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Polo-like kinase 1 induces epithelial-to-mesenchymal transition and promotes epithelial cell motility by activating CRAF/ERK signaling

  1. Jianguo Wu
  2. Andrei I Ivanov
  3. Paul B Fisher
  4. Zheng Fu  Is a corresponding author
  1. Virginia Commonwealth University School of Medicine, United States
Research Article
Cite this article as: eLife 2016;5:e10734 doi: 10.7554/eLife.10734
8 figures and 2 videos

Figures

Figure 1 with 3 supplements
Ectopic expression of PLK1 in RWPE-1 cells promotes cell motility.

(A) Cell lysates were prepared from the indicated PCa cell lines and subjected to Western blots in order to detect the level and activity of PLK1 protein using anti−PLK1 and anti−PLK1(pT210) antibodies, respectively. β-actin was used as a loading control. (B) RWPE-1 cells were infected with lentivirus encoding Flag-PLK1 (PLK1) or empty vector (EV). The protein levels of PLK1, AR, PSA, and β-actin were determined by Western blot. C4-2B cells with high endogenous PLK1 expression were included for comparison. (C) Control RWPE-1 and RWPE-1–PLK1 cells were subjected to a wound healing assay. The figure shows representative images as well as calculated percentage of wound closure during 48 hr of cell migration. Scale bar, 500 µm. (D) In vitro Matrigel invasion assay. The figure shows representative images of invaded cells and quantification of the relative number of cells that invaded over 48 hr. The data are presented as the mean ± s.e.m. *p<0.01, two-tailed Student’s t-test. Scale bar, 100 µm. (EG) Time-lapse video microscopy motility experiments to monitor random migration of control and PLK1-overexpressing RWPE-1 cells. The trajectories of individual cells of different experimental groups (E), track distance (F), and velocity of cell migration (G) are shown. Horizontal bars in F-G represent median and interquartile range. Each dot represents a single-cell measurement. Thirty cells per experimental group were measured. *p<0.01, two-tailed Mann-Whitney rank sum tests.

https://doi.org/10.7554/eLife.10734.003
Figure 1—figure supplement 1
Androgen receptor (AR) mRNA is expressed in RWPE-1 cells.

Total RNA from control RWPE-1 (EV) and RWPE-1–PLK1 (PLK1) cells was subjected to real-time RT-PCR. AR mRNA levels were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) and are expressed as relative expression values. The data are presented as the mean ± s.e.m. *p<0.05, two-tailed Student’s t-test.

https://doi.org/10.7554/eLife.10734.004
Figure 1—figure supplement 2
Ectopic expression of PLK1 in RWPE-1 cells induces cellular transformation and tumorigenicity.

(A) The effect of PLK1 upregulation in RWPE-1 cells on anchorage-independent growth in soft agar. RWPE-1–PLK1 (PLK1) cells and vector control (EV) cells were plated in soft agar and grown for 21 days. The data are presented as the mean ± s.e.m. *p<0.01, two-tailed Student’s t-test. (B, C) Control RWPE-1 or PLK1-overexpressing cells (1×106) were injected subcutaneously into the flanks of NSG mice. Seven mice were used per group. Tumor growth was observed for 5 weeks. (B) Images of primary tumors found in NSG mice subcutaneously engrafted with RWPE-1–PLK1 cells. The table below summarizes the tumor incidence in the 2 groups. (C) Representative images of H & E staining and PSA IHC of primary tumor and lung metastasis. Arrows point to lung micrometastases. Scale bar = 500 μm.

https://doi.org/10.7554/eLife.10734.005
Figure 1—figure supplement 3
Ectopic expression of PLK1 in PrEC cells promotes cell migration and invasion.

(A) Modulated PLK1 levels in PrEC cells. PrEC cells were infected with lentivirus encoding Flag-PLK1 (PLK1) or control vector (EV). PLK1 protein levels were determined by Western blot. (B) A migration assay was performed and a quantitative analysis is shown. (C) The in vitro invasiveness of those cells was measured using Matrigel-coated Transwell chambers. The data are presented as the mean ± s.e.m. *p<0.01, two-tailed Student’s t-test.

https://doi.org/10.7554/eLife.10734.006
Figure 2 with 2 supplements
Overexpressing PLK1 in RWPE-1 cells induces EMT.

