipid–protein interactions. From this point of view, particularly intriguing is the connection

“ABERRANTGLYCOSYLATION”

alterations ofcarbohydrate epitopes

(glycolipids/glycoproteins)in tumors/tumor cells

Changes inGLYCOSPHINGOLIPID

METABOLISMin tumors/tumor cells

NEOPLASTICTRANSFORMATION

Fig. 34.1 Aberrant glycosylation and neoplastic transformation

64534 Glycosphingolipids Modulating Tumor Phenotype

between GSLs and another hydrophobic adapter membrane protein, caveolin-1, which plays multiple roles as a suppressor of tumor growth and metastasis in ovarian, breast, and colon human carcinomas.

A growing body of evidences indicates that caveolin-1 influences the develop-ment of human cancers. Caveolin-1 expression inhibits in vivo tumor growth, metastasis development, and invasiveness in metastatic mammary tumor cells and promotes cell–cell adhesion in ovarian carcinoma cells by a mechanism involving inhibition of nonreceptor Src tyrosine kinases [3]. Caveolin-1 might act as a mem-brane adapter that couples the integrin subunits to Src kinases [4]. The control of tumor–host interactions could be linked to the function of a signaling complex among GSLs, caveolin-1, and membrane proteins belonging to signaling pathways controlling tumor cell adhesion and motility. This notion has been recently sup-ported by the observation that caveolin-1 overexpression in human melanoma cells deeply alters GSL membrane organization, with consequent alteration of leading-edge structure [5]. This allows for the hypothesis of a novel, caveolin-1-dependent role of GSL in regulating tumor cell motility and invasiveness [6].

34.2 GSLs Controlling Tumor–Host Interactions

Tumors arise from a precise series of steps, characterized by a self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death, limitless replicative potential, sustained angiogenesis, and tissue inva-sion and metastasis. After the initial tumorigenic events triggered by genetic muta-tions of oncogenes and tumor suppressor genes occurring within tumor cells, tumor–host interactions are of crucial importance in all the steps leading to cancer disease progression. The stromal compartment of a tumor comprises a variety of host cells, including endothelial cells, fibroblasts, and inflammatory cells. Host-derived cells infiltrated into the tumor tissue interact with tumor cells and are sub-sequently conscripted by tumor cells to produce an array of both soluble and insoluble factors that stimulate tumor growth, angiogenesis within the tumor mass, and metastasis. Another aspect critical for tumor progression is the interaction of tumor cells with the extracellular matrix, which is implicated in the release of cells from the tumor mass and in subsequent extravasation and metastatic invasion.

In the interactions between tumor cells and the surrounding microenvironment, the relevant interface is represented by the surface of the tumor cell, the site where interactions between the cell and the extracellular environment are organized and transduced into signals able to modify the cell properties influencing the tumor phenotype. In all these aspects, the structural and functional properties of the cell membrane play a prime role. Sphingolipids, and in particular GSLs, which are typi-cal components of the plasma membrane asymmetrically enriched in the external leaflet, mediate several aspects of the interactions between a cell and the extracel-lular environment or other cells. They are known as cell surface antigens, as mediators of cell–cell recognition and cell adhesion, and as modulators of several aspects of


the signal transduction processes involved in cell proliferation, differentiation, and oncogenesis. Several lines of evidence point to the importance of GSLs in the for-mation and progression of tumors [2, 7] (Fig. 34.2). Their expression and metabo-lism are dramatically changed during neoplastic transformation and tumor progression. Deep changes in sphingolipid expression occur in several cells and tissues during neoplastic transformation, and overall alterations in the structures of carbohydrate epitopes associated with both GSL and glycoprotein antigens (“aber-rant glycosylation”) are a common feature of tumors and tumor cell lines [8–11]. Moreover, the accumulation of high levels of specific GSLs in specific types of cancer was reported: GD3, GD2, and GM3 gangliosides in human and mouse mela-noma; GD2 in neuroblastoma; Gg3 in human and mouse lymphoma; fucosyl-GM1 in small cell lung carcinoma; and globo-H in breast and ovarian carcinomas. It has been shown that tumor cell lines with higher tumorigenic potential are character-ized by higher ganglioside levels [12, 13] and that artificially induced increase in cellular ganglioside levels enhanced the ability to form tumors. Correspondingly, a correlation between some carbohydrate structures and survival rates in human patients with cancer has been observed [2, 7]. Another aspect of GSL-dependent interactions between tumor and host environment has been suggested by the finding that tumor cells shed significant amounts of selected ganglioside species, mostly in

Inhibition ofimmune system(GSL shedding)

Cell adhesion,motility,

recognition

Cell proliferation(modulation ofgrowth factor

receptors)

INVASION/

METASTASIS

TUMOR

PROGRESSION

TUMORGROWTH

Altered expressionof GSL

in tumors

Fig. 34.2 Consequences of aberrant GSL expression on tumor cell biology


the form of vesicles [14–16], and that serum ganglioside levels in patients with cancer are higher than in healthy individuals [14–16]. Tumor-derived gangliosides released in the host environment might play a role in eluding aggression by the host immune system.

Several hypotheses have been made about the possible functional role(s) of aber-rant GSL expression in defining the surface properties of tumor cells. GSLs in tumor cells have been implicated in the mediation of cell adhesion, motility, and recognition through GSL-binding proteins and/or GSL–GSL interaction [2, 7, 17]. GSL might contribute to the modulation of integrin-dependent interactions of tumor cells with the extracellular matrix, as well as with host cells present in the stromal compartment of the tumor. GSL might also be involved in selectin- or galectin-dependent adhesion of tumor cells to endothelial cells, a crucial step in the extravasation of circulating tumor cells and in the initiation of metastasis. At least in some cases, trans-GSL–GSL interactions are also important in determining the motility and metastatic potential of tumor cells, while in others, GSLs at the tumor cell surface have antiadhesive properties. For example, GM3–GM3 interaction-mediated repulsion of tumor cells was implicated in the release of cells from the tumor mass, playing a role in the initiation of metastasis.

The specific biological function of a GSL in these events depends on the oligo-saccharide and hydrophobic moiety structure of the GSL, as well as on the cellular context considered. In the case of the GM3 ganglioside, by far the most abundant ganglioside in the human body, several studies have shown that its cellular levels are correlated in multiple ways with control of tumor cell motility, invasiveness, and survival. In bladder cancer, GM3 is highly expressed in noninvasive, superficial tumors compared with invasive tumors, as a result of the upregulation of relevant glycosyltransferases [8, 10, 18]. Artificially increased cellular levels of GM3, obtained by incubation in the presence of exogenous GM3 [10] or by pharmaco-logical treatment with brefeldin A [18, 19], were accompanied by a strong reduc-tion in tumorigenic activity and/or the invasive potential of human tumor cell lines. Our group recently showed that a phenotypic variant of the A2780 human ovarian carcinoma cell line, characterized by increased GM3 synthase expression [6], exhibited reduced motility and proliferation [20]. The stable overexpression of GM3 synthase (SAT-I) in a mouse bladder carcinoma cell line reduced cell prolif-eration, motility, and invasion, with a concomitant increase in the number of apop-totic cells [11]. High expression levels of GM3 with concomitant expression of the tetraspanin CD9 in colorectal [21, 22] and bladder [18] cancer cells inhibited Matrigel- and laminin-5-dependent cell motility. In Chinese hamster ovary (CHO) mutants, the coexpression of CD9 and GM3 is essential for the downregulation of cell motility.

A well-characterized example of GSL–GSL interaction controlling the motility and metastatic potential of tumor cells has been studied in B16 melanoma cells. Adhesion of these cells (expressing high levels of GM3) to endothelial cells, which express LacCer and Gg3, is mediated by a GM3–LacCer or GM3–Gg3 interaction and leads to enhanced B16 cell motility and thereby initiates metastasis [23, 24]. In these cells, GM3 is closely associated with signaling proteins, such as c-Src, Rho,


and Ras, within sphingolipid-enriched membrane domains, and binding with Gg3 or anti-GM3 antibody stimulates focal adhesion kinase phosphorylation and c-Src activity.

Thus, it is clear that the roles of GSL in modulating the properties of tumor cells are multiple and heterogeneous. The glycosynapse concept, which will be dis-cussed later in this review, provides a novel unifying mechanistic interpretation, suggesting that many cell properties can be regulated by GSL through supermo-lecular membrane signaling complexes.

34.3 Tumor Cell Phenotype Is Controlled by Glycosynaptic Membrane Domains

At the molecular level, control of the properties of tumor cells by gangliosides requires a complex supermolecular membrane organization that defines highly spe-cialized detergent-insoluble lipid domains that likely represent a specialized form of lipid raft. This has been extensively documented for GM3 and, more recently, for GM2. The term “glycosynapse” has been proposed by Hakomori [7, 25, 26] to generally describe a membrane microdomain involved in carbohydrate-dependent adhesion. Carbohydrate-dependent adhesion in a glycosynapse, occurring through GSL–GSL interactions or through GSL-dependent modulation of adhesion protein receptors (such as integrins), leads to signal transduction events, reflecting deep changes in the motility and invasiveness of tumor cells. As mentioned above, in the case of GM3-dependent adhesion of melanoma cells, it has been shown that GM3 is closely associated with c-Src, Rho, and Ras within GSL-enriched membrane domains, and binding with the Gg3 or anti-GM3 antibody stimulates focal adhesion kinase phosphorylation and c-Src activity. This molecular assembly defines a clas-sically Triton X-100-insoluble GSL-enriched microdomain (termed by Hakomori as “glycosynapse 1”), which can be isolated and separated from a caveolin-containing low-density membrane fraction in B16 cells [27]. A similar association among a sialoglycolipid and c-Src and other related signaling molecules was observed for GM3 in neuroblastoma cells [28], for disialylgalactosylgloboside in renal carci-noma cells [29], and for monosialyl-Gb5 in breast carcinoma cells [30].

