Defining the ability of HPV oncogenes to promote mutagenesis

HPV感染必须持续数年至数十年才能导致宫颈癌。在这段时间里，宿主基因组获得 促进肿瘤发生的突变。HPV癌基因(HPVE6和E7)是否发挥活性(通过以下方式引起突变 破坏DNA修复)和/或被动(通过降解肿瘤抑制因子，如P53和Rb)在获得 这些突变？虽然已经确定HPV癌基因会降解肿瘤抑制基因，但有证据表明，它们 也可能会积极地促进基因组的不稳定。在病毒生命周期中，HPVE6和E7诱导 DNA损伤信号和招募修复因子到病毒复制的位置。我们的团队和其他人已经证明了HPV 癌基因还改变了细胞修复DNA双链断裂(DSB)的方式。我们发布的数据和提供的数据 在本提案中提供了进一步细节，表明HPV癌基因将DSB修复从无错误机制转移 (同源重组或HR)到容易出错(微同源介导的末端连接或MMEJ)机制。这些 数据表明，HPVE6和E7使DSB修复更具突变性，但这一点尚未得到证实。其他 重大问题也依然存在。HPV癌基因如何促进MMEJ向修复的转变？做HPV吗？ 癌基因促进典型的MMEJ，还是在HPV癌基因阻止HR完成后导致向MMEJ的转换？如果 HPV使DSB修复更具突变性，是普遍的增加还是非等位基因同源性的存在(通过 重复的Alu元素)增强了HPV介导的突变？同样，如果HPV癌基因使DSB修复发生突变 他们对这一进程的贡献是否平等？目标1将解决关于HPV癌基因如何 使用活细胞显微镜和固定细胞显微镜相结合的方法，促进MMEJ的使用以及该途径是如何启动的。目标2 将定义HPV癌基因使DSB更具突变性的程度，以及它们的突变性 Alu元素的存在进一步加强了这一点。AIM 2将使用一种新的报告结构和液滴数字聚合酶链式反应， 以及深度测序方法和新颖的生物信息管道来实现这些目标。这些方法 都是由我们的研究团队开创的。这些创新方法将有助于更好地理解 推动宫颈癌发展的基本过程。鉴于宫颈癌是最常见的第四种癌症 女性罹患癌症，每90秒就有人死亡，这一信息很重要。中解决的知识差距 这项建议也与理解为什么大多数HPV感染不会导致宫颈癌和 开发方法来防止那些这样做的人。最后，鉴于宫颈癌通常是用基因毒性药物治疗的 药物(如顺铂或放射治疗)，了解HPV如何改变DNA修复可能导致识别 加强这些干预措施的方法。例如，如果HPV癌基因通过延迟进入MMEJ促进MMEJ 途径，参与MMEJ末端步骤的蛋白质的小分子抑制物可能导致肿瘤特异性 增强放射毒性。

HPV infections must persist for years to decades to cause cervical cancer. During this time, the host genome acquires mutations that promote tumorigenesis. Do HPV oncogenes (HPV E6 and E7) play an active (causing mutations by disrupting DNA repair) and/or a passive (via degradation of tumor suppressors like p53 and RB) role in the acquisition of these mutations? While it is established that HPV oncogenes degrade tumor suppressors, there is evidence that they may also actively promote genome destabilization. During the viral life cycle, HPV E6 and E7 induce a hyperactivation of DNA damage signaling and recruit repair factors to sites of viral replication. Our group and others have shown that HPV oncogenes also change how cells repair double stranded breaks in DNA (DSBs). Our published data and data presented in this proposal provide further details that show that HPV oncogenes shift DSB repair from error free mechanisms (homologous recombination or HR) to error prone (microhomology-mediated end joining or MMEJ) mechanisms. These data suggest that HPV E6 and E7 makes DSB repair more mutagenic, but this has yet to be demonstrated. Other substantial questions also remain. How do HPV oncogenes promote the shift towards repair by MMEJ? Do HPV oncogenes promote canonical MMEJ or does the switch to MMEJ result after HPV oncogenes block completion of HR? If HPV makes DSB repair more mutagenic, is the increase universal or does the presence of non-allelic homology (via repetitive Alu elements) augment HPV-mediated mutagenesis? Similarly, if HPV oncogenes make DSB repair mutagenic do they contribute equally to the process? Aim 1 will address mechanistic question regarding how HPV oncogenes promote the use of MMEJ and how the pathway is initiated, using a combination of live and fixed cell microscopy. Aim 2 will define the extent that HPV oncogenes make DSB more mutagenic and the extent that their mutagenic properties are further enhanced by the presence of Alu elements. Aim 2 will use a novel reporter construct and droplet digital PCR, along with deep sequencing approaches and novel bioinformatic pipelines to accomplish these goals. These approaches have been pioneered by our research team. These innovative approaches will facilitate an improved understanding of the fundamental processes that drive cervical cancer development. Given that cervical cancers are the 4th most common cancer in women and kill someone every 90 seconds, this information is important. The knowledge gaps addressed in this proposal are also relevant to understanding why most HPV infections do not result in cervical cancer and for developing methods to prevent those that do. Finally, given that cervical cancers are often treated with genotoxic agents (e.g., cisplatin or radiotherapy), understanding how HPV changes DNA repair could lead to the identification of ways to augment these interventions. For example, if HPV oncogenes promote MMEJ via a delayed entry into the pathway, small molecule inhibitors of proteins involved in terminal MMEJ steps would likely result in a tumor-specific enhancement of radiotoxicity.