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.
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