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

描述（由申请人提供）：前列腺癌在男性中很常见，但新的有效的治疗方案有限。当代癌症研究的挑战是阐明新的因素，这些因素如果被阻断，可以阻止肿瘤的进展。mir是这些新靶标分子的新候选物。本拨款申请的目的是鉴定和验证控制前列腺肿瘤发生性和转移的mir。这类miRs是有用的生物标记物，不仅可以识别导致疾病的改变，还可以作为阻止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， miR表达的变化可能改变许多下游事件。MiR阵列已经对整个前列腺肿瘤进行了筛查，但产生了相互矛盾的结果。在某种程度上，这是由于没有考虑到前列腺肿瘤是异质性的，并且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。整个肿瘤的筛查使得很难确定哪些mir真正调节疾病进展。许多被提出的肿瘤抑制miRs或oncomir的功能尚未得到实验验证。为了解决这一问题，我们正在使用激光捕获显微解剖（LCM）对福尔马林固定石蜡包埋（FFPE）或冷冻前列腺肿瘤活检切片，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，我们已经验证了miR-17-3p在体外和体内作为前列腺肿瘤抑制因子并靶向蛋白vimentin。目的1将明确miR17-3p对原位肿瘤发生和转移的影响，并阐明其他miR17-3p靶点。结合生物信息学和生物化学的方法，我们有初步的数据表明IGF-1R是miR17-3p的靶标。目的2将鉴定和实验验证控制肿瘤进展的其他肿瘤抑制miRs和oncomir。对FFPE或冷冻活检样本进行LCM分析，可以确定在间质、良性腺上皮、PNI和高级别PIN中差异表达的mir，作为肿瘤形成的前兆。对不同病理分期（T2-T3/T4）和Gleason模式（G3-G5）的肿瘤细胞进行分析，将确定哪些mir在肿瘤变得更具侵袭性时表达差异。通过稳定地改变适当前列腺细胞系中的miR表达，然后监测其对体外细胞行为和体内雄性胸腺裸鼠肿瘤生长的后续影响，将验证这些假定的肿瘤抑制miRs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miRs，从而影响前列腺癌细胞的行为。除了扩大我们对特异性miRs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，可以识别导致转移性疾病的生物标志物，并为对抗前列腺癌提供新的治疗药物。

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.