Molecular mechanism and relevance of microRNAs in DSB repair pathway choice

 描述（由申请人提供）：不断暴露于环境和内源性DNA损伤剂的分裂细胞可能由于不正确的修复而转化为肿瘤。相反，放射和化学治疗剂通过诱导不可修复的DNA损伤来根除肿瘤。癌细胞通常通过操纵DNA修复机制而对治疗产生抗性。因此，我们需要深入了解DNA修复及其相关因素，以便深入了解癌症的原因并提高癌症治疗的疗效。双链DNA断裂（DSB）对细胞健康至关重要，因为单个未修复的DSB足以诱导细胞凋亡。两种主要的机制不同的途径，同源重组（HR）和非同源末端连接（NHEJ）已经进化到修复DSB。DSB修复途径之间的选择的决定性因素是DNA末端保护（NHEJ所必需的）和DNA末端切除（HR所必需的）之间的竞争。DSB末端切除必须限于细胞周期的S/G2期，因为HR需要存在完整的姐妹染色单体。因子如53 BP 1的消耗允许在G1期切除DNA末端，从而损害DSB修复并引起基因组不稳定性。同样，HR蛋白BRCA 1的丢失允许易错NHEJ途径在整个细胞周期中占主导地位，可能导致肿瘤发生。然而，BRCA 1的缺失提供了一个治疗机会，因为这些肿瘤对聚（ADP-核糖）聚合酶（PARP）的抑制剂非常敏感，并且对铂类药物也很敏感。令人惊讶的是，BRCA 1突变肿瘤中53 BP或相关因子（RIF 1，PTIP）的缺失使它们对PARP抑制剂具有抗性，同时恢复HR。因此，调节HR和NHEJ蛋白最佳表达的因子对于途径选择至关重要，并且可能在癌症治疗中具有重要意义。我们发现一类新的基因表达调节因子，microRNA（miRNA）下调DSB修复蛋白，并影响特定的修复途径。miRNA是一种丰富的非编码小RNA，通常抑制基因表达并在肿瘤中异常表达。我们最近观察到，miRNA介导的HR途径的抑制，特别是在G1期可能是基因组稳定性的关键。基于这些观察结果，我们进行了独立的功能筛选以鉴定调节HR途径（miR-1231、miR-876- 3 p、miR-221* 和miR-185*）和NHEJ途径（miR-142- 5 p、miR-502、miR-622和miR-597）的候选miRNA。在这里，我们将调查的假设，miRNA调节DSB修复途径的选择，通过维持细胞周期时相特异性表达的HR和NHEJ因子。我们将测试这样的想法，即miRNA诱导的特异性DSB修复因子的适度减少可能对常染色质与异染色质/基因间位点中的DSB修复产生差异性影响。最后，我们将研究候选miRNA在原发性卵巢肿瘤和淋巴瘤中的临床相关性，即miRNA表达与对PARP抑制剂/铂类治疗的反应和总体预后相关。

 DESCRIPTION (provided by applicant): A dividing cell constantly exposed to environmental and endogenous DNA damaging agents can transform into a tumor due to incorrect repair. Conversely, radiation and chemotherapeutic agents eradicate tumors by inducing irreparable DNA damage. Cancer cells often develop resistance to therapy by manipulating the DNA repair machinery. Therefore we need to have an in-depth understanding of DNA repair and the factors involved, both for gaining insight into the cause of cancer and to enhance the efficacy of cancer therapy. Double stranded DNA breaks (DSBs) are critical for cell health as a single unrepaired DSB is sufficient for inducing apoptosis. Two major mechanistically distinct pathways, homologous recombination (HR) and non-homologous end joining (NHEJ) have evolved to repair DSBs. A decisive factor in the choice between DSB repair pathways is in the competition between DNA end protection (necessary for NHEJ) and DNA end resection (necessary for HR). DSB end resection must be restricted to S/G2 phases of the cell cycle, as HR requires the presence of an intact sister chromatid. Depletion of factors such as 53BP1 allows DNA end resection in the G1 phase, thereby impairing DSB repair and causing genomic instability. Likewise, loss of the HR protein, BRCA1 allows the error-prone NHEJ pathway to dominate throughout the cell cycle potentially leading to tumorigenesis. However, loss of BRCA1 provides a therapeutic opportunity as these tumors are exquisitely sensitive to inhibitors of poly (ADP-ribose) polymerase (PARP), and are also susceptible to platinum-based drugs. Surprisingly, loss of 53BP, or associated factors (RIF1, PTIP) in BRCA1 mutant tumors make them resistant to PARP inhibitors with restoration of HR. Therefore factors that regulate optimal expression of HR and NHEJ proteins are crucial for pathway choice, and may have significant relevance in cancer therapy. We discovered that a new class of gene expression regulators, microRNA (miRNA)s down-modulate DSB repair proteins, and influence specific repair pathways. MiRNAs are abundant small non-coding RNAs that typically dampen gene expression and are aberrantly expressed in tumors. We recently observed that miRNA-mediated suppression of the HR pathway specifically in the G1 phase maybe critical for genomic stability. Building on these observations we conducted independent functional screens to identify candidate miRNAs that regulate the HR-pathway (miR-1231, miR-876-3p, miR-221*and miR-185*) and the NHEJ pathway (miR-142-5p, miR-502, miR-622, and miR-597). Here we will investigate the hypothesis that miRNAs regulate the choice of DSB repair pathways by maintaining the cell cycle phase specific expression of HR and NHEJ factors. We will test the idea that a miRNA-induced, modest decrease in specific DSB repair factors may differentially impact the repair of DSBs in euchromatin versus heterochromatin/ intergenic loci. Finally we will investigate clinical relevance of the candidate miRNAs in primary ovarian tumors and lymphomas, that is, correlate miRNA expression with response to PARP inhibitors/platinum based therapy and overall prognosis.