Abstract 4995: Loss of the lung-specific tumor suppressor Gprc5a causes persistent activation of Stat3 leading to increased survival signaling in normal and malignant lung epithelial cells

Abstract

Abstract The G protein-coupled receptor, family C, group 5, member A (GPRC5A), a retinoic acid induced gene expressed primarily in human and mouse lung tissue. Our previous studies demonstrated that mice with a deletion of the Gprc5a gene develop spontaneous lung adenomas and adenocarcinomas. Since Gprc5a is expressed preferentially in epithelial cells in lung tissues, we isolated epithelial cells from tracheas of Gprc5a+/+ and Gprc5a−/− mice and used them for an analysis of phenotypic and genotypic differences that might explain the mechanisms of Gprc5a actions. We found that normal Gprc5a−/− cells can survive better than Gprc5a+/+ cells in basal medium deprived of supplemented growth factors. Furthermore, the Gprc5a−/− cells but not the Gprc5a+/+ cells were able to form colonies in semi-solid medium. To explore possible mechanisms that underlay the distinct phenotypes of the above cells, we compared and contrasted the survival signaling pathway mediated by the Signal Transducer and Activator of Transcription 3 (STAT3), which is triggered by certain cytokines and growth factors to regulate the expression of genes implicated in cell growth differentiation, survival, inflammation and transformation. Increased Stat3 activity by phosphorylation has been identified in a variety of tumors including non-small cell lung cancers (NSCLCs) and it has been linked to inflammation and tumor progression. The constitutive activity (phosphorylation of tyrosine 705) of Stat3 and the expression of several Stat3 regulated genes including survivin, Bcl-XL, and Mcl1 at both the mRNA and protein levels was higher in the Gprc5a−/− cells compared to normal Gprc5a+/+ cells. Stat3 activation was triggered by autocrine Leukemia inhibitory factor (Lif), a member of the interleukine-6 (IL-6) family of cytokines, as revealed after adding neutralizing antibodies against different Stat3 activating cytokines to the growth medium. Although Gprc5a+/+ cells produced and secreted sufficient amounts of Lif into their conditioned medium, their response to Lif was transient, whereas the Gprc5a−/− cells showed a prolonged response evidenced by a persistent Stat3 activation. Lung cancer cells isolated from an adenocarcinorma of Gprc5a−/− mice exhibited Lif-mediated Stat3 activation similar to the normal Gprc5a−/− cells. Blocking Stat3 activation using AG490, an inhibitor of Stat3 signaling, or using a dominant negative Stat3 (mutant Y705F) in the Gprc5a−/− normal and cancer cells increased apoptosis and inhibited anchorage-independent growth. These results demonstrate that Stat3 activation may be important for the early stages of lung tumorigenesis in Gprc5a−/− mice and by implication that the tumor suppressive effects of Gprc5a are mediated, at least in part, by suppression of Stat3 signaling. Supported in part by the Samuel Waxman Cancer Research Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4995.