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. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Mol Cancer Ther. 2014 Oct 24;14(1):246–258. doi: 10.1158/1535-7163.MCT-14-0447

Elevated LIM kinase 1 in non-metastatic prostate cancer reflects its role in facilitating androgen receptor nuclear translocation

Katerina Mardilovich 1, Mads Gabrielsen 1, Lynn McGarry 1, Clare Orange 2, Rachana Patel 1, Emma Shanks 1, Joanne Edwards 3, Michael F Olson 1,*
PMCID: PMC4297197  EMSID: EMS60760  PMID: 25344584

Abstract

Prostate cancer (PC) affects a large proportion of the male population, and is primarily driven by androgen receptor (AR) activity. First-line treatment typically consists of reducing AR signaling by hormone-depletion, but resistance inevitably develops over time. One way to overcome this issue is to block AR function via alternative means, preferably by inhibiting protein targets that are more active in tumors than in normal tissue. By staining PC tumor sections, elevated LIM kinase 1 (LIMK1) expression and increased phosphorylation of its substrate cofilin were found to be associated with poor outcome and reduced survival in patients with non-metastatic PC. A LIMK selective small molecule inhibitor (LIMKi) was used to determine whether targeted LIMK inhibition was a potential PC therapy. LIMKi reduced PC cell motility, as well as inhibiting proliferation and increasing apoptosis in androgen-dependent PC cells more effectively than in androgen-independent PC cells. LIMK inhibition blocked ligand-induced AR nuclear translocation, reduced AR protein stability and transcriptional activity, consistent with its effects on proliferation and survival acting via inhibition of AR activity. Furthermore, inhibition of LIMK activity increased αTubulin acetylation and decreased AR interactions with αTubulin, indicating that the role of LIMK in regulating microtubule dynamics contributes to AR function. These results indicate that LIMK inhibitors could be beneficial for the treatment of PC both by reducing nuclear AR translocation, leading to reduced proliferation and survival, and by inhibiting PC cell dissemination.

Introduction

Prostate cancer (PC) is the most commonly diagnosed malignancy and second leading cause of cancer deaths in American men (1). At the molecular level, PC development and progression is driven primarily by activity of the androgen receptor (AR), a steroid hormone receptor typically localized in the cytoplasm in the absence of hormone stimulation (2). In the presence of ligand, androgen receptors translocate to the nucleus to activate the transcription of target genes that control cell cycle progression, cell growth and survival. As a result, the first line of therapy in PC has been to decrease AR activity by hormone-depletion (3). Unfortunately, hormone-ablation therapy often leads to the development of castration-resistant PC (CRPC) that may quickly progress to metastatic disease with high mortality rates (4). Therefore, a major goal is to identify potential targets for the development of PC therapies that target AR function in a hormone-independent manner. Such treatments might not only delay the progression of PC to CRPC, but could possibly also be used for the treatment of CRPC, which maintains and relies upon active AR (4).

Targeting the microtubule cytoskeletal network is one approach that has been used to achieve the goal of reducing AR signaling. Docetaxel, a microtubule (MT) stabilizing drug commonly used for the treatment of PC, has been shown to exert its cytotoxic effect on PC cells by inhibiting MT-mediated AR nuclear translocation in addition to its direct anti-mitotic activity (5, 6). Two major issues with docetaxel treatment are that resistance develops over time, and its general anti-mitotic and microtubule stabilizing actions result in strong side effects, including alopecia, neutropenia and anemia. Therefore, an appealing objective for future PC drug development is to identify alternative microtubule regulators, which if inhibited would affect AR function with low non-specific cytotoxicity. In particular, if this target were more active in PC, its inhibition would improve drug selectivity for PC tumors over normal tissue and contribute to a greater therapeutic window.

Although best known as regulators of actin-myosin cytoskeletal dynamics (7), LIM kinases 1 and 2 (LIMK1 and LIMK2) also contribute to the regulation of the MT cytoskeleton (8-10). LIM kinases are highly homologous serine/threonine kinases that are activated by RhoA/ROCK, Rac/PAK, and Cdc42/MRCK signaling pathways (7). The most well-characterized LIMK substrates are cofilin proteins, which are inhibited for their actin-severing activities when phosphorylated on serine 3 (11). There have been previous reports of elevated LIMK1 expression in PC (12-14), where it was postulated to have a role in promoting metastasis (15). However, there have been no previous studies that systematically evaluated the expression levels of LIMK1, LIMK2 or phosphorylation of their common substrate cofilin as an indicator of kinase activity in primary PC tumor samples accompanied by analysis of PC clinical outcomes.

We undertook immunohistochemical analysis of a PC tissue microarray (TMA) comprised of 164 primary PC and 23 benign hyperplasia samples from 94 individual patients (16), and identified significant associations of elevated LIMK1 expression and phosphorylation of nuclear Cofilin with reduced survival of patients with non-metastatic PC. Moreover, elevated levels of LIMK1 and cytoplasmic phospho-Cofilin were both associated with significantly higher lymphovascular invasion. To pharmacologically evaluate whether LIMK could be a potential PC drug target, we tested a potent and selective LIMK inhibitor (LIMKi) (17). LIMK inhibition reduced PC cell motility, suggesting that blocking LIMK activity could be beneficial as an anti-metastatic therapeutic target in PC. Interestingly, we observed a cytotoxic effect of LIMK inhibition that was significantly greater in androgen-dependent PC cells than in androgen-independent cells. Treatment of androgen-dependent PC cells with LIMKi reduced AR nuclear translocation and transcriptional activity by altering microtubule dynamics that facilitate AR interactions with αTubulin, thus inhibiting cell proliferation. Therefore, in addition to a potential role in promoting metastasis, changes in LIMK1 and LIMK2 expression and/or activity might contribute to AR function in PC via regulation of MT cytoskeletal dynamics. These results justify further investigation of LIM kinases as potential targets for PC therapy.

