A Major Nutritional Effect on Intestinal Stem Cells and Tumors

我们已经解剖了西式净化饮食（NWD 1）对肠道和结肠肿瘤发生的影响。 NWD 1概括了小鼠摄入的关键营养素，每种营养素的水平在流行病学上与以下疾病的高风险有关： 西方发达国家的肠癌。我们确定NWD 1可以放大和加速肠道 在许多小鼠遗传模型中，无论遗传病因学和由单一或 多种基因驱动此外，当长期喂养野生型小鼠时，我们和其他人确定NWD 1 导致散发性肠道和结肠肿瘤。在这些散发性肿瘤发生前至少1年， 组织病理学正常粘膜中上皮细胞成熟和潜在分子途径的改变。 这导致了研究肠干细胞的细胞和分子生物学如何被NWD 1改变， 我们报道了令人惊讶的发现，NWD 1损害了肠上皮细胞中Lgr 5 hi细胞的干细胞功能， 体内平衡和肿瘤发生，但这是通过动员Bmi 1+细胞作为干细胞来补偿的， 这些功能。此外，遗传和饮食实验表明，维生素D暴露和Vdr 信号在这些作用中起重要作用，与Clevers报道的干细胞表达特征强烈一致。 表明Vdr信号传导参与Lgr 5 hi干细胞功能。此外，我们注意到， 干细胞已经使用饮食建立维生素D水平远高于美国人口的范围。这 提出了关于干细胞在体内平衡和肿瘤发生中起何种作用以及如何起作用的重要问题。 自上次审查（提交出版）以来，广泛的新数据确立了饮食的深刻影响。 暴露：i）对Lgr 5 hi和Bmi 1+细胞（RNAseq）的编程; ii）对这些细胞中DNA修复的能力; iii）在这些隐窝细胞中积累的突变的后续程度、谱和特征 区室（FACS分离的单细胞的测序）。目的1提出了饮食诱导 Lgr 5 hi干细胞编程、功能和表观遗传改变的变化可能异常长寿， 因此，干细胞可以作为“哨兵细胞”，以监测饮食干预的能力， 这些变化作为潜在的风险标志和干预目标。这将提供基本数据， 关于干细胞功能调节中基因和途径的协调， 相对风险，以及如何优化干预策略。初次提交的目标2主要是 完成，目的2是修改调查的假设，改变DNA修复是重要的， 当条件性Apc时，Lgr 5+和Bmi 1+人群对肿瘤发生的差异饮食效应 突变特异性地靶向这些细胞群体中的每一个。Aim 2还决定了饮食对 Apc突变的性质以及肿瘤中整个基因组的突变。目标3确定影响 饮食对肿瘤组织病理学和编程的肿瘤上皮细胞时，肿瘤是从 Lgr 5+或Bmi 1+干细胞区室。RNAscope和一种新的转录成像方法 确定Lgr 5+和Bmi 1+干细胞对肠道稳态和肿瘤发生的相对贡献， 不同的饮食条件。

We have dissected impact of a western style purified diet (NWD1) on intestinal and colonic tumorigenesis. NWD1 recapitulates mouse intake of key nutrients each at their levels epidemiologically linked to higher risk for intestinal cancer in western developed countries. We established that NWD1 amplifies and accelerates intestinal tumors in many mouse genetic models, regardless of genetic etiology and aggressiveness driven by single or multiple genetic drivers. Moreover, when fed to wild-type mice long-term, we and others established that NWD1 causes sporadic intestinal and colon tumors. At least 1 year before these sporadic tumors develop, there is altered maturation of epithelial cells and underlying molecular pathways in the histopathologically normal mucosa. This led to investigating how the cell and molecular biology of intestinal stem cells are altered by the NWD1, We reported the surprising discovery that NWD1 compromises stem cell functions of Lgr5hi cells in intestinal homeostasis and tumorigenesis, but that this is compensated for by mobilization of Bmi1+ cells as stem cells for these functions. Moreover, genetic and dietary experiments identify decreased vitamin D exposure and Vdr signaling as important in these effects, consistent with Clevers' reported stem cell expression signature strongly implicating Vdr signaling in Lgr5hi stem cell functions. Further, we noted that mouse experiments on intestinal stem cells have used diets establishing vitamin D levels well above the range of that for the US population. This raises significant questions regarding which and how stem cells function in homeostasis and tumorigenesis. Extensive new data since the prior review (submitted for publication) establish profound effects of dietary exposures: i) on programming of Lgr5hi and Bmi1+ cells (RNAseq); ii) on capacity for DNA repair in these cells; iii) on subsequent extent, spectrum and signature of mutations that accumulate in cells of these crypt compartments (sequencing of FACS isolated single cells). Aim 1 addresses the hypothesis that dietary induced changes in Lgr5hi stem cell programming, function, and epigenetic alterations can be exceptionally long lived, and therefore that stem cells can be used as “sentinel cells” to monitor the ability of dietary intervention to modulate these alterations as potential markers of risk and targets for intervention. This will provide fundamental data regarding coordination of genes and pathways in regulation of stem cell function, identification of markers of relative risk, and how intervention strategies can be optimized. Aim2 of the initial submission has been largely accomplished, and Aim 2 is revised to investigate the hypothesis that altered DNA repair is important in differential dietary effects on tumorigenesis from the Lgr5+ and Bmi1+ populations when conditional Apc mutations are targeted specifically to each of these cell populations. Aim2 also determines the impact of diet on the nature of mutations in Apc and throughout the genome in the tumors that arise. Aim 3 determines impact of diet on tumor histopathology and programming of tumor epithelial cells when tumors are initiated from either the Lgr5+ or Bmi1+ stem cell compartments. RNAscope and a novel method of transcriptional imaging then determine relative contribution of Lgr5+ and Bmi1+ stem cells to intestinal homeostasis and tumorigenesis under different dietary conditions.