Myopodin in Invasive Prostate Cancers

描述（由申请人提供）：前列腺癌是一种具有相当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普遍存在，但只有一小部分前列腺癌具有侵袭性、转移性并危及生命。因此，确定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该疾病死亡率的关键。最近，我们利用“差分减法链”（differential subtraction chain），发现了一个在侵袭型前列腺癌中经常被删除的新基因myopodin。无论Gleason分级如何，myopodin的部分或完全缺失与前列腺癌的高侵袭、转移或临床复发率相关。序列分析表明，myopodin与synaptopodin具有显著的同源性，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间接触。有趣的是，超过90%的myopodin部分缺失位于与synaptopodin同源的区域。myopodin在PC-3或LNCaP细胞中过表达，在矩阵穿越分析中使这些细胞系的侵袭性降低3倍或更多，在体内肿瘤生长中降低10倍。myopodin的过表达诱导肌动蛋白捆绑并延缓PC-3的迁移。酵母双杂交和共免疫沉淀分析表明，myopodin与酶合蛋白结合，酶合蛋白是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myopodin在调节细胞运动，维持前列腺上皮正常细胞-细胞接触的完整性，抑制前列腺癌细胞的侵袭方面发挥重要作用，并提出以下具体目的：1)在PC-3、Du145和LNCaP细胞中过表达myopodin，并通过锚定独立生长、基质横贯、横贯膜侵袭、转移和金属蛋白酶检测检测这些细胞的侵袭性变化；2)通过构建缺失突变体阵列，确定myopodin调控侵袭所必需的基序和最小域，并测试其调控侵袭的能力；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/zyxin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.