Materials and Methods

Tissue microarray and immunohistochemistry

The PC TMA, previously described in (16), was comprised of primary PC tumor samples. Within the TMA cohort, 49 samples had available metastasis data, which were typically identified by a bone scan. Survival was defined as disease-specific, patients who died from intercurrent disease were censored in the analysis. Immunohistochemical staining of TMA slides was performed as described previously (18) using antibodies against LIMK1, LIMK2 and p-Cofilin (18, 19). TMA slides were scanned and staining intensities were scored using the SlidePath application (Leica Biosystems). The staining was scored low if the sample histoscore was below or equal to the median histoscore for the entire cohort, or high, if above.

Statistical Analysis

Survival differences were determined using Log Rank (Mantel-Cox) test. Mann-Whitney test was used to compare correlation between protein expression and lymphovascular invasion, in Statistical Package for Social Sciences software (SPSS, Version19). All other indicated statistical analyses were performed in Prism5 (GraphPad) software. F-test was used to compare LIMKi EC50 values.

Cell lines and antibodies

Cell lines were cultured in RPMI media, supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine (GIBCO®). LNCaP-AI cells were cultured in phenol red-free RPMI with 10% charcoal-stripped serum (CSS) and 2 mM L-glutamine (GIBCO®). The LNCaP, DU145, PC3, RWPE-1 cell lines were from ATCC, LNCaP-AI were made in the lab of Hing Leung (Beatson Institute for Cancer Research, Glasgow UK) and gifted to us, CWR22 cells were from Thomas Pretlow (Case Western Reserve University, Cleveland OH, USA). All cell lines were obtained at the beginning of the study in April 2012 and authenticated using the GenePrint 10 system STR multiplex assay (Promega) that amplifies 9 tetranucleotide repeat loci and Amelogenin gender determining marker. Antibodies: Santa Cruz, AR (N-20, sc-816), LIMK2 (H-78, sc-5577); Cell Signaling, p-Cofilin (Ser3, 3311), LIMK1 (3842); Abcam, Cofilin (ab54532), LIMK1 (ab55414); Sigma, α-Tubulin (clone DM1A, T9026); Millipore, MMP-1 (04-1112); Leica, MMP-10 (NCL-MMP10).

Cytotoxicity assays

Cells were plated in 96-well plates at 2,000 cells/well in triplicate and treated the next day without changing the media. Drugs were serial-diluted in DMSO, before diluting equal amounts in media at 2× of the final concentration. Then 100 μl of drug-containing media was added to each well that already contained 100 μl of cells. Cells were treated for 72 hours, fixed with 4% paraformaldehyde, and stained with 250 ng/ml DAPI. Plates were imaged on a High Content Imaging Operetta system (PerkinElmer) and nuclei in each well were quantified using Harmony® High Content Imaging and Analysis Software (PerkinElmer). For cytotoxicity of RWPE-1 cells, CellTiter-Glo® (Promega) Luminescent Cell Viability assay was used according to manufacturer’s instructions. The effect of each treatment was calculated as percent change in cell number relative to DMSO-treated control. EC50 values were calculated from dose-response curves, constrained between 0 to 100%, using Prism 5 (GraphPad) software.

Caspase 3/7 activity

Cells were plated and treated as for the cytotoxicity assay, or transfected with siRNA as in colony formation assay, and plated in 96-well plates. Caspase activity was measured 72-hours after inhibitor treatment or siRNA transfection using Caspase-Glo®3/7 Assay system (Promega) following manufacturer’s instructions.

Sub-G1 quantification

Cells were plated in 6-well plates at 5×105 cells/well and treated the next day with DMSO or 10 μM LIMKi for 24 hours. Percent cells with sub-G1 DNA content were measured and analyzed as described (20).

siRNA knock-down and colony formation assay

LNCaP or CWR22 cells were transiently transfected with ON-TARGETplus SMART pool siRNAs (Thermo Scientific) against LIMK1 or LIMK2, or a non-targeting control (NTC) siRNA, using DreamFect™ Gold transfection reagent (OZ Biosciences) and plated in 24-well plates at 104 cells/well in duplicate. Twenty-four hours after transfection, media was changed to 1 ml per well of growing media and cells were incubated for 24 hours for lysis and knock-down analysis by Western blotting, or incubated for 7 days for colony formation. To quantify colony formation, cells were fixed in methanol and stained with 0.2% (v/v) Crystal Violet solution. Staining was quantified with an ODYSSEY® infrared imaging system (LI-COR).

Co-immunoprecipitation and Western Blotting

LNCaP cells were incubated in phenol red-free RPMI with CSS for 24 hours, and then treated with LIMKi, Docetaxel, or DMSO in the presence of 1 nM dihydrotestosterone (DHT) for 24 hours. Cells were lysed with 500 μl of lysis buffer (1× Tris-buffered saline (TBS), 1% Triton-X, 1 nM EDTA, 0.2 mM Na3VO4, 20 mM NaF, 1 mM PMSF, and cOmplete Mini protease inhibitor cocktail (Roche)) per 10 cm plate. Lysates were cleared by 10-minute centrifugation and pre-cleared with Protein-A agarose beads (Life Technologies). After pre-clearing, beads were removed, and then lysates were incubated with anti-AR antibody at 1:25 dilution for 2 hours at 4°C, then 30 μl Protein-A agarose beads were added for 1 additional hour. Beads were washed 3 times with TBS and boiled with pre-warmed 1% SDS. To quantify the amount of immunoprecipitated protein, IP samples and total cell lysates (1% of IP) were boiled with loading dye and Western blotted (20) with anti-AR and α-Tubulin antibodies. Quantification of Western blots was performed directly without signal amplification or X-ray film using infra-red emitting secondary antibodies and detection with an ODYSSEY® infrared imaging system (LI-COR).

