Phosphatase of regenerating liver-1 promotes cell migration and invasion and regulates filamentous actin dynamics

Abstract

The phosphatases of regenerating liver (PRLs) are a unique family of plasma membrane-associated protein tyrosine phosphatases that have been hypothesized to be involved in metastatic cancer. How PRLs control cancer cell migration, invasion, and proliferation remains largely unknown. In the current study, we demonstrate a role for PRL-1 in the regulation of filamentous actin dynamics, which could promote cell metastatic processes. Human A549 non-small-cell lung cancer cells stably expressing wild-type PRL-1 exhibited a 60% increase in migration and a 3-fold increase in invasion. Cells expressing catalytic mutants of PRL-1 (C104S and D72A) lacked increased cell migration and invasion, indicating that these phenotypic changes required PRL-1 phosphatase activity. In contrast, PRL-1 small interfering RNA decreased in vitro lung cancer cell migration and invasion. The cadherin-catenin complex and dynamic filamentous actin are believed to control cellular invasiveness. Expression of wild-type PRL-1, but not phosphatase-inactive PRL-1 (C104S or D72A), decreased E-cadherin, vinculin, and paxillin expression. Ectopic expression of wild-type PRL-1 increased RhoA levels, which have an important role in actin filament assembly and stabilization of focal adhesion, and decreased activated Cdc42 and Rac. The Rho-associated protein kinase inhibitor, (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride (Y-27632), decreased RhoA activity, actin filament levels, and cellular migration and invasion in PRL-1-expressing cells. These results suggest that PRL-1 could be a productive cancer therapeutic target and support further efforts to identify its substrates.

Fig. 1.

Expression of PRL-1 protein and mRNA in human cancer cell lines. A, endogenous PRL-1 protein expression was examined by Western blotting, and mRNA levels were determined by RT-PCR in seven cancer cell lines and one normal lung cell line, Beas2B. B, endogenous PRL-1 protein expression levels were normalized to β-tubulin protein levels that were determined by Western blotting. PRL-1 mRNA expression levels were normalized to β-actin mRNA levels that were determined with Fujifilm Multi Gauge software (version 3.0), as described under Materials and Methods. The quantitative assessments of protein and mRNA levels are expressed as mean ± S.D. of three independent experiments.

Fig. 2.

PRL-1 promotes cell migration and invasion. A, based on Western blotting, the stable ectopic protein expression of Myc-tag PRL-1 WT and catalytic mutants in A549 cells was approximately the same as that for GAPDH. B, PRL-1 promotes cell motility in a scratch wound-healing assay. A monolayer of A549 cells stably expressing PRL-1 was scratched with a sterile micropipette tip. The number of cells in the denuded zone was determined after the indicated time (0 or 72 h) by inverted microscopy. Quantitative assessment of the mean number of cells in the denuded zone is expressed as the mean ± S.D. The experiments were repeated three times. ∗∗, P < 0.01 compared with vector control. B, PRL-1 promotes cell invasion and migration. Cell invasion of stably transfected A549 cells was assessed at 24 h using Matrigel invasion chambers, as described under Materials and Methods. Three fields in each well were counted and the mean percent invasion through the Matrigel matrix membrane was determined relative to the migration through the control membrane. The bar graph presents the mean relative values obtained from three independent determinations (±S.D.). ∗∗, P < 0.01 compared with the vector control.

Fig. 3.

Depletion of PRL-1 inhibits cell migration and invasion. A, Western blotting detects PRL-1 protein levels at 24 h after siRNA transfection. Protein expression levels are relative to GAPDH. B, reverse transcription-PCR detection of PRL-1 mRNA levels 24 h after siRNA transfection in A549 cells. mRNA levels are relative to β-actin. C, PRL-1 siRNA inhibits cell migration in the scratch wound-healing assay. Transiently siRNA transfected A549 cell monolayers were disrupted with a sterile micropipette tip. The number of cells in the denuded zone was determined at the indicated times (0 or 72 h) by inverted microscopy. Quantitative assessment of the mean number of cells in the denuded zone is expressed as mean ± S.D. The experiment was repeated three times. ∗, P < 0.05; ∗∗, P < 0.01, compared with scrambled control. B, PRL-1 siRNA inhibits cell invasion and migration. Cell invasion of transiently siRNA transfected A549 cells was assessed at 24 h using Matrigel invasion chambers, as described under Materials and Methods. Three fields in each well were counted, and the mean percent invasion through the Matrigel matrix membrane was determined relative to the migration through the control membrane. The bar graph presents the mean relative values obtained from three independent determinations (±S.D.). ∗, P < 0.05; ∗∗, P < 0.01, compared with the scrambled control.

Fig. 4.

PRL-1 regulates cell adhesion. A, cell lysates (40 μg) were analyzed by Western blotting in epithelial markers, E-cadherin, vinculin, and paxillin. GAPDH was used as a loading control. B, relative expression is average fold mean ± S.D. The protein level changes from three independent experiments normalized to vector. ∗, P < 0.05; ∗∗, P < 0.01, compared with vector control.

Fig. 5.

PRL-1 regulates the GTP-Rho family. A, PRL-1-activated RhoA. A549 cells stably expressing PRL-1 (WT), catalytic mutant (C104S and D72A), and empty vector were assessed for the level of active RhoA using a Rho activation assay kit. PRL-1 inactivates GTP-Cdc42 and GTP-Rac. A549 cells stably expressing PRL-1 (WT), catalytic mutant (C104S and D72A), and empty vector were assessed for the level of GTP-Cdc42 or GTP-Rac using a Cdc42/Rac activation assay kit. B, PRL-1-expressing WT and catalytic mutants were compared with empty vector. The data are expressed as means ± S.D. of Rho and Cdc42/Rac activation assays of three independent experiments. ∗, P < 0.05; ∗∗, P < 0.01, compared with the vector control.

Fig. 6.

PRL-1 activity is interrupted by Y-27632. A, A549 cells stably expressing PRL-1 (WT) were cultured in BME with 10% FBS with either 0.2% DMSO alone or 5 or 15 μM Y-27632 for 12 h. The cellular extracts were analyzed by Western blotting using Myc-Tag PRL-1, RhoA, Cdc42, Rac, and GAPDH as indicated. The expression levels of GTP-RhoA were assessed using a Rho activation assay kit. GAPDH was used as a loading control. B, PRL-1 with Y-27632 inhibits cell migration in the scratch wound-healing assay and cell invasion using Matrigel invasion chambers. ∗∗, P < 0.01 compared with WT. C–E, PRL-1 effects on cell shape and actin stress fiber after treatment with Y-27632. A549 cells stably expressing WT PRL-1 were cultured in BME with 10% FBS for 24 h. The cells were incubated in a 5% CO₂ incubator with either vehicle alone (C), 5 μM Y-27632 for 30 min (D), or 5 μM Y-27632 for 3 h at 37°C (E). The cells were then stained for actin stress fibers.