Rad9-Based Mouse Model of Prostate Carcinogenesis

描述(由申请人提供)： 当细胞暴露在破坏DNA的物理或化学试剂中时，有害影响可能随之而来，包括突变、癌症或死亡。然而，修复损伤、稳定基因组和中和有害影响的机制是可用的。我们从酵母、小鼠和人中分离并鉴定了一个进化上保守的基因Rad9，它对于维持基因组稳定和促进细胞对DNA损伤的抵抗至关重要。基于这些以及其他发现，我们研究了Rad9在癌症发生中的作用，并发现了与前列腺癌特别重要的联系。我们的结果包括观察到Rad9在四个常用的前列腺癌细胞系CWR22、DU145、LNCaP和PC-3中显著过表达，相对于正常的人前列腺癌前病变细胞和永生化的非致瘤细胞系PWRE-1。我们还发现，siRNA降低Rad9蛋白水平的程度与注射到裸鼠体内后这些细胞的致瘤性降低的程度有关。此外，非致癌永生化的PWRE-1细胞可以在裸鼠体内形成异常生长，如果这些细胞被设计成过度生产Rad9的话。此外，我们在339例前列腺癌中有153例(45.1%)检测到高水平的Rad9蛋白，但在52例正常前列腺组织中只有2例检测到低丰度。相对较高的Rad9蛋白水平通常与更晚期的疾病相关。因此，我们的结果表明，Rad9在前列腺癌的发生中起着重要的作用。这个项目的重点是假设RAD9蛋白的异常高丰度会导致前列腺癌。主要的实验目标是建立和表征一种新的前列腺癌小鼠模型，该模型基于RAD9在该器官中的异常高表达。为此，我们有初步数据表明，已经构建了这样一条老鼠线。这项研究的具体目的集中在：1)检测前列腺中高水平的RAD9的亲代和转基因小鼠的前列腺组织中自发和辐射诱导的前列腺组织异常的形成和肿瘤的形成与年龄的关系；2)确定高水平的RAD9与自发和电离辐射诱导的转移性前列腺癌的关系；3)明确高水平的Rad9介导前列腺癌的能力与其反式作用功能下游靶基因的表达以及与前列腺癌密切相关的其他基因的表达之间的关系，以开始了解Rad9介导前列腺癌发生的机制基础。最终，该项目将提供一个独特的模型来确定前列腺癌发生的分子机制，并作为一个体内系统来阐明环境致癌的风险。与公共卫生相关：这项提案关注的是RAD9在前列腺癌发生中的作用。其目标是设计并鉴定一种独特的小鼠模型，该模型基于前列腺癌中高水平的Rad9蛋白，展示了这种癌症的遗传易感性。将评估前列腺疾病的自发性和辐射诱导的发展。因此，这个项目很重要，因为结果可能会影响人类健康，无论是在前列腺癌的治疗方面，还是在了解遗传和环境在致癌方面的相互作用方面。

DESCRIPTION (provided by applicant): When cells are exposed to physical or chemical agents that damage DNA, deleterious effects can ensue, including mutation, cancer or death. However, mechanisms are available to repair the damage, stabilize the genome and neutralize harmful effects. We isolated and characterized an evolutionarily conserved gene called Rad9, from yeast, mouse and human, which is critical for maintaining genomic stability and promoting cellular resistance to DNA damage. Based on these as well as other findings, we examined the role of Rad9 in carcinogenesis, and found a significant link in particular to prostate cancer. Our results include the observation that Rad9 is dramatically overexpressed in four commonly used prostate cancer cell lines, CWR22, DU145, LNCaP and PC-3, relative to normal human prostate PrEC cells and the immortalized but nontumorigenic cell line PWRE-1. We also found that the degree to which siRNA reduces Rad9 protein levels correlates with the extent of decrease in tumorigenicity of these cells after injection into nude mice. Moreover, nontumorigenic immortalized PWRE-1 cells can form aberrant growths in nude mice if the cells are engineered to overproduce Rad9. In addition, we detected high Rad9 protein levels in 153 of 339 (45.1%) human prostate adenocarcinomas, but just low abundance in 2 of 52 normal prostate tissue controls. Relatively higher levels of Rad9 protein were in general associated with more advanced disease. Our results thus indicate that Rad9 plays a functional role in prostate carcinogenesis. This project focuses on the hypothesis that aberrantly high abundance of Rad9 protein leads to prostate cancer. The major experimental goal is to create and characterize a novel mouse model of prostate carcinogenesis, based on aberrant high expression of Rad9 specifically in that organ. To this end, we have preliminary data indicating that such a line of mouse has been constructed. Specific aims of the study focus on 1) examining prostate tissue of parental, as well as transgenic mice with high levels of Rad9 in prostate with respect to spontaneous as well as radiation-induced formation of prostate tissue abnormalities and tumorigenesis as a function of age; 2) determine the relationship between high levels of Rad9 and spontaneous as well as ionizing radiation-induced metastatic prostate cancer; and 3) defining the relationship between the ability of high levels of Rad9 to mediate prostate disease and expression of downstream target genes of the transactivity function of Rad9 as well as expression of other genes well-established to be intimately involved in prostate cancer, to begin to understand the mechanistic basis for Rad9 mediated prostate carcinogenesis. In the end, this project will provide a unique model to define molecular mechanisms of prostate carcinogenesis, and serve as an in vivo system for elucidating risk o environmental carcinogenesis. PUBLIC HEALTH RELEVANCE: This proposal focuses on the role of Rad9 in mediating prostate carcinogenesis. The goal is to engineer and characterize a unique mouse model demonstrating genetic predisposition to this kind of cancer, based on high levels of Rad9 protein in prostate. The spontaneous as well as radiation-induced development of prostate disease will be assessed. As such, this project is important since the results could impact on human health, both in terms of treatment for prostate cancer and understanding the interplay of genetics and the environment with respect to carcinogenesis.