LIMK1 antibody validation

LIMK1 peptide (ab158818 (Abcam) was spotted on PVDF membranes at 50, 25 or 5 ng in duplicate, and membranes were blocked and blotted as for Western Blotting with LIMK1 primary antibody ab55414 (Abcam) or LIMK1 antibody that has been pre-incubated with 4 μg/ml LIMK1 peptide or non-specific control Cofilin peptide (amino acids 1-20). After incubation, membrane was developed as for a Western blot. Immunohistochemistry was performed as described above using LIMK1 antibody with or without pre-incubation with LIMK1 competitor peptide or Cofilin peptide as a non-specific control.

Luciferase assay

CWR22 cells were plated in 96-well plates at 2×104 cells/well in triplicates and transfected the next day with p(ARE)3-Luc (21) and CMV-driven Renilla luciferase plasmids at 10:1 ratio using X-tremeGENE HP transfection reagent (Roche) following manufacturer’s instructions. Next day, cells were incubated in phenol red-free RPMI with CSS with DHT or vehicle control (ethanol), in the presence of indicated drugs for 24 hours. Luciferase activity was measured using Dual-Luciferase® Reporter Assay System (Promega), following manufacturer’s protocol. Firefly Luciferase measurements were normalized to Renilla Luciferase values for transfection control and plotted as fold change of DMSO-treated control.

Real-time PCR

CWR22 cells were pre-treated with 3 μM LIMKi, or DMSO vehicle control in charcoal-stripped serum RPMI without phenol-red for 24 hours, then treated with 1 nM DHT in the presence of the drugs for additional 24 hours. RNA was extracted using RNeasy® Mini kit (Qiagen) and reverse-transcribed using QuantiTect® Reverse Transcription kit (Qiagen) according to manufacturer’s instructions. PSA gene expression was quantified using DyNAmo™ HS SYBR™ Green qPCR kit (Thermo Scientific™) and Applied Biosystems® 7500 Fast Real-Time System (Life Technologies) using primers against PSA (F: 5′-GCAGCATTGAACCAGAGGAG-3′, R: 5′-AGAACTGGGGAGGCTTGAGT-3′) and 18S (F: 5′-GTAACCCGTTGAACCCCATT-3′, R: 5′-CCATCCAATCGGTAGTAGCG-3′) as an internal control.

Immunofluorescence

LNCaP cells grown on coverslips were incubated in phenol red-free RPMI with CSS for 24 hours, then treated with indicated drugs for 24 hours, before 2-hour stimulation with 1nM DHT. Cells were fixed and stained with specific antibodies as described (22). Images were taken using a Zeiss710 laser-scanning confocal microscope and analyzed with ZEN2010 (Zeiss) software.

Nuclear AR quantification

Nuclear AR (volume per nucleus and percent cells with nuclear AR) were quantified using Volocity®3D Image Analysis software (Perkin Elmer).

Acetylated Tubulin quantification

Immunofluorescence staining intensity of acetylated αTubulin was quantified in ImageJ software using fixed intensity threshold, and normalized to total αTubulin immunofluorescence intensity levels.

Cell Fractionation

LNCaP cells were incubated in phenol-red free RPMI with CSS for 24 hours, then treated with 1 nM DHT in the presence of indicated drugs for 24 hours. After treatment, cells were lysed and fractionated using NE-PER® Nuclear and Cytoplasmic Extraction kit (Fisher Scientific), according to manufacturer’s instructions.

Migration assays

PC3 and DU145 cells were plated in 96-well plates in quadruplicates at 4×104 cells/well and treated the next day as in cytotoxicity assay, for 24 hours. After treatment, cell monolayers were scratched, washed, and monitored on INCUCYTE™ Kinetic Imaging System (ESSEN BioScience) by continuous imaging every two hours for 24 hours. Percent wound confluence was quantified using INCUCYTE™ software analysis (ESSEN BioScience). LNCaP cells were plated in glass-bottom 6-well dishes, allowed to attach overnight, then medium was changed to CSS supplemented phenol-red free RPMI for 24 hours, followed by treatment with 1 nM DHT with 5 μM LIMKi or DMSO and imaged at three images per well on Nikon time-lapse imagining system for 20 hours. Cell velocities were calculated using ImageJ software for 30 cells per well within each experiment.

Results

LIMK expression and activity in prostate cancer

LIMK1 has previously been reported to be elevated in limited samples of human PC (12-14); however, its association with patient outcome had not been determined. In addition, neither LIMK2 expression nor phosphorylation of the common substrate cofilin had been previously characterized in PC. Therefore, we investigated how LIMK1 and LIMK2 expression and activity varied in PC, and determined how observed variations were associated with patient outcomes using a TMA comprised of 164 primary PC samples and 23 benign hyperplasia samples from 92 individual patients (16) (demographic, clinicopathological and outcome characteristics of the patients detailed in Supplementary Table S1). Using antibodies validated by peptide competition for LIMK1 (Supplementary Fig. S1A) or by staining tissues from knockout mice for LIMK2 (18) or Serine 3 phosphorylated Cofilin (p-Cofilin) (19), TMA samples were stained, scanned and scored for staining intensities using an automated algorithm (23). Staining intensities of each protein were correlated with the histopathological data of the tumor samples. Examples of low and high staining for LIMK1, LIMK2, nuclear and cytoplasmic p-Cofilin are shown in Fig. 1A. There was a significant association between high LIMK1 expression and poor survival in patients diagnosed with non-metastatic prostate cancer (p=0.035; Fig. 1B, Supplemental Table S2), but not in the metastatic disease group (Fig. 1C, Supplemental Table S2). Analysis of LIMK2 levels showed a similar trend of high LIMK2 expression associated with poor patient survival in the non-metastatic group (p=0.151; Fig. 1B, Supplemental Table S3). However, high LIMK2 was significantly associated with increased survival in the metastatic disease group (p=0.048; Fig. 1C, Supplemental Table S3). Since cofilin has both nuclear import and export signals to facilitate nuclear-cytoplasmic shuttling (24), we analyzed nuclear or cytoplasmic p-Cofilin levels in the TMA samples. Similar to LIMK1, high nuclear p-Cofilin levels showed a significant association with poor patient survival in the non-metastatic group (p=0.034; Fig. 1B, Supplemental Table S4). Although the role of cytoplasmic Cofilin has largely been associated with regulation of cytoskeleton dynamics (25), nuclear cofilin may contribute to nuclear actin regulation, gene transcription and mitosis (26, 27). Together, these results indicate that elevated LIMK expression and activity are associated with increased mortality in non-metastatic PC. Although LIMK1 and cofilin phosphorylation were not associated with patient mortality in metastatic PC (Supplemental Tables S2, S4, S5), elevated LIMK1 and cytoplasmic p-Cofilin were significantly associated with increased lymphovascular invasion (Fig. 1D), a clinical characteristic of more aggressive tumors and potential marker of progression to metastatic disease (28). There were no significant associations between LIMK1, LIMK1 or cofilin phosphorylation with patient age, Gleason score, prostate specific antigen elevation or relapse (Supplemental Table S6).

