A role for LIM kinase in cancer invasion

Abstract

In this study, we show that LIM kinase 1 (LIMK1), a critical regulator of actin dynamics, plays a regulatory role in tumor cell invasion. We found that the level and activity of endogenous LIMK1 is increased in invasive breast and prostate cancer cell lines in comparison with less invasive cells. Overexpression of LIMK1 in MCF-7 and in MDA-MB-231 human breast cancer cell lines increased their motility, whereas the specific ROCK and Rho inhibitors Y-27632 and C3, respectively, attenuated this effect. In addition, inhibition of LIMK1 activity in the MDA-MB-231 cells by expression of dominant-negative LIMK1 resulted in decreased motility and formation of osteolytic bone lesions in an animal model of tumor invasion. This study shows an important role for LIMK1 signaling in invasion of cancer, demonstrating its potential as a therapeutic molecular target to decrease metastasis.

Fig. 1.

Immunoblot and immunofluorescence analyses of endogenous LIMK1 expression in cell lines. (A Upper) Endogenous LIMK1 expression in various cell lines (lanes 1–9), and after alkaline phosphatase treatment of cell lysates from transformed and invasive cancer cell lines (lanes 10–12). (Lower) HSP70 expression to indicate loading of proteins. Lane 1, mouse olfactory epithelial cells; lane 2, monkey COS-7 cells; lane 3, 293T cells; lane 4, mouse NIH 3T3 fibroblasts; lanes 5 and 10, Ras-transformed NIH 3T3 fibroblasts; lane 6, breast cancer MCF-7 cells; lane 7, prostate cancer LNCaP cells; lanes 8 and 12, invasive prostate cancer PC-3 cells; lanes 9 and 11, invasive breast cancer MDA-MB-231 cells. (B) LIMK1 and vinculin expression in MCF-7 (Upper) and MDA-MB-231 cells (Lower), and LIMK1 localization in both cell-types after a 30-min treatment with 10 μM Y-27632 (Right). (C) LIMK1 and F-actin expression in nontreated MCF-7 and MDA-MB-231 cells. (Bars = 20 μm.)

Fig. 2.

LIMK1 expression and activity in MDA-MB-231 transfectants. (A) Immunoblots of cell lysates prepared from mock, wtLIMK1, and DN-LIMK1 MDA-MB-231 transfectants probed with rat monoclonal anti-LIMK1 (Upper) or β-actin antibodies, as loading control (Lower). The fold-change in the level of LIMK1 protein compared with the mock transfectants is indicated above Upper. The positions of endogenous and overexpressed tagged-LIMK1 proteins are indicated by double and single arrowheads, respectively. (B) Immunoblots of cell lysates prepared from mock, wtLIMK1, and DN-LIMK1 MDA-MB-231 transfectants probed with anti-phospho-cofilin (Upper) and pancofilin (Lower) antibodies. The fold-change in phosphorylated cofilin level is indicated above Upper. The data are representative of three independent experiments.

Fig. 3.

The invasiveness of LIMK1 MDA-MB-231 and MCF-7 transfectants. Cells from each cell line (5 × 10⁵ cells) were added onto the upper chamber of Matrigel-coated PET membrane. (A) For MDA-MB-231 mock transfectants or overexpressing wtLIMK1 (LIMK1), cells were left untreated or were treated with Y-27632 (10 μM) and added directly to the assay system, or pretreated for 24 h with 10 μg/ml C3. The number of migrating cells was determined after 6h.(B) Migration of MDA-MB-231 DN-LIMK1 or mock transfectants. (C) MCF-7 transfectants expressing wtLIMK1 (LIMK1) or V14RhoA and Δ4ROCK were analyzed after 20 h. The numbers at the bottom of each panel indicate the clone's number. (D) Immunoblots of cell lysates prepared from mock and wtLIMK1 MCF-7 transfectants probed with rat monoclonal anti-LIMK1 (Upper) and β-actin (Lower) antibodies. The fold-change in the level of LIMK1 protein compared with the mock transfectants is indicated above Upper. The positions of endogenous and overexpressed tagged-LIMK1 proteins are indicated by double and single arrowheads, respectively. (E) Immunoblots of cell lysates prepared from mock and wtLIMK1 MCF-7 transfectants probed with anti-phospho-cofilin (Upper) and pan-cofilin (Lower) antibodies. The fold-change in phosphorylated cofilin compared with the mock transfectants is indicated above Lower. All data are expressed as the number of migrated cells and are the average of three independent experiments. Error bars indicate SD. *, P < 0.01 to mock transfectants; #, P < 0.01 to Y (-) each transfectant.

Fig. 4.

Phosphorylated MLC-20 levels in MCF-7 transfectants. Immunoblots of cell lysates prepared from mock, wtLIMK1, V14RhoA, and Δ4ROCK MCF-7 transfectants probed with anti-phospho-MLC-20 antibodies. The position of phosphorylated MLC-20 (PMLC20) is indicated by arrows.

Fig. 5.

Osteolytic lesions caused by LIMK1-MDA-MB-231 cells in nude mice. The number and area of osteolytic lesions in nude mice bearing wtLIMK1-MDA-MB-231 (clone 3; 11 mice), DN-LIMK1 (clone 3; 11 mice), and mock (14 mice) transfected cells. The number and area of osteolytic lesions were scored 3 wk after cell inoculation on the radiographs by using quantitative image analysis.