Another type of glycosynapse requires the presence of CD9 or other members of the tetraspan membrane protein superfamily (“tetraspanins”). Tetraspanins are inte-gral membrane proteins with four transmembrane stretches that have been described in association with integrin receptors [31]. The best characterized tetraspanin, CD9, is a highly hydrophobic molecule that strongly interacts with GSL (a “proteolipid”) [32]. Tetraspanin CD9 and integrin-a3 or -a5 are colocalized within a distinct low-density, Brij 98-insoluble, glycolipid-enriched domain. The presence of high levels of GM3 positively modulated CD9/integrin association and downstream signal transduction controlling cell motility. The function of the GM3/tetraspanin/integrin signaling complex (“glycosynapse 3”) has been extensively studied by Hakomori’s group (Fig. 34.3).


glycerophospholipid

sphingomyelin

cholesterol

ganglioside

acyl-anchor

integrintetraspanin

Src

CskY

627

αβ

Fig. 34.3 The glycosynapse. The glycosynapse is a membrane microdomain involved in carbo-hydrate-dependent adhesion. In type-3 glycosynapse, GSLs (GM3 or GM2) complexed with tet-raspan membrane proteins (CD9 or CD82) and integrin receptor subunits participate in the control of cell motility. The GM3/CD9/integrin signaling complex formed in the presence of high cellular GM3 levels inhibits cell motility by recruiting an Src inhibitor, Csk, thus keeping Src in a less-active state

The association between CD9 and integrin in the CHO mutant cell line ldlD14 (deficient in UDP-Gal-4-epimerase) has been demonstrated by coimmunoprecipita-tion experiments where cells were grown in the presence of galactose, allowing GM3 synthesis, or by supplementing cells with exogenous GM3. In the latter case, the amount of a3- or a5-integrin associated with the anti-CD9 immunoprecipitate was quantitatively dependent on the concentration of added GM3. Colocalization of CD9, a3-integrin, and GM3 in intact ldlD14 cells was observed by laser scanning confocal microscopy in the presence, but not in the absence, of galactose [32]. The formation of a3/CD9/GM3 complexes strongly inhibited the laminin-5-dependent motility in ldlD14 cells. On the other hand, it has been shown that CD9/GM3 com-plexes are essential for the regulation of integrin-mediated cell adhesion and signal transduction in oncogenic transformation, suggesting a crucial role for GM3 com-plexed with CD9 and integrin-a3b1 or -a5b1 in the control of tumor cell motility and invasiveness. v-Jun-transformed mouse and chicken embryo fibroblasts were characterized by lower GM3 levels and downregulated GM3 synthase messenger RNA levels with respect to the nontransformed counterparts [33]. Reversion of v-Jun oncogenic phenotype could be achieved by enhanced GM3 synthase gene transfec-tion. During phenotypic reversion of v-Jun-transformed cells induced by GM3 syn-thase transfection, the association of the CD9 and a5b1 complex was increased.

In a noninvasive cell line (KK47), which originated from superficial human bladder cancer, GM3 levels were higher than in the invasive YTS1 human bladder cancer cell line. Knock down of CD9 or pharmacologically achieved GM3 depletion in KK47 cells induced the phenotypic conversion to invasive variants. On the other hand, exogenous GM3 addition induced phenotypic reversion of the highly invasive


and metastatic cell line YTS1 to low-motility variants. The changes in cell motility were strictly correlated with the association of CD9 with a3-integrin. This interac-tion was higher in noninvasive than in highly invasive cells and was modulated by the cellular levels of GM3. CD9/a3-integrin association was reduced by GM3 depletion in KK47 and conversely enhanced by exogenous GM3 addition in YTS1 cells. The GM3 levels in the glycosynapse control not only CD9/a3-integrin association but also the activation state of c-Src through Csk translocation. c-Src is present in higher amounts in the glycosynapse fraction in YTS1 cells, and it is activated in cells with low GM3 levels and high invasive potential (YTS1 or GM3-depleted KK47). On the other hand, exogenous addition of GM3 to YST1 cells caused Csk translocation to the detergent-insoluble fraction and consequent inactivation of c-Src, influencing cell motility [34]. Preliminary results from our group showed that a phenotypic variant of the A2780 human ovarian carcinoma cell line, characterized by increased GM3 syn-thase expression [6], had reduced motility and proliferation and was associated with higher levels of c-Src phosphorylated at Tyr527, the target of Csk.

All these data support the notion that the integrin signaling machinery is local-ized within sphingolipid-enriched membrane areas and/or modulated by GSL-containing membrane complexes. Recently, it has been shown that integrin signaling can be controlled by other kinds of GSL/tetraspanin complexes. GM2 ganglioside complexed with a different tetraspanin, CD82, inhibits the activation of Met tyrosine kinase induced by hepatocyte growth factor [35]. This suggests that glycosynapse-like assemblies might be a general paradigm controlling cell motility via tyrosine kinase signaling.

The minimal requirements for glycosynapse structural and functional integrity have been assessed by experiments on reconstituted membranes [36]. Reconstituted membranes containing GM3, SM, PC, and cholesterol – with proportions closely resembling those observed in a glycosynapse fraction separated by anti-GM3 anti-body from a total detergent-resistant fraction from B16 cells – were prepared, and mouse recombinant c-Src was incorporated into the reconstituted membranes. When reconstituted membranes were allowed to adhere on Gg3- or anti-GM3-coated dishes (“GM3-dependent adhesion”), c-Src phosphorylation was observed as it occurred with natural glycosynapses separated from melanoma cells. Activation did not occur when GM3 was replaced with GM1, GD1a, or LacCer.