Figure 1. Association of LIMK1, LIMK2 and p-Cofilin with prostate cancer patient outcome.

Figure 1

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A, Human prostate cancer samples were stained with antibodies against LIMK1, LIMK2 or phosphorylated serine 3 cofilin (p-Cofilin). Representative images of Low and High staining for each protein are shown at 10× magnification, inset at 20× magnification, scale bar = 100 μm. B, Kaplan-Meier survival curves showing association of LIMK1, LIMK2, nuclear and cytoplasmic p-Cofilin levels with patient survival with non-metastatic and C, metastatic prostate cancer (blue line – low protein levels, green line – high protein levels). D, Expression levels of LIMK1, LIMK2, nuclear and cytoplasmic p-Cofilin in prostate cancer tumors scored positive or negative for lymphovascular invasion. E, LIMK1 mRNA expression in human prostate cancer tumors and normal gland tissue. Statistical significance was tested by unpaired Student’s t-test with Welch’s correction. F, LIMK1 copy number variations in normal prostate and adenocarcinoma samples determined by The Cancer Genome Atlas (http://tcga-data.nci.nih.gov/tcga/). Statistical significance was tested by unpaired Student’s t-test.

Analysis of publicly available gene expression data using Oncomine (29) revealed significantly elevated LIMK1 mRNA expression levels in prostate carcinoma relative to normal gland tissue (Fig. 1E) (30). Furthermore, analysis using the cBio Cancer Genomics Portal (31) of 85 prostate adenocarcinoma tumor samples revealed significantly increased LIMK1 expression or amplification in 14 cases (16%), with 5/12 patients (42%) having elevated LIMK1 expression undergoing biochemical recurrence in contrast to 16/68 patients (24%) without alterations in LIMK1 expression (32). In addition, disease-free survival trended towards being worse for patients with altered LIMK1 expression (Supplementary Figure S1B). Analysis of LIMK1 gene copy number by The Cancer Genome Atlas (http://tcga-data.nci.nih.gov/tcga/) indicated that there was a significant increase in prostate adenocarcinoma samples relative to normal prostate tissue (Fig. 1F). These results are consistent with increased mRNA contributing to elevated LIMK1 protein in PC. Together, our findings indicate that elevated LIMK expression and activity are associated with early stage PC growth and progression.

LIMKi decreases PC cell motility

Given the previous reports of elevated LIMK1 expression in metastatic PC (12-15) and our findings that blocking LIMK activity reduces breast cancer cell invasiveness (33), we sought to determine whether pharmacological inhibition of LIMK activity reduce PC cell motility. We initially compared LIMK1, LIMK2 and cofilin Serine3 phosphorylation in several PC cells lines, and found that cofilin phosphorylation was highest in parental androgen-dependent LNCaP cells, with lower levels in an androgen-independent LNCaP variant (LNCaP-AI), derived by continuous culture in hormone-depleted media (16), as well as AR-negative PC3 and DU145 cells (Fig. 2A). Combined LIMK1 and LIMK2 levels were highest in LNCaP cells, with lower levels in the androgen-independent LNCaP-AI, PC3 and DU145 PC cells. Treatment of LNCaP cells with 1 or 10 nM dihydrotestosterone (DHT) did not further increase LIMK1 or LIMK2 protein levels (Supplementary Fig S1C).

Figure 2. LIMKi inhibits migration of PC cell lines.

Figure 2

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A, LNCaP, LNCaP-AI, PC3, and DU145 cell lines were analyzed for protein levels of LIMK1, LIMK2 and p-Cofilin by Western blot. B, LNCaP cells were treated with increasing LIMKi concentrations for 24 hours and analyzed by Western blot for changes in p-Cofilin levels. C, Cells were plated at high density in 96-well plates and treated with DMSO vehicle control or 10 μM LIMKi for 24 hours before wounding of the cell monolayer. Images show confluence mask (black region) for PC3 cells at 0 and 24-hour time points. Cell migration was quantified by measuring percent wound confluence (y-axis) over time (x-axis) relative to an initial confluence mask for D, PC3 and E, DU145 cells. Results show average of three independent experiments performed in quadruplicate, ± SEM. Statistical significance was tested by unpaired Student’s t-test. F, Random migration of LNCaP cells was determined by single cell tracking over 20 hours using ImageJ. Five independent determinations were made of the motility of 30 cells in each condition. Boxes are upper and lower quartiles, whiskers indicate minimum and maximum values. Statistical analysis was by one-way ANOVA followed by post-hoc Tukey multiple comparison test.

To investigate the role of LIMK activity in PC cell motlity, we utilized a potent selective small molecule inhibitor (N-{5-[2-(2,6-Dichloro-phenyl)-5-difluoromethyl-2H-pyrazol-3-yl]-thiazol-2-yl}-isobutyramide (compound 3 in (17); hereafter termed LIMKi) that equipotently inhibits LIMK1 and LIMK2. We confirmed that LIMKi effectively reduced p-Cofilin in LNCaP cells after 24-hour treatment (Fig. 2B), as we previously observed for other cell lines including MDAMB231 breast cancer cells (22), NMuMG mouse mammary epithelial cells (34) and U2OS human osteosarcoma cells (20).

