LCM Analysis and Mouse Models to Validate miRs in Prostate Tumor Progression

描述（由申请人提供）：前列腺癌在人类中很常见，但新的有效治疗选择有限。当代癌症研究的挑战是阐明新的因素，如果被阻断，可能会阻止肿瘤的进展。miR是这些新靶分子的新候选者。这项资助申请的目的是鉴定和验证控制前列腺致瘤性和转移的miR。这种miR不仅是鉴定导致疾病的改变的有用生物标志物，而且是阻断肿瘤进展的靶点或药物。由于miR显示组织特异性表达模式并靶向数百种mRNA，因此miR表达的变化可以改变许多下游事件。MiR阵列筛选已经在整个前列腺肿瘤上完成，这产生了相互矛盾的结果。部分原因是没有考虑到前列腺肿瘤是异质性的，在疾病进展的不同阶段包含许多不同的细胞类型和肿瘤细胞。整个肿瘤筛查使得难以确定哪些miR真正调节疾病进展。许多提出的肿瘤抑制miR或oncomiRs的功能尚未通过实验验证。为了解决这个问题，我们正在使用福尔马林固定石蜡包埋（FFPE）或冷冻活检切片的人前列腺肿瘤的激光捕获显微切割（LCM），以确定哪些miR在实际肿瘤细胞中差异表达，并可能导致疾病进展。通过这种方法，我们已经证实了miR-17- 3 p在体外和体内作为前列腺肿瘤抑制因子发挥作用，并靶向蛋白质波形蛋白。目的1将定义miR 17 - 3 p对原位肿瘤发生和转移的影响，并阐明其他miR 17 - 3 p靶点。使用生物信息学和生物化学方法的组合，我们有初步的数据，IGF-1 R是由miR 17 - 3 p靶向。目的2将鉴定和实验验证控制肿瘤进展的其他肿瘤抑制miR和oncomiRs。FFPE或冷冻活检样本的LCM分析将确定在基质、良性腺上皮、PNI和高级别PIN中差异表达的miR作为瘤形成的前奏。不同病理阶段（T2-T3/T4）和Gleason模式（G3-G5）的肿瘤细胞的分析将确定哪些miR随着肿瘤变得更具侵袭性而差异表达。通过稳定改变适当前列腺细胞系中的miR表达，随后监测对雄性无胸腺裸鼠体外细胞行为和体内肿瘤生长的后续影响，将验证这些推定的肿瘤抑制剂miR或oncomiR的作用。这些努力将鉴定促进致瘤性的oncomiR和抑制致瘤性的肿瘤抑制miR，从而影响前列腺癌细胞行为。除了扩大我们对特定miR在前列腺癌进展中的作用的理解外，这项研究还具有很高的潜力来确定有助于转移性疾病的生物标志物，并提供新的治疗药物来对抗前列腺癌。 公共卫生相关性： 虽然前列腺癌是男性中最常见的实体瘤类型，但促进肿瘤进展的遗传改变仍然不明确，并且一旦致瘤细胞离开前列腺的范围，就不存在真实的治疗，因为相关的靶分子是未知的。本研究的目的是通过LCM分析人前列腺肿瘤的FFPE或冷冻活检样品，然后进行功能验证，鉴定控制前列腺致瘤性和转移的miR。这种miR不仅可以识别导致疾病进展的早期改变，而且可以成为开发新药以阻断肿瘤进展的靶点。

DESCRIPTION (provided by applicant): Prostate cancer is common in man yet new and effective treatment options are limited. The challenge of contemporary cancer research is elucidate new factors, which if blocked, could block tumor progression. MiRs are novel candidates for these new target molecules. The purpose of this grant application is to identify and validate miRs that control prostate tumorigenicity and metastasis. Such miRs are useful biomarkers not only to identify alterations leading to disease, but targets or drugs to block tumor progression. Since miRs display tissue-specific expression patterns and target hundreds of mRNAs, changes in miR expression could alter numerous downstream events. MiR array screens have been done on whole prostate tumors, which have generated conflicting results. In part this is due to the failure to consider that prostate tumors are heterogeneous and contain many different cell-types and tumor cells at various stages of disease progression. Whole tumor screens make it difficult to determine which miRs are truly regulating disease progression. The function of many proposed tumor suppressor miRs or oncomiRs has NOT been verified by experimentation. To address this issue we are using Laser Capture Microdissection (LCM) of Formalin-Fixed Paraffin Embedded (FFPE) or frozen biopsy sections of human prostate tumors to determine which miRs are differentially expressed in actual tumor cells and could be causative to disease progression. By this approach we have verified that miR-17-3p functions in vitro and in vivo as a prostate tumor suppressor and targets the protein, vimentin. Aim 1 will define the impact of miR17-3p to orthoptopic tumorigenicty and metastasis, and elucidate additional miR17-3p targets. Using a combination of bioinformatics and biochemical approaches we have preliminary data that IGF-1R is targeted by miR17-3p. Aim 2 will identify and experimentally validate other tumor suppressor miRs and oncomiRs that control tumor progression. LCM analysis of FFPE or frozen biopsy samples will define miRs that are differentially expressed in stroma, benign glandular epithelium, PNI, and high-grade PIN as a prelude to neoplasia. Analysis of tumor cells of different pathologic stages (T2-T3/T4) and Gleason patterns (G3-G5) will determine which miRs are differentially expressed as tumors become more aggressive. The role of these putative tumor suppressor miRs or oncomiRs will be validated by stably altering miR expression in appropriate prostate cell lines followed by monitoring the subsequent effect on cell behavior in vitro and tumor growth in vivo in male, athymic nude mice. These efforts will identify oncomiRs, which promote tumorigenicity, and tumor suppressor miRs, which suppress tumorigenicity, thereby affecting prostate cancer cell behavior. In addition to expanding our understanding of the role of specific miRs in prostate cancer progression, this study has high potential to identify biomarkers that contribute to metastatic disease and offer new therapeutic agents to combat prostatic carcinoma. PUBLIC HEALTH RELEVANCE: Although prostate cancer is the most common type of solid tumor in men, genetic alterations that promote tumor progression remain ill defined, and no real treatment exists once the tumorigenic cell leaves the confines of the prostate, since relevant target molecules are unknown. The purpose of this study is to identify by LCM analysis of FFPE or frozen biopsy samples of human prostate tumors followed by functional validation, miRs that control prostate tumorigenicity and metastasis. Such miRs not only identify early alterations contributing to disease progression, but become targets for the development of new drugs to block tumor progression.