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

项目总结： 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(TNBCs)中发现。有必要 增加对tnbc中mtp53信号的理解，以便确定检测策略 并治疗这种疾病。非裔美国女性中的TNBC比白人女性更常见 妇女，因此拟议的工作将使所有参加TNBC的妇女受益，包括AA 人口比例过高。这项申请建议确定生物角色 与PARP1相关的TNBC相关的功能增益癌基因mtP53 R273H (这里简称PARP)蛋白、MCM2-7解旋酶和复制DNA。P53 Arg 273 替换为His等热点P53错义突变蛋白，是众所周知的 在TNBCs中作为致癌蛋白发挥作用，但它们的功能在作用之外是异常的 转录辅助因子还不是很清楚。R273Hmtp53在高水平存在 在许多TNBCs中的染色质上，并作为辅助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还与 用PARP和MCMS复制DNA。我们的研究揭示了这种新奇的复制- 我们称之为mtP53-PARP-MCM染色质轴。我们的初选和 已发表的数据报道，mtp53 R273H的敲除减少了染色质相关的MCM2-7 和PARP。在这种情况下敲除mtp53会降低结合的PARP抑制物(PARPI) 他唑巴联合DNA损伤剂替莫唑胺介导的细胞死亡，证明 Mtp53表达影响癌细胞对协同PARPI和DNA的敏感性 损伤介导的细胞杀伤。我们的初步和已发表的数据显示核mtp53 R273H与PARP和MCMS共同作用，mtp53 R273H与EDU直接作用于 新生的复制叉子。此外，我们检测到高mtP53 R273H，高PARP蛋白，以及 表达R273Hmtp53的患者来源的异种移植组织中高PARP酶活性。我们 在一例初发肿瘤的TNBC中也检测到mtp53和PARP的高相关性 基因芯片筛选及肿瘤基因组图谱反相分析 蛋白质数据集。因此，我们认为了解染色质相关的生物学 线粒体P53-PARP-MCM复合体关于复制DNA的研究领域将具有 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轴是一个创新的 蛋白质共同作用的途径和机制关系 与复制相关的活动将帮助我们更好地了解这种疾病。我们的假设 在复制应激期间，稳定的mtp53通过氨基与复制的DNA相互作用 来自其羧基末端区域的酸。因此，mtp53-PARP-MCM轴是 允许增加蛋白质复合体的招募和对PARP的敏感性 抑制剂与DNA损伤剂相结合。我们将通过以下几点来检验这一假设 三个目标。 目的1：阐明与TNBC相关的致癌基因mtp53的作用和机制。 PARP-MCM轴。 目的2：提高我们对mtp53和PARP共表达的认识 异种移植小鼠模型的成像平台及多民族人的筛选 乳房组织微阵列使所有患有TNBC的妇女受益。 目的3：验证mtp53-PARP-MCM轴对PARPI结果的驱动作用 在原位小鼠模型中。 完成这项工作将提供关于mtp53如何与PARP和PARP交互对话的机械见解 MCM蛋白在细胞培养和异种移植模型中对染色质的影响，并将提示潜在的 利用新型显像剂检测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.