Abstract
M3 muscarinic receptor (M3R) activation promotes colon cancer cell proliferation, migration, and invasion in vitro. Although over-expression of CHRM3, the gene encoding M3R, is reported in primary colon cancers, expression of M3R itself has not been studied in colon neoplasia. We compared M3R expression in normal colon to colon adenomas, and primary and metastatic colon cancers. Compared to adjacent normal colon, CHRM3 expression was increased up to 128-fold in 10 of 18 consecutive surgical cancer specimens (56%) and associated with metastatic spread (P < 0.05). To analyze M3R protein expression we interrogated 29 consecutive paraffin-embedded colon adenocarcinomas and adjacent normal colon using a specific anti-M3R antibody and immunoperoxidase staining. This revealed weak M3R expression in normal colonocytes, primarily on basolateral surfaces. In contrast, in 25 of 29 cancer tissues (86%) we observed both cytoplasmic and plasma membrane over-expression of M3R; compared to normal epithelium, mean M3R staining intensity was increased more than two-fold in colon cancer (P < 0.001). M3R staining was also increased in 22 colon adenomas compared to adjacent normal colon (P < 0.001). In contrast, M3R staining intensity was not increased in lymph node or liver metastases. These findings suggest M3R expression plays an important role in early progression and invasion of colon neoplasia but is less important once tumors have spread.
Highlights
Of five known muscarinic receptor subtypes, three that stimulate cellular signaling by means of phospholipid turnover (M1R, M3R, and M5R) are conditional oncogenes when expressed in cells capable of proliferation [1]
Since in vitro studies show that M3R activation strongly induces expression of matrix metalloproteinase-1 (MMP1), an enzyme that degrades extracellular matrix [16] and whose expression correlates with advanced colon cancer stage, metastasis and poor prognosis [17, 18], we explored the association of CHRM3/M3R and MMP1 expression in primary colon cancers
There was, a significant relationship between the level of CHRM3 expression and the presence of colon cancer metastases; whereas metastases were absent in all 8 cancers lacking over-expression of CHRM3, metastatic disease was present in 5 of 10 individuals (50%) in whom CHRM3 was over-expressed in the primary tumor (P = 0.04)
Summary
Of five known muscarinic receptor subtypes, three that stimulate cellular signaling by means of phospholipid turnover (M1R, M3R, and M5R) are conditional oncogenes when expressed in cells capable of proliferation [1]. In animal www.impactjournals.com/oncotarget models relevant to sporadic and genetic human colon cancer, M3R activation stimulates colon cancer growth [13] and M3R deficiency attenuates tumor formation [14, 15]. These findings support an important role for M3R expression and activation in the progression of colon neoplasia
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