Effects of Arsenic on Human Prostate Stem Cells and Prostate Cancer Risk

描述（由申请人提供）：拟议工作的总体目标是确定发育或成人暴露于无机砷（iAs）是否会通过重新编程人类前列腺干细胞增加前列腺癌（PCa）风险。先前的研究表明，来自自然来源（如饮用水）的环境iAs暴露会增加PCa的发病率。最近的研究结果还表明，iAs直接改变干细胞并增强转化，并且对PCa细胞系的研究支持这可能发生在前列腺中。进一步界定 iAs在正常人前列腺干细胞中的作用，确定人暴露的敏感性窗口，并将iAs干细胞暴露与PCa发育联系起来，已经使用来自无病人前列腺的原代上皮细胞和人胚胎干细胞（hESC）建立了新的人前列腺干细胞模型。重要的是，已经建立了使用hESC或成人前列腺干细胞与大鼠诱导间充质混合的体内模型，以产生具有正常人前列腺上皮的嵌合前列腺组织。这些原始方法可以直接解决有关iAs暴露和干细胞重编程事件的几个未解决的问题，并可能将iAs与人类PCa生长联系起来。为实现这一目标，提出了三个具体目标。具体目标1：描述iAs在调节正常人前列腺和胚胎干细胞自我更新和分化为上皮细胞谱系中的作用。为了实现这一点，将利用成人前列腺干细胞的FACS和前列腺球测定以及hESC的体外定向分化来研究一系列iAs剂量在干扰正常前列腺发育和生长中的作用。具体目标二：通过鉴定DNA甲基化修饰和基因表达的变化，阐明前列腺干细胞重编程的分子基础。与特定目标1平行，将进行前列腺球DNA甲基化组、DNA羟甲基化组和转录组的分析，以鉴定干细胞中的全基因组标记以及所得的As基因表达特征。具体目标3：使用新型体内嵌合前列腺模型确定As暴露是否引发致癌作用，作为共致癌物或促进人前列腺上皮中的PCa进展。来自正常男性、PCa患者或hESC的成人前列腺干细胞将用于产生在裸鼠中作为肾移植物生长的嵌合组织。在iAs单独暴露或与类固醇作为共致癌物暴露后，将监测正常上皮的致癌作用。将在暴露于iA的PCa干细胞衍生移植物中检查PCa进展。最后，iAs重编程基因的异常表达将在从PCa患者构建的组织微阵列中进行评估，以确定翻译相关性。目前的方法使用新鲜的人体标本与体内模型结合国家的最先进的表观基因组技术是一个显着的进步，目前的方法，无疑将提供重要的新的和有用的信息，有关人类前列腺健康。总之，这些研究将产生关于iAs如何增加PCa风险的新信息，并为iAs暴露评估提供基本框架。 公共卫生相关性：流行病学研究表明，饮用水中的iAs水平与前列腺癌发病率和死亡率之间存在很强的正相关性。拟议研究的总体目标是确定早期生活或成人As暴露是否通过靶向人类前列腺干细胞而增加前列腺癌风险，这反过来又有助于前列腺癌的发作和进展。

DESCRIPTION (provided by applicant): The overall goal of the proposed work is to determine whether developmental or adult exposures to inorganic arsenic (iAs) increase prostate cancer (PCa) risk by reprogramming human prostate stem cells. Prior studies have revealed that environmental iAs exposure from natural sources (e.g. drinking water) increases PCa incidence. Recent findings also indicate that iAs directly alters stem cells and augments transformation and studies with PCa cell lines support that this may occur in the prostate gland. To further delineate iAs action(s) in normal human prostate stem cells, determine windows of sensitivity for human exposures and link iAs stem cell exposures to PCa development, novel human prostate stem cell models have been established using primary epithelial cells from disease-free human prostates and human embryonic stem cells (hESC). Importantly, in vivo models using either hESC or adult prostate stem cells mixed with rat inductive mesenchyme have been produced to generate chimeric prostate tissues with normal human prostate epithelium. These original approaches are in place to directly address several unresolved issues regarding iAs exposures and stem cell reprogramming events and to potentially link iAs to growth of human PCa. To accomplish this three specific aims are proposed. Specific Aim 1: Delineate the effects of iAs in modulating normal human prostate and embryonic stem cell self-renewal and differentiation to epithelial cell lineages. To accomplish this, FACS and prostasphere assay of adult human prostate stem cells and directed differentiation of hESC in vitro will be utilized to interrogate te actions of a range of iAs doses in perturbing normal prostate development and growth. Specific Aim 2: Elucidate the molecular underpinnings of As reprogramming of prostate stem cells by identifying DNA methylation modifications and resultant changes in gene expression. In parallel with Specific Aim 1, analysis of prostasphere DNA methylome, DNA hydroxymethylome and transcriptome will be undertaken to identify genome-wide marks in stem cells with resultant As gene expression signatures. Specific Aim 3: Determine whether As exposure initiates carcinogenesis, acts as a co-carcinogen or promotes PCa progression in human prostate epithelium using novel in vivo chimeric prostate models. Adult prostate stem cells from normal men, PCa patients or hESC will be used to generate chimeric tissues grown as renal grafts in nude mice. Carcinogenesis in normal epithelium will be monitored after iAs exposures alone or with steroids as co-carcinogens. PCa progression will be examined in PCa stem cell-derived grafts exposed to iAs. Finally, aberrant expression of iAs-reprogrammed genes will be evaluated in tissue microarrays constructed from PCa patients for translational relevance. The present approaches using fresh human specimens with in vivo models combined with state-of-the-art epigenomic technologies are a marked advance over current approaches that will undoubtedly provide significant new and useful information pertaining to human prostate health. Together, these studies will generate novel information on how iAs increases PCa risk and provide a rationale framework for iAs exposure assessment. PUBLIC HEALTH RELEVANCE: Epidemiology studies have shown a strong positive association between levels of iAs in drinking water and prostate cancer incidence and mortality. The overall goal of the proposed studies is to determine whether early-life or adult As exposures increase prostate cancer risk by targeting human prostate stem cells which in turn contribute to prostate cancer onset and progression.