(A) Representative phase-contrast images of control RWPE-1 and RWPE-1–PLK1 cells. Scale bar = 50 μm. (B, C) mRNA and protein expression of different EMT markers in RWPE-1–PLK1 cells (PLK1) and vector control cells (EV) were examined by real-time RT-PCR (B) and Western blot (C), respectively. The data are presented as the mean ± s.e.m. *p<0.01, two-tailed Student’s t-test. (D) RWPE-1 cells were infected with lentivirus expressing wild-type PLK1 (WT), constitutively active T210D (TD), or kinase-dead K82M (KM) PLK1 mutants. Expression of EMT markers was examined by immunoblotting. (E) The architecture of adherens junctions (E-cadherin and β-catenin labeling), tight junctions (JAM-A), and the actomyosin cytoskeleton (myosin IIB) in control and PLK1-overexpressing cells was evaluated by immunolabeling and confocal microscopy. Arrows indicate disrupted adherens junctions and tight junctions, whereas arrowheads point to basal stress fibers in PLK1-overexpressing cells. Scale bar, 20 µm.

https://doi.org/10.7554/eLife.10734.009
Figure 2—figure supplement 1
The effect of PLK1 overexpression on EMT is independent of its cell cycle function.

RWPE-1–PLK1 (PLK1) cells and vector control (EV) cells were left either unsynchronized (Asyn) or synchronized at G1, S, and M phases. The expression of EMT markers in these cells was examined by Western blotting.

https://doi.org/10.7554/eLife.10734.010
Figure 2—figure supplement 2
Overexpression of PLK1 in PrEC cells induces EMT.

(A) Representative phase-contrast images of control PrEC (EV) and PrEC-PLK1 (PLK1) cells. Scale bar = 50 μm. (B, C) EMT marker mRNA and protein expression in PrEC cells (PLK1) and vector control cells (EV) was examined by real-time RT-PCR (B) and Western blot (C), respectively. The data are presented as the mean ± s.e.m. *p<0.01, two-tailed Student’s t-test.

https://doi.org/10.7554/eLife.10734.011
Figure 3 with 4 supplements
Downregulation of PLK1 reverses EMT and inhibits the motility of metastatic PCa cells.

(A) Two metastatic PCa cell lines (DU145 and C4-2B) were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#1) or serve as a control (shCTL). Wild-type (WT) or kinase-defective (KM) PLK1 were then re-expressed in PLK1 knockdown cells. The expression of PLK1 protein and EMT markers was determined by immunoblotting. In the PLK1 immunoblot, the lower bands show endogenous PLK1 expression and the upper bands show exogenous PLK1 expression. (B) Representative phase-contrast images of control, PLK1 knockdown cells, and PLK1 knockdown cells with re-expression of WT or KM PLK1. Scale bar = 50 μm. (C) The architecture of adherens junctions (E-cadherin) and tight junctions (JAM-A) in control cells and cells with PLK1 manipulation was evaluated by immunolabeling and confocal microscopy. Scale bar, 20 µm. (D, E) The effects of PLK1 knockdown on planar migration and invasion of PCa cells were measured by the wound healing assay (D) and Matrigel invasion assay (E), respectively. The data are presented as the mean ± s.e.m. *p<0.01, two-tailed Student’s t-test. Scale bar, 500 µm (D), and 100 µm (E).

https://doi.org/10.7554/eLife.10734.012
Figure 3—figure supplement 1
Partial knockdown of PLK1 in PCa cells has minimal effect on cell cycle progression and cell death.

(A) The effect of PLK1 downregulation on cell cycle distributions was analyzed by flow cytometric analysis. (B) The effect of PLK1 downregulation on apoptosis was determined by staining with annexin V and PI solution, followed by flow cytometric analysis.

https://doi.org/10.7554/eLife.10734.013
Figure 3—figure supplement 2
Downregulation of PLK1 results in EMT reversion and reduction of migration of metastatic PCa cells.

(A) Three metastatic PCa cell lines (PC3, C4-2B, and DU145) were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#2) or serve as a control (shCTL). The expression of PLK1 protein and EMT markers was determined by immunoblotting. (B) Representative phase-contrast images of control and PLK1 knockdown cells. Scale bar = 50 μm. (C) The architecture of adherens junctions (E-cadherin) and tight junctions (JAM-A) in control and PLK1 downregulated cells was evaluated by immunolabeling and confocal microscopy. Scale bar, 20 µm. (D, E) The effects of PLK1 knockdown on planar migration and invasion of PCa cells were measured by wound healing assay (D) and Matrigel invasion assay (E), respectively. The data are presented as the mean ± s.e.m. *p<0.01, two-tailed Student’s t-test. Scale bar, 500 µm (D), and 100 µm (E). (F) The effect of PLK1 downregulation on cell proliferation was determined by MTS assay as described in 'Materials and methods'.

https://doi.org/10.7554/eLife.10734.014
Figure 3—figure supplement 3
Validation of the role of PLK1 in induction of EMT in the ARCaP cell culture model.

