Abstract: The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.
Notes: Feature paper of the issue with interview in Proteomics podcast http://www.wiley-vch.de/goproteomics/index.php?page=podcast_archiv2 (Nov 2008)
Abstract: Melanoma progression is associated with changes in adhesion receptor expression, in particular upregulation of N-cadherin which promotes melanoma cell survival and invasion. Plasma membrane lipid rafts contribute to the compartmentalization of signaling complexes thereby regulating their function, but how they may affect the properties of adhesion molecules remains elusive. In this study, we addressed the question whether lipid rafts in melanoma cells may contribute to the compartmentalization of N-cadherin. We show that a fraction of N-cadherin in a complex with catenins is associated with cholesterol/sphingolipid-rich membrane microdomains in aggressive melanoma cells in vitro and experimental melanomas in vivo. Partitioning of N-cadherin in membrane rafts is not modulated by growth factors and signaling pathways relevant to melanoma progression, is not necessary for cell-cell junctions' establishment or maintenance, and is not affected by cell-cell junctions' and actin cytoskeleton disruption. These results reveal that two independent pools of N-cadherin exist on melanoma cell surface: one pool is independent of lipid rafts and is engaged in cell-cell junctions, while a second pool is localized in membrane rafts and does not participate in cell-cell adhesions. Targeting to membrane rafts may represent a previously unrecognized mechanism regulating N-cadherin function in melanoma cells.
Abstract: The emergence of omics technologies allowing the global analysis of a given biological or molecular system, rather than the study of its individual components, has revolutionized biomedical research, including cardiovascular medicine research in the past decade. These developments raised the prospect that classical, hypothesis-driven, single gene-based approaches may soon become obsolete. The experience accumulated so far, however, indicates that omic technologies only represent tools similar to those classically used by scientists in the past and nowadays, to make hypothesis and build models, with the main difference that they generate large amounts of unbiased information. Thus, omics and classical hypothesis-driven research are rather complementary approaches with the potential to effectively synergize to boost research in many fields, including cardiovascular medicine. In this article we discuss some general aspects of omics approaches, and review contributions in three areas of vascular biology, thrombosis and haemostasis, atherosclerosis and angiogenesis, in which omics approaches have already been applied (vasculomics).
Abstract: During tumor progression, cancer cells undergo dramatic changes in the expression profile of adhesion molecules resulting in detachment from original tissue and acquisition of a highly motile and invasive phenotype. A hallmark of this change, also referred to as the epithelial-mesenchymal transition, is the loss of E- (epithelial) cadherin and the de novo expression of N- (neural) cadherin adhesion molecules. N-cadherin promotes tumor cell survival, migration and invasion, and a high level of its expression is often associated with poor prognosis. N-cadherin is also expressed in endothelial cells and plays an essential role in the maturation and stabilization of normal vessels and tumor-associated angiogenic vessels. Increasing experimental evidence suggests that N-cadherin is a potential therapeutic target in cancer. A peptidic N-cadherin antagonist (ADH-1) has been developed and has entered clinical testing. In this review, the authors discuss the biochemical and functional features of N-cadherin, its potential role in cancer and angiogenesis, and summarize the preclinical and clinical results achieved with ADH-1.
Abstract: Vascular integrins are essential regulators and mediators of physiological and pathological angiogenesis, including tumor angiogenesis. Integrins provide the physical interaction with the extracellular matrix (ECM) necessary for cell adhesion, migration and positioning, and induce signaling events essential for cell survival, proliferation and differentiation. Integrins preferentially expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, are considered as relevant targets for anti-angiogenic therapies. Anti-integrin antibodies and small molecular integrin inhibitors suppress angiogenesis and tumor progression in many animal models, and are currently tested in clinical trials as anti-angiogenic agents. Cyclooxygense-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxans, is highly up-regulated in tumor cells, stromal cells and angiogenic endothelial cells during tumor progression. Recent experiments have demonstrated that COX-2 promotes tumor angiogenesis. Chronic intake of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors significantly reduces the risk of cancer development, and this effect may be due, at least in part, to the inhibition of tumor angiogenesis. Endothelial cell COX-2 promotes integrin alphaVbeta3-mediated endothelial cell adhesion, spreading, migration and angiogenesis through the prostaglandin-cAMP-PKA-dependent activation of the small GTPase Rac. In this article, we review the role of integrins and COX-2 in angiogenesis, their cross talk, and discuss implications relevant to their targeting to suppress tumor angiogenesis.
Abstract: Integrin alpha6beta4 signaling proceeds through Src family kinase (SFK)-mediated phosphorylation of the cytoplasmic tail of beta4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, alpha6beta4 mediates assembly of hemidesmosomes. Here, we report that part of alpha6beta4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of beta4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of alpha6beta4 in lipid rafts, but does not affect alpha6beta4-mediated adhesion or assembly of hemidesmosomes. The fraction of alpha6beta4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective alpha6beta4 does not activate SFK signaling to extracellular signal-regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the alpha6beta4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis.
