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

LCM 分析和小鼠模型验证 miR 在前列腺肿瘤进展中的作用

• 批准号: 8112264
• 负责人: JOY Laurin WARE
• 金额: $ 16.26万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8112264
• 关键词: Address; Affect; Applications Grants; Benign; Biochemical; Bioinformatics; Biological Markers; Biopsy; Biopsy Specimen; Blood; Cell Line; Cells; Clinical; Conflict (Psychology); Coupled; Data; Detection; Development; Diagnostic Neoplasm Staging; Disease; Disease Progression; E2F1 gene; Epithelial; Epithelium; Event; Failure; Formalin; Freezing; Genes; Goals; Heterogeneity; Human; In Vitro; Individual; Malignant neoplasm of prostate; Measures; Messenger RNA; Metastatic to; MicroRNAs; Monitor; Mutation; Neoplasm Metastasis; Neoplasms; Nude Mice; Paper; Paraffin Embedding; Pathologic; Patients; Pattern; Pharmaceutical Preparations; Prostate; Prostate carcinoma; Prostatic Neoplasms; Proteins; RNA; Reporting; Research Personnel; Role; Sampling; Solid Neoplasm; Staging; System; Therapeutic Agents; Tissues; Tumor Cell Line; Tumor Suppressor Proteins; Tumor stage; Tumorigenicity; Validation; Vimentin; Xenograft procedure; advanced disease; anticancer research; base; cancer cell; cell behavior; cell stroma; cell type; combat; effective therapy; in vivo; laser capture microdissection; male; man; men; mouse model; neoplastic cell; novel; novel therapeutics; restoration; subcutaneous; therapeutic target; tumor; tumor growth; tumor progression; tumorigenic

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.

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，如果阻断这些因素，就可能阻止肿瘤的进展。 MiR 是这些新靶分子的新候选者。本次拨款申请的目的是鉴定和验证控制前列腺致瘤性和转移的 miR。此类 miR 是有用的生物标志物，不仅可以识别导致疾病的改变，还可以识别阻止肿瘤进展的靶标或药物。由于 miR 显示组织特异性表达模式并靶向数百种 mRNA，因此 miR 表达的变化可能会改变许多下游事件。 MiR 阵列筛选已对整个前列腺肿瘤进行，但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的，并且包含许多不同的细胞类型和处于疾病进展不同阶段的肿瘤细胞。整个肿瘤筛查使得很难确定哪些 miR 真正调节疾病进展。许多提出的肿瘤抑制 miR 或 oncomiR 的功能尚未通过实验验证。为了解决这个问题，我们使用福尔马林固定石蜡包埋 (FFPE) 的激光捕获显微切割 (LCM) 或人类前列腺肿瘤的冷冻活检切片来确定哪些 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发挥作用，并靶向蛋白质波形蛋白。目标 1 将定义 miR17-3p 对原位致瘤性和转移的影响，并阐明其他 miR17-3p 靶点。通过结合生物信息学和生化方法，我们获得了 miR17-3p 靶向 IGF-1R 的初步数据。目标 2 将鉴定并通过实验验证其他控制肿瘤进展的肿瘤抑制 miR 和 oncomiR。 FFPE 或冷冻活检样本的 LCM 分析将确定在间质、良性腺上皮、PNI 和高级别 PIN 中差异表达的 miR，作为肿瘤形成的前奏。对不同病理阶段 (T2-T3/T4) 和格里森模式 (G3-G5) 的肿瘤细胞的分析将确定随着肿瘤变得更具侵袭性，哪些 miR 会差异表达。这些推定的肿瘤抑制 miR 或 oncomiR 的作用将通过稳定改变适当前列腺细胞系中的 miR 表达，然后监测随后对雄性无胸腺裸鼠体外细胞行为和体内肿瘤生长的影响来验证。这些努力将鉴定出促进致瘤性的oncomiRs和抑制致瘤性的抑癌miRs，从而影响前列腺癌细胞的行为。除了扩大我们对特定 miR 在前列腺癌进展中的作用的理解之外，这项研究还具有很大的潜力来识别有助于转移性疾病的生物标志物，并提供对抗前列腺癌的新治疗药物。 公共健康相关性：尽管前列腺癌是男性最常见的实体瘤类型，但促进肿瘤进展的基因改变仍然不明确，而且一旦致瘤细胞离开前列腺范围，就没有真正的治疗方法，因为相关靶分子尚不清楚。本研究的目的是通过对人类前列腺肿瘤的 FFPE 或冷冻活检样本进行 LCM 分析，然后进行功能验证，确定控制前列腺致瘤性和转移的 miR。此类 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止肿瘤进展的目标。