Glycosynapses bear distinctive properties with respect to other types of mem-brane microdomains: (1) glycosynapses can be separated as detergent-resistant membrane fractions, but the behavior of different glycosynapses toward different detergents is radically dissimilar. Glycosynapse 1, such as GM3-enriched membrane domain from B16 melanoma or from Neuro2a neuroblastoma cells [28], is insoluble in 1% Triton X-100. Glycosynapse 3, such as the GM3/CD9/integrin complex from human bladder cancer cells, is insoluble in 1% Brij 98 [37] but soluble in 1% Triton X-100. (2) Glycosynapses can be separated from other detergent-resistant microdo-mains and are characterized by a distinctive lipid composition. GM3-enriched mem-brane domains from B16 melanoma and caveolar membranes can be separated by immunoaffinity using anti-GM3 or anti-caveolin antibodies [23]. The immu-noseparated GM3-enriched membrane fraction contained GM3, sphingomyelin, and


cholesterol. In contrast, the caveolar membrane fraction, separated by anti-caveolin antibody, contained no GM3, only a very small quantity of sphingomyelin and glucosylceramide, and a very large quantity of cholesterol [23]. This different lipid composition has a profound functional significance, as indicated by the fact that glycosynapse-dependent biological events (e.g., GM3-dependent adhesion and consequent c-Src and FAK activation in B16 melanoma cells) are not affected by cholesterol-sequestering drugs. On the other hand, they are disrupted by compounds structurally related to GM3, such as lyso-GM3 and sialyl a2→1 sphingosine [36, 38]. (3) The presence of a specific glycolipid is often essential for the biological function of the glycosynapse. Increased association of a3-integrin and CD9 with consequent inhibition of motility in YTS1 bladder cancer cells induced by exoge-nous GM3 addition was not reproduced by the addition of GM1 [34]. Activation of c-Src and FAK with enhanced motility and invasiveness was induced in MCF-7 breast carcinoma cells by the anti-monosialyl-Gb5 monoclonal antibody, but not by antibodies to other GSLs (anti-Gb3, anti-Gb5, and anti-GM2) [30].

34.4 Caveolin-1 Controlling Tumor Progression

Caveolins [39, 40] are a family of 21–24-kDa integral membrane proteins that have been originally described as the main structural protein components of plasma membrane specializations known as caveolae [41, 42]. Caveolin-1 has a hydropho-bic putative membrane-spanning sequence, and it is palmitoylated at the C-terminal domain. Caveolins form high-mass oligomeric complexes, providing a scaffold for caveolin-interacting proteins (including H-Ras [43], c-Src, heterotrimeric G-proteins [43, 44], and growth factor receptors [45, 46]) that can thus be concentrated within caveolin-rich membrane areas [47, 48].

A growing body of evidence indicates that caveolin-1 influences the develop-ment of human cancers. However, the exact functional role of caveolin-1 is still controversial. In certain cell types, antisense inhibition of caveolin-1 expression is sufficient to induce the oncogenic transformation. Targeted downregulation of caveolin-1 in NIH-3T3 cells activates MAPK and stimulates anchorage-independent growth [49]. As demonstrated in caveolin-1 null mice, loss of caveolin-1 accelerates tumorigenesis and metastasis formation [50]. Caveolin-1 is highly expressed in normal ovary, but it is markedly downregulated in human ovarian carcinoma. Immunohistochemistry confirmed that caveolin-1 is present in normal ovarian epithelium and in benign serous adenomas, mainly as a membrane-associated pro-tein with prevalent basolateral distribution, but it is downregulated in carcinomas (where it is characterized by an even, cytoplasmic distribution) and is completely absent in mucinous adenomas [51]. The CAV-1 gene is downregulated in human tumors derived from ovary, breast, and colon, but it is upregulated in tumor samples from kidney, prostate, and stomach. The caveolin-1 protein is present at high levels in immortalized human ovarian epithelial cells, in benign serous adenomas, and in noninvasive ovarian cancer cells, but not in four highly aggressive ovarian carcinoma


cell lines [51]. Caveolin-1 expression is lost in human mammary carcinoma cell lines [49], and expression of caveolin-1 in a highly metastatic carcinoma cell line suppressed in vivo metastasis formation and reduced in vitro invasion into the extracellular matrix. Decreased invasion of caveolin-1-expressing cells was accom-panied by a reduction in metalloprotease secretion and gelatinolytic activity, and reduced ERK1/2 signaling, in response to growth factors [50]. Caveolin-1 poten-tially restrains tumor cell growth and metastatic potential. Caveolin reexpression inhibited tumor cell growth [52] and reduced tumorigenicity [53] in human breast cancer and colon carcinoma cell lines; negatively affected in vivo tumor growth, metastasis development, and invasiveness in mammary metastatic tumor cells; and promoted cell–cell adhesion in ovarian carcinoma cells by a mechanism involving inhibition of Src kinase [3]. In OVCAR-3 human ovarian carcinoma cells, caveolin-1-induced expression reduced colony formation by 90% and significantly increased the number of apoptotic cells. The proapoptotic effects of caveolin-1 might depend on the PI-3 kinase/Akt pathway [51].

This evidence suggests a role for caveolin-1 as a suppressor of tumor growth and metastasis in ovarian, breast, and colon human carcinomas. However, the function of caveolin-1 might be entirely different in other tissues. Depending on the cellular context, opposing functions might exist, resulting in tumor progression promotion rather than inhibition. As mentioned above, transformed cells usually contain reduced or no caveolin, but caveolin-1 expression is increased in tumor samples from kidney, prostate, and stomach compared to normal tissues [51], and reexpres-sion is found in some advanced adenocarcinomas. Elevated expression of caveolin-1 is associated with progression in prostate, colon, breast, and lung carcinoma. Remarkably, induced reexpression of caveolin-1 in less-invasive, caveolin-negative lung cancer cell lines enhanced the invasive capability [54]. Caveolin-1 protects prostate cancer cells from c-Myc-induced apoptosis [55]. Thus, engagement of caveolin-1 as a tumor metastasis promoter or tumor metastasis suppressor is strongly determined by the specific cellular context and, at the molecular levels, by the signaling molecules interacting with the caveolin and by the signaling pathways affected and regulated by caveolin.

