): This proposal is directed to establishing the role of the gastrin-releasing p e p tide receptor (GRP-R) in causing the proliferation of non-antral adenocarcinomas of the stomach. GRP-R are not normally expressed by mucosal epithelial cells lining the gastrointestinal (GI) tract except in the antrum of the stomach. However, the applicants show that 40-50 percent of non-antral gastric cancers aberrantly express GRP-R, and provide data showing that these receptors can be activated by 2 different mechanisms. 1), GRP-R are often mutated such that they become constitutively active. This finding provides a non-mechanism whereby aberrant GRP-R expression alone can cause proliferation. 2), Immunohistochemical studies show that many, but not all tumors aberrantly e x press both receptor and ligand. Thus the proliferation of gastric adenocarcinomas may occur secondary to autocrine activation by GRP. Three separate approaches will be used to further elucidate the role of GRP/GRP-R in gastric cancer proliferation. I. Prospectively, RNA will be e x t racted from all patients with newly diagnosed non-antral gastric adenocarcinoma at the time of initial endoscopic examination. GRP-R expression will be determined by RTPCR, and mutations screened for using the novel technique of conformational fragment length polymorphism (CFLP) analysis. Mutated GRP-R will be re-created by site-directed mutagenesis, and their functional consequence determined in transiently transfected CHO-KI cells. II. Retrospectively, the applicants will study 168 consecutive patients with gastric adenocarcinomas who underwent operative resection of their tumor between 1980-96. The paraffin blocks of these tumors have been collected and will be evaluated for both GRP and GRP-R expression by immunohistochemistry using specific antibodies. Initial and follow-up clinical data exists for these patients, allowing the applicants to correlate aberrant ligand and receptor expression with tumor stage, patient survival, and response to chemotherapy. To conclusively establish the role of GRP-R expression in gastric cancer, they will study a recently developed GRP-R """"""""knock out"""""""" mouse. Knock-out mice, which have been genetically rendered incapable of expressing GRP-R. will be evaluated alongside wild type mice fed N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), a gastric cancer-inducing agent, and the propyl derivative PNNG, which only causes intestinal metaplasia. These studies will allow the applicants to determine the contribution of the GRP-R to the development and progression of gastric adenocarcinomas, and potentially will lead to therapeutic advances in the treatment of this disease.
