Overexpression of ubiquitin E3 ligase WWP1 as an oncogenic factor in the prostate

描述（由申请人提供）：前一个资助期的目标是绘制、克隆和表征前列腺癌13 q21的肿瘤抑制基因。在包括7篇已发表和2篇已提交的9篇论文中，我们将转录因子KLF 5确定为13 q21基因的强有力候选者，并证明了癌细胞中KLF 5的三种机制，包括基因组缺失、转录下调和过度蛋白降解。此外，我们发现KLF 5是TGF β信号通路中不可或缺的组成部分;并且泛素化和降解KLF 5的泛素E3连接酶WWP 1通常在前列腺癌中通过8 q21处的拷贝数增加而过表达，这是癌细胞中KLF 5过度降解的原因。此外，已经显示VWVP 1通过诱导Smad 2、Smad 4和I型TGF β受体的降解来负调节TGF β信号传导途径。因此，我们假设上皮细胞中E3连接酶WWP 1的过度表达导致KLF 5和TGF β信号通路的其他组分的过度蛋白质降解，从而使细胞对TGF β在细胞增殖中的抑制作用产生抗性。因此，细胞变得更容易受到其他因素诱导的致癌作用。我们将在三个具体目标中进一步检验和验证这一假设。1)为了评估人前列腺癌中WWP 1和相关分子的分子改变，将检查临床癌症标本的拷贝数增加、过表达和WWP 1突变以及KLF 5和TGF β途径其他组分的表达变化。这些改变将与前列腺癌的临床病理特征相关。2)为了检查在KLF 5和TGF β的情况下WWP 1在细胞生长中的功能，将检查表达不同水平WWP 1的细胞的生长、细胞周期进展、肿瘤发生和基因表达变化。3)为了使用遗传修饰的小鼠测试WWP 1过表达在致癌作用中的作用，WWP 1将在小鼠的前列腺中特异性过表达。这些小鼠将与NKX3.1和PTEN敲除小鼠杂交。将在这些小鼠中分析表型和分子变化。这些研究的完成可能会表明WWP 1是一种可用于开发生物标志物和治疗靶点以及了解前列腺癌生物学的分子。

DESCRIPTION (provided by applicant): The goals of the previous funding period were to map, clone, and characterize a tumor suppressor gene at 13q21 in prostate cancer. In 9 papers including 7 published and 2 submitted, we identified the transcription factor KLF5 as a strong candidate for the 13q21 gene, and demonstrated three mechanisms that inactivate KLF5 in cancer cells, including genomic deletion, transcriptional downregulation, and excessive protein degradation. In addition, we found that KLF5 is an indispensable component of the TGFbeta signaling pathway; and that a ubiquitin E3 ligase that ubiquitinates and degrades KLF5, WWP1, is often overexpressed via copy number gain at 8q21 in prostate cancer, which is responsible for excessive degradation of KLF5 in cancer cells. Furthermore, it has been shown that VWVP1 negatively regulates the TGFbeta signaling pathway by inducing the degradation of Smad2, Smad4, and TGFbeta receptor type I. We therefore hypothesize that overexpression of the E3 ligase WWP1 in epithelial cells causes excessive protein degradation of KLF5 and other components of the TGFbeta signaling pathway, and thus makes cells resistant to the inhibitory effect of TGFbeta in cell proliferation. As a result, cells become more susceptible to other factor-induced carcinogenesis. We will further test and validate this hypothesis in three specific aims. 1) To assess molecular alterations of WWP1 and related molecules in human prostate cancer, clinical cancer specimens will be examined for copy number gain, overexpression, and mutations of WWP1 and for expression change in KLF5 and other components of the TGFbeta pathway. The alterations will be correlated with clinicopathological features of prostate cancer. 2) To examine the function of WWP1 in cell growth in the context of KLF5 and TGFbeta, cells expressing different levels of WWP1 will be examined for growth, cell cycle progression, tumorigenesis, and gene expression changes. 3) To test the role of WWP1 overexpression in carcinogenesis using genetically modified mice, WWP1 will be specifically overexpressed in the prostates of mice. Such mice will be crossed with NKX3.1 and PTEN knockout mice. Phenotypic and molecular alterations will be analyzed in these mice. Completion of these studies will likely show WWP1 to be a molecule useful for developing biomarkers and therapeutic targets as well as for understanding prostate cancer biology.