(A) The level and activity of PLK1 protein in ARCaPE and ARCaPM cells were examined by Western blotting analysis using anti-PLK1 and anti-PLK1(pT210) antibodies, respectively. β-actin was used as a loading control. (B) ARCaPE cells were transiently transfected with a PLK1 expression construct. The expression of PLK1 protein was determined by immunoblotting. The effects of PLK1 overexpression on EMT marker expression, morphological changes, assembly of intracellular junctions, and invasiveness were determined as described in Figures 12. (C) The endogenous PLK1 in ARCaPM cells was downregulated by transfecting with 2 different siRNAs targeting endogenous PLK1 (si#1 and si#1). The expression of PLK1 protein was determined by immunoblotting. The effects of PLK1 downregulation in ARCaPM cells on EMT marker expression, morphology, intracellular junctions, and invasiveness were determined as described in Figures 12.

https://doi.org/10.7554/eLife.10734.015
Figure 3—figure supplement 4
PLK1 contributes to physiological EMT events.

RWPE-1 and ARCaPE cells were treated with a combination of TGF-β1 (4 ng/mL) and EGF (50 ng/mL) in the presence and absence of BI2536 (1 nM) for 5 days. The EMT marker expression and morphological alteration were determined as described in Figures 12. The phosphorylation of FoxM1 (a PLK1 substrate) at S724 was used as a readout for the PLK1 activity.

https://doi.org/10.7554/eLife.10734.016
Figure 4 with 1 supplement
Both ZEB1 and ZEB2 play a causal role in PLK1-induced EMT and increased motility of prostate epithelial cells.

(A) Expression of different EMT-inducing transcription factors was examined in control and PLK1-overexpressing RWPE-1 cells by quantitative real-time RT-PCR. mRNA expression of genes of interest was normalized by the level of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA and is presented as relative expression. The data are presented as the mean ± s.e.m. *p<0.05, two-tailed Student’s t-test. (B) The levels of ZEB1 and ZEB2 proteins in control and PLK1-overexpressing cells were examined by immunoblotting. (CF) The effects of either individual shRNA-mediated downregulation of ZEB1 and ZEB2, or their dual knockdown in PLK1-induced EMT (C); cell-cell junctional disassembly (D); planar cell migration (E); and cell invasion (F) were determined as described in Figures 12. N.S.: no significant difference. Scale bar, 20 µm (D), 500 µm (E), and 100 µm (F).

https://doi.org/10.7554/eLife.10734.017
Figure 4—figure supplement 1
Elevated PLK1 levels result in activation of ERK, and upregulation of ZEB1, ZEB2, and Fra1 in PrEC cells.

Protein levels of ZEB1 and ZEB2, Fra1, and activated and total ERK1/2 in PrEC (EV) and PrEC-PLK1 (PLK1) cells were determined by Western blotting.

https://doi.org/10.7554/eLife.10734.018
ERK1/2 activation is essential for PLK1-induced EMT and increased motility of prostate epithelial cells.

(A) The effect of PLK1 overexpression on activation (phosphorylation) of ERK1/2 and MEK1/2 was determined by immunoblotting. (BE) Control (EV) and PLK1-overexpressing (PLK1) RWPE-1 cells were treated for 24 hr with either vehicle (DMSO) or RO5126766 (10 mM). The effects of MEK inhibition on PLK1-dependent induction of EMT (B), junctional disassembly (C), accelerated planar cell migration (D), and Matrigel invasion (E) were determined as described in Figures 1 and 2. The data are presented as the mean ± s.e.m. *p<0.05, two-tailed Student’s t-test. NS: no significant difference. Scale bar, 20 µm (C), 500 µm (D), and 100 µm (E).

https://doi.org/10.7554/eLife.10734.019
Fra1 is a critical mediator of PLK1-induced EMT and accelerated motility of prostate epithelial cells.

(A, B) mRNA and protein expression of Fra1 were examined in control (EV) and PLK1-overexpressing (PLK1) RWPE-1 cells by real-time RT-PCR (A) and immunoblotting (B), respectively. The data are presented as the mean ± s.e.m. *p<0.05, two-tailed Student’s t-test. (CF) The effects of Fra1 depletion on PLK1-induced EMT (C), planar cell migration (D), Matrigel invasion (E), and epithelial junctional disassembly (F) were determined as described in Figures 1 and 2. NS: no significant difference. Scale bar, 500 µm (D), 100 µm (E), and 20 µm (F).

https://doi.org/10.7554/eLife.10734.020
CRAF plays an essential role in PLK1-induced EMT and accelerated motility of prostate epithelial cells.

