| Abstract |
Melanoma is a frequent and therapy resistant human malignancy with a rising incidence rate, worldwide (Houghton et al., 2002). Malignant melanoma arises in the epidermis either in situ or in an invasive form, progressing from radial growth phase to vertical growth phase through enhanced invasion into the dermis (Lever, 2005). With malignant progression tumor cells partly through autocrine and paracrine production of growth factors and cytokines actively modulate their microenvironment in a tumor-favourable way to enable successful invasion (Selvamurugan et al., 2002; Hakamori et al., 2002; Nikitovic et al., 2006). The dermis extracellular matrix (ECM) consists of different types of collagens, glycoproteins, hyaluronan (HA) and proteoglycans (PGs) (Wang et al., 2003).
Members of the small leucine-rich proteoglycans (SLRPs) family were found to participate in both the structural and the functional organization of the skin (Wang et al., 2003; Carrino et al., 2000). Previously it was shown that the SLRP lumican significantly affects the mechanical properties of the skin and that the lack of its expression results in skin fragility and laxity (Carrino et al., 2000; Vuillermoz et al., 2005). The role of lumican has been investigated in the growth and metastasis of several cancers, without being fully elucidated (Naito, 2005). Specifically it was demonstrated that human colorectal cancer cells (Lu et al., 2002), cervical squamous carcinoma cells (Naito, 2002) and pancreatic adenocarcinoma cells express lumican mRNA and different glycosylated types of its protein (Lu, 2002). In contrast, lumican in breast cancer tissues is expressed in fibroblasts adjacent to cancer cells but not in cancer cells (Leygue et al., 1998). Furthermore, it has been demonstrated that reduced expression of lumican is associated with poor outcome in node-negative invasive
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breast cancer (Troup et al., 2003) while high expression level of lumican is associated with a high pathological tumor grade, low estrogen receptor levels in the cancer tissue, and younger age of patients (Leygue et al., 1998). Pancreatic myofibroblasts of the tumor stroma were also found to strongly overexpress lumican (Koninger et al., 2004). An in vitro/in vivo study showed that lumican expression by transfected B16F1 mouse melanoma cells resulted in inhibition of hypodermal melanoma growth in syngenic mice, but failed to change the B16F1 cell differentiation, growth rate and adhesion properties (Vuillermoz et al2004). The above reports suggest that lumican may have a role both in the host reaction of the peritumoral cells as well as in tumor growth. The expression of lumican by human melanoma cells has not, to our knowledge, been reported. The aim of the present study was to investigate and to characterize lumican expression at the mRNA, protein and carbohydrate level, in two human malignant melanoma cell lines (WM9 and M5) and normal neonatal human melanocytes (HEMN).
Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan (PGs) form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.
The members of the fibroblast growth factor (FGF) family (Eswarakumar, Lax, & Schlessinger, 2005) function through four distinct, high-affinity cell surface receptors with intrinsic tyrosine kinase activity, designated FGFR1–4 (Basilico & Moscatelli, 1992). The binding of FGFs to the extracellular ligand domain of FGFRs induces receptor dimerization, activation and autophosphorylation of multiple
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tyrosine residues in the cytoplasmic domain of the receptor molecule (Jaye, Schlessinger, & Dionne, 1992), leading to the stimulation of intracellular signaling pathways that control cell proliferation, differentiation, migration or survival (Eswarakumar et al., 2005). Human melanomas most uniformly constitutively express basic fibroblast growth factor (FGF-2). As these cancer cells simultaneously express FGFRs their growth is stimulated by FGF-2 in an autocrine manner (Rodeck, 1993; Rodeck et al., 1991; Yeoman, 1993). The biological significance of this autocrine loop has been demonstrated using anti-sense oligonucleotides that targeted FGF-2 or by inactivating FGF-2 with anti-FGF-2 antibodies resulting in an inhibition of melanoma cell proliferation both in vitro and in vivo (Beckcer, Meier, & Herlyn 1989; Rofstad & Halsor, 2000; Wang & Becker, 1997). On the other hand, increased expression of FGF-2 stimulates the transformed melanoma phenotype demonstrating that FGF-2-dependent signaling is essential in melanoma tumor progression (Meier et al., 2000).
FGF-2 has a strong binding affinity for heparin/heparan sulfate glycosaminoglycans (GAGs) which when covalently bound in to core proteins form HS proteoglycans (HSPGs). Indeed, membrane HSPGs have been characterized as low-affinity binding sites for FGFs that do not transmit a biological signal but rather function as accessory molecules that regulate FGF binding and the activation of the occupied signaling receptors (Yayon, Klagsbrun, Esko, Leder, & Ornitz, 1991). Their removal from the cell surface impairs responsiveness to this growth factor (Rapraeger, Krugka, & Olwin, 1991; Yayon et al., 1991). In some cases extracellular HSPGs act as substitutes for their cell-associated counterparts and correctly present the bound FGF-2 to its respective receptors (Aviezer, Iozzo, Noonan, & Yayon, 1997; Delehedde, Deudon, Boilly, & Hondermarck, 1996). A rising amount of evidence has
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lately implicated chondroitin sulfate-containing proteoglycans (CSPGs) to participate in FGF-2 signaling (Bao et al., 2004; Molteni, Modrowski, Hott, & Marie, 1999; Smith et al., 2007). CSPGs were shown to modulate the binding of FGF-2 to FGFRs, either autonomously in the role of low affinity binding sites (Smith et al., 2007) or in cooperation with HS chains (Deepa, Yamada, Zako, Goldberger, & Sugahara, 2004). Therefore, the content and the fine structure of both HS and CS chains might modulate FGF-2 binding and FGFRs activation in a cell type specific manner.
