Dietary Risk for Colon Cancer in the Mouse

描述(申请人提供)：“散发性”结肠癌是指美国和其他发达国家90%的结肠癌。我们已经证明，这可以通过给C57BL/6小鼠喂食一种结合了美国和其他西方国家结肠癌的多种营养风险因素的“新西式饮食”(NWD1)来模拟：1)NWD是根据营养密度制定的，以反映大部分人群消耗的较高脂肪和较低的钙和维生素D3水平；2)对照组C57BL/6小鼠发生肿瘤的时间为其生命的2/3，类似于大多数人类散发性结肠癌发生之前生命的2/3(即50-60岁)；3)肿瘤的发病率和频率与普通人群相同；4)将饮食中的钙和维生素D3提高到与人类结肠癌风险较低相关的水平，可防止肿瘤的发展。我们报道，在C57BL/6小鼠中，在肿瘤形成之前，喂食NWD1 6个月，在大肠和小肠的扁平、组织正常的粘膜中，基因表达有细微但显著的变化，这些变化与突变的APC等位基因遗传引起的变化显著重叠，这种变化可以通过提高钙和维生素D3(NWD2)来防止。因此，微扰与肿瘤形成的可能性密切相关。最后，我们使用了一种新的转录成像方法来证明在Apc1638N/+小鼠中，APC在调节肠道细胞成熟的动态方面是单倍不足的，这一肠道内稳态的扰动也可能是由饮食诱导的更高的风险引起的。我们的假设是，散发性结肠癌的发展是一个随机过程，其概率取决于肠粘膜对营养摄入模式的适应。目的1，确定高风险饮食如何改变Lgr5+肠道干细胞的数量和位置分布，这些细胞分配到不同的谱系，Wnt和Notch信号的作用及其相互作用在黏膜内稳的破坏，以及这些变化是否被钙和维生素D3的升高所阻止。这些实验使用了新的方法学，包括：一种标记了肠道干细胞并允许对其后代进行特定谱系追踪的小鼠遗传品系；一种根据细胞沿隐窝轴的位置进行细胞分离的方法；以及在单细胞水平上原位转录激活特定基因的独特成像。目的2采用了一个更接近于人类散发性结肠癌的模型，即APC基因的条件/诱导突变将被用来确定喂养6个月的饲料如何在动物适应营养因素后的生命周期中引入杂合子或纯合子APC突变，从而使粘膜为癌前病变和肿瘤病变的发展做准备。目的3扩展了我们已发表的基因表达数据和最近生成的代谢数据，表明饮食和遗传风险都会导致正常粘膜细胞早期向糖酵解转变，特别是在祖细胞/增殖细胞室，远在这种转变之前，已知的结肠肿瘤的特征。为了回应先前综述中的评论，我们将抑制2-脱氧-D葡萄糖的糖酵解，并确定这如何调节饮食对干细胞、细胞成熟途径、谱系特异性分配和炎症反应的影响。增加了目标4，这也是为了回应对最初申请的审查中的具体意见。在这一目标中，我们描述了饮食对结肠和小肠炎症和细胞因子产生的影响。此外，基于我们最近关于IL12在调节巨噬细胞和结肠肿瘤上皮细胞之间的重要调节串扰中的作用的报道，以及我们的数据，该细胞因子由NWD1升高，但这可通过提高NWD2中的钙和维生素D来防止，我们将使用一种新的IL12中和单抗来确定这是否消除或调节饮食对粘膜肿瘤相关变化的影响。

DESCRIPTION (provided by applicant): "Sporadic" colon cancer refers to >90% of colon cancer in the US and other developed countries. We have shown that this can be modeled by feeding C57Bl/6 mice a "new western- style diet" (NWD1) that combines a number of nutrient risk factors for colon cancer in the US and other western countries: 1) the NWD is formulated on the basis of nutrient density to reflect the higher fat and lower calcium and vitamin D3 levels consumed by large segments of the population; 2) tumors develop in control C57Bl/6 mice fed the diet for 2/3 of their life-span, similar to the 2/3 of the lifespan (ie 50-60 years) before most human sporadic colon cancer develops; 3) tumors develop at the same incidence and frequency as in the general population; 4) elevating calcium and vitamin D3 in the diet to levels associated with lower risk for colon cancer in the human prevents tumor development. We reported that in C57Bl/6 mice fed the NWD1 for 6 months, well before tumors form, there are subtle but significant alterations in gene expression in the flat, histologically normal mucosa of the large and small intestines that significantly overlap with alterations caused by inheritance of a mutant Apc allele, and that can be prevented by elevating calcium and vitamin D3 (NWD2). Thus, the perturbations track with probability of tumor formation. Finally, we have used a novel method of transcriptional imaging to demonstrate that Apc is haploinsufficient in the Apc1638N/+ mouse for regulating the dynamics of intestinal cell maturation, a perturbation of intestinal homeostasis that may also be caused by dietary induced higher risk. Our hypothesis is that development of sporadic colon cancer is a stochastic process the probability of which is determined by adaptation of the intestinal mucosa to patterns of nutrient intake. Aim 1, determines how the higher risk diet alters the number and positional distribution of Lgr5+ intestinal stem cells, allocation of these cells to different lineages, the role of Wnt and Notch signaling and their interaction in perturbations of mucosal homeostasis, and if these are prevented by elevation of calcium and vitamin D3. These experiments make use of novel methodology, including: a mouse genetic strain in which intestinal stem cells are marked and that also allows lineage specific tracing of their progeny; a method of cell isolation as a function of cell position along the crypt-luminal axis; and unique imaging of transcriptional activation of specific genes at the single cell level in situ. Aim 2 employs a model that more closely recapitulates human sporadic colon cancer in that conditional/inducible mutation of the Apc gene will be used to determine how the diets fed for 6 months prime the mucosa for development of preneoplastic and neoplastic lesions upon introduction of heterozygous or homozygous Apc mutation later in the animal's life-span following adaptation to nutritional factors. Aim 3 extends our published gene expression data and recently generated metabolomic data suggesting that both dietary and genetic risk for intestinal cancer cause an early shift towards glycolysis in cells of the normal appearing mucosa, specifically in the progenitor/proliferating cell compartment, long before such shifts that are known to characterize colon tumors. In response to comments made in the prior review, we will inhibit glycolysis with 2-deoxy-D glucose and determine how this modulates the dietary effects on stem cells, cell maturation pathways, and lineage specific allocation, and on inflammatory responses. Aim 4 has been added, again in response to specific comments in the review of the original application. In this aim we characterize the effects of the diets on inflammation and cytokine production of the colon and small intestine. Moreover, based on our recent reports dissecting the role of IL12 in mediating an important regulatory cross-talk between macrophages and colon tumor epithelial cells, and our data that this cytokine is elevated by the NWD1, but that this is prevented by elevating calcium and vitamin D in NWD2, we will use a novel neutralizing monoclonal Ab to IL12 to determine whether this eliminates or modulates the effects of diet on tumor associated alterations in the mucosa.