Gastrin-releasing peptide receptor upregulation in human lung adenocarcinoma

4405 Gastrin-releasing peptide (GRP) is a mitogen for lung epithelial cells and initiates signaling through a G protein-coupled receptor, gastrin-releasing peptide receptor (GRPR). Previous work from our laboratory has shown that GRP induces Akt activation through a c-Src-mediated transactivation of the epidermal growth factor receptor (EGFR) in non-small cell lung carcinoma (NSCLC) cells. In this study, we investigated the level of GRPR protein expression in human lung tissue microarrays (TMA) consisting of 26 cases of NSCLC (19 adenocarcinomas, 3 squamous cell carcinomas, 4 designated as “other”) as well as matched normal lung tissue from 25 cases and positive and negative lymph nodes from 7 and 9 cases, respectively. Immunostaining was carried out using a GRPR antibody recognizing the 2 nd extracellular domain of the human form (MBL Intl., Woburn, MA), which in an immunoblot gives a single band migrating at 50kD. GRPR immunostaining was scored by ranking the intensity of staining from 0-3 and the percentage of positive cells from 0-5, with the sum of the two rankings accounting for the total staining score. For all cases combined, the median total GRPR staining score was 3.50 in normal lung compared to 5.38 in tumor tissue (P = 0.0002, Mann-Whitney test). The difference in GRPR staining between adenocarcinoma tissue (median 5.6) and matched normal tissue (median 3.38) was significant (P = 0.0001, Mann-Whitney test), while the difference in GRPR staining score between squamous cell and other tumor types combined was not significantly different from their matched normal lung. Lymph nodes that scored positive for tumor infiltration by H & E staining had a significantly higher median GRPR staining score (5.65) compared to nodes scored negative for tumor infiltration (score 1.25, P = 0.005, Mann-Whitney test). For adenocarcinoma cases alone, the median GRPR score in positive nodes was 5.8, compared to 0 in negative nodes. These results suggest that GRPR protein expression is increased in human lung adenocarcinomas compared to its surrounding normal lung tissue. Furthermore, we have shown that GRP can rescue NSCLC cells exposed to gefitinib through release of amphiregulin and activation of Akt. Amphiregulin release is blocked by expression of a dominant negative Src. Pretreatment of NSCLC cells with 100 nM GRP resulted in a 5-fold increase in the IC 50 of gefitinib. This protective effect was mimicked by pre-treatment of cells with amphiregulin and reversed by inhibition of AKT using the PI-3kinase inhibitor LY294002. These results suggest that GRPR and/or EGFR autocrine pathways may modulate therapeutic responses to EGFR inhibitors, and that GRPR may be a novel target for therapy in NSCLC either alone or in combination with EGFR inhibitors. This work was supported by the National Cancer Institute Lung SPORE grant P50 CA090440 to J.M.S.