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

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， 占发达国家病例的90%以上。我们将利用一项重要的技术进步 最近由共同研究者Vijg报道的单细胞全基因组测序， 检测罕见突变事件，以确定Lgr 5+、Bmi 1+和 作为小鼠年龄和饮食两者的函数的“正常”粘膜中的转运扩增区室。此外，本发明还 我们将确定这些突变的原因和后果。我们利用衰老的老鼠和 一种独特的营养驱动的野生型小鼠散发性结肠和肠道肿瘤模型， 散发性结肠癌的随机发展（在暴露于 主要的人类饮食风险因素的2/3的主机寿命）。该模型已在3个不同的 实验室，包括我们的，和病理报告。我们报道了结肠和肠道肿瘤的发生， 该模型包括炎症、异位和升高的Wnt信号传导、改变的谱系分配和 转移到粘膜中的糖酵解。新的初步数据显示，肠道中DNA损伤的积累 与炎症反应有关的隐窝，以及肠道Lgr 5+干细胞的后代保留在 喂食促肿瘤饮食的小鼠的隐窝更长。这些数据补充了来自Vijg的报告， 随着年龄的增长，作为DNA复制的结果，肠粘膜中的突变负荷。 基本原理是，肠和结肠粘膜中的干细胞是肿瘤启动的靶点， 事件，应该作为港口的基因组的完整性，维持肠道内稳态。我们假设 当这失败时，祖细胞群体中突变的积累增加了 散发性结肠和肠肿瘤发生。因此，我们将1）确定每个LGR 5+中的细胞如何， Bmi 1+和转运放大区室积累突变作为年龄和饮食的函数， 肿瘤发展; 2）通过下一代测序鉴定单细胞中的突变和突变基因座， 最先进的生物信息学管道; 3）确定增加的原因和后果 这些群体中的突变负荷;以及4）确定哪些突变可能在这些群体中被选择。 最终出现的散发性肿瘤。 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。初次提交的审查人 很热情针对他们的意见，我们：a）澄清了必要的顺序， 生物信息资源; B）从阿尔伯特·爱因斯坦癌症中心获得配套资金， 单细胞的基因组测序，而不是外显子组测序; c）增加人员对数据的承诺 分析; d）提供了新的初步数据，即高风险饮食导致Lgr 5+干细胞延迟退出 来自肠隐窝的后代; e）基于新的初步结果增加了新的实验。数据，将 炎症反应的积累DNA损伤和保留干细胞后代在 crypt; 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， 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，NCI TCGA努力定义遗传改变的领导者， 人类结肠肿瘤和小鼠和人类基因组测序项目的关键贡献者。他 将担任科学顾问和TCGA的联络人。

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 recapitulates 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 replicated 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.