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。在所有人类头颈部鳞状细胞癌(SCCHN)中，约75%与烟草和/或酒精有关。近几十年来，人类SCCHN的总存活率(五年内约为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道癌模型。我们将使用这个4-硝基氧化喹啉(4-NQO)致癌模型来测量酒精(乙醇)对口腔癌变发生率和表观遗传学变化的影响。我们将检验我们的假设，即酒精可能通过促进表观遗传变化，如组蛋白修饰或CpG胞嘧啶上更大的DNA甲基化，导致基因沉默，从而导致舌基底层上皮干/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学变化，因为乙醇通过抑制视黄醇产生视黄酸(RA)来降低细胞中维甲酸(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学变化具有重要作用。该应用的具体目的是：(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，并通过免疫组织化学方法评估Cyclin D1；RAR22；p16；SFRP 1，2，4和5；以及Nanog在正常上皮干/祖细胞和肿瘤“癌干/祖细胞”中的表达；(2)评估在4-NQO致癌过程中，口腔组织基底层(如舌头)上皮细胞的表观遗传学变化。我们将通过流式细胞仪从舌基底层提纯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)和芯片技术测量表观遗传学标志物；以及(3)确定抑制特定表观遗传修饰的药物在酒精存在的致癌过程中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，同样主要使用芯片和芯片技术。这些拟议的实验将为酒精影响癌症发生的分子机制提供重要的见解。他们还将测试干细胞/祖细胞的表观遗传变化在SCCHN发展中的重要性，并探索改变表观遗传变化的药物治疗作用的机制。建议的实验将有助于确定干细胞/祖细胞在口腔癌变过程中上皮细胞的作用。我们将获得有价值的信息，了解致癌物、酒精和药物(如ZeBularine和5-aza-2‘-deoxcytidine)如何影响干细胞/祖细胞的表观基因组，这些药物已被证明在其他致癌模型中既能抑制DNA甲基转移酶，又能降低肿瘤发病率。)

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. )