As a major regulator of cytoskeleton dynamics, LIMK1 has been implicated in PC cell migration and invasion (12, 14, 15). Given that maximal inhibition of cofilin phosphorylation was achieved at 10 μM LIMKi (Fig. 2B), we tested this concentration on DU145 and PC3 cell migration. Cells were plated in a dense monolayer in 96-well plates, then treated the next day with 10 μM LIMKi for 24 hours. After treatment, PC3 cell migration was analyzed in an automated scratch wound-healing assay, with images acquired every two hours for 24 hours (Fig. 2C). After creating an initial mask, wound confluence was measured by determining the percentage of wound area that is occupied by cells over time. There was a marked effect of LIMKi on 2D migration of PC3 cells over time (Fig. 2D, left graph) and at the 24 hour experimental endpoint (Fig. 2D, right graph). LIMKi had a more moderate inhibitory effect on the 2D-migration of DU145 cells over time (Fig. 2E, left graph), which was significant at the 24 hour experimental endpoint (Fig.2E, right graph). These results suggest that LIMK inhibition could have the therapeutic benefit of reducing PC dissemination.

Sensitivity of androgen-dependent prostate cancer cells to LIMK inhibition

In addition to testing how LIMKi affected DU145 and PC3 scratch wound closure, we initially tested LNCaP cells but found that they did not migrate in this assay format. As an alternative, we used single cell tracking to measure random migration velocity. Treatment with 1 nM DHT significantly increased migration velocity, which was significantly reversed by 5 μM LIMKi (Fig. 2F), indicating a role for LIMK activity in androgen-induced motility. Expression of the matrix metalloproteinases MMP-10 (35) and MMP-1 (36), which had previously been implicated in prostate tumor growth, were not affected by DHT or LIMKi treatment (Supplementary Fig S1D). Interestingly, when LNCaP cells were treated with 10 μM LIMKi we readily observed reduced LNCaP cell numbers. To quantify the effect of LIMKi on proliferation, we treated LNCaP or LNCaP-AI cells in 96-well plates with half-log serial dilutions of LIMKi. After 72 hours, surviving cells were fixed, stained with DAPI, and nuclei numbers quantified with an Operetta High Content Imaging System. LNCaP cells were significantly more sensitive to LIMKi than LNCaP-AI cells (Fig. 3A, left panel). However, the two cell lines did not differ in their sensitivities to Adriamycin, Actinomycin D or Camptothecin (Fig. 3A), all of which exert their cytotoxicity independent of LIMK inhibition. In addition, 10 μM LIMKi treatment induced a substantial increase in the percentage of LNCaP cells with sub-G1 DNA content (i.e. <2N), indicative of the induction of apoptosis, but not in LNCaP-AI cells (Fig. 3B). Moreover, evaluation of the LIMKi rank order of potency on cell number for various PC cell lines revealed androgen-dependent LNCaP and CWR22 cells to be >2-8 times more sensitive than androgen-independent LNCaP-AI, DU145 and PC3 cells (Table 1). Similarly, using a cell viability assay that measures ATP levels, immortalized RWPE-1 normal prostate epithelial cells had an EC50 for LIMKi of 6.67 μM, similar to the values determined for androgen dependent CWR22 cells (Table 1). Consistent with these results, activity of the apoptosis executioner Caspases 3 and 7 were induced by 10 μM LIMKi in LNCaP and CWR22 cells over 3-fold relative to untreated control cells, but not in androgen-independent LNCaP-AI, DU145 and PC3 cells (Fig. 3C). These results indicate that androgen-dependent PC cells, which had the highest relative levels of LIMK expression and Cofilin phosphorylation, were more sensitive to LIMK inhibition than androgen-independent PC cells, suggesting that LIMK activity may contribute to AR function and activity.

Figure 3. LIMK inhibitor blocks survival of androgen-dependent PC cell lines.

Figure 3

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A, LNCaP (red) and LNCaP-AI (green) cell lines were treated with half-log serial dilutions of LIMKi, Adriamycin, Actinomycin D, and Camptothecin (left to right) for 72 hours. Drug cytotoxicity was measured as percent survival relative to DMSO treated control (100%). Graphs show combined results of three independent experiments performed in triplicate ± SEM. B, DNA content of LNCaP or LNCaP-AI cells treated with 10 μM LIMKi (red line) or DMSO vehicle (black line) for 72 hours as analyzed by propidium iodide staining followed by flow cytometry. Graph shows the results from four independent experiments ± SEM. Statistical significance was tested by two-way ANOVA, p value indicates significant effect of cell type on drug response C, Caspase3/7 activity for each treatment was measured and normalized to DMSO alone-treated control. Graph shows combined results of three independent experiments performed in triplicate ± SEM. Statistical significance was tested by one-way ANOVA and post-hoc Newman-Keuls multiple comparison test, groups of conditions differing significantly from each other as indicated. D, LNCaP or CWR22 cells were transfected with non-targeting control (NTC), LIMK1, LIMK2, or LIMK1+LIMK2 siRNA oligos as indicated, and western blotted after 48 hours. Graphs show combined results of three independent experiments performed in duplicate ± SEM. E, Caspase3/7 activity for each treatment was measured and normalized to untransfected control cells. Graph shows combined results of three independent experiments performed in triplicate ± SEM. Statistical significance was tested by one-way ANOVA and post-hoc Dunnett’s multiple comparison test, conditions differing significantly from non-targeting control as indicated. F, Colony formation of LNCaP or CWR22 cells after 7 days following transfection with indicated siRNA oligos. Graphs show combined results of three independent experiments performed in duplicate ± SEM. Statistical significance was tested by one-way ANOVA and post-hoc Dunnett’s multiple comparison test, groups of conditions differing significantly from each other as indicated.

Table 1. Antiproliferative effect of LIMKi on PC cell lines.

