Roles of EGFR and miR-143/miR-145 in Western diet-promoted colonic tumorigenesis

描述(由申请者提供)：虽然西方饮食与结肠癌风险增加有关，但这些饮食影响的分子基础在很大程度上仍不清楚。偶氮甲烷(AOM)和APC+/min小鼠模型模拟了人类结肠癌的许多特征，包括西方饮食促进肿瘤的作用。我们发现西方饮食上调了表皮生长因子受体(EGFR)的配体。此外，EGFR对于肿瘤的促进也是必需的。几种EGFR配体被脂筏相关的金属蛋白酶ADAM17从膜结合的前配体中释放出来。我们最近的研究表明，ADAM17被microRNA-145(miR-145)下调，而EGFR效应因子K-ras则被miR-143抑制。这些共转录的miRNAs在人类结肠癌中下调。我们最近发现，在AOM和APC+/min肿瘤中，EGFR信号下调miR-143和miR-145。此外，这些miRNA的减少对于EGFR有丝分裂的作用是必要的。根据我们的数据，我们假设ADAM17的上调以及miR-143和miR-145的下调在西方饮食诱导的肿瘤促进中发挥了重要作用。我们提出了几个目标来解决这一假设：目标1：阐明饮食促进的结肠肿瘤发生中对ADAM17的需求。我们假设ADAM17的抑制或缺失将抑制饮食相关的肿瘤促进作用。我们将使用1a)条件性ADAM17缺失小鼠的AOM模型；1b)含有新型ADAM17药物抑制剂INCB3619的APC+/MIN模型来剖析ADAM17在饮食促进的肿瘤形成中的作用；1c)脂筏的体外研究以剖析脂肪酸对结肠癌细胞中ADAM17的影响。目的：探讨miR-143和miR-145在饮食促进的结肠肿瘤发生中的作用。我们假设这些miRNAs的丢失对于饮食诱导的肿瘤促进是必要的。我们将使用APC+/MIN小鼠与2a)表达Villin启动子调控的前miR-143和前miR-145的转基因小鼠，或与2b)miR-143缺失小鼠或2c)miR-145缺失小鼠杂交，以揭示这些miRNAs在饮食促进的肿瘤发生中的作用。在目标2d)中，我们将研究与ADAM17调控和/或饮食相关肿瘤发生有关的其他miRNAs，包括miR-1、-31、-148和-152。目的3：探讨西方饮食对miR-143和miR-145基因表达的调节及其与肿瘤发生的关系。我们假设西方饮食和恶性转化抑制转录，而肿瘤性转化也会扰乱加工。我们将3a)在活体模型中评估ADAM17、饮食和肿瘤阶段对miR-143、-145的原始、前期和成熟水平的影响；3b)通过突变缺失分析EGFR和脂肪酸对miR-143/-145启动子活性的影响，以确定顺式调控元件；3c)确定在小鼠加工能力强的YAMC和加工非加工能力的CT26结肠癌细胞中与生物素化miR-143或miR-145存在差异的蛋白质，以发现失调控的加工因子。我们的建议将阐明ADAM17的作用，并测试一个新的假设，即EGFR和这些miRNAs形成一个自我放大的环路，驱动饮食促进的肿瘤发生。

DESCRIPTION (provided by applicant): While Western diets are implicated in increased colon cancer risk, molecular underpinnings of these dietary effects remain largely unknown. The azoxymethane (AOM) and Apc+/min mouse models mimic many features of human colon cancer, including tumor promotion by Western diet. We showed that Western diet up-regulated ligands for epidermal growth factor receptors (EGFR). Furthermore, EGFR was required for tumor promotion. Several EGFR ligands are released from membrane-bound pro-ligands by the lipid-raft-associated metalloproteinase ADAM17. Our recent studies indicate that ADAM17 is down-regulated by microRNA-145 (miR-145), whereas K-ras, an EGFR effector is suppressed by miR-143. These co-transcribed miRNAs are down-regulated in human colon cancer. We recently showed that EGFR signals downregulate miR-143 and miR-145 in AOM and Apc+/min tumors. Furthermore, these miRNA reductions are necessary for EGFR mitogenic effects. Based on our data we hypothesize that ADAM17 up-regulation and miR-143 and miR-145 down-regulation play essential roles in Western diet-induced tumor promotion. We propose several aims to address this hypothesis: Aim 1: Elucidate the requirement for ADAM17 in diet-promoted colonic tumorigenesis. We hypothesize that ADAM17 inhibition or deletion will suppress diet-related tumor promotion. We will use 1a) the AOM model in conditional ADAM17-deleted mice; 1b) the Apc+/min model with a novel ADAM17 pharmacological inhibitor INCB3619 to dissect the role of ADAM17 in diet-promoted tumorigenesis; 1c) in vitro studies of lipid rafts to dissect fatty acid effects on ADAM17 in colon cancer cells. Aim 2: To determine contributions of miR-143 and miR-145 in diet-promoted colonic tumorigenesis. We hypothesize that loss of these miRNAs is necessary for diet-induced tumor promotion. We will employ Apc+/min mouse interbred with 2a) transgenic mice expressing villin-promoter regulated pre-miR-143 and pre-miR-145; or with 2b) miR-143 null mice or with 2c) miR-145 null mice to uncover the role of these miRNAs in diet- promoted tumorigenesis. In aim 2d), we will examine other miRNAs implicated in ADAM17 regulation and/or diet-related tumorigenesis, including miR-1, -31, -148, and -152. Aim 3: Determine the regulation of miR-143 and miR-145 by Western diet and tumorigenesis. We hypothesize that Western diet and malignant transformation suppress transcription, while neoplastic transformation also deranges processing. We will 3a) assess effects of ADAM17, diet and neoplastic stage on pri-, pre- and mature levels of miR-143, -145 in in vivo models; 3b) dissect EGFR and fatty acid effects on miR-143/-145 promoter activity using mutant deletions to identify cis regulatory elements; 3c) Determine proteins differentially co-associating with biotinylated miR-143 or miR-145 in murine processing-competent YAMC and processing-incompetent CT26 colon cancer cells to discover deregulated processing factors. Our proposal will clarify the role of ADAM17 and test a novel hypothesis that EGFR and these miRNAs form a self-amplifying loop that drives diet-promoted tumorigenesis.