Figure 2
From: Muscarinic receptor agonist-induced βPix binding to β-catenin promotes colon neoplasia
ARHGEF7/βPix and CHRM3/M3R overexpression in colon cancer is associated with nuclear βPix expression. (a) βPix (ARHGEF7) and M3R (CHRM3) overexpression in colorectal cancer. UALCAN server analysis47 reveals ARHGEF7 (left panel) and CHRM3 (right panel) mRNA transcripts are overexpressed in colon cancer (n = 286 patients) compared with normal colon (n = 41 patients). For normal colon, maximum, upper quartile, median, lower quartile, and minimum values, respectively, are 24.615, 20.3421, 18.244, 16.709, and 11.871 for ARHGEF7 and 3.645, 2.055, 1.272, 1.069, and 0.678 for CHRM3. For colon cancer, these values, respectively, are 46.986, 29.116, 21.734, 17.029 and 8.098 for ARHGEF7 and 10.027, 4.072, 2.528, 1.094, and 0.02 for CHRM3. (b) Patterns of ARHGEF7 and CHRM3 co-expression in colon cancer. mRNA was measured by qPCR in 17 colon cancers and adjacent normal colon tissue. CHRM3 and ARHGEF7 expression were increased greater than two-fold in 47% and 71% of cancers, respectively; both genes were co-overexpressed in 41% of cancers. (c) ARHGEF7 and CHRM3 gene expression are increased in colon cancer. ARHGEF7 and CHRM3 mRNA expression was significantly increased in cancer (bars represent means ± SE normalized to β-2 microglobulin; *p < 0.05 compared to adjacent normal colon). (d) βPix protein expression in colon cancer. βPix protein expression was increased up to 100-fold in 16 of 21 colon cancers (T) compared to adjacent normal colon (N). Immunoblots were arranged in order of decreasing βPix protein expression in tumors. Original uncut immunoblots are shown in Supplemental Materials. (e) Representative M3R and βPix immunostaining. Arrows show nuclear βPix staining. Size bars, 100 μm. (f) M3R and βPix staining was significantly increased in cancer compared to adjacent normal colon (***, p < 0.001). Bars, means ± SE of 29 samples. (g) Correlation between M3R and nuclear βPix immunostaining in normal colon (◯, N = 29) and cancer (⬤, N = 29) (numbers in symbols represent multiplicity of samples with same result); Pearson correlation coefficient = 0.44, p < 0.05. Dashed line = ‘best fit’. MW, molecular weight.
