The central hypothesis to be tested in this proposal is that bile acids induce colon cancer cell proliferation by interaction with M3 muscarinic receptors (M3R), thereby causing transactivation of epidermal growth factor receptors (EGFR). To elucidate post-receptor signaling that results in bile acid-induced colon cancer cell proliferation and to determine the requirement for, and mechanism of, transactivation of EGFR the following Specific Aims are proposed: 1. To elucidate further post-receptor signal transduction pathways which mediate cholinergic agonist-induced transactivation of EGFR and stimulate colon cancer cell proliferation. 1a. For use in the proposed studies, in addition to cells that naturally express M3R and EGFR, colon cancer cells will be transfected with cDNA clones for M3R and/or EGFR. 1b. The mechanism of cholinergic agonist-induced cell proliferation will be studied using immunoblotting to probe for activated proteins in the p44/42 MARK, p38 MARK and JNK pathways, and by examining the roles of phospholipase C and protein kinase C activation and Ca2+ mobilization.1c. The requirement for cholinergic agonist-induced transactivation of EGFR will be confirmed by examining EGFR phosphorylation and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants.2. To determine the requirement for co-expression of M3R and EGFR and delineate the molecular mechanisms whereby bile acids regulate colon cancer cell proliferation. 2a. Bile acid-induced post-receptor signaling and the requirement for co-expression of M3R and EGFR will be determined using radioligand binding assays, immunoblotting for activated p44/42 MARK, p38 MARK and JNK cascade proteins, and by using EGFR inhibitors, antisense oligonucleotides, and dominant negative mutants. 2b. Bile acid-induced activation and expression of transcription factors (p90RSK, p38 MARK and JNK) and genes (CREB, NF-kappaB, c-Fos and c-Jun) related to colon cancer proliferation will be elucidated. 3. To determine the molecular mechanism in human colon cancer cells of bile acid-induced transactivation of EGFR. 3a) Expression and release of EGFR ligands, including HB-EGF, will be determined using immunoblots, northern blots, and RT-PCR, and the dependence of bile acid-induced EGFR transactivation on release of EGFR ligands will be determined using EGFR antibodies, metalloproteinase inhibitors, and specific antibodies and inhibitors for EGFR. 3b) Colon cancer cell metalloproteases will be identified by immunoblotting and in situ hybridization and the role of these enzymes in mediating bile acid-induced HB-EGF release will be determined by using metalloprotease inhibitors and antisera, and by knocking down metalloprotease expression. 3c) The mechanism whereby bile acids activate metalloproteases will be determined by exploring the roles of PKC, Ca2+, and Src in bile acid-induced HB-EGF release.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA107345-05
Application #
7625247
Study Section
Clinical and Integrative Gastrointestinal Pathobiology Study Section (CIGP)
Program Officer
Salnikow, Konstantin
Project Start
2005-06-28
Project End
2011-05-31
Budget Start
2009-06-01
Budget End
2011-05-31
Support Year
5
Fiscal Year
2009
Total Cost
$213,890
Indirect Cost
Name
University of Maryland Baltimore
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Rachakonda, Vikrant; Jadeja, Ravirajsinh N; Urrunaga, Nathalie H et al. (2015) M1 Muscarinic Receptor Deficiency Attenuates Azoxymethane-Induced Chronic Liver Injury in Mice. Sci Rep 5:14110
Cheng, Kunrong; Xie, Guofeng; Khurana, Sandeep et al. (2014) 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. Mol Cancer 13:77
Peng, Zhongsheng; Heath, Jonathon; Drachenberg, Cinthia et al. (2013) Cholinergic muscarinic receptor activation augments murine intestinal epithelial cell proliferation and tumorigenesis. BMC Cancer 13:204
Khurana, Sandeep; Jadeja, Ravirajsinh; Twaddell, William et al. (2013) Effects of modulating M3 muscarinic receptor activity on azoxymethane-induced liver injury in mice. Biochem Pharmacol 86:329-38
Chahdi, Ahmed; Raufman, Jean-Pierre (2013) The Cdc42/Rac nucleotide exchange factor protein ?1Pix (Pak-interacting exchange factor) modulates ?-catenin transcriptional activity in colon cancer cells: evidence for direct interaction of ?1PIX with ?-catenin. J Biol Chem 288:34019-29
Peng, Zhongsheng; Raufman, Jean-Pierre; Xie, Guofeng (2012) Src-mediated cross-talk between farnesoid X and epidermal growth factor receptors inhibits human intestinal cell proliferation and tumorigenesis. PLoS One 7:e48461
Khurana, Sandeep; Raina, Hema; Pappas, Valeria et al. (2012) Effects of deoxycholylglycine, a conjugated secondary bile acid, on myogenic tone and agonist-induced contraction in rat resistance arteries. PLoS One 7:e32006
Xie, Guofeng; Peng, Zhongsheng; Raufman, Jean-Pierre (2012) Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation. Am J Physiol Gastrointest Liver Physiol 302:G1006-15
Raufman, Jean-Pierre; Shant, Jasleen; Xie, Guofeng et al. (2011) Muscarinic receptor subtype-3 gene ablation and scopolamine butylbromide treatment attenuate small intestinal neoplasia in Apcmin/+ mice. Carcinogenesis 32:1396-402
Khurana, Sandeep; Raufman, Jean-Pierre; Pallone, Thomas L (2011) Bile acids regulate cardiovascular function. Clin Transl Sci 4:210-8

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