Muscarinic Receptors Regulate Colon Cancer Stem Cell Function and Invasiveness

在退伍军人中，结肠癌和直肠癌的发病率排名第三，仅次于前列腺癌和肺癌；大约 被诊断患有结肠癌的退伍军人中有三分之一将死亡，主要死于转移性疾病。然而， 结肠癌扩散的分子事件仍然不确定，目前晚期结肠癌的治疗方法 疾病是有限的，只能提供短暂的好处。迫切需要更有效、更持久的治疗方法。使用 之前的VA优点支持，我们发现M3 M受体(M3R)的激活促进结肠癌的进展 通过许多不利的行为。我们的新发现揭示了两种M受体亚型M1R和M3R编码 通过CHRM1和CHRM3，在结肠癌连续体和结肠癌干细胞中过表达 (CCSCs)驱动肿瘤韧性、复发和转移。这些发现表明同时以M1R为目标 而M3R直接可能具有巨大的翻译潜力。因此，我们建议检验核心假设，即 选择性靶向M受体亚型可减轻结肠癌的进展和对治疗的抵抗。 要做到这一点，我们必须填补有关M1R和M3R在结肠中表达如何调控的关键知识空白 并确定M1R和M3R在推动癌症进展中的单独作用。我们的新数据显示 随着肿瘤的进展，与M3R相比，M1R的表达逐渐减少；在大多数结肠中 肿瘤M3R水平远远超过M1R，我们在肿瘤侵袭的CCSCs中检测不到M1R 边缘。值得注意的是，我们确定了M1R表达和两个microRNAs之间的相互关系，miR- 107和miR-103，通过计算分析预测与CHRM1 3‘-UTR相互作用。这些发现是 与miR-107/-103抑制晚期癌症CHRM1 mRNA翻译一致，从而抑制M1R 级别。由于miR107/-103还可以扩增针对CHRM3的β-连环蛋白信号，这些miRNAs可能间接地 提高M3R水平。M3R水平的增加也可能是由于miR-30-5p表达减少所致，miR-30-5p可以 在结肠癌中与CHRM3 3‘-UTR相互作用，并与M3R水平呈负相关。有趣的是，我们的 新的发现还表明，结肠癌也表达环状(CIRC)RNA，cTFRC，能够‘海绵’miR-107/- 103，从而通过降低游离miR-107和miR-103-WE水平间接调节M1R和M3R水平 建议利用CircRNA的治疗潜力。基于这些新的发现，我们建议使用 复杂的体外、体外和体内模型，以执行细致的，重点研究后 通过这些非编码RNA对M1R和M3R表达的转录调节，并了解如何调节M1R M3R水平影响重要的临床前终点-肿瘤的生长、扩散、对化疗的反应-以及 免疫疗法和动物生存。为了实现这些目标，我们提出了两个全面的具体目标： 目的1：严格检测选择性耗竭和灭活M1R和M3R对结肠癌的影响 进展、对化疗和免疫治疗的反应，以及生存。 目标2：确定miR-107、-103和-30-5p作为M1R和M3R水平的调节因子的确切作用 结肠癌进展的生物标志物，以及CircRNA海绵的治疗潜力。 由于毒扁豆碱受体可被已获FDA批准用于其他适应症的抑制剂选择性地下药，我们 相信在动物模型中证明了调节M受体活性的治疗益处 模拟人类疾病将刺激此类药物用于治疗结肠癌的临床试验。辨别一部小说 与结肠癌进展相关的microRNA/CircRNA/M受体轴将是一项重大的概念进展 我们新提出的针对miR-107/-103的CircRNA海绵就是其治疗前景的例证。

In Veterans, cancers of the colon and rectum rank third in incidence behind those of the prostate and lung; about one-third of Veterans diagnosed with colon cancer will die, primarily from metastatic disease. However, the molecular events underlying the spread of colon cancer remain uncertain and current treatments for advanced disease are limited and provide only transient benefit. More effective, durable treatments are urgently needed. With previous VA Merit support, we found M3 muscarinic receptor (M3R) activation promotes colon cancer progression by many adverse actions. Our new findings reveal that two muscarinic receptor subtypes, M1R and M3R encoded by CHRM1 and CHRM3, are overexpressed early in the colon cancer continuum and in colon cancer stem cells (CCSCs) which drive tumor resilience, relapse, and metastasis. These findings suggest concurrently targeting M1R and M3R directly may have great translational potential. Accordingly, we propose to test the central hypothesis that selectively targeting muscarinic receptor subtypes attenuates colon cancer progression and resistance to therapy. To do so, we must fill key gaps in knowledge regarding how M1R and M3R expression is regulated in colon neoplasia and identify the individual roles of M1R and M3R in driving cancer progression. Our new data reveal that as neoplasia progresses, M1R expression is progressively reduced compared to M3R expression; in most colon cancers M3R levels greatly exceed those of M1R and we could not detect M1R in CCSCs at the tumor invasive edge. Notably, we identified a reciprocal relationship between M1R expression and that of two microRNAs, miR- 107 and miR-103, predicted by computational analysis to interact with the CHRM1 3’-UTR. These findings are consistent with miR-107/-103 suppressing CHRM1 mRNA translation in advanced cancer, thereby damping M1R levels. As miR-107/-103 can also amplify β-catenin signaling, which targets CHRM3, these miRNAs may indirectly augment M3R levels. Increased levels of M3R may also result from reduced expression of miR-30-5p which can interact with the CHRM3 3’-UTR and has an inverse relationship with M3R levels in colon cancer. Intriguingly, our new findings also show colon cancers also express a circular (circ)RNA, cTFRC, capable of ‘sponging’ miR-107 / - 103; and, thus indirectly regulating M1R and M3R levels by reducing levels of free miR-107 and miR-103 – we propose to leverage the therapeutic potential of circRNAs. Building on these new findings, we propose to use sophisticated in vitro, ex vivo, and in vivo models to perform a meticulous, focused investigation of the post- transcriptional regulation of M1R and M3R expression by these non-coding RNAs and learn how modulating M1R and M3R levels impacts important preclinical endpoints – tumor growth, spread, response to chemo- and immunotherapy, and animal survival. To accomplish these goals, we propose two comprehensive Specific Aims: Aim 1: Rigorously test the effects of selectively depleting and inactivating M1R and M3R on colon cancer progression, responses to chemo- and immunotherapy, and survival. Aim 2: Define the exact roles of miR-107, -103, and -30-5p as regulators of M1R and M3R levels and biomarkers for colon cancer progression, and the therapeutic potential of circRNA sponges. As muscarinic receptors are selectively druggable by inhibitors already FDA-approved for other indications, we believe demonstrating the therapeutic benefits of modulating muscarinic receptor activity in animal models that mimic human disease will spur clinical trials of such agents repurposed to treat colon cancer. Identifying a novel microRNA/circRNA/muscarinic receptor axis linked to colon cancer progression will be a major conceptual advance whose therapeutic promise is exemplified by our newly proposed circRNA sponges for miR-107/-103.