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

描述(申请人提供)：前一个资助阶段的目标是定位、克隆和鉴定前列腺癌中13q21的肿瘤抑制基因。在7篇已发表和2篇提交的9篇论文中，我们确定了转录因子KLF5是13q21基因的有力候选者，并证明了三种使KLF5在癌细胞中失活的机制，包括基因组缺失、转录下调和蛋白质过度降解。此外，我们发现KLF5是TGFbeta信号通路中不可缺少的组成部分；泛素化和降解KLF5的泛素E3连接酶WWP1在前列腺癌中经常通过8q21的拷贝数增加而过度表达，这是导致KLF5在癌细胞中过度降解的原因。此外，已有研究表明，VWVP1通过诱导Smad2、Smad4和TGFbeta I型受体的降解来负向调节TGFbeta信号通路。因此，我们假设E3连接酶WWP1在上皮细胞中的过度表达导致KLF5和TGFbeta信号通路的其他成分的过度蛋白质降解，从而使细胞抵抗TGFbeta在细胞增殖中的抑制作用。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上进一步检验和验证这一假设。1)为了评估WWP1及相关分子在前列腺癌中的分子变化，我们将检测临床肿瘤标本中WWP1的拷贝数增加、过度表达和突变，以及KLF5和TGFbeta通路的其他组成部分的表达变化。这些改变将与前列腺癌的临床病理特征相关联。2)为了在KLF5和TGFbeta的背景下研究WWP1在细胞生长中的作用，我们将检测表达不同水平WWP1的细胞的生长、细胞周期进展、肿瘤形成和基因表达的变化。3)为了利用转基因小鼠测试WWP1过表达在致癌中的作用，将在小鼠的前列腺中特异性地过表达WWP1。这些小鼠将与NKX3.1和PTEN基因敲除小鼠杂交。将分析这些小鼠的表型和分子变化。这些研究的完成可能会显示WWP1是一个对开发生物标记物和治疗靶点以及了解前列腺癌生物学有用的分子。

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.