This proposal is an extension of ongoing program of development of new functional gene discovery methodologies for identification of cancer-related genes. In the previous phase, it was primarily focused on detection of new candidate tumor suppressor and drug sensitivity genes. Recent methodological advancements made it possible now to focus on prospective molecular anticancer treatment targets (ACTTs) defined as cellular factors, the repression of which leads to selective killing or sensitization to treatment of tumor cells. Candidate ACTT search is based on a combination of three powerful techniques - microarray gene expression analysis, genetic suppressor element (GSE) methodology and newly developed selection-subtraction approach (SSA). These techniques are integrated in one technological pipeline that allows identification of genetic events making tumor cells tolerant to oncogenic Ras. The search for ACTTs will start from collection of a pool of candidate sequences representing genes that become upregulated in a series of populations of human fibroblasts rescued from Ras-mediated growth arrest by different cooperating genetic elements identified in the course of preliminary studies. GSE library will be constructed from the selected sequences and screened, using SSA technique, for the GSEs capable of killing Ras-transformed but not normal fibroblasts. Identified genes will be validated as prospective ACTTs by using siRNA constructs against each individual candidate that will be tested for their specific cytotoxicity for tumor cells. Relevance of identified candidate ACTTs to naturally occurring tumor cells will be characterized and the program will be further extended towards development of small molecules with anticancer properties acting by suppression of identified ACTT. The success of the proposed study will indicate development of a powerful target discovery tool that will be applicable for ACTT identification in other types of cancer.