Diet and Alcohol Induced Epigenetic Changes in Oral Cavity Carcinogenesis Model

描述（申请人提供）：头颈癌是全球第六大常见癌症。烟草和/或酒精与大约75%的人类头颈部鳞状细胞癌（SCCHN）有关。近几十年来，人类SCCHN的总生存率（5年内约为50%）没有太大变化，因此迫切需要新的治疗方法。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。我们将验证我们的假设，即酒精可能通过促进表观遗传变化，如组蛋白修饰或CpG胞嘧啶DNA甲基化，从而导致基因沉默，从而促进舌基底层上皮干细胞/祖细胞的癌变。我们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RA来降低细胞中视黄酸（RA）的水平，我们已经证明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。该申请的具体目的是：(1)在我们的小鼠口腔癌变模型中测量酒精对口腔癌发病率的影响，并评估细胞周期蛋白D1等分子标志物的表达；RAR22;p16;SFRP 1、2、4、5；免疫组化检测正常上皮干细胞/祖细胞与肿瘤“癌干细胞/祖细胞”的nanog差异；(2)评估4-NQO致癌过程中口腔组织（如舌）基底层上皮细胞的表观遗传变化，无论是否随后给予酒精。我们将利用FACS技术纯化舌基底层的正常细胞和具有癌干/祖细胞特性的细胞，并利用染色质免疫沉淀（ChIP）和ChIP- ChIP技术测量表观遗传标记；(3)确定在酒精存在的癌变过程中，抑制特定表观遗传修饰的药物是否会影响口腔癌的发病率和/或影响基底层干/祖细胞的表观遗传学，同样主要使用ChIP和ChIP- ChIP方法。这些拟议的实验将为酒精影响癌变的分子机制提供重要的见解。他们还将测试干细胞/祖细胞表观遗传变化在scchn发育中的重要性，并探索修饰表观遗传变化的药物治疗机制。本实验将有助于确定干细胞/祖细胞在口腔癌变过程中在上皮中的作用。我们将获得关于干细胞/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'-脱氧胞苷）影响的有价值的信息，这些药物已被证明可以抑制DNA甲基转移酶并在其他致癌模型中降低肿瘤发生率。

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.