Muscarinic Receptors Regulate Colon Cancer Stem Cell Function and Invasiveness

毒蕈碱受体调节结肠癌干细胞功能和侵袭性

• 批准号: 10260301
• 负责人: JEAN-PIERRE RAUFMAN
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10260301
• 关键词: 3' Untranslated Regions; 3-Dimensional; Advanced Malignant Neoplasm; Affect; Animal Model; Animals; Attenuated; Automobile Driving; Binding Sites; Biological Assay; Biological Markers; Bioluminescence; CHRM1 gene; Cell Culture Techniques; Cells; Clinical Trials; Colon; Colon Carcinoma; Colonic Neoplasms; Computer Analysis; Computer Models; Data; Diagnosis; Disease; Equilibrium; Event; Exonuclease; FDA approved; Fluorouracil; Genetic Transcription; Genetic Translation; Genomic DNA; Goals; Growth; Human; Immune checkpoint inhibitor; Immunotherapeutic agent; Immunotherapy; In Vitro; Incidence; Individual; Interstitial Collagenase; Intervention; Investigation; Knowledge; Learning; Link; Luciferases; Lung; Malignant Neoplasms; Messenger RNA; MicroRNAs; Molecular; Mothers; Mus; Muscarinic Acetylcholine Receptor; Muscarinic M3 Receptor; Neoplasm Metastasis; Neoplasms; Neurons; Organoids; Pattern; Porifera; Post-Transcriptional Regulation; Property; Prostate; RNA; Receptor Activation; Rectal Cancer; Regulation; Relapse; Reporter; Reporting; Research; Resistance; Role; Signal Transduction; Testing; Therapeutic; Translations; Untranslated RNA; Untranslated Regions; Veterans; Work; Xenograft Model; Xenograft procedure; advanced disease; beta catenin; cancer cell; cancer stem cell; chemotherapy; colon cancer progression; colon cancer treatment; design; drug repurposing; established cell line; human disease; in vivo; in vivo Model; inhibitor/antagonist; innovation; intervention effect; novel; overexpression; oxaliplatin; pre-clinical; prognostic; programmed cell death protein 1; promoter; receptor; receptor expression; resilience; response; stem cell differentiation; stem cell function; stem cell proliferation; therapy resistant; tumor; tumor growth; tumor progression

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.

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。关于 三分之一被诊断患有结肠癌的退伍军人将死亡，主要死于转移性疾病。然而， 结肠癌扩散的分子事件仍不确定，目前晚期结肠癌的治疗方法 疾病是有限的并且只能提供短暂的益处。迫切需要更有效、更持久的治疗方法。和 之前的 VA Merit 支持，我们发现 M3 毒蕈碱受体 (M3R) 激活可促进结肠癌进展 通过许多不良行为。我们的新发现揭示了两种毒蕈碱受体亚型 M1R 和 M3R 编码 CHRM1 和 CHRM3 在结肠癌连续体和结肠癌干细胞的早期过表达 （CCSC）驱动肿瘤恢复、复发和转移。这些发现表明同时针对 M1R 直接和M3R可能具有巨大的转化潜力。因此，我们建议检验中心假设： 选择性靶向毒蕈碱受体亚型可减轻结肠癌的进展和对治疗的抵抗力。 为此，我们必须填补有关结肠中 M1R 和 M3R 表达如何调节的知识的关键空白 肿瘤形成并确定 M1R 和 M3R 在驱动癌症进展中的各自作用。我们的新数据表明 随着肿瘤的进展，与 M3R 表达相比，M1R 表达逐渐减少；在大多数结肠中 癌症中 M3R 水平大大超过 M1R，并且我们无法在肿瘤浸润处的 CCSC 中检测到 M1R 边缘。值得注意的是，我们确定了 M1R 表达与两种 microRNA（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 在晚期癌症中抑制 CHRM1 mRNA 翻译，从而抑制 M1R 一致 水平。由于 miR-107/-103 还可以放大针对 CHRM3 的 β-catenin 信号传导，因此这些 miRNA 可能间接 增强 M3R 水平。 M3R 水平增加也可能是由于 miR-30-5p 表达减少所致，这可以 与 CHRM3 3'-UTR 相互作用，并与结肠癌中的 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；因此，通过降低游离 miR-107 和 miR-103 的水平来间接调节 M1R 和 M3R 水平 – 我们 建议利用 circRNA 的治疗潜力。基于这些新发现，我们建议使用 复杂的体外、离体和体内模型，对术后进行细致、集中的研究 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 和 M3R 水平影响重要的临床前终点——肿瘤生长、扩散、对化疗和化疗的反应 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： 目标 1：严格测试选择性消耗和灭活 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：定义 miR-107、-103 和 -30-5p 作为 M1R 和 M3R 水平调节剂的确切作用 结肠癌进展的生物标志物以及 circRNA 海绵的治疗潜力。 由于毒蕈碱受体可以通过 FDA 批准用于其他适应症的抑制剂进行选择性药物治疗，我们 相信在动物模型中证明调节毒蕈碱受体活性的治疗益处 模拟人类疾病将促进此类药物重新用于治疗结肠癌的临床试验。识别小说 与结肠癌进展相关的 microRNA/circRNA/毒蕈碱受体轴将是一个重大概念进展 我们新提出的 miR-107/-103 的 circRNA 海绵证明了其治疗前景。