The molecular changes associated with the transition of melanoma cells from radial growth phase (RGP) to vertical growth phase (VGP, metastatic phenotype) are not very well-defined. Recent work from this laboratory demonstrated that this transition is associated with loss of expression of the AP-2 transcription factor. We showed that lack of expression of AP-2 in metastatic melanoma cells resulted in deregulation of the c-KIT and MCAM/MUC18 genes, both of which are involved in the progression of human melanoma. Moreover, inactivation of w.t. AP-2 in primary cutaneous melanoma cells by dominant-negative AP-2 (AP-2B gene) resulted in an increase in their tumorigenicity and metastatic potential in vivo due to upregulation of MMP-2. Loss of AP-2 expression was also observed in advanced primary of melanoma patients indicating that loss of AP-2 is a crucial event in the progression of human melanoma. In an effort to identify other target genes regulated by AP-2, here we provide evidence of an inverse correlation between the expression of AP-2 and the thrombin receptor (PAR-1) in metastatic melanoma cells. Regulation of PAR-1 by AP-2 in melanoma cells is demonstrated in vitro and in vivo, thus providing a unique link between the coagulation system and the progression of human melanoma. Recent evidence suggests that thrombin receptor plays an important role in metastasis of human melanoma. PAR-1 is a unique G-coupled protein receptor that belongs to the protease activated receptor family. Activation of PAR-1 can result in upregulation of gene products involved in adhesion (integrins), invasion (MMP's) and angiogenesis (IL-8, VEGF, uPA, PDGF and bFGF). The hypothesis to be tested in this proposal is that loss of AP-2 results in upregulation of PAR-1 which correlates with the malignant phenotype of human melanoma. To test this hypothesis we now propose to: 1) determine how AP-2 regulates PAR-1 expression; 2) to analyze AP-2 and PAR-1 expression in tumor specimens from melanoma patients and; 3) to study how activation of PAR-1 contributes to the metastatic phenotype in human melanoma. Understanding the mechanisms by which PAR-1 is activated may lead to new techniques to inhibit melanoma invasion and metastasis.
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