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

描述（由申请人提供）：对于美国男性和女性，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便胆汁酸谱和浓度的变化联系起来。目前的研究是由我们意外的观察结果开始的，即胆汁酸与毒蕈碱受体相互作用。在大多数结肠癌中过度表达的M3毒蕈碱受体（M3 R）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M3 R功能性相互作用，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）的反式激活。通过ERK的受体后信号传导刺激人结肠癌细胞增殖。总的来说，这些观察结果将M3 R鉴定为预防和/或治疗结肠癌的新型治疗靶点。该修订R 01申请的中心假设是体内M3 R表达和活化介导胆汁酸促进肠肿瘤形成。在用前致癌物[氧化偶氮甲烷（AOM）]处理的小鼠和具有apc基因突变的小鼠（ApcMin/+小鼠）中，我们的初步研究结果表明，M3 R缺乏减少了肠道肿瘤数量。修订后的研究计划的主要目标是使用这些独特的小鼠结肠癌模型来确定M3 R表达对结肠肿瘤形成的重要性，以确定胆汁酸诱导的M3 R活化促进致癌的分子机制，并确定M3 R活化的药理学抑制模拟m3 r基因消融，从而减轻肠肿瘤形成。为了检验我们的中心假设并实现这些目标，我们提出了三个有针对性的具体目标：目标1。确定M3 R表达对小鼠肠肿瘤的关键作用。 目标二。确定胆汁酸诱导的M3 R活化通过ERK介导的基因转录促进肠肿瘤形成。 目标3。确定M3 R的药理学抑制模拟m3 r基因消融的作用并减少肠肿瘤形成。该修订后的申请包括支持我们的中心假设的强有力的初步数据，定义M3 R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。基于我们支持M3 R在结肠肿瘤形成中的关键作用的挑衅性初步数据，确定用抗毒蕈碱受体药物阻断M3 R活化是否模拟在M3 R缺陷小鼠中观察到的肿瘤数量减少是及时和重要的。结果将建立M3 R的关键作用，并通过促进药物策略的发展来促进人类健康，以通过减弱毒蕈碱受体活化来预防和治疗结肠肿瘤。同时，关于毒蕈碱受体在介导胆汁酸作用和肿瘤形成中的作用的基础知识将推进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，结肠癌是发病率和死亡率的主要原因;在美国，每年约有15万人被诊断出患有结肠癌，5万人死于结肠癌。晚期结肠癌的治疗效果有限，需要新的方法。预计对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（例如患有家族性腺瘤性息肉综合征的人群）中的结肠癌，并治疗这种重要的，经常致命的疾病，不能用手术治愈。

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.