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.
OBJECTIVE
In small cell lung cancer cells (SCLC), various autocrine stimuli lead to the parallel activation of G(q/11) and G(12/13) proteins. Although the contribution of the G(q/11)-phospholipase C-beta cascade to mitogenic effects in SCLC cells is well established, the relevance of G(12/13) signaling is still elusive. In other tumor entities, G(12/13) activation promotes invasiveness without affecting cellular proliferation. Here, we investigate the role of G(12/13)-dependent signaling in SCLC.


METHODS
We used small hairpin RNA-mediated targeting of G(alpha)(12), G(alpha)(13), or both in H69 and H209 cells and analyzed the effects of G(alpha)(12) and/or G(alpha)(13) knockdown on tumor cells in vitro, tumor growth in vivo, and mitogen-activated protein kinase (MAPK) activation.


RESULTS
Lentiviral expression of small hairpin RNAs resulted in robust and specific G(alpha)(12) and G(alpha)(13) knockdown as well as markedly inhibited proliferation, colony formation, and bradykinin-promoted stimulation of cell growth. Analyzing the activation status of all three major MAPK families revealed nonredundant functions of G(alpha)(12) and G(alpha)(13) in SCLC and a marked p42/p44 activation upon G(alpha)(12)/G(alpha)(13) knockdown. In a s.c. tumor xenograft mouse model, G(alpha)(12) or G(alpha)(13) downregulation led to decreased tumor growth due to reduced tumor cell proliferation. More importantly, G(alpha)(12)/G(alpha)(13) double knockdown completely abolished H69 tumorigenicity in mice.


CONCLUSIONS
G(alpha)(12) and G(alpha)13) exert a complex pattern of nonredundant effects in SCLC, and in contrast to other tumor types, SCLC cell proliferation in vitro and tumorigenicity in vivo critically depend on G(12/13) signaling. Due to the complete abolishment of tumorgenicity in our study, RNAi-mediated double knockdown may provide a promising new avenue in SCLC treatment.
Department of Pharmacology and Toxicology, Philipps-University School of Medicine, Marburg, Germany.
Marius
M
Grzelinski
Olaf
O
Pinkenburg
Thomas
T
Büch
Maike
M
Gold
Stefanie
S
Stohr
Hermann
H
Kalwa
Thomas
T
Gudermann
Achim
A
Aigner
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D055752:Small Cell Lung Carcinoma / Q000378:metabolism* / Q000473:pathology; D014162:Transfection
D044365:GTP-Binding Protein alpha Subunits, G12-G13