As mentioned above, it has been shown recently that caveolin-1 promotes cell–cell adhesion in ovarian carcinoma cells by a mechanism involving inhibition of Src kinases. Nonreceptor tyrosine kinases of the Src family are involved in several cell functions, such as mitogenic response of growth factors [56–63], fibroblast cell migration, and epithelia cell scattering [60, 64, 65], as well as in cancers [66, 67]. Src kinases, located on the inner face of membranes, segregate in the specific mem-brane domains defined by sphingolipids and usually enriched in caveolin. Src kinase localization in caveolae and/or sphingolipid-enriched domains seems to be instrumental for growth factor-induced Src-dependent mitogenic response [64]. Src kinases are activated and involved in cancer progression and metastasis in most human carcinomas. We showed that c-Src is in a less-active state in low-motility human ovarian carcinoma cell lines expressing high levels of GM3 ganglioside and caveolin-1 [20]. The C-terminal Src kinase, Csk, is the main negative regulator of c-Src and other related kinases [67]. Csk is a cytosolic enzyme that may need an


intermediary protein for location in the Src kinase’s vicinity. Several candidates have been described, such as paxillin, a Csk-binding protein/phosphoprotein asso-ciated with GSLs (CBP/PAG), and caveolin-1. Interactions of Src with caveolin-1 have important consequences. Caveolin-1 seems to act as a membrane adapter that couples integrin receptors to Src kinases [4] (Fig. 34.4).

Src induces phosphorylation of caveolin-1 at Tyr14, which is responsible for the rearrangement of caveolin-1 within the cell [68–70]. Caveolin-1 phosphorylation is involved in the regulation of the docking of Csk, the negative regulator of Src, sug-gesting a mechanism for the negative regulation of Src activity by phosphorylated caveolin [71]. Moreover, phosphorylated caveolin is recruited to lipid-enriched membrane domains upon integrin receptor disengagement, inhibiting the internal-ization of these specialized membrane areas and the signaling events downstream from the integrin receptor [72–74].

34.5 Caveolar and Noncaveolar Domains at the Cell Surface

Caveolae were morphologically described over 50 years ago [75, 76]. More recently, it has been shown that caveolae [41, 42] are characterized by the presence of 21–24-kDa integral polypeptides, termed caveolins [39, 40], as their main struc-tural protein components (Fig. 34.5).

Caveolins form high-mass oligomeric complexes, providing a scaffold for caveolin-interacting proteins (including H-Ras [43], c-Src, heterotrimeric G-proteins [43, 44], and growth factor receptors [45, 46]) that can thus be concentrated within caveolae membranes [47, 48]. Mainly based on this observation, it has been hypoth-esized that caveolae may act as specialized plasma membrane structures able to

glycerophospholipid

sphingomyelin

cholesterol

ganglioside

acyl-anchor

Fyn

integrin

αβ

ERK

RAS

Fig. 34.4 A caveolin-1-containing sphingolipid-enriched domain. Caveolin-1 can be associated independently with sphingolipid-enriched membrane domains by its localization in caveolae. Multiprotein complexes organized by caveolin-1 in the presence of GSL might transduce signals from integrin receptors to Src kinases, influencing cell motility. A caveolin-1-dependent signaling complex rich in GSL could be classified as a novel, tetraspanin-independent type of glycosynapse


glycerophospholipid

sphingomyelin

cholesterol

ganglioside

acyl-anchorcaveolin

Fig. 34.5 A caveola. The caveola is a specialized membrane domain characterized by the pres-ence of an oligomeric caveolin-1 scaffold and by a clear flask-shaped morphology. Caveolae have been speculated to serve as multimolecular signaling sites. However, their enrichment in GSL is still debated, and they are clearly distinguishable from glycosynapses

assemble and coordinate the functions of signal-transducing protein complexes, in which caveolin should play a pivotal role as a scaffolding molecule [48, 77–80]. The presence of caveolins as specific scaffolding proteins represents the distinctive fea-ture of caveolae membranes [39, 81, 82]. Caveolin-1 transfection in cell lines lack-ing caveolin and caveolae resulted in the formation of morphologically distinguishable caveolae in some cell types (e.g., lymphocytes and Fischer rat thyroid cells) [83, 84] but not in others (e.g., the human prostate cancer cell line LNCaP) [84]. Caveolin-1 thus seems an essential component of caveolae, even if its presence on the cytoplas-mic face of caveolae was somehow challenged by the finding that caveolin-1 is rather present in intramembrane particles interacting with the caveolae surface [85]. However, it is quite clear that caveolin-1 is also found at many other intracellular locations, including the trans-Golgi network and in other organelles [86, 87], and caveolin-1 antibodies also bind to flat membrane portions [88].

