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

描述（由申请人提供）：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仍然很大程度上未知。氧化偶氮甲烷 (AOM) 和 Apc /min 小鼠模型模仿了人类结肠癌的许多特征，包括西方饮食促进肿瘤的发生。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对于肿瘤促进是必需的。脂筏相关金属蛋白酶 ADAM17 从膜结合前配体中释放多种 EGFR 配体。我们最近的研究表明，ADAM17 被 microRNA-145 (miR-145) 下调，而 EGFR 效应子 K-ras 被 miR-143 抑制。这些共转录的 miRNA 在人类结肠癌中下调。我们最近发现 EGFR 信号下调 AOM 和 Apc /min 肿瘤中的 miR-143 和 miR-145。此外，这些 miRNA 的减少对于 EGFR 促有丝分裂作用是必要的。根据我们的数据，我们假设 ADAM17 上调以及 miR-143 和 miR-145 下调在西方饮食诱导的肿瘤促进中发挥重要作用。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 抑制或缺失将抑制与饮食相关的肿瘤生长。我们将在条件性 ADAM17 缺失小鼠中使用 1a) AOM 模型； 1b) 采用新型 ADAM17 药理学抑制剂 INCB3619 的 Apc /min 模型，以剖析 ADAM17 在饮食促进的肿瘤发生中的作用； 1c) 脂筏体外研究，以剖析脂肪酸对结肠癌细胞中 ADAM17 的影响。目标 2：确定 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 miRNA 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) 表达绒毛启动子调节的 pre-miR-143 和 pre-miR-145 的转基因小鼠杂交；或使用 2b) miR-143 缺失小鼠或 2c) miR-145 缺失小鼠来揭示这些 miRNA 在饮食促进的肿瘤发生中的作用。在目标 2d) 中，我们将检查与 ADAM17 调节和/或饮食相关肿瘤发生有关的其他 miRNA，包括 miR-1、-31、-148 和 -152。目标 3：确定西方饮食和肿瘤发生对 miR-143 和 miR-145 的调节。我们假设西方饮食和恶性转化抑制转录，而肿瘤转化也会扰乱加工。我们将 3a) 在体内模型中评估 ADAM17、饮食和肿瘤阶段对 miR-143、-145 的初级、前期和成熟水平的影响； 3b) 使用突变体缺失来剖析 EGFR 和脂肪酸对 miR-143/-145 启动子活性的影响，以鉴定顺式调控元件； 3c) 确定小鼠加工能力强的 YAMC 和加工能力不强的 CT26 结肠癌细胞中与生物素化 miR-143 或 miR-145 差异共关联的蛋白质，以发现失调的加工因子。我们的提案将阐明 ADAM17 的作用，并测试一个新的假设，即 EGFR 和这些 miRNA 形成一个自我放大环，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.