All cell lines were treated with half-log serial dilutions of LIMKi for 72 hours. EC50 values were measured from dose-response curves from three independent experiments performed in triplicates.

Cell line LIMKi EC50, μM p value, F test (compared to LNCaP)
LNCaP 1.33 -
CWR22 6.97 <0.0001
LNCaP-AI 11.11 <0.0001
DU145 11.46 <0.0001
PC3 12.68 <0.0001
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To validate the on-target effects of LIMKi (17), we analyzed the effect of individual or combined LIMK1 and LIMK2 siRNA-mediated knock-down (Fig. 3D) on the induction of apoptosis by measuring Caspase 3/7 activity. In both LNCaP and CWR22 cells, LIMK2 or combined LIMK1 +LIMK2 knockdown induced significant Caspase activity (Fig. 3E). Furthermore, proliferation of androgen dependent LNCaP and CWR22 cell lines were strongly inhibited by the simultaneous knock-down of both LIMK1 and LIMK2 in a colony-formation assay. (Fig. 3F), similar to the effect of LIMKi. These results indicate that LIM kinases have roles in supporting proliferation of androgen-dependent PC cells.

LIMK inhibition targets AR activity in androgen-dependent PC cells

AR is a steroid hormone receptor that translocates into the nucleus upon ligand binding to regulate transcription of downstream target genes that promote prostate cell growth, proliferation and survival. Given the observed effects of LIMKi on androgen-dependent PC cell number and apoptosis, we sought to determine whether LIMKi treatment affected AR subcellular localization. Following 24-hour incubation in hormone-depleted media (37) to reduce background AR signaling, LNCaP cells were stimulated with 1 nM Dihydrotestosterone (DHT) for 2 hours. Representative confocal immunofluorescence images show increased nuclear AR staining following DHT stimulation, quantified either by determining the percentage of cells with detectable nuclear AR (Fig. 4A, upper panels) or by measuring the mean volume of AR staining per nucleus (Fig. 4A, lower panels), both of which were reduced by treatment with 3 μM LIMKi or 4 nM Docetaxel (Dcxl), a standard PC chemotherapy drug previously shown to inhibit AR nuclear translocation (38). Furthermore, the effect of LIMKi and Docetaxel on DHT-induced AR nuclear translocation was supported by cellular fractionation following the same treatment used for immunofluorescence experiments. A representative Western blot shows that DHT-induced nuclear AR accumulation was reduced by 1 and 3 μM LIMKi or 4 nM Docetaxel (Fig. 4B). Combined results from three independent experiments revealed that 3 μM LIMKi had a comparable inhibitory effect on DHT-induced nuclear accumulation of AR as 4 nM Docetaxel (Fig. 4B). Interestingly, inhibition of p-Cofilin by 4 nM Docetaxel was comparable to that observed for 3 μM LIMKi, suggesting that Docetaxel inhibits LIMK activity as a direct or indirect off-target, which may additionally contribute to its anti-proliferative mechanism of action in PC.

Figure 4. Treatment with LIMKi reduces AR function.

Figure 4

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A, LNCaP cells were stimulated with 1 nM DHT for two hours after being hormone-starved and treated with DMSO vehicle, 3 μM LIMKi, or 4 nM Docetaxel (Dcxl) for 24 hours. Cells were fixed and stained with fluorescent antibodies against AR (green), αTubulin (red), or stained with DAPI (blue). Representative confocal immunofluorescence images (upper panels) and 3-dimensional reconstructions from z-stacks (lower panels) are shown. Percentage cells with nuclear AR (upper graph) and mean AR volume (μm3) per nucleus (lower graph) were quantified with Volocity software. Combined results from three independent experiments ± SEM are shown. Statistical significance was tested by unpaired Student’s t-test between indicated conditions. B, LNCaP cells were treated as above. Cells were fractionated and nuclear lysates analyzed by Western blot for changes in nuclear AR. Total cell lysates of the same cells were analyzed by Western blot for changes in p-Cofilin. Graph shows quantification results of nuclear AR levels normalized to nuclear fraction loading control, plotted as percent change compared to unstimulated cells. Average results of three independent experiments ± SEM are shown. C, LNCaP cells were treated with 3 μM LIMKi or DMSO for 24 hours, and then incubated with 20 ng/ml cycloheximide (CHX) for indicated times. Representative Western blot shows changes in AR protein levels during translational block. AR levels were normalized to αTubulin and plotted as percent change relative to a no-cycloheximide treatment control. Graph shows results of three independent experiments ± SEM. Statistical significance was tested by two-way ANOVA, indicated p value reports significance of drug treatment. D. CWR22 cells were transiently transfected with p(ARE)3-luciferase reporter and CMV-driven Renilla luciferase constructs. 24 hours after transfection, cells were stimulated with DHT and treated with DMSO vehicle, 3 μM or 10 μM LIMKi, 10 μM Bicalutamide for 24 hours. Promoter activity was measured as relative luciferase units and normalized to the activity of unstimulated cells. Results show average values from three independent experiments performed in triplicate ± SEM. E, PSA expression relative to 18S RNA in CWR22 cells pre-treated with 3 μM LIMKi, or DMSO vehicle for 24 hours, then treated with 1 nM DHT in the presence of vehicle or LIMKi for additional 24 hours. Graph shows average results for four independent experiments ± SEM.

Due to the inhibitory effect of LIMKi on AR nuclear accumulation, we tested the possibility that LIMK inhibition might affect AR protein stability. LNCaP cells were treated with 3 μM LIMKi or vehicle (DMSO) control for 24 hours, followed by inhibition of de novo protein translation with 20 ng/mL cycloheximide (CHX) for the indicated times. By measuring AR protein levels at each time point by Western blotting, we observed less remaining AR protein in the presence of LIMKi compared to DMSO-treated controls, consistent with LIMKi treatment reducing AR protein stability (Fig. 4C).

