Age and Diet: Major interacting factors that drive sporadic intestinal cancer

描述（由申请人提供）：年龄和饮食是常见散发性结肠癌的两个最明确的危险因素，在发达国家，超过90%的病例与年龄和饮食有关。我们将利用最近由共同研究者Vijg报道的单细胞全基因组测序的重要技术进步，该技术进步可以独特地检测罕见的突变事件，以确定“正常”粘膜中Lgr 5+，Bmi 1+和Transit Amplifying隔室中的每个突变负荷和谱作为小鼠年龄和饮食的函数。此外，我们将确定这些突变的原因和后果。我们利用衰老小鼠和野生型小鼠中散发性结肠和肠道肿瘤的独特营养驱动模型，其重现了散发性结肠癌的随机发展（在暴露于主要人类饮食风险因素2/3的宿主寿命后，25%的小鼠中有1-2个肿瘤）。这种模式已被复制。 在3个不同的实验室进行了艾德检查，包括我们的实验室，并报告了病理学。我们报道了在该模型中结肠和肠肿瘤发生包括炎症、异位和升高的Wnt信号传导、改变的谱系分配和粘膜中糖酵解的转变。新的初步数据显示，肠道隐窝中DNA损伤的积累与炎症反应有关，并且肠道Lgr 5+干细胞的后代在喂食促肿瘤饮食的小鼠中保留在隐窝中的时间更长。这些数据补充了Vijg的报告，即随着年龄的增长，肠粘膜中的突变负荷增加，这是DNA复制的结果。 基本原理是，肠和结肠粘膜中的干细胞是肿瘤起始事件的靶点，应该作为维持肠内稳态的基因组完整性的港湾。我们假设，当这失败时，祖细胞群体中突变的积累增加了散发性结肠和肠道肿瘤发生的概率。因此，我们将1）确定Lgr 5+、Bmi 1+和转运扩增区室中的每一个中的细胞如何在肿瘤发展之前作为年龄和饮食的函数积累突变; 2）通过下一代测序和最先进的生物信息学管道鉴定单细胞中的突变和突变基因座; 3）确定这些群体中突变负荷增加的原因和后果;和4）确定在最终出现的散发性肿瘤中可能选择哪些突变。 所有的老鼠品系都在掌握之中，许多必要的杂交品种也在培育之中。最初提交的评论者热情高涨。针对他们的评论，我们已经：a）澄清了必要的测序和生物信息学资源; B）从阿尔伯特·爱因斯坦癌症中心获得了匹配资金，以允许单细胞的全基因组测序，而不是外显子组测序; c）增加了人员对数据分析的承诺; d）提供了新的初步数据，即高风险饮食导致Lgr 5+干细胞后代从肠隐窝中延迟退出; e）根据新的初步结果增加了新的实验。数据，将炎症反应与DNA损伤的积累和干细胞后代在隐窝中的保留联系起来; f）描述了我们的数据，建立了饮食诱导的肠道微生物组改变作为肿瘤发生风险的潜在贡献者; g）增加了2名长期合作者作为顾问：Sellers，组织病理学专家; Kucherlapati博士是NCI TCGA努力定义人类结肠肿瘤遗传改变的领导者，也是小鼠和人类基因组测序项目的关键贡献者。他将担任TCGA的科学顾问和联络员。

DESCRIPTION (provided by applicant): Age and diet are the two most clearly recognized risk factors for common sporadic colon cancer, responsible for >90% of cases in developed countries. We will make use of an important technical advance for whole genome sequencing of single cells recently reported by co-investigator Vijg that can uniquely detect rare mutational events to define the mutational load and spectrum in each of the Lgr5+, Bmi1+ and Transit Amplifying compartments in the "normal" mucosa as a function of both mouse age and diet. Further, we will determine the cause and consequences of these mutations. We make use of aging mice and a unique nutritionally driven model of sporadic colon and intestinal tumors in wild-type mice that recapitulate the stochastic development of sporadic colon cancer (1-2 tumors in 25% of the mice following exposure to major human dietary risk factors for 2/3 of the host lifespan). This model has been replicat ed in 3 different laboratories, including ours, and pathology reported. We reported that colon and intestinal tumorigenesis in this model encompasses inflammation, ectopic and elevated Wnt signaling, altered lineage allocation and a shift to glycolysis in the mucosa. New preliminary data show accumulation of DNA damage in intestinal crypts linked to the inflammatory response, and that progeny of intestinal Lgr5+ stem cells are retained in the crypt longer in mice fed the tumor promoting diet. These data complement reports from Vijg of increased mutational load in the intestinal mucosa with age as a consequence of DNA replication. The rationale is that stem cells in the intestinal and colonic mucosa, which are targets for tumor initiating events should serve as harbors of genomic integrity that maintain intestinal homeostasis. We hypothesize that when this fails, accumulation of mutations in progenitor cell populations increases the probability of sporadic colon and intestinal tumorigenesis. Therefore, we will 1) determine how cells in each of the Lgr5+, Bmi1+ and transit amplifying compartments accumulate mutations as a function of both age and diet before tumors develop; 2) identify the mutations and mutated loci in single cells by next generation sequencing and a bioinformatic pipeline that are state-of-the-art; 3) determine causes and consequences of increased mutational load in these populations; and 4) determine which mutations are likely selected for in the sporadic tumors that eventually arise. All mouse strains are in hand and many necessary crosses are bred. Reviewers of the initial submission were enthusiastic. In response to their comments, we have: a) clarified the necessary sequencing and bioinformatic resources; b) secured matching funds from the Albert Einstein Cancer Center to permit whole genome, rather than exome, sequencing of single cells; c) increased commitment of personnel for data analysis; d) provided new preliminary data that the high risk diet causes delayed exit of Lgr5+ stem cell progeny from the intestinal crypts; e) added new experiments, based on new prelim. data, to link the inflammatory response to the accumulation of DNA damage and the retention of stem cell progeny in the crypt; f) described our data establishing dietary induced alterations of the intestinal microbiome as a potential contributor to risk for tumorigenesis; g) added 2 long-time collaborators as advisors: Sellers, an expert in histopathology; Kucherlapati, a leader in the NCI TCGA effort to define genetic alterations in human colon tumors and a key contributor to both the mouse and human genome sequencing projects. He will serve as a scientific advisor and liaison with the TCGA.