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通过重新编程人类前列腺干细胞来确定发育或成人暴露于无机砷(IAS)是否会增加前列腺癌(PCA)的风险。先前的研究表明，来自自然来源(如饮用水)的环境IAS暴露会增加前列腺癌的发病率。最近的发现还表明，IAS直接改变干细胞并增强转化，对PCa细胞系的研究支持这可能发生在前列腺癌中。为了进一步描绘 通过对正常人前列腺干细胞的IAS作用(S)，确定人类暴露的敏感窗口，并将IAS干细胞暴露与前列腺癌的发育联系起来，使用来自无疾病的人前列腺原代上皮细胞和人类胚胎干细胞(HESC)建立了新型的人前列腺干细胞模型。重要的是，使用hESC或成年前列腺干细胞与大鼠诱导间充质混合的体内模型已经被制造出来，以产生与正常人类前列腺上皮嵌合的前列腺组织。这些原始的方法是为了直接解决有关IAS暴露和干细胞重编程事件的几个悬而未决的问题，并潜在地将IAS与人类PCa的生长联系起来。为了实现这一目标，本文提出了三个具体目标。具体目标1：阐明IAS在调节正常人类前列腺和胚胎干细胞自我更新和向上皮细胞分化中的作用。为了实现这一目标，我们将利用流式细胞仪、成人前列腺干细胞的PROSTASHER分析和hESC的体外定向分化来研究不同剂量的IAS在干扰正常的前列腺发育和生长方面的作用。具体目标2：通过识别DNA甲基化修饰和由此导致的基因表达变化，阐明前列腺干细胞AS重新编程的分子基础。在具体目标1的同时，将进行前期DNA甲基组、DNA羟甲基组和转录组的分析，以确定干细胞中的全基因组标记，并将其作为基因表达特征。具体目标3：使用新的体内嵌合前列腺模型，确定AS暴露是否启动了人前列腺上皮的癌变，作为辅助致癌物或促进了前列腺癌的进展。来自正常人、前列腺癌患者或hESC的成年前列腺干细胞将被用来产生嵌合组织，作为裸鼠的肾移植。在IAS单独暴露或与类固醇共同致癌后，将监测正常上皮的癌变情况。PCa进展将在暴露于IAS的PCa干细胞来源的移植物中进行检测。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翻译相关性评估。目前的方法使用新鲜的人体标本与活体模型结合最先进的表观基因组学技术是相对于现有方法的显著进步，这无疑将提供与人类前列腺健康有关的重要的新的和有用的信息。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暴露评估提供一个基本框架。 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关系。拟议研究的总体目标是确定早期或成年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，而后者反过来又有助于前列腺癌的发生和发展。

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.