Diet and Alcohol Induced Epigenetic Changes in Oral Cavity Carcinogenesis Model

描述（由申请人提供）：头颈癌是全球第六大常见癌症。烟草和/或酒精涉及大约75%的头颈部鳞状细胞癌（SCCHN）。人类SCCHN的总生存率（5年内约为50%）在近几十年来没有太大变化，因此迫切需要新的SCCHN治疗方法。我们建立了一种新的致癌物诱导的小鼠口腔和食管癌模型。我们将使用这个4-硝基喹啉氧化物（4-NQO）致癌模型来测量酒精（乙醇）对口腔致癌发生率和表观遗传变化的影响。我们将测试我们的假设，即酒精可能有助于通过促进表观遗传变化，如组蛋白修饰或CpG胞嘧啶更大的DNA甲基化，从而导致基因沉默的舌基底层上皮干/祖细胞的致癌作用。我们的假设的一个推论是，乙醇导致异常的表观遗传变化，因为乙醇通过抑制视黄醇产生视黄酸（RA）降低细胞中RA的水平，我们已经表明，RA是一种信号分子，在干细胞分化过程中启动表观遗传变化中起主要作用。本申请的具体目的是：（1）在我们的小鼠口腔癌发生模型中测量酒精对口腔癌发生率的影响，并通过免疫组织化学评估正常上皮干/祖细胞与来自肿瘤的“癌干/祖细胞”中分子标记物如细胞周期蛋白D1、RAR 22、p16、SFRP 1、2、4和5以及nanog的表达;（2）评估在4-NQO致癌过程中，在随后给予和不给予酒精的情况下，口腔组织（例如舌头）基底层中上皮细胞的表观遗传变化。我们将通过流式细胞术从舌基底层中纯化正常细胞和具有癌症干/祖细胞特性的细胞，并通过染色质免疫沉淀（ChIP）和ChIP-Chip技术测量表观遗传标记;和（3）确定抑制特异性表观遗传修饰的药物是否影响口腔癌的发病率和/或影响基底层干/干细胞的表观遗传学。祖细胞在致癌过程中在酒精的存在下，再次主要使用ChIP和ChIP-Chip方法。这些拟议的实验将提供重要的见解酒精影响致癌的分子机制。他们还将测试干/祖细胞中表观遗传变化在SCCHN发展中的重要性，并探索修饰表观遗传变化的药物治疗作用的机制。本实验将有助于进一步明确干/祖细胞在口腔上皮癌变过程中的作用。我们将获得关于干/祖细胞的表观基因组如何受到致癌物、酒精和药物（如zebularine和5-aza-2 '-deoxycytidine）影响的有价值的信息，这些药物已被证明可以抑制DNA甲基转移酶并降低其他致癌模型中的肿瘤发病率。) 公共卫生相关性：人类口腔癌是世界上最常见的癌症之一，长期生存率很低。拟议的研究将集中在酒精增加口腔癌发病率的机制上。

DESCRIPTION (provided by applicant): Head and neck cancer is the sixth most common cancer worldwide. Tobacco and/or alcohol are involved in approximately 75% of all human squamous cell carcinomas of the head and neck (SCCHN). The overall survival rate for human SCCHN (approximately 50% in five years) has not changed very much in recent decades, so there is an urgent need for new approaches for SCCHN treatment. We have developed a novel carcinogen induced murine oral cavity and esophageal carcinogenesis model. We will use this 4-nitroquinoline oxide (4-NQO) carcinogenesis model to measure the effects of alcohol (ethanol) on the incidence of oral cavity carcinogenesis and on epigenetic changes. We will test our hypothesis that alcohol may contribute to carcinogenesis in epithelial stem/progenitor cells of the basal layer of the tongue by promoting epigenetic changes, such as histone modifications or greater DNA methylation at CpG cytosines, which leads to gene silencing. A corollary of our hypothesis is that ethanol leads to aberrant epigenetic changes because ethanol lowers the levels of retinoic acid (RA) in cells via inhibition of RA production from retinol, and we've shown that RA is a signaling molecule which has a major role in initiating epigenetic changes during stem cell differentiation. The specific aims of the application are: (1) to measure the effects of alcohol on the incidence of oral cavity cancer in our murine oral cavity carcinogenesis model, and to assess the expression of molecular markers such as cyclin D1; RAR22; p16; SFRP 1,2,4, and 5; and nanog in normal epithelial stem/progenitor cells versus "cancer stem/progenitor cells" from the tumors by immunohistochemistry; (2) to assess epigenetic changes in epithelial cells in the basal layer of oral cavity tissues, such as the tongue, during 4-NQO carcinogenesis, with and without subsequent alcohol administration. We will purify both normal cells from the basal layer of the tongue and cells with properties of cancer stem/progenitor cells by FACS and measure epigenetic markers by the chromatin immunoprecipitation (ChIP) and the ChIP-Chip techniques; and (3) to determine if drugs that inhibit specific epigenetic modifications influence the incidence of oral cavity cancer and/ or influence the epigenetics of basal layer stem/progenitor cells during the carcinogenesis process in the presence of alcohol, again primarily using ChIP and ChIP-Chip approaches. These proposed experiments will provide important insights into the molecular mechanisms by which alcohol influences carcinogenesis. They will also test the importance of epigenetic changes in stem/progenitor cells in the development of SCCHNs and explore the mechanisms by which drugs that modify epigenetic changes act therapeutically. The proposed experiments will help to establish the roles of stem/progenitor cells in the epithelium during the oral cavity carcinogenesis process. We will gain valuable information about how the epigenome of stem/progenitor cells is influenced by carcinogens, alcohol, and drugs, such as zebularine and 5-aza-2'-deoxycytidine, which have been shown both to inhibit DNA methyltransferases and to reduce tumor incidence in other carcinogenesis models. ) PUBLIC HEALTH RELEVANCE: Human oral cavity cancer is one of the most common cancers in the world, with a poor long-term survival rate. The proposed research will focus on the mechanisms by which alcohol increases the incidence of oral cavity cancer.