ABB  Vol.5 No.1 , January 2014
Cell-type specific and non-redundant anti-proliferative effects of shRNA-mediated Galpha12- and Galpha13 knockdown in lung cancer cell lines

In small cell lung cancer cells, various autocrine stimuli lead to the parallel activation of Gq/11 and G12/13 proteins. The contribution of the Gq/11-PLC-β cascade to the mitogenic effects in SCLC cells is well established, but the relevance of G12/13 signaling is less explored. While in prostate and breast cancer, G12/13 activation has been shown previously to promote invasiveness without being involved in cellular proliferation, previous data from our group indicate anti-proliferative effects of G12/13 knockdown in small cell lung cancer (SCLC) cells. To further investigate the role of G12/13-dependent signaling in lung tumor cells, we employed shRNA-mediated targeting of Gα12, Gα13, or both, in SCLC and NSCLC cell lines. Lentiviral expression of shRNAs resulted in specific Gα12 and Gα13 knockdown. Of note, upon single knockdown of one family member, no counter-upregulation of the other one was observed. Interestingly, inhibition of proliferation was cell line dependent. In cell lines where knock-down led to antiproliferation, single knockdown of either Gα12 or Gα13 was sufficient to impair proliferation and double knockdown of Gα12 and Gα13 tended not to further increase anti-proliferative effects. Likewise, when single knockdown was insufficient for an inhibition of proliferation, no effects were observed in double knockdowns. Taken together, these findings indicate that both Gα12 and Gα13 affect cellular proliferation individually and interference with one family member is sufficient for anti-tumor effects.

Cite this paper
Büch, T. , Grzelinski, M. , Pinkenburg, O. , Gudermann, T. and Aigner, A. (2014) Cell-type specific and non-redundant anti-proliferative effects of shRNA-mediated Galpha12- and Galpha13 knockdown in lung cancer cell lines. Advances in Bioscience and Biotechnology, 5, 73-80. doi: 10.4236/abb.2014.51011.
[1]   Govindan, R., Page, N., Morgensztern, D., Read, W., Tierney, R., Vlahiotis, A., Spitznagel, E.L. and Piccirillo, J. (2006) Changing epidemiology of small-cell lung cancer in the United States over the last 30 years: Analysis of the surveillance, epidemiologic, and end results database. Journal of Clinical Oncology, 24, 4539-4544.

[2]   Heasley, L.E. (2001) Autocrine and paracrine signaling through neuropeptide receptors in human cancer. Oncogene, 20, 1563-1569.

[3]   Gudermann, T. and Roelle, S. (2006) Calcium-dependent growth regulation of small cell lung cancer cells by neuropeptides. Endocrine-Related Cancer, 13, 1069-1084.

[4]   Grzelinski, M., Pinkenburg, O., Buch, T., Gold, M., Stohr, S., Kalwa, H., Gudermann, T. and Aigner, A. (2010) Critical role of G(alpha)12 and G(alpha)13 for human small cell lung cancer cell proliferation in vitro and tumor growth in vivo. Clinical Cancer Research, 16, 1402-1415.

[5]   Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W.S. and Khvorova, A. (2004) Rational siRNA design for RNA interference. Nature Biotechnology, 22, 326-330.

[6]   Elbashir, S.M., Harborth, J., Weber, K. and Tuschl, T. (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 26, 199213.

[7]   Smrcka, A.V. (2013) Molecular targeting of Galpha and Gbetagamma subunits: A potential approach for cancer therapeutics. Trends in Pharmacological Sciences, 34, 290-298.

[8]   Wu, J., Xie, N., Zhao, X., Nice, E.C. and Huang, C. (2012) Dissection of aberrant GPCR signaling in tumorigenesis—A systems biology approach. Cancer Genomics & Proteomics, 9, 37-50.

[9]   Entschladen, F., Zanker, K.S. and Powe, D.G. (2011) Heterotrimeric G protein signaling in cancer cells with regard to metastasis formation. Cell Cycle (Georgetown, Tex), 10, 1086-1091.

[10]   Spiegelberg, B.D. and Hamm, H.E. (2007) Roles of G-protein-coupled receptor signaling in cancer biology and gene transcription. Current Opinion in Genetics & Development, 17, 40-44.

[11]   Hepler, J.R. and Gilman, A.G. (1992) G proteins. Trends in Biochemical Sciences, 17, 383-387.

