TY - JOUR
T1 - 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
AU - Cheng, Kunrong
AU - Xie, Guofeng
AU - Khurana, Sandeep
AU - Heath, Jonathon
AU - Drachenberg, Cinthia B.
AU - Timmons, Jennifer
AU - Shah, Nirish
AU - Raufman, Jean Pierre
N1 - Funding Information:
We thank Dr. Jürgen Wess, Molecular Signaling Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, for kindly providing knockout mice used in this work and for helpful suggestions and insights during the course of this work. We thank Xue-Min Gao, University of Maryland School of Medicine, for technical assistance. This work was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to J-P. Raufman) and by National Institutes of Health grant numbers R01 CA107345, R01 CA120407, R21 DK093406 (to J-P. Raufman), K08 DK080843 (to G. Xie) and K08 DK081479 (to S. Khurana). J. Timmons and N. Shah were supported by National Institutes of Health grant number T32 DK067872 (to J-P. Raufman).
PY - 2014/4/3
Y1 - 2014/4/3
N2 - Background: M3 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.Methods: We 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.Results: Azoxymethane-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.Conclusions: Our 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.
AB - Background: M3 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.Methods: We 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.Results: Azoxymethane-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.Conclusions: Our 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.
KW - Azoxymethane
KW - Chrm1
KW - Chrm3
KW - Colon cancer
KW - M1R
KW - M3R
KW - Mouse models
KW - Muscarinic receptors
KW - ZNF277
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U2 - 10.1186/1476-4598-13-77
DO - 10.1186/1476-4598-13-77
M3 - Article
C2 - 24694019
AN - SCOPUS:84897573934
VL - 13
JO - Molecular Cancer
JF - Molecular Cancer
SN - 1476-4598
IS - 1
M1 - 77
ER -