963. MITF as a Potential Molecular Target for Gene Therapy for Human Melanoma; a Synergistic Inhibitory Effect of Inactivation of Both MITF and BRAFV600E on Melanoma Growth

Abstract

Top of pageAbstract MITF (microphthalmia-associated transcription factor) is a family of transcriptional factors controlling melanocyte differentiation. However, the roles of MITF on the cell proliferation have been controversial. Previous works have suggested that MITF negatively regulates cell proliferation or apoptosis in melanocytes, which is mediated by induction of p16INK4a, p21 or Bcl-2. But a recent study showed gene amplification of MITF in melanoma tissues (10-21%) associated with increase of the MITF protein. Furthermore, MITF gene amplification was a prognostic factor for survival, and several in vitro studies suggested that the MITF protein is attributed to increased cell proliferation, suggesting that MITF is an oncogene. Presently, the role of MITF in melanoma and melanomagenesis is not clear. We have attempted to investigate the role of MITF in malignant phenotypes of melanoma cells by using HIV-mediated RNA interference (RNAi) in a panel of melanoma cell lines with different status of the MITF expression to clarify the significance of MITF as a target for gene therapy, and to find any factors affecting sensitivity for the MITF inactivation. Furthermore, we have attempted to evaluate any additional effects by simultaneous suppression of both MITF and BRAFV600E on the melanoma growth, since BRAFV600E, the most frequent form of human BRAF mutation, is attributed to the enhanced MAPK signaling and several malignant phenotypes of melanoma. We have constructed two short hairpin RNA (shRNA) HIV vectors for MITF, and one control shRNA HIV vector (GL3B; for firefly luciferase). An HIV vector expressing two shRNAs for MITF and BRAFV600E was also constructed by tandemly aligning two shRNA expression cassettes in one HIV vector. Seven melanoma cell lines were infected with these shRNA HIV vectors, and the effects of MITF RNAi on the cell growth and apoptosis were evaluated. In six of 7 melanoma cell lines with substantial expression of the MITF protein, the MITF RNAi induced significant inhibition of cell growth, associated with G1 arrest. One melanoma cell line, A375, without any detectable expression of MITF was resistant to the MITF RNAi. One melanoma cell line showed significant apoptotic changes represented by the increase of subG1 population (7.23-7.60%) and increase of cleaved caspase-3 and caspase-9, following the MITF RNAi. Interestingly, the MITF RNAi induced G1 arrest in most melanoma cell lines tested, however, no clear correlation was observed between the protein levels of cdk inhibitors and G1 arrest. Finally, three melanoma cell lines with the simultaneous inhibition of both MITF and BRAFV600E, showed synergistic inhibition of cell growth, which was associated with more G1 arrest. Our results suggest that MITF could be a molecular target for gene therapy for most human melanoma, and the synergistic therapeutic effects of MITF and BRAFV600E indicate the possibly more potent gene therapy for melanoma by simultaneous down-regulation of these molecules.