To test the effect of LIMKi on AR transcriptional activity, we used a Luciferase reporter construct comprised of three consensus Androgen Receptor-response Element (ARE) repeats (21) in a transcription-reporter assay. LNCaP cells could not be adequately transfected for the assay, so androgen-dependent CWR22 cells were used. Cells were transfected with p(ARE)3-luciferase reporter and Renilla luciferase control plasmids and then treated with 1 or 10 nM DHT for 24 hours in the presence of 3 or 10 μM LIMKi, DMSO vehicle control or 10 μM of the anti-androgen Bicalutamide. These experiments revealed that 10 μM LIMKi reduced DHT-induced AR transcriptional activity to a similar extent as 10 μM Bicalutamide (Fig. 4D). Statistical analysis by two-way ANOVA revealed a significant effect of LIMKi treatments on DHT-induced luciferase activity (p=0.046). Expression of endogenous prostate specific antigen (PSA) mRNA was induced by 1 nM DHT treatment could be effectively reversed by 3 μM LIMKi (Fig. 4E). Taken together, these experiments indicate that inhibition of LIMK activity reduced AR nuclear accumulation, stability and transcriptional activity in androgen-dependent PC cells, which would contribute to the anti-proliferative and pro-apoptotic effects of LIMKi treatment (Fig. 3).

LIMK inhibition affects microtubule stability and AR-αTubulin interactions

AR has been previously shown to interact directly with microtubules, while disruption of MT dynamics leads to reduced nuclear AR translocation (38). In addition to being a major regulator of actin dynamics, LIMK has also been shown to regulate MT dynamics (8-10). Therefore, we tested the effect of LIMKi on the levels and distribution of acetylated αTubulin (Ace-αTub), which is associated with increased stabilization and altered dynamics of the microtubule network (39). To visualize changes in MT structure and αTubulin acetylation, we performed confocal immunofluorescence microscopy on LNCaP cells treated with 10 μM LIMKi for 24 hours. Representative Z-plane optical slices as well as maximum projection images show a noticeable change in overall MT structure (Fig. 5A). Acetylation of αTubulin with increasing concentration of LIMKi was also detected by immunofluorescence treatment (Fig. 5B); which, when combined results from three independent experiments were quantified and normalized to total αTubulin, revealed increased tubulin acetylation with increasing LIMKi concentrations (Fig. 5B). Western blot analysis showed a similar trend of increased acetylated αTubulin over total αTubulin with increasing LIMKi concentrations (Fig. 5C). To determine if the LIMKi-induced increase in αTubulin acetylation affected AR-αTubulin association, LNCaP cells were treated identically as for AR nuclear localization analysis in Figure 4, and AR-αTubulin interactions analyzed by co-immunoprecipitation. There was a reduction in αTubulin associated with AR immunoprecipitated from cells treated with LIMKi (Fig. 5D); 3 μM LIMKi had a similar effect to 4 nM Docetaxel treatment (Fig. 5D). These results indicate that LIMK inhibition attenuates AR nuclear translocation by increasing αTubulin acetylation with consequent effects on MT dynamics, and by reducing AR interaction with αTubulin.

Figure 5. LIMKi increases αTubulin acetylation and reduces AR-αTubulin interactions.

Figure 5

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A, LNCaP cells were treated with DMSO vehicle, or 10 μM LIMKi for 24 hours, fixed and stained with fluorescent antibodies against total αTubulin. Narrow Z-plane images were taken at 0.5 μm intervals, and distance of each image from the starting point is indicated. B, Fluorescence staining intensity of acetylated αTubulin (Ace-αTub, green) was quantified and normalized to total αTubulin (red) for each field. Graph shows average quantification results for three independent experiments (each using three fields per treatment) ± SEM. Statistical significance was tested by one-way ANOVA and post-hoc Newman-Keuls multiple comparison test, conditions differing significantly from each other as indicated. C, LNCaP cells were treated with DMSO vehicle or increasing LIMKi concentrations for 24 hours. Cells were lysed and αTubulin acetylation levels determined by Western blot. Acetylated αTubulin (Ace-αTub) was normalized to total αTubulin levels and plotted as fold change over DMSO-treated control. Graph shows results of three independent experiments ± SEM. Statistical significance was tested by one-way ANOVA and post-hoc Dunnett’s multiple comparison test, condition differing significantly from DMSO only control as indicated. D, LNCaP cells were treated with DMSO vehicle, 3 μM or 10 μM LIMKi, or 4 nM Docetaxel (Dcxl) for 24 hours. Cells were lysed and AR was immunoprecipitated (IP). Amount of co-immunoprecipitated αTubulin was determined by Western blot. Total cell lysates (1% of the IP amount) were analyzed by Western blot. Graph shows αTubulin levels normalized to AR levels in each IP sample from three independent experiments ± SEM.

Discussion

In this report, we established a correlation between poor survival in patients with non-metastatic PC and increased LIMK1 and nuclear phospho-Cofilin levels (Fig. 1B, Supplemental Tables S2, S4). In addition, elevated LIMK1 and phosphorylated cytoplasmic Cofilin were associated with increased lymphovascular invasion (Fig. 1D). Associated with these expression patterns, we identified a role for LIM kinases in metastatic PC cell motility and in promoting AR function in PC via their contribution to regulating microtubule dynamics. Intriguingly, inhibition of LIMK activity with a selective small molecule inhibitor had greater cytotoxic effects on androgen-dependent than on androgen-independent PC cells (Fig. 3 and Table 1). Treatment of androgen-responsive cells with LIMKi decreased DHT-induced AR nuclear translocation, protein stability and transcriptional activity (Fig. 4). Impaired nuclear translocation of AR in the presence of LIMKi was likely the product of decreased AR-αTubulin interactions and increased αTubulin acetylation, indicative of increased MT stability (Fig. 5). Taken together, our results indicate that LIM kinases positively regulate AR function in AR-dependent PC, promoting disease development and progression to an early locally invasive state. In addition, these studies provide pharmacological evidence, with mechanism-of-action detail, showing that LIMK inhibition has potential as a therapeutic approach for the treatment of PC

The finding of LIMK1 association with poor patient survival in the non-metastatic PC group is consistent with a previous observation of correlation between expression of the LIMK upstream regulator RhoA with poor patient survival at early stages of PC and PC lymph node invasion (40). LIMK1 has been previously reported to be upregulated in PC tumors and cell lines, where it was postulated to have a role in metastasis largely due to the generally held view of LIMK1 as a promoter of cell motility and migration (12-14). These studies, however, did not examine LIMK1 expression levels in non-metastatic versus metastatic tumor samples, nor was the link between LIMK1 expression with patient outcome determined for either group.

