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

描述(申请人提供)：前列腺癌在男性中很常见，但新的和有效的治疗选择有限。当代癌症研究的挑战是阐明新的因素，如果这些因素被阻止，可能会阻止肿瘤的发展。MIR是这些新的靶分子的新候选者。这项赠款申请的目的是识别和验证控制前列腺癌发生和转移的MIR。这种MIR不仅是识别导致疾病的改变的有用生物标记物，而且是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，并靶向数百个mRNAs，miR表达的变化可能会改变许多下游事件。对整个前列腺肿瘤进行了MIR阵列筛查，但产生了相互矛盾的结果。部分原因是没有考虑到前列腺癌是异质性的，在疾病发展的不同阶段包含许多不同的细胞类型和肿瘤细胞。完整的肿瘤筛查使确定哪些MIR真正控制疾病进展变得困难。许多已提出的抑癌基因miRs或oncomiRs的功能尚未得到实验验证。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)或冷冻活检切片的激光捕获显微解剖(LCM)来确定哪些miRs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，我们在体外和体内验证了miR-17-3p作为前列腺癌抑制因子的功能，并以波形蛋白为靶点。目标1将定义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化方法，我们有初步数据表明，IGF-1R是miR17-3p的靶标。AIM 2将鉴定和实验验证控制肿瘤进展的其他肿瘤抑制因子miRs和oncomiRs。对FFPE或冰冻活检样本的LCM分析将在间质、良性腺上皮、PNI和高级别PIN中差异表达的miRs定义为肿瘤发生的前奏。对不同病理阶段的肿瘤细胞(T2-T3/T4)和Gleason模式(G3-G5)的分析将确定哪些miR在肿瘤变得更具侵袭性时存在差异表达。这些假定的肿瘤抑制因子miRs或oncomiRs的作用将通过稳定改变适当的前列腺细胞系中miR的表达来验证，然后监测随后对雄性裸鼠体外细胞行为和体内肿瘤生长的影响。这些努力将确定促进致瘤性的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。除了扩大我们对特定miRs在前列腺癌进展中的作用的理解外，这项研究还具有识别与转移性疾病有关的生物标志物的高潜力，并提供新的治疗药物来对抗前列腺癌。 公共卫生相关性：尽管前列腺癌是男性最常见的实体肿瘤类型，但促进肿瘤进展的基因改变仍未得到明确定义，而且一旦肿瘤细胞离开前列腺范围，就没有真正的治疗方法，因为相关的靶分子尚不清楚。本研究的目的是通过对人前列腺肿瘤的FFPE或冰冻活检样本进行LCM分析，然后进行功能验证，确定控制前列腺癌发生和转移的MIR。这种MIR不仅识别导致疾病进展的早期改变，而且成为阻止肿瘤进展的新药开发的靶点。

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.