Divergent effects of muscarinic receptor subtype gene ablation on murine colon tumorigenesis reveals association of M3R and zinc finger protein 277 expression in colon neoplasia

Abstract

BackgroundM3 and M1 subtype muscarinic receptors are co-expressed in normal and neoplastic intestinal epithelial cells. In mice, ablating Chrm3, the gene encoding M3R, robustly attenuates intestinal tumor formation. Here we investigated the effects of Chrm1 gene ablation, alone and in combination with Chrm3 ablation.MethodsWe used wild-type, Chrm1-/-, Chrm3-/- and combined Chrm1-/-/Chrm3-/- knockout (dual knockout) mice. Animals were treated with azoxymethane, an intestine-selective carcinogen. After 20 weeks, colon tumors were counted and analyzed histologically and by immunohistochemical staining. Tumor gene expression was analyzed using microarray and results validated by RT-PCR. Key findings were extended by analyzing gene and protein expression in human colon cancers and adjacent normal colon tissue.ResultsAzoxymethane-treated Chrm3-/- mice had fewer and smaller colon tumors than wild-type mice. Reductions in colon tumor number and size were not observed in Chrm1-/- or dual knockout mice. To gain genetic insight into these divergent phenotypes we used an unbiased microarray approach to compare gene expression in tumors from Chrm3-/- to those in wild-type mice. We detected altered expression of 430 genes, validated by quantitative RT-PCR for the top 14 up- and 14 down-regulated genes. Comparing expression of this 28-gene subset in tumors from wild-type, Chrm3-/-, Chrm1-/- and dual knockout mice revealed significantly reduced expression of Zfp277, encoding zinc finger protein 277, in tissue from M3R-deficient and dual knockout mice, and parallel changes in Zfp277 protein expression. Notably, mRNA and protein for ZNF277, the human analogue of Zfp277, were increased in human colon cancer compared to adjacent normal colon, along with parallel changes in expression of M3R.ConclusionsOur results identify a novel candidate mouse gene, Zfp277, whose expression pattern is compatible with a role in mediating divergent effects of Chrm3 and Chrm1 gene ablation on murine intestinal neoplasia. The biological importance of this observation is strengthened by finding increased expression of ZNF277 in human colon cancer with a parallel increase in M3R expression. The role of zinc finger protein 277 in colon cancer and its relationship to M3R expression and activation are worthy of further investigation.

High expression of muscarinic acetylcholine receptor 3 predicts poor prognosis in patients with pancreatic ductal adenocarcinoma

“…Our findings suggested that M3 in PDAC could play a significant role in the invasive process in vivo. Recent investigations into the functional role of M3 in carcinomas have demonstrated that the expression of M3 augmented cell motility on extracellular matrix components4–7 and promoted tumor growth in mice16,17 in addition to its effect on cancer cell proliferation 18. These findings support our hypothesis that M3 in PDAC could play a role in the invasive process and its expression could be a more malignant phenotype.…”

Interacting post-muscarinic receptor signaling pathways potentiate matrix metalloproteinase-1 expression and invasion of human colon cancer cells

“…This may be considered speculative as acetylcholine is a non-selective M3R agonist. Nonetheless, we believe this conclusion is justified based on the strong body of evidence that M3R regulates these signaling cascades in human colon cancer cells [13, 46, 48], the identification of M3R as the primary M3R subtype expressed in both HT-29 and H508 colon cancer cells [17], and the key finding in animal models that M3R deficiency robustly attenuates the development of colon neoplasia whereas M1R deficiency has no effect [11, 18, 20]. …”

“…Of the five muscarinic receptor subtypes, M1R, M3R, and M5R are conditional oncogenes when expressed in cells capable of proliferation [8]. M3R are expressed widely in the gut and both M3R and CHRM3 , the gene encoding its expression, are over-expressed in colon cancer [7, 9–11]. We previously showed that M3R activation governs key hallmarks of colon cancer progression including cell proliferation, survival, migration, and invasion [12–17].…”

Muscarinic receptor signaling and colon cancer progression

“…We showed divergent effects of MR subtype gene ablation on murine colon tumorigenesis. [ 45 ] Although AOM-treated M3R-deficient mice had fewer and smaller colon tumors than control WT mice, reductions in colon tumor number and size were not observed in M1R-deficient and dual M1R/M3R-deficient mice. Microarray and real-time PCR analyses revealed a possible role for zinc finger protein (Zfp) 277 expression in mediating these different phenotypes.…”