The Role of Myopodin in Invasive Prostate Cancers

描述（由申请人提供）：前列腺癌是一种具有相当大的生物异质性和临床侵袭性的疾病。虽然前列腺癌在老年人群中很常见，但只有一小部分前列腺癌会变成侵袭性、转移性和危及生命的。因此，识别将前列腺癌从相对惰性疾病转变为侵袭性疾病的基因和潜在机制是降低疾病死亡率的关键。最近，我们利用“差减链”技术发现了一个新的基因，命名为“肌足蛋白”，它在侵袭型前列腺癌中频繁缺失。无论Gleason分级如何，肌足蛋白的部分或完全缺失与前列腺癌的高侵袭率、转移率或临床复发率相关。序列分析表明，肌足蛋白与突触足蛋白具有显著的同源性，突触足蛋白是一种在神经元和肾足细胞中表达的蛋白质，负责形成这些细胞的生理细胞-细胞接触。有趣的是，超过90%的肌足蛋白部分缺失位于与突触足蛋白同源的区域。在PC-3或LNCaP细胞中过表达肌足蛋白使这些细胞系的侵袭性在基质胶遍历分析中降低3倍或更多，并且在体内肿瘤生长中降低10倍。肌足蛋白的过度表达诱导PC-3中肌动蛋白成束并阻碍迁移。酵母双杂交和免疫共沉淀分析表明，肌足蛋白结合zyxin，肌动蛋白细胞骨架重组，细胞运动和有丝分裂的关键调节蛋白。在我们的提案中，我们假设肌足蛋白在调节细胞运动性、维持前列腺上皮正常细胞-细胞接触的完整性以及抑制前列腺癌细胞的侵袭性方面起重要作用，并提出以下具体目标：1）在PC-3中过表达肌足蛋白，Du 145和LNCaP细胞，并随后通过贴壁非依赖性生长分析、基质胶横贯分析、膈肌侵袭分析、转移测定和金属蛋白酶测定; 2）通过构建缺失突变体阵列来确定对于调节侵袭必需的肌足蛋白的基序和最小结构域，并测试它们调节侵袭的能力; 3）通过关注肌足蛋白转化细胞的细胞运动性和细胞骨架重组相关分析来鉴定肌足蛋白/zyxin相互作用的功能意义; 4）通过对肌足蛋白表达细胞基因表达的综合分析，鉴定可能介导肌足蛋白生物学功能的潜在效应基因。

DESCRIPTION (provided by applicant): Prostate cancer is a disease with considerable biological heterogeneity and clinical aggressiveness. Although prostate cancer is prevalent among elderly population, only a small proportion of prostate cancer become invasive, metastatic and life-threatening. Thus, identifying genes and the underlying mechanisms that convert prostate cancer from a relatively indolent disease to an aggressive one hold the key for reducing the mortality of the disease. Recently, by using "differential subtraction chain", we identified a novel gene named "myopodin" that is frequently deleted in aggressive type of prostate cancer. Regardless of Gleason grade, deletions of myopodin, either partial or complete, correlate with high rate of invasion, metastasis or clinical relapse of prostate cancer. Sequence analysis indicates that myopodin shares significant homology with synaptopodin, a protein expressed in neurons and kidney podocytes and responsible for forming physiological cell-cell contact for these cells. Interestingly, over 90% of the partial deletions of myopodin are located in the region sharing homology with synaptopodin. Overexpression of myopodin in PC-3 or LNCaP cells reduced the invasiveness of these cell lines by 3 fold or more in matrigel traverse analysis, and by 10 fold in tumor growth in vivo. Overexpression of myopodin induces actin bundling and retards migration in PC-3. Yeast two-hybrid and co-immunoprecipitation analyses indicate that myopodin binds zyxin, a critical regulatory protein in actin cytoskeleton reorganization, cell motility and mitosis. In our proposal, we hypothesize that myopodin plays an important role in regulating cell motility, maintaining the integrity of normal cell-cell contacts of prostate epithelium, and inhibits the invasiveness of prostate cancer cells, and propose the following specific aims: 1) to overexpress myopodin in PC-3, Du145 and LNCaP cells and subsequently to test the changes of invasiveness of these cells through anchorage independent growth assay, matrigel traverse analysis, diaphragm invasion analysis, metastasis assay and metalloproteinase assays; 2) to define the motifs and minimal domain of myopodin essential for regulation of invasion by constructing arrays of deletion mutants and test their ability to regulate invasiveness; 3) to identify the functional significance of myopodin/zyxin interaction by focusing on cell motility and cytoskeleton re-organization related analysis on myopodin transformed cells; and 4) To identify potential effector genes that might mediate the biological function of myopodin through a comprehensive analysis of gene expression of myopodin expressing cells.