Abstract: New blood vessel formation, a process referred to as angiogenesis, is essential for embryonic development and for many physiological and pathological processes during postnatal life, including cancer progression. Endothelial cell adhesion molecules of the integrin family have emerged as critical mediators and regulators of angiogenesis and vascular homeostasis. Integrins provide the physical interaction with the extracellular matrix necessary for cell adhesion, migration and positioning, and induction of signaling events essential for cell survival, proliferation and differentiation. Antagonists of integrin alpha V beta 3 suppress angiogenesis in many experimental models and are currently tested in clinical trials for their therapeutic efficacy against angiogenesis-dependent diseases, including cancer. Furthermore, interfering with signaling pathways downstream of integrins results in suppression of angiogenesis and may have relevant therapeutic implications. In this article we review the role of integrins in endothelial cell function and angiogenesis. In the light of recent advances in the field, we will discuss their relevance as a therapeutic target to suppress tumor angiogenesis.
Abstract: We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses alpha(V)beta(3)- (but not alpha(5)beta(1)-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE(2) and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE(2) accelerated alpha(V)beta(3)-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE(2) effects. alpha(V)beta(3)-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked alpha(V)beta(3)-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued alpha(V)beta(3)-dependent spreading in the presence of NS398 or, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. alpha(5)beta(1)-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE(2), 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE(2) accelerates alpha(V)beta(3)-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces alpha(V)beta(3)-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins alpha(V)beta(3) by COX-2/PGE(2) during tumor angiogenesis and inflammation.
Abstract: Ligation of the alpha(6)beta(4) integrin induces tyrosine phosphorylation of the beta(4) cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of beta(4) that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr(1440), or secondarily Tyr(1422), interacts with the SH2 domain of Shc, whereas Tyr(1526), or secondarily Tyr(1642), interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by alpha(6)beta(4). In addition, mutation of beta(4) Tyr(1526), which binds to the PTB domain of Shc, but not of Tyr(1422) and Tyr(1440), which interact with its SH2 domain, abolishes the activation of ERK by alpha(6)beta(4). Phenylalanine substitution of the beta(4) tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of alpha(6)beta(4) in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of beta4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type beta(4). This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of beta(4) containing phenylalanine substitutions at Tyr(1422) and Tyr(1440), at Tyr(1526) and Tyr(1642), or at all four tyrosine phosphorylation sites. These results suggest that beta(4) Tyr(1526) interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.
Abstract: We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.
Abstract: Caveolin-1 functions as a membrane adaptor to link the integrin alpha subunit to the tyrosine kinase Fyn. Upon integrin ligation, Fyn is activated and binds, via its SH3 domain, to Shc. Shc is subsequently phosphorylated at tyrosine 317 and recruits Grb2. This sequence of events is necessary to couple integrins to the Ras-ERK pathway and promote cell cycle progression. These findings reveal an unexpected function of caveolin-1 and Fyn in integrin signaling and anchorage-dependent cell growth.
Abstract: The low-affinity nerve growth factor receptor p75NTR belongs to a membrane receptor superfamily whose members, in certain cell types, are able to transduce an apoptotic signal. To investigate the effect of p75NTR expression in neuroblastoma cells, we transfected the p75NTR cDNA into SK-N-BE cells, a neuroblastoma cell line that lacks expression of both p75NTR and TrkA. Cell clones expressing elevated levels of p75NTR showed a high degree of cell death by apoptosis, even in serum-supplemented medium. Moreover, the level of apoptosis correlated directly with the expression level of the receptor, indicating that p75NTR could activate the cell death program by itself. Clones expressing p75NTR showed a dramatic increase of cell death when switched into serum-free medium; these cultures rapidly extinguished. This apoptotic effect was greatly inhibited by NGF treatment. Our results support the hypothesis that p75NTR, when it is not bound by NGF, may play a role in neuronal selection during embryonic development and suggest that neuroblastomas may arise from immature neuroblasts that escape programmed cell death. Therefore, the loss of p75NTR expression in developing neural crest cells might be a primary event in the genesis of neuroblastoma.
Abstract: The retinoid N-(hydroxyphenyl) retinamide (4-HPR) appears to be a promising tool for chemoprevention of breast carcinoma, and clinical trials to evaluate its effect are in progress. However, its action on tumor cells has remained largely undefined. We report here that 4-HPR induced apoptosis and/or differentiation in breast cancer cell lines, independent of hormone receptor status and retinoic acid receptor expression, although it was slightly more efficient in inhibiting proliferation of estrogen receptor-positive cells. 4-HPR up-modulated expression of several differentiation markers (class 1 HLA, laminin, and beta 1 integrin chain) and down-regulated expression of molecules associated with tumor progression, including the p185/HER2 oncoprotein, the epidermal growth factor receptor, and the M(r) 67,000 laminin receptor. These data suggest that 4-HPR could exert a beneficial effect by inhibiting cell proliferation and modulating breast tumor aggressiveness.