The role of LIMK2 in PC has largely been overlooked. Our results indicate that LIMK2 expression in non-metastatic PC is similar to that of LIMK1, with a trend of elevated LIMK2 associated with poor patient survival and increased lymphovascular invasion. However, these observations did not achieve statistical significance in the number of samples in the TMA dataset. However, the strong trend of association with poor survival in the non-metastatic patient cohort and lymphovascular invasion for all three markers, LIMK1, LIMK2, and p-Cofilin, and the requirement for combined LIMK1 and LIMK2 knockdown by siRNA for full anti-proliferative effects (Fig. 3E) leads us to propose that both LIMK1 and LIMK2 contribute to development and progression of early stage PC, which is often characterized as AR-positive and hormone-dependent. Indeed, a role of LIM kinase signaling in early AR-positive and hormone-dependent PC is consistent with the greater sensitivity of androgen-dependent than androgen-independent PC cell lines to LIMKi in cell proliferation and apoptosis detection assays (Fig. 3, Table 1).

The greater sensitivity of androgen-dependent PC cell lines and RWPE-1 prostate epithelial cells to LIMKi supports the conclusion that LIMKi targets AR function (Fig. 4 and Table 1). We observed an inhibitory effect of LIMKi on AR nuclear translocation (Fig. 4A-B) and decreased AR-αTubulin interaction that were associated with increased tubulin acetylation (Fig. 5). LIM kinases are important regulators of actin cytoskeleton dynamics (7). Given that AR nuclear translocation has been previously reported to depend on the filamentous actin cross-linking protein filamin (21), an additional possibility is that LIMK inhibition may affect AR translocation via effects on the actin cytoskeleton. This double effect on both actin and microtubule cytoskeletal networks that contribute to AR nuclear accumulation may explain the apparent selective advantage for elevated LIMK1 expression in the poor-outcome non-metastatic PC patients and the increased sensitivity of androgen-dependent PC cell lines to LIMKi.

Ligand-bound AR is rapidly degraded by a proteasome-mediated degradation pathway if sequestered in the cytoplasm (41, 42). The inhibition of nuclear AR accumulation in the presence of LIMKi was accompanied by a more rapid decrease in AR protein levels (Fig. 4C), possibly due to the accumulation of ligand-bound AR in the cytoplasm, which would subsequently be targeted for degradation. This inhibitory effect on protein stability likely contributes to the LIMKi cytotoxic mechanism of action in androgen-dependent PC cells.

We found a significant association between elevated LIMK1 expression and poor survival in non-metastatic PC, while LIMK2 expression had a similar trend in the same patient group (Fig. 1). However, we also observed that elevated LIMK2 expression in metastatic PC was associated with better patient outcome. These differences could indicate a unique role for LIMK2 in cancer. We previously reported that progressively decreased LIMK2 expression due to promoter methylation was associated with poor prognosis in colorectal cancer patients, while Limk2 deletion increased tumor burden in a colitis-associated colorectal cancer mouse model (18). We determined that was due to a role of LIMK2 in restraining gastrointestinal stem cell proliferation. Based on these observations, a possibility is that there is selection for decreased LIMK2, but not LIMK1, expression in advanced PC to promote the expansion of stem-like tumor cells. Consistent with our observations, analysis using the cBio Cancer Genomics Portal (31) of mRNA levels in 85 prostate adenocarcinoma tumor samples revealed significantly decreased LIMK2 relative to normal tissue in 27 cases (31%) (32).

Our results suggest that there may be potential for the use of LIMK inhibitors as an AR-dependent PC targeted therapy. Moreover, the effect of LIMKi on PC cell migration (Fig. 2) suggests that LIMK inhibitors might have an additional benefit of limiting cancer spread as well as targeting tumor growth. Although AR-negative PC cell lines showed a relatively weak response to LIMKi in cytotoxicity assays (Fig. 3 and Table 1), LIMK inhibitors may target CRPC cells that still rely on AR for survival. Recent research has shown that CRPC cells are reliant on AR signaling, and adapt to hormone depletion therapy by increasing AR expression and/or up-regulating androgen production (43, 44). This hypothesis is supported by the successful treatment of CRPC with taxanes, such as docetaxel that target AR function or abiraterone, which inhibits 17 α-hydroxylase activity to reduce androgen production (45). These findings support the notion that blocking AR activity is the prime target for treatment of both drug-naïve PC and CRPC. As general MT-targeting drugs, taxanes have high overall toxicity because their target is essential in normal cells, and is not differentially expressed in tumor cells. Given the elevated expression of LIM kinases in the non-metastatic poor outcome patient group (Fig. 1), our data supports the idea that inhibition of LIM kinases could have tumor selective effects while leaving normal tissues relatively unaffected. In addition, the finding that LIMK2 knockdown sensitized neuroblastoma cells to taxane treatment (46) suggests that the treatment of CRPC with docetaxel could be made more effective through combination therapy with LIMK inhibitors.

Supplementary Material

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Acknowledgements

Thanks to Hing Leung (Beatson Institute for Cancer Research, Glasgow UK) for advice and gifts of prostate cancer cell lines and test tumor sections.

Financial Support

This research was supported a grant from Cancer Research UK (A12966) to M.F. Olson.

Footnotes

The authors have no conflicts of interest to disclose.

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