Differential expression of PGs on the surface of human melanoma cells is characterized by altered experimental metastatic potential thus demonstrating that GAGs/PGs participate in melanoma progression (Timar et al., 1995). Furthermore, both cell-associated and extracellular HSPGs were shown to alter melanoma cell FGF-2 signaling to increase melanoma metastatic potential (Delehedde et al., 1996; Reiland, Kempf, Roy, Denkins, & Marcheti, 2006). On the other hand, it is well known that FGF-2 may regulate the synthesis and distribution of GAGs/PGs by different cancer cell lines (Tzanakakis, Hjerpe, & Karamanos, 1997) and thus it may modulate the expression of GAGs/PGs in an autocrine manner in the human melanoma cell model.
The aim of the present study was to examine the possible participation of various glycosaminoglycans (GAGs), i.e. chondroitin sulfate, dermatan sulfate and heparin on basal and FGF-2-induced growth of WM9 and M5 human metastatic melanoma cells. Exogenous glycosaminoglycans mildly inhibited WM9 cell’s proliferation, which was abolished by FGF-2. Treatment with the specific inhibitor of the glycosaminoglycan sulfation, sodium chlorate, demonstrated that endogenous GAGs/PGs production is required for both basal and stimulated by FGF-2 proliferation of these cells. Heparin capably restored their growth, and unexpectedly
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exogenous chondroitin sulfate to WM9 and both chondroitin sulfate and dermatan sulfate to M5 cells allowed FGF-2 mitogenic stimulation. Furthermore, in WM9 cells the degradation of membrane-bound chondroitin/dermatan sulfate stimulates basal growth and even enhances FGF-2 stimulation. The specific tyrosine kinase inhibitor,
genistein completely blocked the effects of FGF-2 and glycosaminoglycans on melanoma proliferation whereas the use of the neutralizing antibody for FGF-2 showed that the mitogenic effect of chondroitin sulfate involves the interaction of FGF-2 with its receptors. Both the amounts of chondroitin/dermatan/heparan sulfate and their sulfation levels differed between the cell lines and were distinctly modulated by FGF-2. In this study, we show that chondroitin/dermatan sulfate-containing proteoglycans, likely in cooperation with heparan sulfate, participate in metastatic melanoma cell FGF-2-induced mitogenic response, which represents a novel finding and establishes the central role of sulfated glycosaminoglycans on melanoma growth.
Furthermore, FGF-2-overexpressing melanocytes exhibit marked proliferation, upwards migration, cluster formation and type IV collagen expression within the epidermal compartment, stimulating early radial growth phase melanoma, demonstrating that FGF-2 signaling is essential in melanoma progression (Meier et al., 2003). The interactions between the extracellular matrix (ECM) and the cancer cells regulate their adhesive/migratory properties and thus define their invasive phenotypes (Cavallaro and Christofori, 2001). These adhesion-dependent signals are mediated by clustering of adhesion receptors such as integrins and cell surface proteoglycans (PGs), organization of the actin cytoskeleton, and congregation of signaling adaptors and enzymes into specialized morphological structures, including focal adhesions, focal complexes, and fibrillar adhesions (Yamada and Geiger, 1997). Importantly, changes in the expression or function of these adhesion molecules have
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been documented in the progression of primary melanoma (Satyamoorthy and Herlyn, 2002).
Syndecan-4 is a member of a family of HS-containing transmembrane proteoglycans (syndecans 1–4), that are characterized by short cytoplasmic domains containing a variable region that is unique for each family member and divergent extracellular domains (Simons and Horowitz, 2001). This PG has been established as a key adhesion molecule, unique among the syndecan family members to localize to sites of cell-matrix adhesions, including focal adhesions (Woods and Couchman, 1994, 2001; Couchman and Woods, 1999; Petit and Thiery, 2000). The HS chains of
syndecan-4 specifically attach to the heparin-binding site of FN, affecting focal adhesion formation on this substrate, which in turn regulates cell migration (Midwood et al., 2006). FN, a high molecular weight multidomain glycoprotein of the ECM (Pankov and Yamada, 2002) which contains multiple binding sites including those for sulphated GAGs and cell surface integrin receptors, was chosen as the specific adhesion substrate. Expression of FN has been correlated to melanoma progression (Jaeger et al., 2007) and specific regulation of tumor FN expression by constitutive ERK/MAP kinase signaling has been shown, indicating that self-production of this matrix component may promote melanoma cell invasion (Gaggioli and Sahai, 2007). FGF-2 is established as a key modulator of melanoma progression however, its effects on melanoma cell adhesion and migration capabilities are not well known.
In this study we showed that M5 human metastatic melanoma cells’ ability to migrate was significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their
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amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduced the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration. These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4 dependent mechanism.
Summarizing, the results of this PhD thesis demonstrated the crucial role of PGs and GAGs on the basic and FGF-2 induced cell proliferation, adhesion and migration of melanoma cells and normal melanocytes.
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Βιοχημική και ανοσολογική μελέτη της δράσης των αυξητικών παραγόντων στη βιοσύνθεση εκκρινόμενων Chondroitin και dermatan surfate πρωτεογλυκανών σε κυτταρικές σειρές μελανώματος και φυσιολογικών μελανοκυττάρων
