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 the expression of the cell surface adhesion molecule MCAM/MUC18, which belongs to the immunoglobulin superfamily, directly correlates with the metastatic potential of human melanoma cells. While none of the MCAM/MUC18-negative cell lines examined have been found to be metastatic, transfection of such cells with MCAM/MUC18 gene rendered them metastatic in nude mice. In addition, we have recently demonstrated that the progression of melanoma is associated with loss of expression of the c-KIT proto-oncogene tyrosine kinase receptor. Furthermore, re-expression of the c-KIT receptor in highly metastatic cell lines inhibited their tumorigenicity and metastatic potential in nude mice. Indeed, exposure of c-KIT-positive melanoma cells in vitro and in vivo to stem cell factor (SCF), the ligand for c-KIT, triggered apoptosis of these cells but not of normal melanocytes. The mammalian transcription factor AP-2 is a sequence-specific DNA-binding protein expressed in neural crest lineages and regulated by retinoic acid. Our preliminary data indicate that both MCAM/MUC18 and c-KIT gene expression is highly regulated by AP-2. In addition, other genes that are involved in the progression of melanoma such as MMP-2, bel-2, E-cadherin, and p21/WAF-1 are also regulated by the transcription factor AP-2. Our laboratory has recently made the observation that while several nonmetastatic melanoma cell lines express AP-2, the highly metastatic cell lines do not express this transcription factor. Therefore, we hypothesize that loss of AP-2 expression may be a crucial event in the development of malignant melanoma. We are therefore proposing the three Specific Aims: 1) to provide a direct evidence for the involvement of AP-2 in the acquisition of the metastatic phenotype by transfecting metastatic cells with w.t. AP-2 and primary melanoma cells with a dominant negative AP-2B genes and subsequently analyze their tumorigenicity and metastatic potential in nude mice. 2) To study the effect of AP-2 and AP-2B on the expression of the above mentioned genes. 3) To evaluate the status of AP-2 expression and function in tumor specimens from patients in well-characterized melanoma database. These experiments will generate valuable information on the progression of melanoma, and help to develop new molecular staging markers and a common target for anti-tumor/metastasis therapy.
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