Role of the DNA Damage Response in Prostate Cancer Initiation

本项目研究NKX3.1在保护前列腺上皮细胞免受DNA损伤中的作用，这可能为其在肿瘤抑制中的功能提供关键机制，NKX3.I是位于8 p21的前列腺特异性同源框基因，8 p21是人类前列腺癌中最常见的染色体丢失的靶点。NKX3.1也是一种肿瘤抑制因子，其降低的蛋白质表达足以使前列腺上皮细胞生长失调并引起上皮内瘤形成;此外，Nkx3.1单倍不足导致小鼠前列腺上皮发育不良。与其在人前列腺癌中作为肿瘤抑制因子的作用一致，我们已经表明NKX3.I同源结构域中的失活突变与前列腺癌家族中的早发性前列腺癌共分离。在最近的研究中，我们已经表明NKX3.1增强DNA损伤后的细胞存活，并影响DNA断裂识别的最早事件，包括YH 2AX灶的形成和ATM的磷酸化。 基于我们的假设，即NKX3.1缺失导致肿瘤发生的关键机制是通过其对DNA损伤反应的影响，我们的研究将确定NKX3.I在体外DNA损伤反应中的机制作用，以及其对体内DNA损伤的影响。我们还将确定NKX3.I是否介导前列腺细胞形成特征性TMPRSS 2- ERG染色体重排的易感性，该重排发生在约50%的前列腺癌中。综上所述，我们的三个目标将描绘NKXS.I在不同背景下的DNA损伤反应中的作用，从而提供对肿瘤抑制的这一关键机制的全面调查。在目标1中，我们的实验将定义NKX3.1和ATM之间功能性相互作用的动力学，这种相互作用发生在DNA损伤的几分钟内。在目的2中，我们将确定Nkx3.I基因拷贝数是否影响前列腺上皮，特别是前列腺上皮干细胞中的体内DNA损伤反应。最后，在目标3中，使用我们已经衍生的LNCaP细胞的独特系，我们将确定NKX3.1是否影响TMPRSS 2-ERG基因重排的频率。这些拟议的研究将与整个计划项目高度整合。值得注意的是，Aim 2中的实验包括与Core A合作的定量免疫染色分析，Aim 2中关于前列腺干细胞的工作将与Michael Shen（项目1）合作进行，整个项目与Cory Abate-Shen（项目2）的细胞衰老中Nkx3.1的分子分析有关。

This project investigates the role of NKX3.1 in protection against DNA damage in the prostate epithelium, which may provide a key mechanism for its function in tumor suppression, NKX3.I is a prostate specific homeobox gene located on 8p21, the target for the most frequent chromosomal loss in human prostate cancer. NKX3.I is also a tumor suppressor for which reduced protein expression is sufficient to deregulate prostate epithelial cell growth and cause Intraepithelial neoplasia; moreover, Nkx3.1 haploinsufficiency results in prostate epithelial dysplasia in mice. Consistent with its role as a tumor suppressor in human prostate cancer, we have shown that an Inactivating mutation In the NKX3.I homeodomain cosegregates with early onset prostate cancer in a prostate cancer family. In recent studies, we have shown that NKX3.1 enhances cell survival after DNA damage and affects the earliest events in recognition of DNA breaks, including formation of YH2AX foci and phosphorylation of ATM. Based on our hypothesis that a key mechanism by which NKX3.1 loss contributes to tumor initiation is through its impact on the DNA damage response, our studies will define the mechanistic role of NKX3.I in the DNA damage response in vitro, as well as its impact on DNA damage in vivo. We also will determine whether NKX3.I mediates the susceptibility of prostate cells to formation of the characteristic TMPRSS2- ERG chromosomal rearrangement that occurs in approximately 50% of prostate cancers. Taken together, our three aims will delineate the role of NKXS.I in the DNA damage response in distinct contexts, thereby providing a comprehensive investigation of this key mechanism of tumor suppression. In Aim 1, our experiments will define the dynamics of the functional interaction between NKX3.1 and ATM, which occurs within minutes of DNA damage. In Aim 2, we will determine whether Nkx3.I gene copy number affects the DNA damage response in vivo in the prostate epithelium, and particularly in prostate epithelial stem cells. Finally, in Aim 3, using unique lines of LNCaP cells that we have derived, we will determine whether NKX3.1 affects the frequency of TMPRSS2-ERG gene rearrangements. These proposed studies will be highly integrated with the overall program project. Notably, the experiments in Aim 2 include quantitative immunostaining analyses in collaboration with Core A, the work in Aim 2 on the prostate stem cells will be performed in collaboration with Michael Shen (Project 1), and the entirety of this project is linked to the molecular analyses of Nkx3.1 in cellular senescence by Cory Abate-Shen (Project 2).