The Role of the Mutant p53-PARP-MCM Pathway in Triple Negative Breast Cancer

项目摘要： 突变型p53-PARP-MCM通路在三阴性乳腺癌中的作用 突变型p53（mtp53）在80%的三阴性乳腺癌（TNBC）中发现。有必要 增加对TNBC中mtp53信号传导的理解，以确定检测策略 并治疗疾病。TNBC在非洲裔美国妇女中比在白色妇女中更常见 因此，拟议的工作将使包括AA在内的所有TNBC妇女受益 人口过剩。本申请提出确定以下的生物学作用： 与PARP1相关的TNBC相关的功能获得性致癌mtp53 R273 H （本文称为PARP）蛋白、MCM2 - 7解旋酶和复制DNA。p53 Arg 273 和其他热点p53错义突变体蛋白，是众所周知的， 在TNBC中作为致癌蛋白起作用，但它们的功能除了作为异常的致癌蛋白外， 转录辅因子还不清楚。R273H mtp53以高水平存在 在许多TNBC的染色质上，并作为辅因子激活固醇的转录 生物合成基因和染色质重塑因子，但令人难以置信的是，R273H也共同关联 与PARP和MCMs在DNA复制上的关系我们的研究发现了这种新的复制- 我们称之为mtp53-PARP-MCM染色质轴。我们的初步和 已发表的数据报道，敲低mtp53 R273 H可降低染色质相关的MCM2 - 7 和PARP。在这种情况下，mtp53的敲低降低了组合PARP抑制剂（PARPi） talazoparib加DNA损伤剂替莫唑胺介导的细胞死亡，表明 mtp53表达影响癌细胞对协同PARPi加DNA的敏感性 损伤介导的细胞杀伤。我们的初步和公开数据显示，核mtp53 R273 H与PARP和MCMs共缔合，mtp53 R273 H在100 ℃时与EdU直接缔合。 新生复制叉。此外，我们检测到高mtp53 R273 H，高PARP蛋白， 在表达R273H mtp53的患者来源的异种移植物组织中的高PARP酶活性。我们 还在初步肿瘤中的TNBC中检测到mtp53和PARP的高度共关联 微阵列筛选以及通过分析癌症基因组图谱反相 蛋白质数据集。因此，我们考虑了解染色质相关的生物学 mtp53-PARP-MCM复合物对DNA复制的影响将是一个研究领域， 对我们对TNBC的理解、检测和治疗选择产生持续的长期影响 在不同的女性群体中。TNBC中的mtp53-PARP-MCM轴是一种创新的 途径和阐明的机制关系的蛋白质一起工作， 复制相关的活动将有助于我们更好地了解这种疾病。我们的假设 稳定的mtp53在复制应激期间使用氨基与复制DNA相互作用， 酸从其羧基末端区域。因此，mtp53-PARP-MCM轴是 参与允许增加蛋白质复合物的募集和对PARP的敏感性 抑制剂与DNA损伤剂组合。我们将通过以下方式检验这一假设 三个目标。 目的1：阐明TNBC相关致癌基因mtp53- PARP-MCM轴。 目的2：增加我们对mtp53和PARP共表达的理解， 异种移植小鼠模型中的成像平台和多种族人类的筛选 乳腺组织微阵列，使所有TNBC女性受益。 目的3：确定影响PARPi结局的mtp53-PARP-MCM轴的驱动作用 在原位小鼠模型中。 完成这项工作将提供关于mtp53如何与PARP交叉会谈的机制见解， MCM蛋白在细胞培养和异种移植模型中染色质上的作用，并将告知 使用新型显像剂检测mtp53和PARP的核共表达作为信息 TNBC的生物标志物。

Project Summary: The Role of the Mutant p53-PARP-MCM Pathway in Triple Negative Breast Cancer Mutant p53 (mtp53) is found in 80% of triple negative breast cancers (TNBCs). There is a need to increase understanding of mtp53 signaling in TNBC in order to determine strategies to detect and treat the disease. TNBCs are more common in African American women than in white women and therefore the proposed work will benefit all women with TNBC including the AA over-represented population. This application proposes to determine the biological role of TNBC-associated gain-of-function oncogenic mtp53 R273H that is associated with PARP1 (herein referred to as PARP) protein, MCM2-7 helicases and replicating DNA. The p53 Arg 273 substituted with His, and other hotspot p53 missense mutant proteins, are well known to function as oncogenic proteins in TNBCs, but their functions outside of acting as aberrant transcription co-factors are not clearly understood. The R273H mtp53 is present in high levels on chromatin in many TNBCs and works as a co-factor to activate the transcription of sterol biosynthesis genes and chromatin remodeling factors, but incredibly R273H also co-associates with PARP and MCMs on replicating DNA. Our studies have uncovered this novel replication- associated pathway and we call it the mtp53-PARP-MCM chromatin axis. Our preliminary and published data reports that knockdown of mtp53 R273H reduces chromatin associated MCM2-7 and PARP. Knockdown of mtp53 in this setting decreases combined PARP inhibitor (PARPi) talazoparib plus DNA damaging agent temozolomide-mediated cell death, demonstrating that mtp53 expression influences the sensitivity of cancer cells to synergistic PARPi plus DNA damage mediated cell killing. Our preliminary and published data showed nuclear mtp53 R273H co-associates with PARP and MCMs and mtp53 R273H directly associates with EdU at nascent replication forks. Furthermore, we detected high mtp53 R273H, high PARP protein, and high PARP enzymatic activity in patient derived xenograft tissue expressing R273H mtp53. We also detected high co-association of mtp53 and PARP in TNBC in a preliminary tumor microarray screen as well as through analysis of The Cancer Genome Atlas reverse phase protein data sets. As a result, we consider understanding the biology of chromatin-associated mtp53-PARP-MCM complexes on replicating DNA to be an area of inquiry that will have a sustained long-term influence on our understanding, detection, and treatment choices for TNBC in diverse populations of women. The mtp53-PARP-MCM axis in TNBC is an innovative pathway and elucidating the mechanistic relationship of the proteins working together in replication-associated activities will help us to better understand the disease. Our hypothesis is that stable mtp53 interacts with replicating DNA during replication stress using amino acids from its carboxy-terminal regions. As a consequence, the mtp53-PARP-MCM axis is engaged allowing increased recruitment of protein complexes and sensitivity to PARP inhibitors combined with DNA damaging agents. We will test this hypothesis by the following three aims. Aim 1: Elucidate the role and mechanism of the TNBC-associated oncogenic mtp53- PARP-MCM axis. Aim 2: Increase our understanding of co-expression of mtp53 and PARP using novel imaging platforms in xenograft mouse models and screening of multiethnic human breast tissue microarrays to benefit all women with TNBC. Aim 3: Validate the driver role of the mtp53-PARP-MCM axis influencing PARPi outcomes in orthotopic mouse models. Completing this work will provide mechanistic insights on how mtp53 cross-talks with PARP and MCM proteins on chromatin in cell culture and xenograft models, and will inform the potential of using novel imaging agents to detect nuclear co-expression of mtp53 and PARP as informative biomarkers for TNBC.