Oncogene c-Myc-dependent pathways in cutaneous melanoma

Roswell Park Cancer Institute Corp, Buffalo NY

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Abstract

DESCRIPTION (provided by applicant): Malignant melanoma is 1 of the most aggressive types of human cancer. Its ability to metastasize, in combination with resistance to conventional anticancer chemotherapy, makes melanoma extremely difficult to cure and consequently, the median survival of metastatic melanoma patients is 8.5 months. Molecular mechanisms underlying melanoma progression are not completely understood, and good markers associated with specific stage(s) of tumor progression are yet to be determined. Elevated expression of the oncogene C-MYC was reported in melanoma, although a controversy remains as to what stage(s) this event occurs. We discovered that high amounts of C-MYC are maintained at the protein level in the majority of metastatic melanoma samples and melanoma cell lines. The overall hypothesis of our proposal is that viability of malignant melanoma depends on high levels of C-MYC. We demonstrated that high C-MYC levels and protein stability in several melanoma cell lines could not be fully accounted for by activation of MAPK-ERK pathway, and that inhibition of factors involved in proteosomal degradation of C-MYC is likely to participate in upregulation of C-MYC levels and protein stability in these cells. Expression analysis identified that among proteins promoting C-MYC proteolitic degradation, B56a subunit of PP2A phospahtase complex was underexpressed in all tested melanoma cell lines compared to normal melanocytes. Additionally, inhibition of PP2A-B56a subunit in melanoma cells caused upregulation of c-MYC levels. To identify MYC-target genes rate-limiting for melanoma cell viability, we stably inhibited C-MYC in several human melanoma cell lines by lentivirus-based shRNA. Depletion of C-MYC resulted in apoptosis and/or inhibition of proliferation accompanied by decreased amounts of intracellular deoxyribonucleoside triphopshates (dNTPs) and downregulation of several rate-limiting enzymes involved in dNTP metabolism. Importantly, apoptotic/cytostatic effects caused by C-MYC depletion were partially alleviated by supplementing cell media with precursors of nucleotide biosynthesis. We therefore propose to i) determine whether elevated C-MYC protein levels are associated with a specific stage of melanoma progression and ii) functionally characterize the role of PP2A-B56a in C-MYC-dependent melanocytic transformation and in C-MYC-mediated maintenance of viability of melanoma cells. iii) functionally characterize involvement of the identified enzymes in MYC-mediated control of the nucleotide metabolism and iv)identify rate-limiting steps of nucleotide metabolism affected in MYC-depleted cells using methods of biochemical analysis.