Synergistic DNA repair genes and sensitivity to radiation therapy in prostate cancer

前列腺癌中的协同 DNA 修复基因和放射治疗敏感性

• 批准号: 10291608
• 负责人: Junya Tomida
• 金额: $ 44.65万
• 依托单位: UNIVERSITY OF NORTH CAROLINA CHARLOTTE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10291608
• 关键词: Affect; Basic Science; Benign; Binding; Binding Sites; Biochemical; Biological Assay; Cancer Patient; Cell Line; Cells; Cellular biology; Chemoresistance; Chemotherapy and/or radiation; Combined Modality Therapy; Complement; Confocal Microscopy; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA-Directed DNA Polymerase; Defect; Diagnosis; Double Strand Break Repair; EXO1 gene; Embryo; Environment; Enzymes; Excision; Fiber; Fibroblasts; Functional disorder; Funding; Gene Combinations; Gene Targeting; Health Sciences; Hypersensitivity; Immunoblotting; Immunohistochemistry; In Vitro; Knock-out; Knockout Mice; Malignant Neoplasms; Malignant neoplasm of prostate; Measures; Mediating; Metastatic Prostate Cancer; Methods; Monitor; Mutation; Nonhomologous DNA End Joining; Normal Cell; Pathway interactions; Patients; Poly(ADP-ribose) Polymerases; Polymerase; Predisposition; Prostate Cancer therapy; Prostatic Neoplasms; Protein Isoforms; Quality of life; Radiation; Radiation therapy; Regulation; Research; Research Training; Resistance; Roentgen Rays; Role; Testing; Therapeutic; Tissues; Universities; VP 16; anticancer research; cancer therapy; career; castration resistant prostate cancer; complex data; experience; genome sequencing; graduate student; homologous recombination; improved; inhibitor/antagonist; mRNA Expression; mutant; novel; nuclease; prevent; prognostic; prostate cancer cell; prostate cancer cell line; repaired; response; therapy resistant; tumor; undergraduate student; whole genome

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.

项目摘要/摘要 在美国，每年有超过33,000名患者死于前列腺癌(PCA)，估计有190,000名新患者 病例已确诊。这些患者中有10-20%被诊断为转移性前列腺癌(MPCA)。MPCA和 转移性去势耐药前列腺癌(MCRPC)对已有的治疗方法和 对患者的生活质量有深远影响的进步。MCRPC仍然是致命的。 我们团队首次描述了新的DNA修复基因FAM35A，其状态可能对肿瘤有很大影响 很大比例的前列腺癌患者对放射治疗敏感。这个R15项目将确定一个新的 前列腺癌中FAM35A相关耐药/增敏机制的研究我们的中心假设是1) FAM35A调节切除抑制和2)FAM35A和DNA聚合酶theta(POLQ)拮抗 同源重组(HR)途径。我们将通过以下两个具体目标进行调查： 目的1：探讨FAM35A介导的肿瘤切除抑制机制。虽然我们的FAM35A-Complex 数据包括BLM作为结合伙伴，目前尚不清楚FAM35A如何阻止切除酶的活性 (BLM-DNA2和BLM-EXO1)和/或阻止BLM与DNA2或EXO1的相互作用。我们将测试我们的 工作假说FAM35A通过与DNA和/或BLM结合来阻止核酸酶活性。我们的 实验方法将结合生化和细胞生物学方法确定FAM35A的切除 抑制活性。首先，我们将a)确认两个FAM35A亚型的体外DNA结合活性，以及b) 确定FAM35A中的BLM结合位点。利用FAM35A基因敲除细胞系与突变体互补 FAM35A，然后我们将使用克隆分析来评估DNA损伤敏感性；监测DNA损伤 使用共聚焦显微镜进行反应；并使用DNA纤维分析来测量切除。目标2：阐明 FAM35A参与POLQ KO PCA中DNA损伤反应的机制。我的工作 假设FAM35A/POLQ的缺失会导致c-和ALT-NHEJ通路的完全功能障碍， 导致HR过度激活。1)测试FAM35A缺陷对DNA损伤剂的影响 在没有POLQ的情况下，我们将使用三个不同的FAM35A缺失的PCa细胞系进行克隆分析 POLQ状态。2)检测DNA对FAM35A缺失的POLQ KO PCA细胞DNA修复活性的影响 损伤剂，我们将使用共聚焦显微镜和免疫印迹来监测DNA损伤反应 用DNA损伤诱导剂。我们将进行DSB诱导的HR分析来测量HR的活动。 这些结果有望阐明DNA修复功能障碍在前列腺癌和前列腺癌耐药/敏化中的作用 促进发现放射治疗和预后靶点。主要的积极影响将是 阐明DNA修复途径在FAM35A和POLQ中的作用。这一区域项目将使 为追求健康和基础科学职业的本科生和研究生提供研究培训。