In the 1990s, the concept of “lipid rafts” or “sphingolipid- and cholesterol-enriched membrane domains” as restricted membrane areas that specialized in spe-cific functions evolved from the confluence of experimental results coming from very heterogeneous research areas. Knowledge on the segregation or clustering of specific membrane components (GSLs, glycosylphosphatidylinositol [GPI]-anchored proteins) within the cell membrane and on the existence of caveolae [75, 76, 89] seemed to be unified by the observation that both segregated membrane components and caveolae components behave as detergent-insoluble complexes. Lipid mem-brane domains and caveolae share another property: both are sites for the clustering of proteins, and this could be a mechanism for regulating cell signaling and other cellular events (such as endocytosis [90–92] and entry of pathogens [93]).

Sphingolipid-enriched membrane domains share several properties with caveolae (i.e., a peculiar lipid composition, resistance to solubilization by cold nonionic detergents, and a low buoyancy on sucrose density gradients), even if several pieces


of evidence suggest that caveolae and sphingolipid-enriched membrane domains can exist independently.

In all cell types so far investigated, cell sphingolipids, together with cholesterol and saturated phosphatidylcholine, are associated with a low-density membrane fraction that can be prepared by flotation on sucrose gradient after cell lysis in the presence of Triton X-100 or other nonionic detergents [94]. This fraction is usually regarded as a lipid membrane domain-enriched fraction. When this procedure has been applied to cells expressing caveolins, caveolins have been found to partition into the same Triton-insoluble, low-buoyancy membrane fractions [95], and these fractions have been often considered to represent isolated caveolae fractions. However, the relationship between caveolae and lipid membrane domains is much more complicated. Detergent-insoluble domains are also found in cells lacking caveolin expression and caveolae structures, including several neuronal cell types (indeed, for many authors, the postulated equivalence between caveolae and lipid membrane domains has been so much emphasized that terms such as “caveolae-like domains” or “noncaveolar domains” were coined to describe lipid membrane domains in the absence of caveolins or caveolae) [28, 96–102]. Moreover, caveolin-1 may exist in lipid membrane domains without the formation of caveolae. For example, in MDCK cells, caveolae are present only on the basolateral, but not on the apical, surface. Caveolin-1 in these cells is present in caveolae at the basolateral membrane but as detergent-insoluble, cholesterol-dependent complexes at the apical membrane [103]. In the human prostate cancer cell line LNCaP, where caveolin-1 transfection did not correspond to the formation of caveolae, detergent-resistant lipid membrane domains exist independently from the expression levels of caveolin [84]. The immortalized murine gonadotropin-releasing hormone neuronal cell lines GN11 and GT1 represent immature, migrating neurons and nonmigrating, fully differentiated cells, respectively [104]. The presence of caveolin-1 polypep-tide was reported in the GN11 neuronal-like cell line, while, in contrast, GT1 cells are totally devoid of caveolin [105]. GT1 cells are characterized by a very high GSL level (mainly represented by GM3 ganglioside) compared with GN11 cells. From both cell lines, it was possible to prepare a Triton-insoluble, low-density membrane fraction similarly enriched in sphingolipids that, in the case of caveolin-expressing GN11 cells, was also rich in caveolin [105].

Thus, it is clear enough that caveolae and lipid membrane domains can exist independently. A growing number of experimental data support this concept. Fluorescence microscopy and electron microscopy (EM) studies showed that GPI-anchored proteins are not constitutively concentrated in caveolae. Their caveolar clustering only occurs upon multimerization triggered by cross-linking [106]. When a plasmalemmal fragment population was obtained by a detergent-free silica coating procedure from rat lung vasculature and a caveolae fraction was further purified by specific immunoisolation using anticaveolin-coated magnetic beads, this caveolae fraction was devoid of a number of proteins involved in signal trans-duction that are usually recovered in the detergent-resistant (lipid membrane domain) fraction [107]. Caveolae and caveolin-1 are involved in the modulation of growth factor receptor signaling [46], and epidermal growth factor receptor (EGFR)


is localized within a caveolin-rich fraction in A431 cells. However, EGFR-containing membrane fragments can be separated from caveolae [108, 109]. The noncaveolar EGFR-enriched membrane domain behaves as a classic lipid membrane domain, i.e., it is Triton X-100 insoluble, enriched in GM1 ganglioside, and sensitive to the manipulation of cholesterol levels [110, 111].

The question to be answered is whether caveolae or caveolin-containing membrane domains are or are not lipid membrane domains. This question is, of course, not merely semantic but involves the possible role of lipids as structural and functional components of caveolae-based signaling complexes within the plasma membrane. Many papers report on the relationship between caveolae/caveolins and cholesterol. Caveolae are disassembled by cholesterol-binding drugs [39]. EM experiments with cholesterol-binding probes showed that caveolae are enriched in cholesterol [112]. Caveolin-1 binds free cholesterol and cholesterol-containing artificial phospholipid liposomes [113–115]. Caveolin oligomerization in cell mem-branes depends on the cholesterol level [116]. Similarly, EM immunogold-labeling experiments using antilipid antibodies or cholera toxin suggested that caveolae are enriched in GM1 gangliosides, neutral glycolipids, and sphingomyelin [88, 116, 117]. However, in the case of neutral glycolipids and sphingomyelin, caveolar local-ization only occurs after antibody cross-linking. The presence of sphingomyelin and ceramide was observed in a Triton X-100 resistant caveolae fraction from human fibroblasts [118], and GM1 was detected by cholera toxin labeling in a purified caveolae fraction prepared from rat lung endothelium [117]. Caveolin-1 has been shown to bind photoreactive derivatives of GM1 and GM3 in A431 and MDCK cells [119, 120]. However, kinetic studies showed that the interaction between GM3 and caveolin-1 in MDCK is a transient process; it occurs shortly after the incorpora-tion of the ganglioside derivative in the plasma, but it is lost after a 24-h chase, suggesting that a redistribution of the ganglioside takes place. Indeed, EM experiments showed that, in these cells, caveolin-1 and GM3 are not localized in the same domain at the steady state [119]. Thus, the information available about the interaction of specific sphingolipids with caveolin and their presence in caveolae is scant, fragmentary, and somehow contradictory. Indeed, the question raised by Fujimoto in 1996 – whether sphingolipids are concentrated in the purified caveola fraction or not – still remains to be answered [88].

