Epigenetic changes in a PhIP-induced prostate cancer model in hCYP1A mice

描述(申请人提供)：前列腺癌是发达国家最常见的恶性肿瘤之一，也是美国男性癌症相关死亡的主要原因。前列腺癌涉及许多危险因素，特别是年龄、种族和饮食。暴露在PhIP中，这是一种高温烹调肉类和鱼类产生的饮食致癌物质，与前列腺癌风险有关。在这项研究中，我们将研究PhIP诱导的前列腺癌模型在细胞色素PYP1a人源化(HCYP1a)中的致癌作用。本研究的目的是确定参与DNA甲基化的主要甲基转移酶DNMT1的诱导机制，并确定DNMT1上调水平所导致的基因启动子DNA高甲基化。根据以前的研究和初步数据，假设DNMT1是由RAS/MAPK/AP-1和/或PI3K/Akt信号通路诱导的，并且DNMT1的上调导致人源化小鼠前列腺中关键的调控和抑癌基因的高甲基化和失活。为了验证这些假说，我们提出了三个具体的目标：(1)阐明在PhIP治疗后1、3、7和14天在hCYP1A小鼠中诱导DNMT1的机制；(2)利用甲基化PCR阵列鉴定关键调控和肿瘤抑制基因的启动子DNA超甲基化导致DNMT1水平升高；以及(3)研究DNMT1在PhIP诱导致癌的早期(1-3天)和晚期(30-40周)的上调之间的关系。拟议的实验将使用标准的组织学(H&E)、免疫组织化学(IHC)和分子技术(Western blotting和定量RT-PCR)以及更专门的方法(激光捕获显微解剖、甲基化特异性PCR和DNA甲基化PCR阵列)。这项研究将揭示DNMT1的诱导机制以及PhIP致癌物引起的表观遗传学变化。确定PhIP处理的hCYP1A小鼠的分子机制和变化将加深我们对前列腺癌发生和癌症发展的理解。

DESCRIPTION (provided by applicant): Prostate cancer is one of the most frequently diagnosed malignancies in developed countries and a leading cause of cancer-related death among men in the United States. Prostate cancer involves numerous risk factors, particularly age, race, and diet. Exposure to PhIP, a dietary carcinogen generated from high temperature cooking of meat and fish, has been associated with prostate cancer risk. In this study, we will investigate carcinogenesis of a PhIP-induced prostate cancer model in CYP1A-humanized (hCYP1A). The objectives are to determine the induction mechanism of DNMT1, a major methyltransferase enzyme involved in DNA methylation, and identify the promoter DNA hypermethylation of genes caused by the upregulated level of DNMT1. Based on previous studies and preliminary data, the hypotheses are that DNMT1 is induced by the Ras/MAPK/AP-1 and/or PI3K/Akt signaling pathway and that the upregulation of DNMT1 causes hypermethylation and inactivation of key regulatory and tumor suppressor genes in the prostate of the humanized mice. To test these hypotheses, three specific aims are proposed: (1) elucidate the mechanism of DNMT1 induction in hCYP1A mice at 1, 3, 7, and 14 days after PhIP treatment; (2) identify promoter DNA hypermethylation of key regulatory and tumor suppressor genes caused the elevated level of DNMT1 using methylation PCR array; and (3) investigate the relationship between DNMT1 upregulation in the early time- points (1 & 3 days) and the late stages (30 & 40 weeks) of PhIP-induced carcinogenesis. The proposed experiments will employ standard histological (H&E), immunohistochemical (IHC), and molecular techniques (Western Blotting & quantitative RT-PCR) as well as more specialized methods (Laser Capture Micro- dissection, Methylation Specific PCR, & DNA methylation PCR array). The study will reveal the induction mechanism of DNMT1 and the epigenetic changes caused by the PhIP carcinogen. Determining the molecular mechanism and alterations in the PhIP-treated hCYP1A mice will enhance our understanding of prostate carcinogenesis and cancer development.