Abstract LB-244: Growth regulation in small cell lung cancer cells through G proteins: Analysis of Gq/11 and G12/13-dependent pathways
Authors: Büch Thomas , Schäfer Eva , Grzelinski Marius , Stohr Stefanie , Pinkenburg Olaf , Gudermann Thomas , Aigner Achim Michael 
Published: February 2011

Background: The activation of G protein-coupled receptors by autocrine factors represents a principal growth regulating signal in small cell lung cancer (SCLC) cells, which relies on the concerted parallel activation of Gq/11- and G12/13-dependent signaling pathways. However, the molecular effectors of these pathways and the relative contributions of either signaling cascade to the cellular phenotype of SCLC cells are not clearly defined.

Materials and Methods: In the present study we further characterised the molecular make-up and the phenotypic implications of Gq/11- and G12/13-dependent signaling in SCLC cells. For this purpose, we employed small hairpin RNA (shRNA)-mediated knockdown of G 12, G 13, or both, in the SCLC cell lines H69, H209, and H510. We analyzed the effects of G 12 and/or G 13 knockdown on mitogenic signaling cascades as well as on proliferation in vitro and tumor growth in vivo. Furthermore, we selectively modulated Gq/11-regulated effectors by employing shRNA-mediated gene knockdown, forced expression of mutant signaling molecules or pharmacological inhibitors. For comparison, a non-small cell lung cancer (A549) and a neuroendocrine cell line (PC-12) were also included.

Results: In SCLC cell lines, lentiviral expression of shRNAs against G 12, G 13, or both, inhibited basal as well as bradykinine-promoted proliferation in vitro. In contrast, G 12 or G 13 knockdown did not exert antiproliferative effects in non-SCLC cells. The analysis of the activation status of all three major MAPK families revealed nonredundant functions of G 12 and G 13 in SCLC cells. In an s.c. SCLC xenograft mouse model, G 12 or G 13 downregulation resulted in reduced tumor growth due to decreased tumor cell proliferation. More strikingly, G 12/G 13 double knockdown completely abolished SCLC tumorigenicity in mice. Regarding Gq/11−-regulated signaling, we identified the non-receptor tyrosine kinase Pyk2 asa master switch which is able to promote either mitogenic or pro-apoptotic signaling. Pyk2 was indispensable to mediate neuropeptide/Ca2+-dependent activation of the Ras-ERK1/2 cascade, and the downregulation of Pyk2 severely inhibited proliferation of SCLC cells. On the other hand, overexpression of Pyk2 strongly induced apoptosis in SCLC, but not in PC12 cells. This pro-apoptotic effect of Pyk2 was independent of its central kinase domain, but dependent on an intact PPPKP717SRP motif responsible for activation of p130Cas-promoted signaling.

Conclusions: The activation of G12/13 and Gq/11−signaling exerts a complex pattern of nonredundant effects in SCLC. In contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G12/13 signaling. The complete abolishment of in vivo tumor growth in our study suggests that RNAi-mediated double knockdown of G 12 and G 13, may provide a promising new avenue in SCLC treatment. Finally, Pyk2 represents a point of convergence of Gq/11-dependent pathways in SCLC cells and is able to promote either mitogenic or pro-apoptotic effects depending on the signaling context.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 LB-244.