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E1AF/PEA3 Activates the Rho/Rho-Associated Kinase Pathway to Increase the Malignancy Potential of Non–Small-Cell Lung Cancer Cells

¹ First Department of Medicine and ² Department of Medical Oncology, Hokkaido University Graduate School of Medicine and ³ Department of Biology, Sapporo Medical University School of Medicine, Sapporo, Japan

Requests for reprints: Ichiro Kinoshita, Department of Medical Oncology, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan. Phone: 81-11-706-5551; Fax: 81-11-706-5077; E-mail: kinoshii{at}med.hokudai.ac.jp.

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.

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