Striking similarities exist between molecular mechanisms driving embryonic liver development and progression of hepatocellular carcinoma (HCC). Bone morphogenetic proteins (BMPs), particularly BMP4, have been proposed to regulate embryonic hepatic development. BMP expression has been observed in neoplasia but the expression and biological role of BMP4 in human HCC are unknown. We found increased BMP4 mRNA and protein in HCC cell lines and tissue samples compared to primary human hepatocytes and corresponding non-tumourous tissue. Hypoxia further induced BMP4 expression in HCC cells, which was abolished by transfection of a dominant negative form of HIF-1 alpha (dnHIF-1 alpha). However, gel shift assays revealed only minor binding activity in nuclear extracts from (hypoxic) HCC cells to a putative hypoxia-response element in the BMP4 promoter. Sequence analysis of the BMP4 promoter revealed two Ets-1 binding sites, and Ets-1 activity was increased in HCC cells under hypoxic conditions. Transfection of dnHIF-1 alpha completely abrogated hypoxia-induced Ets-1 activity as well as BMP4 expression. Overexpression of Ets-1 markedly enhanced BMP4 promoter activity, while antisense Ets-1 almost completely abolished basal as well as hypoxia-induced BMP4 expression. These data demonstrate that Ets-1 activity contributes to baseline expression of the BMP4 gene and is the predominant mediator of the HIF-dependent BMP4 induction under hypoxic conditions. To determine the functional relevance of BMP4 expression, HCC cell lines were treated with antisense BMP4 constructs or siRNA against BMP4. BMP4 suppression resulted in a strong reduction of the migratory and invasive potential and anchorage-independent growth. Furthermore, tube formation assays indicated that BMP4 expressed by HCC cells promotes vasculogenesis. Our findings demonstrate that BMP4 is increased in HCC and promotes HCC progression. Therefore, BMP4 expression may have clinical relevance, and interfering with BMP4 signalling appears as an attractive therapeutic target for this highly aggressive tumour.

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MIA (melanoma inhibitory activity) has been identified as a small protein secreted from malignant melanoma cells, which interacts with extracellular matrix proteins including fibronectin. Here, we show that MIA negatively regulates the activity of the mitogen-activated protein kinase pathway in malignant melanoma. Using far Western blotting and co-immunoprecipitation we searched for MIA-binding cell surface proteins. We found that MIA interacts with integrin ␣4␤1 and ␣5␤1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings also suggest that MIA may play a role in tumor progression and the spread of malignant melanomas via mediating detachment of cells from extracellular matrix molecules by modulating integrin activity. Inhibiting MIA functions in vivo may therefore provide a novel therapeutic strategy for metastatic melanoma disease.We have previously identified MIA (melanoma inhibitory activity), an 11-kDa protein secreted into the tissue culture supernatant from malignant melanoma cells (1). MIA expression in vivo correlates with progressive malignancy of melanocytic tumors (2). Additionally, in recent studies we detected enhanced MIA protein levels specifically in the serum of patients with metastatic melanomas (3). In vitro studies revealed a role for MIA in supporting the invasive and migratory potential of melanoma cells. In vivo studies in two animal model systems confirmed the importance of MIA in melanoma metastasis. MIA expression levels parallel closely the capability of melanoma cells to form metastases in syngeneic animals (4, 5).Three-dimensional analyses of MIA by multidimensional NMR (6 -8) or x-ray crystallography (9) indicate that MIA defines a novel family of secreted proteins that adopt an SH3 domain-like fold in solution. Furthermore, NMR spectra revealed that MIA interacts with peptides matching to type III human fibronectin repeats that are closely related to ␣4␤1 integrin-binding sites (6). These data support a model in which MIA regulates attachment to specific components of the extracellular matrix. Based on these results and on the observation that MIA alters melanoma cell morphology, we determined that MIA treatment results in cell detachment by decreasing interactions between melanoma cells and extracellular matrix molecules (10). The study presented here was performed to find additional MIA-interacting proteins and to identify signaling pathways regulated by MIA. EXPERIMENTAL PROCEDURESCell Lines and Culture Conditions-The melanoma cell lines Mel Im and Mel Ei, have been described in detail previously (11). The cell line Mel Ei was derived from a primary cutaneous melanoma, and the cell line Mel Im was derived from a metastasis of malignant melanoma. To establish fibronectin-deficient fibroblast-like cells, primary embryonic fibroblasts were isolated from E13.5 fibronectin (flox/flox) embryos and immortalized by retroviral transduction of the SV-40 large T antigen and cloned (12). Subsequently, two clonal lines...

Melanoma inhibitory activity (MIA) has been identified as a small protein secreted from malignant melanoma cells, which strongly enhances melanoma cell migration and invasion. Detailed analyses performed by our group showed interaction of MIA with extracellular matrix proteins and integrin a4b1 and a5b1 leading to cellular detachment. In this study, we identified cadherin-7 as a new MIA-binding protein using surface-enhanced laser desorption/ ionization-mass spectrometry technology and co-immunoprecipitation. Cadherin-7 is a classical cell-cell adhesion molecule which was shown to be upregulated in malignant melanoma. We demonstrated enhanced expression of cadherin-7 in primary tumor cells compared to metastatic cells. Upregulation of cadherin-7 expression in metastatic cell lines but also downregulation of expression in cells derived from primary melanomas resulted in reduced cell migration. In addition, we speculate that MIA/cadherin-7 interaction may regulate cell-cell adhesion of malignant melanoma cells influencing the migration of the cells. Interestingly, overexpression of cadherin-7 resulted in a decreased MIA mRNA expression. In addition, MIA effects on cell migration were abrogated in cell clones overexpressing cadherin-7. In conclusion, these findings suggest that cadherin-7 regulates the expression and activity of MIA and the migration of melanoma cells playing a role in tumor development of malignant melanoma. (Cancer Sci 2009; 100: 261-268)

BackgroundThe C-terminal binding protein 1 (CtBP1) is a known co-repressor of gene transcription. We recently revealed that CtBP1 expression is lost in melanoma cells and melanoma inhibitory activity (MIA) expression is subsequently increased. The present study was performed to evaluate a more general role of CtBP1 in human melanoma and identify further CtBP1-regulated target genes.MethodsSequence analysis and expression profile of CtBP1 in melanoma cell lines were done by PCR. Boyden Chamber assays and co-immunoprecipitation were performed to investigate the functional role of CtBP1. Gene expression analysis and micro array data were used to define target genes.ResultsInterestingly, we detected an alternative splice product of CtBP1 with unknown function whose expression is induced at reduction of full length CtBP1. Overexpression of full length CtBP1 in melanoma cells had no effect on cell proliferation but did influence cell migration and invasiveness. To understand the effect of CtBP1 we identified putative LEF/TCF target genes found to be strongly expressed in melanoma using DNA microarray analysis. We focused on fourteen genes not previously associated with melanoma. Detailed analysis revealed that most of these were known to be involved in tumor metastasis. Eleven genes had expression profiles associated with melanoma cell invasiveness.ConclusionIn summary, this study revealed that reduction of CtBP1 expression is correlated with migratory, invasive potential of melanoma cells.