基于调控DNA同源重组修复通路研究扁柏素联合PARP抑制剂治疗卵巢癌的作用及其机制

Ovarian cancer has very poor survival. PARP inhibitors have been approved to treat BRCA mutated or homologous recombination(HR) deficient ovarian cancer. However, PARP inhibitors are not effective to BRCA wild type or HR proficient ovarian cancer. In order to expand the spectrum of ovarian cancer patients who would benefit from PARP inhibitors, especially those that are BRCA wild type and HR proficient, rational combination therapies that lead to HR defects and consequently synergize with PARP inhibitors should be evaluated. .Combination therapy needs to consider toxicity and cost. Natural products with approved safety and from also inexpensive source may provide an effective way to identify synergistic combination. But how natural products affect DNA damage response and repair remains largely unknown. .Our preliminary study developed a high-throughput natural products screen for DNA repair regulators and identified beta-Thujaplicin as a novel HR repair inhibitor. We further explored the mechanism by which it affects HR repair. Our study found that it can inhibit the recruitment of key HR repair protein Rad51 at DSBs and increase the persistent phosphorylation of RPA. .On the basis of our preliminary studies, we hypothesize that beta-Thujaplicin as a novel HR inhibitor can synergize with PARP inhibitors by regulating DNA repair process mediated by HR. .Two specific aims are proposed to test this hypothesis:.Aim 1. Determine the synergistic effects of beta-Thujaplicin and PARP inhibitors combination on ovarian cancer, especially those that are BRCA wild type and HR proficient. .Aim 2: Determine the mechanism how beta-Thujaplicin regulates HR repair, includes,1) Examine the effects of beta-Thujaplicin on DNA end resection, 2) Examine the effects of beta-Thujaplicin on inhibiting the histone deacetylases (HDAC), thus enhance the acetylation and subsequent degradation of critical repair proteins, such as CtIP..These experiments are anticipated to mechanistically characterize the function of beta-Thujaplicin in HR repair. We expect our proposed study will improve the responses of ovarian cancer to PARP inhibitor by reducing HR repair efficiency. The proposed study will provide a poof of principal that using our screening assays to enhance therapeutic efficacy of targeted therapy in cancer treatment.

卵巢癌是女性常见恶性肿瘤，总体生存率低，预后差。PARP抑制剂是卵巢癌治疗上的突破性进展，但它只对BRCA突变型和同源重组修复（HR）缺陷型卵巢癌有效。如何拓宽其治疗谱，提高疗效是亟待解决的关键科学问题。申请者前期研究通过高通量药物筛选平台，甄选出有明确HR抑制功能的天然植物药--扁柏素，并发现它能通过抑制DNA损伤修复蛋白Rad51在双链DNA断裂处的募集，抑制HR修复。申请者预实验还发现扁柏素能增加肿瘤细胞对PARP抑制剂的敏感性。基于前期研究基础和“合成致死”理论，本研究假设，外源性HR功能抑制剂扁柏素联合PARP抑制剂治疗卵巢癌，能克服PARP抑制剂的“选择性”，对BRCA野生型和HR非缺陷型卵巢癌产生协同抗瘤效应。本研究拟在体内外水平检测扁柏素联合PARP抑制剂对卵巢癌的抗瘤效应，阐明扁柏素调节HR修复的分子机制，为治疗BRCA野生型和HR非缺陷型卵巢癌提供新的联合治疗策略。

卵巢癌是全球常见的妇科肿瘤，死亡率仅次于乳腺癌，居女性癌症死亡原因的第五位。但迄今为止，除PARP（聚ADP核糖聚合酶）抑制剂以外，卵巢癌的靶向治疗靶点十分有限。FAM111B（序列相似的家族111成员B基因）与DNA和RNA合成、细胞增殖及凋亡、细胞周期调控和肿瘤转移有关，是扁柏素作用的下游通路基因。本研究明确了扁柏素联合顺铂对卵巢癌细胞不具有协同抗瘤效应，同时也发现其FAM111B转染卵巢癌细胞后对HR修复通路重要蛋白r-H2AX无明显影响。.为研究FAM111B是否在卵巢癌中发挥致病作用及其可能的调控机制，本研究在卵巢癌组织中检测到FAM111B在mRNA水平上保持显著的高表达。在ES-2和A2780细胞系中干扰FAM111B基因表达，发现其在体外显著抑制了细胞的增殖、迁移和侵袭；同时发现FAM111B沉默的动物模型肿瘤生长也显著降低。本研究还发现FAM111B以MYC通路依赖的方式介导恶性生物学行为。综上所述，我们通过体内外实验研究表明，促癌基因FAM111B以MYC依赖的方式促进卵巢癌的恶性进展，包括肿瘤增殖、迁移和侵袭。因此，FAM111B有望成为未来卵巢癌靶向治疗的潜在治疗靶点。