Modeling E2F3 isoform specificity in prostate cancer

描述(由申请人提供)： 前列腺癌是老年VA患者癌症发病率和死亡率的主要原因。雄激素消融治疗最初是成功的，但由于耐去势前列腺癌(CRPC)的发展而复发。目前CRPC的治疗选择有限，这表明需要确定新的治疗靶点。研究表明，E2F3转录因子在前列腺癌中过度表达，并且过度表达与不良的临床预后相关。E2F3基因座编码E2F3a和E2F3b两种异构体，但由于技术原因，以前的研究只检测了E2F3a的表达。我们实验室的初步数据表明，前列腺癌来源的细胞株比未转化的前列腺细胞表达更高水平的E2F3a和E2F3b。比较人前列腺肿瘤和正常前列腺组织中E2F3a和E2F3b的表达，发现部分肿瘤组织中E2F3b和E2F3b的表达升高，提示E2F3b在肿瘤发生中具有独特的作用。此外，我们还发现E2F3b调节microRNA加工核酸酶Disher的表达。以前的研究报告说，在晚期和转移性前列腺癌中，DICER的表达升高。我们的研究将E2F/Rb信号通路与miRNA机制联系起来。我们假设E2F3b通过促进增殖、改变粘附性和运动性来促进前列腺癌的发生。拟议的研究有三个具体目标。在特定的目标1中，我们将利用前列腺癌来源的细胞培养系来确定E2F3b在增殖、细胞黏附和运动中的作用。此外，还将研究E2F3b在内源Dever基因调控中的作用，最后，E2F3异构体在调节miRNA簇中的作用将被研究。在《特定目标2》中提出的研究将集中在转基因小鼠模型上。我们将研究三种前列腺癌模型中E2F3亚型和DICER的表达：1)SV40T抗原，2)高水平的c-myc，3)Nkx3.1和Pten肿瘤抑制因子半合子。这些模型模拟了在人类肿瘤中发现的途径改变。针对特定目标3的研究将利用免疫组织化学、免疫印迹和显微解剖肿瘤组织的定量RT-PCR分析来表征E2F3亚型在人类肿瘤样本、周围组织和BPH对照样本中的表达。补充性研究将确定DICER在肿瘤和非肿瘤组织中的表达特征。最后，E2F3亚型和DICER的表达将与疾病进展和治疗反应相关。这些研究将确定E2F3b或E2F3b靶点是否为阻止前列腺癌进展的潜在治疗靶点。 公共卫生相关性： 前列腺癌是退伍军人管理局医疗保健中诊断出的最常见的癌症，几乎没有治疗选择，目前很难预测哪种治疗方法将有效。最近的研究表明，接触橙剂的退伍军人前列腺癌的发病率高出48%，诊断年龄更早，并加速了疾病的进程。建议的研究将分析E2F3a和b蛋白，我们的研究表明这两种蛋白在肿瘤发展中非常重要。

DESCRIPTION (provided by applicant): Prostate cancer is a major cause of cancer morbidity and mortality of older VA patients. Androgen ablation therapy is initially successful, but relapses occur due to the development of castration resistant prostate cancer (CRPC). Currently treatment options for CRPC are limited, indicating a need for the identification of new therapeutic targets. Studies indicate that the E2F3 transcription factor is overexpressed in prostate cancers, and overexpression correlates with a poor clinical outcome. The E2F3 locus encodes two isoforms, E2F3a and E2F3b, but, due to technical reasons, previously reported studies only examined E2F3a expression. Preliminary data from our laboratory indicates that prostate tumor derived cell lines express higher levels of E2F3a and E2F3b than non-transformed prostate cells. A comparison of human prostate tumors and control benign prostate hypertrophy samples indicates some tumors have elevated E2F3a and b. siRNA mediated decrease of E2F3b in tumor derived cells has a more severe effect on cell physiology than a decrease of E2F3a, indicating that E2F3b has a unique role in tumorigenesis. Additionally we found that E2F3b regulates the expression of the microRNA processing nuclease Dicer. Previous studies reported that Dicer expression is elevated in advanced and metastatic prostate tumors. Our studies link the E2F/RB signaling pathway to the miRNA machinery. We hypothesize that E2F3b promotes prostate tumorigenesis by increasing proliferation, and altering adhesion and motility. The proposed studies have three specific aims. In Specific Aim 1 we will define the role of E2F3b in proliferation, cell adhesion and motility using prostate tumor-derived cell culture lines. Additionally, the role of E2F3b in the regulation of the endogenous Dicer gene will be characterized, and lastly, the role of E2F3 isoforms in regulating miRNA clusters will be examined. Studies proposed in Specific Aim 2 will focus on genetically engineered mouse models. The expression of the E2F3 isoforms and of Dicer will be studied in three models of prostate cancer: 1) SV40 T-Antigen, 2) high levels of c-myc, and 3) Nkx3.1 and Pten tumor suppressor hemizygosity. These models mimic pathway alteration identified in human tumors. The studies in Specific Aim 3 will characterize E2F3 isoform expression in human tumor samples, in surrounding tissue and BPH control samples using immunohistochemical, Western immunoblot, and quantitative RT-PCR analysis of micro-dissected tumor tissue. Complementary studies will characterize the expression of Dicer in tumor and non-tumor tissue. Lastly, the expression of the E2F3 isoforms and Dicer will be correlated with disease progression and response to therapy. The studies will determine if E2F3b or E2F3b targets are potential therapeutic targets to halt prostate tumor progression. PUBLIC HEALTH RELEVANCE: Prostate cancer, the most common cancer diagnosed in the VA healthcare has few treatment options and currently it is difficult to predict which treatments will be effective. Recent studies indicate that veterans that were exposed to Agent Orange have 48% higher rates of prostate cancer, an earlier age of diagnosis and accelerated the disease process. The studies proposed will analyze the E2F3a and b proteins, which our studies indicate are important in tumor development.