Functional analysis of the novel melanoma master-regulators

Abstract

Melanoma is a devastating form of cancer that is especially common in New Zealand and that is incompletely understood. In this project, we are using clinical microarray information about the function of tens of thousands of genes in melanoma patients. We are combining this clinical microarray information with microarray analysis of melanoma cells in the laboratory. In this laboratory analysis we have knocked down the expression of 75 different genes using a method known as siRNA. This clinical and laboratory data is combined mathematically using a method known as gene network analysis. This allows us to identify novel "master-regulators" of melanoma, which are seen as “hubs” in melanoma gene networks. These hubs may drive the uncontrolled growth of melanoma cells, and are likely to be useful as prognostic markers or new drug targets. We have already identified several novel melanoma gene network hubs using this method. The sequences encoding these hubs are cloning into an expression vector, and the candidate hub genes are then screened through a functional screening platform. Since we are interested the clinically important genes, we screen to identify those genes that may play a role in proliferation, sensitivity to drug-induced apoptosis, and survival. We will transfect all the candidate genes (alongside a negative vector only control and a positive control of Bcl-2) into 293T cells (human embryonic kidney cells containing the SV40large T antigen to promote episomal replication of transfected plasmids), and A375 cells (melanoma). We will screen for expressed gene function using assays for viable cell number (MTT), assays for cell cycle progression (flow cytometry), and apoptosis assays. In particular we will use the anti-neoplastic drug etoposide (VP16) to induce apoptosis in cells that have been transfected by these genes and controls, to assess gene product function in modifying the apoptosis induced by VP16. For clinically relevant network hub genes that appear to play a role in controlling proliferation, apoptosis and survival, we will home in further on their mechanism of action (the methods used will depend upon our hypotheses about the gene product's function). We hope some of those hubs may lead to novel diagnostic/prognostic assays and novel drug targets.