The objective of this proposal is to determine the degree to which functional gastrin-releasing peptide receptors (GRP-R) are expressed in colon cancer, and to elucidate the mechanism(s) by which these receptors regulate the differentiation of individual colon cancer cells when expressed in functional form. We have previously shown that the GRP-R acts as a morphogen, critically regulating colon cancer differentiation (Cell Growth Diff 2000; 11: 385). We have also shown that GRP-R mRNA is aberrantly expressed by all human colon cancer cell lines studied but is frequently mutated and rendered pharmacologically non-functional (Mol Pharmacol 2000; 58: 601). In our Preliminary Data we show that the X-linked GRPR gene is also mutated in archived human colon cancers, with mutation number increasing as tumor cells de-differentiate. Poorly differentiated tumor cells contain GRPR gene mutations that cause receptor inactivation whereas this is never seen in well differentiated cells. Thus we propose the mechanistic hypothesis that expression of functional GRP-R within any particular colon cancer primarily regulates tumor cell differentiation; with receptor-inactivating mutations representing a major mechanism allowing tumor cells to de-differentiate. However the extent and effect of these mutations on GRP-R behavior, and the mechanism(s) by which expression of functional receptor modulates tumor cell appearance, have not been determined.
Specific Aim 1 focuses on elucidating the mechanism(s)whereby functional GRP-R modulate tumor cell differentiation. In our Preliminary Data we show that Caco-2 cells variably express GRP-R, which when present regulates normal morphological progression by activating focal adhesion kinase (FAK). Caco-2 cells will be transfected with vectors under control of an inducible promoter allowing for the directed expression of mini-genes. The products of these mini-genes will block specific regions of the GRP-R from binding to any G protein, or block specific G proteins themselves. The ability of each construct to undergo normal morphological progression will then be assessed.
Specific Aim 2 is directed to identifying mutations in the GRPR gene in human colon cancers as a function of the differentiation of individual tumor cells. Archived colon cancers maintained in the GI Tumor Bank will be randomly selected and all cells of defined differentiation removed by laser capture microscopy. The GRPR gene will be isolated, and all mutations identified recreated by site-directed mutagenesis so that they can be evaluated in a transiently transfected cell system. Overall these studies will provide critical information regarding colon cancer differentiation, and shed light on a novel mechanism regulating the inactivation, or desensitization, of a clinically important heptaspanning receptor.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA094346-09
Application #
6867372
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Yassin, Rihab R,
Project Start
1997-09-01
Project End
2008-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
9
Fiscal Year
2005
Total Cost
$293,036
Indirect Cost
Name
University of Illinois at Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Rivera, Claudio A; Ahlberg, Ned C; Taglia, Lauren et al. (2009) Expression of GRP and its receptor is associated with improved survival in patients with colon cancer. Clin Exp Metastasis 26:663-71
Taglia, Lauren; Matusiak, Damien; Benya, Richard V (2008) GRP-induced up-regulation of Hsp72 promotes CD16+/94+ natural killer cell binding to colon cancer cells causing tumor cell cytolysis. Clin Exp Metastasis 25:451-63
Jensen, R T; Battey, J F; Spindel, E R et al. (2008) International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol Rev 60:1-42
Matusiak, Damien; Benya, Richard V (2007) CYP27A1 and CYP24 expression as a function of malignant transformation in the colon. J Histochem Cytochem 55:1257-64
Taglia, Lauren; Matusiak, Damien; Matkowskyj, Kristina A et al. (2007) Gastrin-releasing peptide mediates its morphogenic properties in human colon cancer by upregulating intracellular adhesion protein-1 (ICAM-1) via focal adhesion kinase. Am J Physiol Gastrointest Liver Physiol 292:G182-90
Ruginis, Tom; Taglia, Lauren; Matusiak, Damien et al. (2006) Consequence of gastrin-releasing peptide receptor activation in a human colon cancer cell line: a proteomic approach. J Proteome Res 5:1460-8
Matusiak, Damien; Glover, Sarah; Nathaniel, Rajkumar et al. (2005) Neuromedin B and its receptor are mitogens in both normal and malignant epithelial cells lining the colon. Am J Physiol Gastrointest Liver Physiol 288:G718-28
Matusiak, Damien; Murillo, Genoveva; Carroll, Robert E et al. (2005) Expression of vitamin D receptor and 25-hydroxyvitamin D3-1{alpha}-hydroxylase in normal and malignant human colon. Cancer Epidemiol Biomarkers Prev 14:2370-6
Glover, Sarah; Nathaniel, Rajkumar; Shakir, Lubna et al. (2005) Transient upregulation of GRP and its receptor critically regulate colon cancer cell motility during remodeling. Am J Physiol Gastrointest Liver Physiol 288:G1274-82
Glover, Sarah; Delaney, Melissa; Dematte, Cecile et al. (2004) Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide's morphogenic properties. J Cell Physiol 199:77-88

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