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 sele...