[12]   Chan, A.M., Fleming, T.P., McGovern, E.S., Chedid, M., Miki, T. and Aaronson, S.A. (1993) Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product. Molecular and Cellular Biology, 13, 762-768.

[13]   Xu, N., Bradley, L., Ambdukar, I. and Gutkind, J.S. (1993) A mutant alpha subunit of G12 potentiates the eicosanoid pathway and is highly oncogenic in NIH 3T3 cells. Proceedings of the National Academy of Sciences of the United States of America, 90, 6741-6745.

[14]   Xu, N., Voyno-Yasenetskaya, T. and Gutkind, J.S. (1994) Potent transforming activity of the G13 alpha subunit defines a novel family of oncogenes. Biochemical and Biophysical Research Communications, 201, 603-609.

[15]   Gardner, J.A., Ha, J.H., Jayaraman, M. and Dhanasekaran, D.N. (2013) The gep proto-oncogene Galpha13 mediates lysophosphatidic acid-mediated migration of pancreatic cancer cells. Pancreas, 42, 819-828.

[16]   Ha, J.H., Ward, J.D., Varadarajalu, L., Kim, S.G. and Dhanasekaran, D.N. (2014) The gep proto-oncogene Galpha12 mediates LPA-stimulated activation of CREB in ovarian cancer cells. Cellular Signalling, 26, 122-132.

[17]   Offermanns, S. and Schultz, G. (1994) What are the functions of the pertussis toxin-insensitive G proteins G12, G13 and Gz? Molecular and Cellular Endocrinology, 100, 71-74.

[18]   Kelly, P., Casey, P.J. and Meigs, T.E. (2007) Biologic functions of the G12 subfamily of heterotrimeric g proteins: Growth, migration, and metastasis. Biochemistry, 46, 6677-6687.

[19]   Dhanasekaran, N. and Dermott, J.M. (1996) Signaling by the G12 class of G proteins. Cellular signalling, 8, 235-245.

[20]   Kozasa, T., Hajicek, N., Chow, C.R. and Suzuki, N. (2011) Signalling mechanisms of RhoGTPase regulation by the heterotrimeric G proteins G12 and G13. The Journal of Biochemistry, 150, 357-369.

[21]   Juneja, J., Cushman, I. and Casey, P.J. (2011) G12 signaling through c-Jun NH2-terminal kinase promotes breast cancer cell invasion. PloS one, 6, E26085.

[22]   Radhakrishnan, R., Ha, J.H. and Dhanasekaran, D.N. (2010) Mitogenic signaling by the gep oncogene involves the upregulation of s-phase kinase-associated protein 2. Genes & cancer, 1, 1033-1043.

[23]   Touge, H., Chikumi, H., Igishi, T., Kurai, J., Makino, H., Tamura, Y., Takata, M., Yoneda, K., Nakamoto, M., Suyama, H., Gutkind, J.S. and Shimizu, E. (2007) Diverse activation states of RhoA in human lung cancer cells: Contribution of G protein coupled receptors. Journal of Clinical Oncology, 30, 709-715.

[24]   Rubinson, D.A., Hochster, H.S., Ryan, D.P., Wolpin, B.M., McCleary, N.J., Abrams, T.A., Chan, J.A., Iqbal, S., Lenz, H.J., Lim, D., Rose, J., Bekaii-Saab, T., Chen, H.X., Fuchs, C.S. and Ng, K. (2013) Multi-drug inhibition of the HER pathway in metastatic colorectal cancer: Results of a phase I study of pertuzumab plus cetuximab in cetuximab-refractory patients. Investigational New Drugs.

[25]   Beekman, A., Helfrich, B., Bunn, P.A., Jr. and Heasley, L.E. (1998) Expression of catalytically inactive phospholipase Cbeta disrupts phospholipase Cbeta and mitogenactivated protein kinase signaling and inhibits small cell lung cancer growth. Cancer Research, 58, 910-913.

[26]   Wittau, N., Grosse, R., Kalkbrenner, F., Gohla, A., Schultz, G. and Gudermann, T. (2000) The galanin receptor type 2 initiates multiple signaling pathways in small cell lung cancer cells by coupling to G(q), G(i) and G(12) proteins. Oncogene, 19, 4199-4209.