Role of M3 muscarinic receptors in bile acid-induced colon cancer

描述（由申请人提供）：对于美国的男性和女性来说，结肠癌是癌症死亡的第三大常见原因，也是胃肠道死亡的主要原因。流行病学和动物研究将结肠癌风险与粪胆汁酸谱和浓度的改变联系起来。目前的调查线是由我们意想不到的观察胆汁酸与毒蕈碱受体相互作用发起的。M3毒蕈碱受体（M3R）在大多数结肠癌中过表达，是结肠癌细胞增殖的关键参与者。我们的数据表明，胆汁酸与人结肠癌细胞系上表达的M3R相互作用，从而激活基质金属蛋白酶(MMP)-7，释放HB-EGF，并诱导表皮生长因子受体（EGFR）的转激活。受体后信号通过ERK刺激人结肠癌细胞增殖。总的来说，这些观察结果确定M3R是预防和/或治疗结肠癌的新治疗靶点。本修订后的R01应用的中心假设是M3R在体内的表达和激活介导胆汁酸促进肠道肿瘤的发生。在原致癌物偶氮甲烷（azoxymethane， AOM）处理的小鼠和apc基因突变小鼠（ApcMin/+小鼠）中，我们的初步研究结果表明，m3r缺乏可减少肠道肿瘤数量。修订后的研究计划的主要目标是利用这些独特的小鼠结肠癌模型来确定M3R表达对结肠肿瘤发展的重要性，确定胆酸诱导的M3R激活促进癌变的分子机制，并确定M3R激活的药理学抑制模拟M3R基因的消蚀从而减轻肠道肿瘤。为了验证我们的中心假设并实现这些目标，我们提出了三个重点的具体目标：目标1。建立M3R表达在小鼠肠道肿瘤中的关键作用。目标2。证实胆汁酸诱导的M3R激活通过ERK介导的基因转录促进肠道肿瘤的发生。目标3。建立M3R的药理学抑制模拟M3R基因消融的作用，减少肠道肿瘤的发生。修订后的申请包括支持我们中心假设的强有力的初步数据，明确M3R和胆汁酸促进结肠癌发生机制的重点方法，独特的动物模型，创新的方法，以及一组经验丰富的合作研究者和顾问。基于我们具有挑战性的初步数据支持M3R在结肠肿瘤形成中的关键作用，确定用抗毒蕈碱受体药物阻断M3R激活是否模拟M3R缺陷小鼠中观察到的肿瘤数量减少是及时和重要的。结果将确立M3R的关键作用，并通过促进药物策略的发展，通过减弱毒蕈碱受体激活来预防和治疗结肠肿瘤，从而促进人类健康。同时，获得的关于毒蕈碱受体在介导胆汁酸作用和肿瘤发生中的作用的基础知识将推动癌症生物学的一般领域。公共卫生相关性：对于男性和女性来说，结肠癌是发病和死亡的主要原因；在美国，每年大约有15万人被诊断出结肠癌，其中5万人死于结肠癌。晚期结肠癌的治疗效果有限，需要新的治疗方法。我们期望对M3毒蕈碱受体在肠肿瘤中的关键作用的研究将揭示一种新的、低风险的策略，以预防高危人群（如家族性腺瘤性息肉综合征患者）的结肠癌，并治疗这种重要的、经常致命的疾病，这些疾病无法通过手术治愈。

DESCRIPTION (provided by applicant): For men and women in the United States, colon cancer is the third most common cause of cancer death and the leading gastrointestinal cause of death. Epidemiological and animal studies associate colon cancer risk with alterations in the spectrum and concentration of fecal bile acids. The present line of investigation was initiated by our unanticipated observation that bile acids interact with muscarinic receptors. M3 muscarinic receptors (M3R), which are over-expressed in most colon cancers, are key players in colon cancer cell proliferation. Our data indicate that bile acids interact functionally with M3R expressed on human colon cancer cell lines thereby activating matrix metalloproteinase (MMP)-7, releasing HB-EGF, and inducing transactivation of epidermal growth factor receptors (EGFR). Post-receptor signaling via ERK stimulates human colon cancer cell proliferation. Collectively, these observations identify M3R as a novel therapeutic target to prevent and/or treat colon cancer. The central hypothesis of this revised R01 application is that M3R expression and activation in vivo mediates bile acid promotion of intestinal neoplasia. In mice treated with a procarcinogen [azoxymethane (AOM)] and mice with an apc gene mutation (ApcMin/+ mice), our preliminary findings show that M3R-deficiency reduces intestinal tumor number. Primary goals of the revised research plan are to use these unique murine colon cancer models to establish the importance of M3R expression for development of colon neoplasia, to identify molecular mechanisms whereby bile acid-induced activation of M3R promotes carcinogenesis, and to establish that pharmacologic inhibition of M3R activation mimics m3r gene ablation thereby attenuating intestinal neoplasia. To test our central hypothesis and address these goals we propose three focused Specific Aims: Aim 1. Establish the critical role of M3R expression for murine intestinal neoplasia. Aim 2. Establish that bile acid-induced M3R activation promotes intestinal neoplasia by ERK- mediated gene transcription. Aim 3. Establish that pharmacologic inhibition of M3R mimics the effects of m3r gene ablation and reduces intestinal neoplasia. This revised application includes strong preliminary data supporting our central hypothesis, a focused approach to defining the mechanisms whereby M3R and bile acids promote colon carcinogenesis, unique animal models, innovative methods, and an outstanding group of highly experienced co-investigators and consultants. Based on our provocative preliminary data supporting the key role of M3R in colon tumor formation, it is timely and important to determine whether blocking M3R activation with anti-muscarinic receptor agents mimics the reduction in tumor number observed in M3R-deficient mice. Outcomes will establish the critical role of M3R and advance human health by spurring development of pharmacologic strategies to prevent and treat colon neoplasia by attenuating muscarinic receptor activation. At the same time, fundamental knowledge gained regarding the role of muscarinic receptors in mediating bile acid actions and neoplasia will advance the general area of cancer biology. PUBLIC HEALTH RELEVANCE: For men and women colon cancer is a leading cause of morbidity and mortality; in the US, approximately 150,000 people are diagnosed and 50,000 die from colon cancer each year. Efficacy of treatment for advanced colon cancer is limited and new approaches are needed. It is anticipated that the proposed investigation on the critical role of M3 muscarinic receptors in intestinal neoplasia will reveal a novel, low-risk strategy to prevent colon cancer in high-risk populations (e.g. those with familial adenomatous polyposis syndrome) and to treat this important, frequently deadly disease in those who cannot be cured with surgery.