The role of PARP10 in alleviating replication stress and promoting cellular proliferation and tumorigenesis

项目摘要 鉴定癌细胞优先采用和依赖的分子途径， 是设计新型个性化癌症疗法的关键。PARP 10是一种特征不佳的 PARP家族的成员。我们之前表明PARP 10促进跨损伤合成（TLS）- 在DNA复制过程中介导绕过DNA损伤，从而减轻复制应激。最近， 我们还发现PARP 10是一种新的癌基因。我们发现PARP 10基因被扩增和/或 在包括乳腺癌和卵巢癌在内的大量肿瘤中过度表达， 下调或损失。我们发现PARP 10基因在一个大的细胞中扩增和/或过表达， 包括乳腺癌和卵巢癌在内的人类肿瘤的比例，几乎没有发生下调或 损失此外，我们发现PARP 10在非转化的人上皮RPE 1细胞中过表达， 导致增强的增殖、对复制应激的抗性和增加的异种移植肿瘤形成， 免疫受损的小鼠。在癌症HeLa中敲除PARP 10后发现了相反的表型 细胞这些发现表明PARP 10是一个假定的癌基因，其表达促进肿瘤的发生 形成和生长。诱变性TLS先前已被提出，以促进转化， 允许过度增殖并诱导基因组不稳定性。因此，我们假设PARP 10表达 通过TLS介导的复制停滞结构的旁路抑制复制应激， 使癌细胞过度增殖。我们建议在这里直接测试这一点，在三个具体的目标， 在三个不同的层次上解决假设：目标1将研究PARP 10所采用的机制， 使用生物化学和细胞定位在分子水平上调节PCNA依赖性TLS， 相互作用测定。目标2将在功能上测试这种细胞过程机制的影响，包括 基因组稳定性和DNA复制。目标3将采用小鼠遗传模型来明确地研究 如果Parp 10表达诱导肿瘤形成或促进肿瘤生长。使用最先进的手机 分子和基因组工具（包括：CRISPR/Cas9介导的基因组编辑;分子DNA纤维 梳理以测量叉稳定性;下一代测序方法以测量诱变， 突变负担），我们将在这里研究这种新的致癌功能的分子机制 PARP10。这可能最终导致癌症治疗新靶点的验证。

Project Summary Identification of molecular pathways that are preferentially employed and relied upon by cancer cells compared to normal cells is key to designing novel personalized cancer therapies. PARP10 is a poorly characterized member of the PARP family. We previously showed that PARP10 promotes translesion synthesis (TLS)- mediated bypass of DNA lesions during DNA replication, thereby alleviating replication stress. More recently, we also showed that PARP10 is a novel oncogene. We found that the PARP10 gene is amplified and/or overexpressed in a large number of tumors including breast and ovarian, with very few observed occurrences of downregulation or loss. We found that the PARP10 gene is amplified and/or overexpressed in a large proportion of human tumors including breast and ovarian, with almost no occurrences of downregulation or loss. Moreover, we found that PARP10 overexpression in, non-transformed human epithelial RPE1 cells results in enhanced proliferation, resistance to replication stress, and increased xenograft tumor formation in immunocompromised mice. The opposing phenotypes were found upon knockout of PARP10 in cancer HeLa cells. These findings suggest that PARP10 is a putative oncogene and its expression promotes tumor formation and growth. Mutagenic TLS has been previously proposed to promote transformation by both allowing hyper-proliferation and inducing genomic instability. Thus, we hypothesize that PARP10 expression suppresses replication stress through TLS-mediated bypass of replication arresting structures, thereby allowing hyper-proliferation of cancer cells. We propose here to directly test this, in three specific aims which address the hypothesis at three different levels: Aim 1 will investigate the mechanism employed by PARP10 to modulate PCNA-dependent TLS at the molecular level, using biochemical and cellular localization and interaction assays. Aim 2 will functionally test the impact of this mechanism of cellular processes including genomic stability and DNA replication. Aim 3 will employ a mouse genetic model to unambiguously investigate if Parp10 expression induces tumor formation or promotes tumor growth. Using state-of-the-art cellular, molecular and genomic tools (including: CRISPR/Cas9-mediated genome editing; molecular DNA fiber combing to measure fork stability; next generation sequencing approaches to measure mutagenesis and mutation burden) we will investigate here the molecular mechanisms underlying this novel oncogenic function of PARP10. This may eventually result in validation of a new target for cancer therapy.