E1AF/PEA3 activates the Rho/Rho-associated kinase pathway to increase the malignancy potential of non-small-cell lung cancer cells.
E1AF/PEA3, an Ets family transcription factor, is frequently overexpressed in non-small-cell lung cancers (NSCLCs). Overexpression of E1AF increases motility and invasion of VMRC-LCD and NCI-H226 NSCLC cells, which lack endogenous E1AF expression, and the effect is synergistically increased by hepatocyte growth factor (HGF). The small GTPase Rho/Rho-associated kinase (ROCK) pathway is also involved in motility and invasion. To determine the role of the Rho/ROCK pathway in malignant phenotypes induced by E1AF, we analyzed VMRC-LCD cells transfected with an E1AF expression vector (LCD-E1AF cells) or with empty vector (LCD-vector cells). LCD-E1AF cells had more GTP-bound (active) Rho than LCD-vector cells and Rho activation was synergistically increased by HGF. The Rho activation by E1AF and HGF was also shown in NCI-H226 cells. Phosphorylation of myosin light chain (MLC), a downstream effector of ROCK signaling, was higher in LCD-E1AF cells than in LCD-vector cells, especially under HGF treatment. A specific ROCK inhibitor, Y27632, strongly suppressed MLC phosphorylation, cell motility, and invasion. In nude mice implanted s.c. and intrapulmonarily, LCD-E1AF cells made more local tumors than LCD-vector cells (six of six versus one of seven mice and four of seven versus one of seven mice, respectively). Three of the four mice with lung tumors from LCD-E1AF cells had lymph node metastases whereas the mouse with LCD-vector tumors did not. LCD-E1AF tumors showed higher MLC phosphorylation than LCD-vector tumors. These results suggest that E1AF activates the Rho/ROCK pathway in an HGF-enhanced manner and its activation is important in E1AF-induced motility and invasion as well as tumorigenesis and metastasis in NSCLC cells.
Hakuma
Nobuyuki
N
First Department of Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
Kinoshita
Ichiro
I
Shimizu
Yasushi
Y
Yamazaki
Koichi
K
Yoshida
Koichi
K
Nishimura
Masaharu
M
Dosaka-Akita
Hirotoshi
H
D017500:Adenovirus E1A Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation; D005260:Female; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D050783:Proto-Oncogene Proteins c-ets; D011725:Pyridines / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D054460:rho-Associated Kinases
D017500:Adenovirus E1A Proteins; D000577:Amides; C079853:ETV4 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011725:Pyridines; D014157:Transcription Factors; C065788:transcription factor PEA3; C108830:Y 27632; D017228:Hepatocyte Growth Factor; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases