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J. Biol. Chem., Vol. 283, Issue 10, 6013-6021, March 7, 2008

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Suppression of the Invasive Capacity of Rat Ascites Hepatoma Cells by Knockdown of Slingshot or LIM Kinase^*

Yuji Horita, Kazumasa Ohashi, Mutsuko Mukai, Masahiro Inoue, and Kensaku Mizuno¹

From the Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan and the Department of Biochemistry, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan

Actin cytoskeletal reorganization is essential for tumor cell migration, adhesion, and invasion. Cofilin and actin-depolymerizing factor (ADF) act as key regulators of actin cytoskeletal dynamics by stimulating depolymerization and severing of actin filaments. Cofilin/ADF are inactivated by phosphorylation of Ser-3 by LIM kinase-1 (LIMK1) and reactivated by dephosphorylation by Slingshot-1 (SSH1) and -2 (SSH2) protein phosphatases. In this study, we examined the roles of cofilin/ADF, LIMK1, and SSH1/SSH2 in tumor cell invasion, using an in vitro transcellular migration assay. In this assay, rat ascites hepatoma (MM1) cells were overlaid on a primary-cultured rat mesothelial cell monolayer and the number of cell foci that transmigrated underneath the monolayer in the presence of lysophosphatidic acid (LPA) was counted. The knockdown of cofilin/ADF, LIMK1, or SSH1/SSH2 expression by small interfering RNAs (siRNAs) significantly decreased the LPA-induced transcellular migration of MM1 cells and their motility in two-dimensional culture. Knockdown of LIMK1 also suppressed fibronectin-mediated cell attachment and focal adhesion formation. Our results suggest that both LIMK1-mediated phosphorylation and SSH1/SSH2-mediated dephosphorylation of cofilin/ADF are critical for the migration and invasion of tumor cells and that LIMK1 is involved in the transcellular migration of tumor cells by enhancing both adhesion and motility of the cells.

Received for publication, August 7, 2007 , and in revised form, November 5, 2007.

^* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Science, Sports and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental videos S1–S4 and Figs. S1–S3.

¹ To whom correspondence should be addressed: Dept. of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan. Tel.: 81-22-795-6676; Fax: 81-22-795-6678; E-mail: kmizuno{at}biology.tohoku.ac.jp.

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