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cell muscarinic receptors can regulate cell proliferation: and EGFR is important for regulating colon cancer cell
(1) fecal bile acids are in contact with intestinal epithelial proliferation. [31]
cells for many years (average age for developing colon
cancer is > 50 years); (2) bile acids lack an ester linkage Previously, we found that in H508 cells which over-
[35]
and are not hydrolyzed by tissue cholinesterases that express both M3R and EGFR, but not in SNU-C4
rapidly inactivate ACh; (3) lipophilic lithocholic acid cells that express EGFR but not M3R, ACh stimulated
[28]
derivatives have access to muscarinic receptors in the cell proliferation by approximately 200% compared
[31]
lipid bilayer of cell membranes (i.e. the novel bile acid: to control. In H508 cells, both ACh and EGF
ACh hybrid molecule, lithocholylcholine, interacts with stimulated calcium-dependent EGFR activation (tyrosine
muscarinic receptors on rat aortic strips); (4) neoplastic phosphorylation) and activation of extracellular signal-
[28]
cells commonly lose polarity, thereby expanding regulated kinase 1 and 2 (ERK1/2); MR antagonists
expression of muscarinic receptors, usually restricted to and inhibitors of the mitogen-activated protein kinase
the basolateral membrane, to the entire plasma membrane; (MAPK) phosphorylation blocked these effects. In
and (5) increased tight junction permeability between addition, ACh- and EGF-induced phosphorylation of
neoplastic cells allows access of luminal bile acids to ERK1/2 MAPK and cell proliferation were abolished by
basolateral membrane receptors. We believe that this EGFR inhibitors. However, in Cho cells transfected with
[36]
collective evidence makes a compelling argument that rat M3R, which lack EGFR, ACh-induced ERK1/2 MAPK
muscarinic receptors and MR ligands play an important phosphorylation was not altered by EGFR inhibitors. It
role in intestinal epithelial cell proliferation and CRC was concluded that, in H508 cells, cholinergic ligand
progression. interaction with M3R results in transactivation of EGFR,
thereby stimulating cell proliferation. These results
[31]
Although experimental studies in rodents suggest that indicate that EGFR transactivation is a key mechanism
bile acids are intestinal tumor promoters, [37] the role of underlying MR-mediated intestinal tumorigenesis and
endogenous bile acids in colon carcinogenesis remains cancer progression.
poorly understood. Dawson et al. showed that in
[38]
mice deficient in the ileal apical sodium-dependent bile Cell proliferation and tumorigenesis
acid transporter (ASBT, encoded by SLC10A2), fecal Uncontrolled cell proliferation is a hallmark of
bile acid excretion increased more than 10-fold. To malignancies. We showed that in human colon cancer
examine the development of aberrant crypt foci (ACF), cells ACh-induced activation of M3R stimulates robust
the earliest histological marker of colon neoplasia, but selective matrix metalloproteinase (MMP) gene
we treated WT and Asbt-deficient [Slc10a2 (-/-)] male expression. In H508 human colon cancer cells, ACh
[42]
mice with azoxymethane (AOM), an intestine-selective caused a striking dose- and time- dependent increase
carcinogen. We also used a combination of AOM and in mRNA and protein levels of MMP1, 7, and 10 by
[39]
dextran sodium sulfate to induce colon tumorigenesis. upregulating gene transcription. As a consequence,
Compared to littermate controls, we found that Asbt- ACh stimulated MMP7-dependent cell proliferation by
deficient mice demonstrated significant increases in ACF, transactivating EGFR.
as well as colon tumor number and size. Also, Asbt-
deficient mice had a two-fold increase in the number Using in vivo models, we showed that genetic ablation
of colon adenocarcinomas. Finally, in murine colon of M3R in AOM-treated mice attenuates epithelial
neoplasia, increased fecal bile acids were associated with cell proliferation, and the number of adenomas and
increased expression of M3R and EGFR, and activation adenocarcinomas per mouse colon (65% reduction in
of post-EGFR signaling. These observations indicate the number of adenocarcinomas/colon). Whereas 50%
[43]
that endogenous bile acids also promote intestinal of AOM-treated wild-type (WT) animals had multiple
tumorigenesis. adenocarcinomas/colon, this was not the case with any
M3R-deficient animal. Moreover, in M3R-deficient mice
MR SIGNALING IN CRC AND DISEASE the overall colon tumor volume was reduced by 60%
PROGRESSION compared to that in WT animals. Collectively, these
observations suggest that M3R may play a role in both
Transactivation of EGFR tumor initiation and promotion; that is, both the number
EGFR is commonly over-expressed in many epithelial and size of tumors was reduced in M3R-deficient animals.
malignancies and this feature often indicates a more
aggressive phenotype. Likewise, as observed with MR antagonists are potential therapeutics in CRC. We
[40]
M3R, EGFR is frequently over-expressed in colon showed that M3R gene ablation and treatment with
cancer (in 25-77% of tumors compared to adjacent scopolamine butylbromide, a non-subtype-selective MR
normal mucosa). [40,41] Co-expression of M3R and EGFR inhibitor, attenuated small intestinal neoplasia in Apc Min/+
in many colon cancer cell lines, and over-expression of mice with aberrant beta-catenin signaling. Compared
[44]
these receptors in the majority of colon cancers suggests with Apc Min/+ mice, Apc Min/+ M3R mice showed 70%
-/-
that the functional interaction observed between M3R and 81% reductions in tumor number and volume,
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ June 15, 2016 ¦ 197