Molecular mechanism of diet induced carcinogenesis

描述(由申请人提供)： 长期目标是促进我们对甲基供体饮食不足在肝癌发生中基因表达表观遗传调控中的作用的理解。在最后一次授予期间，我们发现编码蛋白酪氨酸磷酸酶O(PTPRO)的基因在大鼠癌前结节和诱发的原发性肝癌模型中被甲基化和沉默。随后的研究证实，PTPRO是一种真正的肿瘤抑制物。在本研究中，我们将探讨其启动子甲基化是否可以作为特定病因的肝癌及其在肝脏中的功能的生物标志物。我们还将以蛋氨酸腺苷转移酶1a(Mat1a)Null(MAT1KO)小鼠和甲基缺乏饮食为模型系统，探索营养基因相互作用在肝癌发生中的表观遗传调控。在人的原发性肝肿瘤中，Mat1a的频繁丢失导致SAM(所有甲基化反应的辅因子)水平降低，并与肝细胞的去分化有关。MAT1KO小鼠饲喂甲基缺乏饲料后，肝脏SAM水平降低，肝脏损伤易感性增加。该提案的具体目的是：1)利用EpiTyper技术分析大量不同病因的人原发性肝癌中跨越启动子和外显子1的PTPRO CpG岛的甲基化情况，并确定PTPRO在肝癌中的甲基化是否与其表达呈负相关。2)确定PTPRO在调节肝癌细胞侵袭性中的潜在作用及其对药物(阿霉素、5-氟尿嘧啶)诱导的细胞凋亡的增敏作用，并通过底物捕捉试验确定PTPRO的肝脏特异性底物(S)，从而揭示肝癌的潜在治疗靶点。3)与野生型小鼠比较，研究了食用CDAA饲料的MAT1KO小鼠的肝脏病理生理及对肝癌的易感性。4)比较MAT1KO小鼠和野生型小鼠在CDAA饮食诱发肝癌的早期阶段肝脏基因组的甲基化情况，确认候选基因的差异甲基化，并确定这些基因的甲基化状态是否与其表达呈负相关。希望本研究能为我们提供新的见解：(A)PTPRO在肝肿瘤发生中的作用；(B)膳食甲基缺乏与肝细胞癌发生早期DNA甲基化和基因表达变化的关系；(C)Mat1a和SAM在膳食不足诱发肝癌中的作用；(D)为肝癌的早期诊断和表观遗传治疗提供新的分子靶点。这项建议也与最近多个研究所宣布的关于饮食、表观遗传事件和癌症预防的计划非常吻合。肝癌是世界上第五大最常见的癌症，是与癌症相关的死亡的第三大原因，每年的数据超过50万，并且在美国的频率和死亡率(特别是男性)正在增加。本研究将探讨膳食甲基缺乏在肝脏肿瘤发生中的作用。具体地说，这项研究的目的是(A)阐明在肝癌早期阶段甲基化和沉默的特定基因的作用，以响应这种饮食不足，以及(B)确定用于肝癌早期诊断和表观遗传治疗的新的分子靶点。这一建议也与最近宣布的多个研究所关于饮食、表观遗传事件和癌症预防的计划相吻合。

DESCRIPTION (provided by applicant): The long term objective is to advance our understanding of the role of dietary deficiency of the methyl donors in epigenetic regulation of gene expression in hepatocarcinogenesis. In the last granting period, we showed that the gene encoding protein tyrosine phosphatase O (PTPRO) is methylated and silenced in preneoplastic nodules and primary hepatomas induced in a rat model. Subsequent study established that PTPRO is a bona fide tumor suppressor. In the present study we will examine whether its promoter methylation can be used as biomarker for liver cancer of specific etiology and its function in liver. We will also explore epigenetic regulation of nutrient gene interaction in hepatocarcinogenesis using methionine adenosytransferase 1a (Mat1a) null (MAT1KO) mice and methyl deficient diet as a model system. Frequent loss of Mat1a in human primary liver tumors results in decreased SAM (cofactor for all methylation reactions) level and correlates with dedifferentiation of hepatocytes. MAT1KO mice exhibit reduced hepatic SAM level and increased susceptibility to hepatic injury upon feeding methyl-deficient diet. The specific aims of the proposal are: 1) to analyze methylation profile of PTPRO CpG island spanning the promoter and exon1 in a large number of human primary HCCs of different etiology using EpiTyper technology, and determine whether methylation of PTPRO in HCCs inversely correlates with its expression. 2) Determine the potential role of PTPRO in regulating invasiveness of HCC cells and their sensitization to drug (doxorubicin, 5-Fluorouracil)-induced apoptosis, and identify liver-specific substrate(s) of PTPRO by substrate-trapping assay that will unravel potential therapeutic targets for HCC. 3) Investigate liver pathophysiology and susceptibility to hepatocarcinogenesis of MAT1KO mice on CDAA diet compared to the wild type mice. 4) Compare methylation profile of the liver genome of MAT1KO mice with the wild type mice at early stages of CDAA diet-induced hepatocarcinogenesis, confirm differential methylation of the candidate genes and determine whether methylation status of these genes correlates inversely with their expression. It is hoped that this study will provide us with novel insights into (a) role of PTPRO in liver tumorigenesis, (b) relationship between dietary methyl deficiency and changes in DNA methylation and expression of genes at early stages of hepatocarcinogenesis, (c) role of Mat1a and SAM in induction of liver cancer by the deficient diet and (d) additional molecular targets for early stage diagnosis and epigenetic therapy of HCC. This proposal also fits well with the recent program announcement of multiple institutes on diet, epigenetic events and cancer prevention.Liver cancer is the fifth most prevalent cancer in the world and is the third leading cause of cancer-related death with annual date rate exceeding 500,000, and is increasing in frequency and mortality (particularly in men) in the United States. The present study will address the role of dietary methyl deficiency in the initiation of liver tumorigenesis. Specifically, this study is aimed at (a) elucidation of the role of specific genes that are methylated and silenced at early stages of liver cancer in response to this dietary deficiency and (b) identification of novel molecular targets for early diagnosis and epigenetic therapy of liver cancer. This proposal also fits well with the recent program announcement of multiple institutes on diet, epigenetic events and cancer prevention.