(A) The effects of PLK1 overexpression on the levels of total and phosphorylated CRAF were determined by immunoblotting. p/t: indicates densitometric intensity ratio of phosphorylated to total CRAF. (BE) The effect of shRNA-mediated CRAF knockdown on PLK1-induced EMT (B), epithelial junctional disassembly (C), planar cell migration (D), and Matrigel invasion (E) were determined as described in Figures 12. N.S.: no significant difference. Scale bar, 20 µm (C), 500 µm (D), and 100 µm (E).

https://doi.org/10.7554/eLife.10734.021
Figure 8 with 1 supplement
PLK1-mediated phosphorylation of CRAF leads to CRAF activation and stabilization.

(A) Co-immunoprecipitation of endogenous PLK1 and CRAF from HeLa cell lysates obtained under control conditions or following nocodazole treatment to arrest cells at M phase. (B) GST pull-down assay was performed using GST-tagged wild-type (WT) or mutant (MUT) PLK1 PBD (upper panel). Equal loading of kinase substrates is indicated by Coomassie Blue staining (lower panel). (C) Bacterially expressed CRAF was subjected to in vitro kinase assays with constitutively active (TD) or kinase-defective (KM) PLK1 mutants purified from insect cells. The phosphorylation of CRAF was observed by immunoblotting with the indicated antibodies. Ponceau S staining of the blot was used to indicate equal loading of the assays. (D) RWPE-1 cells were infected with lentivirus expressing empty vector (EV), wild-type PLK1 (WT), constitutively active T210D (TD), or kinase-dead K82M (KM) mutants. Total cell lysates were subject to immunoprecipitation with CRAF antibody and then analyzed by immunoblotting. p/t: indicates densitometric intensity ratio of phosphorylated to total CRAF. (E) Immunoblotting analysis of ERK1/2 activation (phosphorylation) in RWPE-1 cells expressing EV or PLK1 WT, TD, or KM mutants. (F) C4-2B cells were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#1) or serve as a control. Wild-type (WT) or kinase-defective (KM) PLK1 were then re-expressed in PLK1 knockdown cells. The levels of phosphorylated and total CRAF, MEK and ERK, Fral, and ZEB1/2 were determined by Western blotting analysis. (G) C4-2B cells were arrested in M phase by nocodazole (0.1 mg/mL) treatment for 16 hr, and then subject to BI 2536 treatment for 30 min. The levels of phosphorylated and total CRAF were determined by Western blotting analysis. (H) Flag-tagged CRAF WT-, CRAF-A/A-, and CRAF-D/E-overexpressing RWPE-1 cells were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblotting with the indicated antibodies. (I) Control- (EV), PLK1 WT-, PLK1 TD- and PLK1 KM-overexpressing RWPE-1 cells were treated with cycloheximide (CHX: 20 μg/ml) for the indicated times, and the CRAF protein levels were monitored by immunoblotting (left panel). Quantification of the endogenous CRAF protein levels relative to β-actin expression is shown in the right panel. The data are presented as the mean ± s.e.m. (J) PLK1 WT-, and PLK1 KM-overexpressing RWPE-1 cells were treated with cycloheximide (CHX: 20 μg/ml) along with vehicle (DMSO), MG132 (2.5μM), or chloroquine (CLQ, 50 mM) for the indicated times, and level of CRAF protein was examined by immunoblotting. (K) Flag-tagged CRAF WT-, CRAF-S621A-, CRAF-S621D-, CRAF-A/A-, and CRAF-D/E-overexpressing RWPE-1 cells were treated with cycloheximide (CHX: 20 μg/ml) for the indicated time and the level of CRAF protein was determined by immunoblotting. (L) A proposed diagram of a novel signaling cascade that mediates PLK1-dependent induction of EMT and cell motility.

https://doi.org/10.7554/eLife.10734.022
Figure 8—figure supplement 1
Downregulation of PLK1 led to dramatic reduction of CRAF phosphorylation at S338, S339, and S621 that was rescued by WT PLK1, but not KM PLK1.

DU145 cells were infected with lentiviral shRNA constructs that target either the 3’-UTR of endogenous PLK1 (shPLK1#2) or serve as a control. WT or KM PLK1 was then re-expressed in PLK1 knockdown cells. The levels of phosphorylated and total CRAF, MEK and ERK, Fral, and ZEB1/2 were determined by Western blotting analysis.

https://doi.org/10.7554/eLife.10734.023

Videos

Video 1
Video showing the random migration of control RWPE-1 cells.
https://doi.org/10.7554/eLife.10734.007
Video 2
Video showing the random migration of RWPE-1−PLK1 cells.
https://doi.org/10.7554/eLife.10734.008

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