The Role of Myopodin in Invasive Prostate Cancers

描述(申请人提供)：前列腺癌是一种具有相当大的生物学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具有侵袭性、转移性和生命危险。因此，识别前列腺癌从一种相对缓慢的疾病转变为一种侵袭性疾病的基因和潜在机制是降低这种疾病死亡率的关键。最近，我们利用“差减链”技术，发现了侵袭型前列腺癌中常见缺失的新基因“myopodin”。无论Gleason分级，Myopodin的部分或完全缺失都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，myopodin与synaptopodin有显著的同源性，synaptopodin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myopodin部分缺失位于与synaptopodin同源的区域。在PC-3或LNCaP细胞中过表达myopodin使这些细胞系的侵袭力在Matrigel横向分析中降低了3倍或更多，在体内肿瘤生长中降低了10倍。Myopodin过表达可诱导PC-3细胞肌动蛋白捆绑并延缓迁移。酵母双杂交和免疫共沉淀分析表明，myopodin与Zysin结合，Zysin是肌动蛋白细胞骨架重组、细胞运动和有丝分裂的关键调控蛋白。在我们的建议中，我们假设myopodin在调节细胞运动、维持正常的前列腺上皮细胞-细胞接触的完整性以及抑制前列腺癌细胞的侵袭性方面发挥重要作用，并提出了以下具体目标：1)在PC-3、Du145和LNCaP细胞中过表达myopodin，并随后通过锚定非依赖性生长分析、Matrigel横向分析、横跨分析、膜片侵袭分析、转移分析和金属蛋白酶检测来检测这些细胞侵袭性的变化；2)通过构建缺失突变体阵列来确定myopodin在侵袭调控中所必需的基序和最小结构域，并测试它们调节侵袭力的能力；3)通过对myopodin转化细胞的细胞运动和细胞骨架重组相关分析，确定myopodin/Zysin相互作用的功能意义；4)通过对myopodin表达细胞基因表达的综合分析，寻找可能介导myopodin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.