However, the role of caveolin-1 as a molecular organizer for membrane multi-protein signaling complexes is indicated by several pieces of evidence. Caveolin-1 has a hydrophobic putative membrane-spanning sequence, and it is palmitoylated at the C-terminal domain. Caveolin-1 is typically associated with sphingolipid-enriched membrane domains or other subtypes of lipid rafts, i.e., with Triton-insoluble, low-buoyancy membrane fractions enriched in (glyco)sphingolipids and cholesterol, even if, as mentioned above, caveolae and noncaveolar lipid domains can exist independently in cells. Caveolin-1 association with sphingolipid-enriched membrane domains seems to be regulated by the cellular levels of ceramide pro-duced by sphingomyelin hydrolysis or de novo synthesis. Caveolin palmitoylation does not affect its association with GSL-enriched membrane domains. However, caveolin palmitoylation is relevant for (1) caveolin interaction with cholesterol and (2) caveolin interaction with other acylated proteins. Molecules belonging to


mitogenic signaling pathways were localized to caveolae, or it has been shown that they physically interact with caveolin-1. In this pathway, caveolin-1 appears to function as a membrane adapter, which couples the integrin subunit to cytosolic Src-family protein tyrosine kinases. Src-family kinases, such as Fyn, Yes, Lck, and Lyn, are thought to reside in cholesterol-enriched domains or assemblies (com-monly referred to as “lipid rafts”) and caveolae by virtue of their dual acylation (myristoyl and palmitoyl residues) [121–126]. This situation is less clear for Src, which has a single myristoyl residue [127]. However, interaction of Src with caveo-lin is well established and has important consequences. In nontransformed cells, caveolin-1 is phosphorylated at Tyr14 in response to growth factor signaling [128]. Caveolin-1 is phosphorylated at Tyr14 under basal conditions in carcinoma cell lines, but not in immortalized ovarian epithelial cells [51]. Src induces phosphory-lation of caveolin-1 at Tyr14 [68–70], which is responsible for the rearrangement of caveolin within the cell (e.g., triggering caveolar endocytosis). Caveolin-1 phos-phorylation is in turn involved in the regulation of the docking of Csk, the negative regulator of Src, suggesting a mechanism of negative regulation of Src activity by phosphorylated caveolin. Src and caveolin-1 appear to be highly interdependent, as Src kinase activity is required for stimulation of caveolar endocytosis [129, 130], and small interfering RNA to c-Src inhibits caveolar endocytosis and increases the accumulation of caveolae at the cell surface [131].

Integrin signaling, responding to cell adhesion to the extracellular matrix and modulating it, is tightly connected with the internalization of caveolae-like mem-brane complexes. When cells are attached to the matrix, integrin receptors nega-tively regulate the internalization of caveolar membrane domains, preventing uncoupling of downstream signaling molecules. In this context, Src-mediated tyrosine phosphorylation of caveolin-1 at Tyr14, consequent to cell detachment from the extracellular matrix, is responsible for a shift of caveolin-1 from focal adhesions to caveolae, which induces the internalization of lipid-enriched mem-brane domains, with consequent inhibition of signaling pathways downstream, to integrin receptors [72–74].

The question that arises is whether the complex supermolecular membrane organization assembled by caveolin-1 – defining a highly specialized detergent-insoluble lipid domain and possibly representing a specialized form or subset of lipid raft or lipid-rich membrane domain – can be controlled in its function by the composition of its lipid environment. Interestingly, a connection has been proposed between caveolin-1 and plasma membrane enzymes that are possibly able to mod-ify the local sphingolipid composition of the plasma membrane. The role of a plasma membrane-associated sialidase active on gangliosides (NEU3) in modifying the cell surface ganglioside composition, with deep consequences for apoptosis in colon cancer, has been highlighted [132]. In colon and renal cancer, this sialidase seemed to be responsible for maintaining high cellular levels of lactosylceramide, which would exert a Bcl-2-dependent antiapoptotic effect, contributing to the sur-vival of cancer cells and consequent tumor progression [133, 134]. In our context, it is important to recall that Neu3 is not randomly distributed at the cellular surface; this ganglioside sialidase has been reported to be associated with Triton X-100-insoluble GSL-enriched membranes [135] and to closely interact with caveolin-1 in


Neu3-transfected COS-1 cells [136]. Moreover, we showed that Neu3 is also able to hydrolyze gangliosides on adjacent cells, indicating that this enzyme is important in sphingolipid-mediated cell–cell recognition, a key event of tumor dissemination [137]. A relevant role in cancer cells has been suggested, also for enzymes involved in the regulation of cellular ceramide levels. Ceramide is the key intermediate in sphingolipid biosynthetic and catabolic pathways. On the other hand, ceramide per se is an important signaling molecule, and some data indicated that ceramide mem-brane levels might be important in modulating the structure and signaling function of sphingolipid-enriched membrane domains and of caveolin-dependent signaling [138]. In particular, it has been suggested that increased ceramide production upon different stimuli might displace caveolin-1 from sphingolipid- and cholesterol-rich membrane complexes [139]. Usually, the production of bioactive ceramide is ascribed to sphingomyelin hydrolysis by sphingomyelinases, and these enzymes have been found in sphingolipid-enriched fractions [1, 40] where, in some cases, they have been reported to interact with caveolin-1.

