Research Project
10291608
PROJECT SUMMARY/ABSTRACT In the U.S, over 33,000 patients die yearly from prostate cancer (PCa), and an estimated 190,000 new cases are diagnosed. 10-20% of these patients are diagnosed with metastatic PCa (mPCa). mPCa and metastatic castration-resistant prostate cancer (mCRPC) acquire resistance to established treatments and progress with profound effects on patient quality of life. mCRPC remains fatal. Our group was the first to describe the new DNA repair gene FAM35A whose status may greatly affect tumor sensitivity to radiation treatment in a large proportion of PCa patients. This R15 project will determine a new mechanism of FAM35A-related treatment resistance/sensitization in PCa. Our central hypothesis is that 1) FAM35A regulates resection inhibition and 2) FAM35A and DNA polymerase theta (POLQ) antagonize the homologous recombination (HR) pathway. We will investigate through the following two specific aims: AIM 1: Determine the mechanism of FAM35A-mediated resection inhibition. Although our FAM35A-complex data includes BLM as a binding partner, it remains unclear how FAM35A blocks activity of resection enzymes (BLM-DNA2 and BLM-EXO1) and/or prevents the interaction of BLM with DNA2 or EXO1. We will test our working hypothesis that FAM35A prevents nuclease activity by binding to DNA and/or by binding to BLM. Our experimental approach will incorporate biochemical and cell biology methods to determine FAM35A?s resection inhibition activity. First, we will a) confirm in vitro DNA-binding activity of the two FAM35A isoforms, and b) determine the BLM binding site in FAM35A. Using FAM35A knockout cell lines complemented with mutant FAM35A, we will then assess DNA damage sensitivity using clonogenic assays; monitor the DNA damage response using confocal microscopy; and measure resection using DNA fiber assays. AIM 2: Elucidate the mechanism by which FAM35A contributes to the DNA damage response in POLQ KO PCa. My working hypothesis is that depletion of FAM35A/POLQ causes complete dysfunction of both c- and alt- NHEJ pathways, resulting in HR hyperactivation. 1) To test how FAM35A deficiency affects DNA damaging agents with and without POLQ, we will perform clonogenic assays using three FAM35A-depleted PCa cell lines of varying POLQ status. 2) To determine DNA repair activity in FAM35A-depleted POLQ KO PCa cells treated with DNA damaging agents, we will use confocal microscopy and immunoblotting to monitor the DNA damage response with DNA damage inducing agents. We will perform DSB-induced HR assays to measure HR activity. Results are expected to clarify the role of DNA repair dysfunction in resistance/sensitization in PCa and facilitate discovery of therapeutic and prognostic targets for radiation. The primary positive impact will be clarification of roles of DNA repair pathway between FAM35A and Polq. This AREA project will enable the research training of undergraduate and graduate students pursuing careers in health and basic sciences.
