The Roles Of Lung Tumor Suppressor GPRC5A In Lung Tumorigenesis

Objectives: To investigate the key signaling pathways for GPRC5A-mediated tumor suppression via Gprc5a-knock out(ko) mouse model, and to explore the roles and biological significance of GPRC5 A in development of NSCLC(Non-Small Cell Lung Cancer).Materials and methods: 1. The differential gene expression profile of the global m RNAs in Gprc5a-/- MTECs(from a Gprc5a-ko mouse) vs Gprc5a+/+ MTECs(from a wide-type mouse) was obtained by Affymatrix analysis. Validation of deregulated signaling pathways in Gprc5a-/- vs Gprc5a+/+ MTECs was performed by RT-PCR and immunoblot analysis. 2. A series of GPRC5 A deletion mutants were constructed for determination of the functional domains of GPRC5 A, such as their effects on EGFR-mediated activation of STAT3, p-STAT3 and STAT3-driven reporter. The interaction between GPRC5 A and EGFR was determined by using co-immunoprecipitation and immunoflorescent assay. 3. Gprc5a-/- MTEC were treated with various inhibitors, such as EGFR tyrosine kinase inhibitors, Gefitinib, Erlotinib and AG1478, and examined for their survival and proliferation by CCK-8 kit. 4. Lung tissues from various groups of mice, were harvested for measurement of EGFR and STAT3 status, by RT-PCR and Westernblot analysis. 5. Stable GPRC5 A transfectants of H1975 and A549 were developed for investigation if overexpression of exogenous GPRC5 A inhibits EGFR signaling in human lung cancer cells; GPRC5 A stable transfectants of A549 and parental cells were subcutaneously injected into nude mice, and tumor sizes were measured as for biological effects of GPRC5 A expression.Results: 1. The results of Affymatrix m RNAs chips analysis showed that EGFR signaling pathway is deregulated in Gprc5a-/- vs Gprc5a+/+ MTECs. 2. The results of RT-PCR and Westernblot showed that the expression and activation of EGFR and STAT3 were much higher in Gprc5a-/- MTECs than in Gprc5a+/+ MTECs; In HEK293 T cells, overexpression of GPRC5 A inhibited the activation of EGFR signaling pathway. 3. The transmembrane(7TM) domain of GPRC5 A is required for inhibition of EGFR signaling, since the tansmembrane deletion mutant((35)7TM) lost the inhibition activity; GPRC5 A physically interacts with EGFR via its transmembrane domains, and these two molecules were colocalized in vivo.4. Gprc5a-/- MTECs were much more sensitive to EGFR inhibitors than Gprc5a+/+ cells, but the susceptibility to other inhibitors was similar. 5. p-EGFR and p-STAT3 in Gprc5a-/- mouse lungs were much higher than those in Gprc5a+/+ mouse lungs 6. Overexpression of GPRC5 A inhibited the activation of EGFR signaling in human lung cancer cells H1975 and A549; and GPRC5 A expression suppressed the tumor growth of human lung cancer cells A549 xenografts in nude mice.Conclusions: 1. EGFR-STAT3 signaling is deregulated in Gprc5a-/- MTECs, and GPRC5 A could negatively regulate EGFR mediated signaling. 2. Transmembrane domain of GPRC5 A is required for inhibition of EGFR signaling, and GPRC5 A can physically interact with EGFR via its transmembrane domain. 3. Gprc5a-/- MTECs are EGFR signaling-dependent. 4. EGFR-STAT3 signaling pathway is aberrantly activated in the lung tissues of Gprc5a-ko mice. 5. Overexpression of GPRC5 A can inhibit the activation of EGFR signaling pathway in human lung cancer cells and the growth of A549 xenografts in nude mice.