In the UDP-Gal-4-epimerase-deficient ldlD14 cell line, it has been shown that caveolin-1 is localized within detergent-insoluble lipid domains under experimental conditions not allowing GM3 synthesis (removal of galactose from the culture medium), but it is relocated outside glycolipid-enriched membrane fractions when GM3 synthesis occurs [140]. In a keratinocyte-derived cell line, GM3 overexpres-sion induced a shift of caveolin-1 to detergent-soluble membrane regions, allowing its functional interaction with the EGFR, which caused inhibition of EGFR tyrosine phosphorylation and dimerization [141]. Recently, Nakashima et al. suggested a cor-relation between the GD3 ganglioside and caveolin-1 in the regulation of signals able to attenuate the malignant properties of human melanoma cells. In these cells, GD3 rather than GM3 seems to play a crucial role in the control of motility. GD3 expres-sion induced tyrosine phosphorylation of p130Cas and paxillin, mediating proinva-sive signals [142]. Overexpression of caveolin-1 in human melanoma cells had no effects on the GSL levels or pattern but caused a marked dispersion of GD3 outside the detergent-insoluble fraction, which corresponded to a deep disorganization of the leading edges [5].

These data suggest that the connection between caveolin-1 and sphingolipid-controlled signaling complexes needs to be further investigated to clarify whether Dr. Hakomori’s concept of glycosynapse as a membrane microdomain involved in carbohydrate-dependent adhesion can be extended to caveolin-1-dependent signal-ing complexes. This would represent a novel unifying concept allowing for a better understanding of the multiple and apparently contradictory functions of caveolin-1 in the control of tumor cell phenotype.

34.6 Summary

After the initial tumorigenic events triggered by genetic mutations of oncogenes and tumor suppressor genes, tumor–host interactions represent a critical feature of each step leading to cancer disease progression. Host-derived cells (endothelial


cells, fibroblasts, and inflammatory cells) infiltrate into tumor tissue, interact with tumor cells, and produce soluble and insoluble factors that stimulate tumor angio-genesis, growth, and metastasis. Another aspect critical for tumor progression is interaction with the extracellular matrix, representing crucial events in the release of cells from the tumor mass, extravasation, and metastatic invasion. In all these aspects, the structural and functional properties of the cell membrane play a prime role. GSLs, typical components of the plasma membrane asymmetrically enriched in the external leaflet, are known as cell surface antigens, as mediators of cell–cell recognition and cell adhesion, and as modulators of several aspects of signal trans-duction processes involved in cell proliferation, differentiation, and oncogenesis. Several lines of evidence indicate the importance of GSL in the formation and progression of tumors, and several hypotheses have been made about the possible functional role(s) of aberrant GSL expression in defining the surface properties of tumor cells. GSLs in tumor cells have been implicated in the mediation of cell adhe-sion, motility, and recognition through GSL-binding proteins and/or GSL–GSL interactions. In particular, GSL might contribute to the modulation of integrin-dependent interactions of tumor cells with the extracellular matrix as well as with host cells present in the stromal compartment of the tumor. A unifying concept about the mechanisms by which GSLs participate in the control of basic tumor cell functions emerged in the past years: GSLs are assembled within membranes with other membrane components, leading to the creation of organized supermolecular structures that function as signaling units. At the molecular level, GM3 ganglioside control on the properties of tumor cells requires a complex supermolecular mem-brane organization that defines highly specialized detergent-insoluble lipid domains. GM3 gangliosides complexed with tetraspanins and integrin-a3b1 or -a5b1 func-tion as a signaling unit (“glycosynapse”), controlling tumor cell motility and invasiveness. GM3 levels in glycosynapses are related to the activation state of c-Src. c-Src is activated in cells with low GM3 levels and high invasive potential, and artificially induced increase in GM3 caused Csk translocation to a detergent-insoluble membrane fraction and consequent inactivation of c-Src, influencing cell motility.

On the other hand, a growing body of evidence indicates that caveolin-1 influ-ences the development of human cancers. Caveolin-1 expression inhibits in vivo tumor growth, metastasis development, and invasiveness in metastatic mammary tumor cells and promotes cell–cell adhesion in ovarian carcinoma cells by a mechanism involving inhibition of nonreceptor Src tyrosine kinases. Furthermore, caveolin-1 might act as a membrane adapter that couples the integrin subunits to Src kinases. The control of tumor cell invasiveness could be linked to the function of a signaling complex among GM3, caveolin-1, and members of the integrin-signaling cassette, leading to inactivation of Src kinase signaling. This allows us to hypothesize a novel, caveolin-1-dependent role of GM3 in regulating tumor cell motility and invasiveness.

Acknowledgments This work was supported by the Mizutani Foundation for Glycoscience, Grant 070002, to Alessandro Prinetti, and by the CARIPLO Foundation, Grant 2006, to Sandro Sonnino.


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