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The molecular basis of adhesion of highly metastatic human melanoma cells to the 33-kD fragment of fibronectin (FN) was elucidated. The results showed that binding of the melanoma cells to FN involves the action of alpha-4-integrin subunits as well as cell surface chondroitin sulfate proteoglycan. Additionally, a region within the 33-kD fragment was identified as a novel heparin binding, cell adhesion-promoting region. The region, termed as FN-C/H-III, acts in coordination with other cell adhesion promoting sites to bind a melanoma cell surface receptor complex.

Abstract Background The tetraspan protein CD63, originally described as a stage-specific melanoma antigen but also present in a number of normal cells, regulates melanoma cell growth in nude mice, motility in serum containing media, and adhesion to several extracellular matrix proteins. CD63 has been reported to associate with β1 and β2 integrins, but the mechanism of signal transduction by CD63 is not clear. This study examined whether CD63 is associated with protein kinase and can transmit signals in melanoma cells. Methods Immunoprecipitation and radiolabeling were used to test for association of protein kinase activity with CD63. Adhesion of cells to monoclonal antibodies immobilized to microtiter plates was used to examine the ability of CD63 to transmit signals. Results CD63 was capable of transmitting a signal in melanoma cells that required extracellular calcium. In the absence of extracellular calcium at the time of binding to the CD63 mAb, the cell was no longer responsive to stimulation by CD63. Immunoprecipitation studies demonstrated protein kinase activity associated with CD63, and phosphoamino acid analysis revealed that most of this protein kinase activity was due to serine kinase activity. Conclusion The current study suggests that serine protein kinase activity associated with CD63 may play a role in signaling by CD63 in melanoma cells.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-α[sub 4]β[sub 1]-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p30[sup cas], an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130[sup cas]. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130[sup cas]. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization. [ABSTRACT FROM AUTHOR]

Molecular heterogeneity in human breast cancer has challenged diagnosis, prognosis, and clinical treatment. It is well known that molecular subtypes of breast tumors are associated with significant differences in prognosis and survival. Assuming that the differences are attributed to subtype-specific pathways, we then suspect that there might be gene regulatory mechanisms that modulate the behavior of the pathways and their interactions. In this study, we proposed an integrated methodology, including machine learning and information theory, to explore the mechanisms. Using existing data from three large cohorts of human breast cancer populations, we have identified an ensemble of 16 master regulator genes (or MR16) that can discriminate breast tumor samples into four major subtypes. Evidence from gene expression across the three cohorts has consistently indicated that the MR16 can be divided into two groups that demonstrate subtype-specific gene expression patterns. For example, group 1 MRs, including ESR1, FOXA1, and GATA3, are overexpressed in luminal A and luminal B subtypes, but lowly expressed in HER2-enriched and basal-like subtypes. In contrast, group 2 MRs, including FOXM1, EZH2, MYBL2, and ZNF695, display an opposite pattern. Furthermore, evidence from mutual information modeling has congruently indicated that the two groups of MRs either up- or down-regulate cancer driver-related genes in opposite directions. Furthermore, integration of somatic mutations with pathway changes leads to identification of canonical genomic alternations in a subtype-specific fashion. Taken together, these studies have implicated a gene regulatory program for breast tumor progression. [ABSTRACT FROM AUTHOR]

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-α[sub 4]β[sub 1]-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p30[sup cas], an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130[sup cas]. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130[sup cas]. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization. [ABSTRACT FROM AUTHOR]

Introduction: Hyaluronan (HA) and its biosynthetic enzymes hyaluronan synthases (HAS2 and HAS3) mediate Matrigel invasion by SW620 colon carcinoma cells. Because matrix metalloproteinases (MMPs) have been implicated in cancer invasion, we hypothesized that changes in HAS expression would alter MMP expression and activity in these cells., Methods: To determine whether an MMP was involved in invasion, Matrigel invasion assays with SW620 cells were performed in the presence or absence of the MMP inhibitors GM6001 or TIMP2. HAS isozymes were inhibited by stably transfecting SW620 cells with vectors that contained antisense HAS2 and/or -3 cDNA; transfection with an empty vector served as a control. MMP-7 transcription was assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR). MMP-7 protein was detected by enzyme-linked immunosorbent assay (ELISA) and enzymatic activity compared using zymography., Results: GM6001 and TIMP2 decreased Matrigel invasion, which confirms that an MMP played a key role in this process. MMP-7 expression was then detected in SW620 cells. Finally, MMP-7 expression, protein, and enzymatic activity were significantly lower in antisense HAS tranfectants than in SW620 or vector control cells., Conclusion: We have demonstrated previously that inhibition of HAS expression and HA production in SW620 colon carcinoma cells inhibits Matrigel invasion. In the studies presented here, we have demonstrated that SW620 cells express high levels of MMP-7 and that inhibition of HAS isozymes dramatically decreases MMP-7 expression, protein, and enzymatic activity. Taken together, these findings suggest that HAS and HA may mediate cellular invasion via changes in MMP-7 expression.

Tumor cell invasion through basement membranes and into stromal tissue are key steps for promoting growth and metastasis. Tumor cells express various extracellular-matrix-degrading enzymes such as matrix metalloproteinases (MMPs) to degrade extracellular matrix components to facilitate tumor migration and invasion. Histological and clinical studies suggest a role for MMP-1 (collagenase-1) in malignant melanoma invasion. In this study, we evaluated MMP-1 in regulating malignant phenotypes of human melanoma cells by generating human melanoma cells stably transfected with pro-MMP-1 cDNA. The transfectants expressed the active form of MMP-1 associated with cells and showed enhanced invasive and growth abilities in type I collagen gel. Furthermore, MMP-1 expression promoted anchorage-independent growth, which was inhibited in the presence of type II transforming growth factor (TGF)-beta receptor:Fc fusion protein that scavenges TGF-beta receptors. Finally, we demonstrated that MMP-1 directly generated active TGF-beta from its latent form. Thus, these results suggest that MMP-1 produced from melanoma cells would play a role in tumor progression by both degrading matrix proteins and generating active growth factors such as TGF-beta in vivo.

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in vitro. To assess HA functionality, HAS expression was inhibited in SW620 cells by transfection with antisense HAS constructs. Decreased HA secretion and retention in the transfectants were confirmed using competitive binding and particle exclusion assays. SW620 cells demonstrated greater invasion through Matrigel than did SW480 cells. Antisense transfection decreased Matrigel invasion by SW620 cells by >60%; addition of exogenous HA restored invasion. Because the cell-surface HA receptor CD44 has been implicated in cancer progression, HA-CD44 interaction was then inhibited by incubation with an anti-CD44 antibody. Anti-CD44 antibody impaired invasion into Matrigel by 95%. Taken together, these data suggest that pericellular HA is critical for colon carcinoma cell invasion and that this invasive capability is dependent on interaction with CD44.