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. Author manuscript; available in PMC: 2015 May 19.
Published in final edited form as: Cancer Res. 2010 Mar 2;70(6):2307–2317. doi: 10.1158/0008-5472.CAN-09-3252

Hsp27 promotes IGF-1 survival signaling in prostate cancer via p90Rsk-dependent phosphorylation and inactivation of BAD

Amina Zoubeidi 1, Anousheh Zardan 1, Romina M Wiedmann 1, Jennifer Locke 1, Eliana Beraldi 1, Ladan Fazli 1, Martin E Gleave 1
PMCID: PMC4437589  NIHMSID: NIHMS687553  PMID: 20197463

Abstract

Hsp27 is highly expressed in castrate-resistant prostate cancer. While its overexpression confers resistance to androgen ablation and chemotherapy, the mechanisms by which Hsp27 inhibits treatment-induced apoptosis are incompletely defined. Castrate-resistance often correlates with increased activity of autocrine and/or paracrine growth/survival stimulatory loops including the MAPK and Akt pathways and insulin-like growth factor (IGF) axis components. Since Hsp27 can be activated by both MAPK and Akt pathways, it is possible that interactions between IGF-1 signaling and Hsp27 phospho-activation function to promote castrate resistant progression. Here we report that Hsp27 expression and phosphorylation levels correlate with IGF-1 signaling and castrate resistant progression in human prostate cancer specimens and cell lines. IGF-1 induces Hsp27 phosphorylation in a time- and dose-dependent manner via p90Rsk, which directly interacts with and phosphorylates Hsp27 in vitro and in vivo. Conversely, p90Rsk inhibition using siRNA or a mutant dominant negative abolishes IGF-1 induced Hsp27 phosphorylation. Hsp27 over-expression increases IGF-1-induced phosphorylation of Erk, p90Rsk and Akt. Conversely, Hsp27 knockdown abrogates IGF-1 induced phosphorylation of Erk, p90Rsk and Akt, thereby destabilizing Bad/14-3-3 complexes and increasing apoptotic rates. These data elucidate interactions between Hsp27 phosphorylation and the IGF-1R signaling pathway and support targeting Hsp27 as a therapeutic strategy for castrate-resistant prostate cancer.

Keywords: Hsp27, IGF-1, survival, MAPK, prostate cancer

Introduction

Prostate cancer is the second leading cause of cancer deaths in North American men, killing more than 40,000 annually. A major challenge for treatment of men with metastatic or recurrent disease is development of treatment resistance after androgen deprivation therapy even when combined with docetaxel chemotherapy. While the majority of patients initially respond to such treatments, most go on to develop lethal castrate-resistant prostate cancer (CRPC). Under conditions of castrate levels of serum testosterone, other growth factors assume a more dominant role in promoting cancer cell survival (1). CRPC is a complex process involving clonal selection (2) and androgen receptor activation by low levels of androgens, receptor mutations or increased co-activator levels (3–5). Additional mechanisms include activation of alternative growth factor pathways like IGF-1 (6, 7) and up-regulation of anti-apoptotic genes including Hsp27 (8).

The IGF axis is an important regulator of growth, survival and metastatic potential in a variety of malignancies and is strongly implicated in prostatic carcinogenesis and CRPC (9, 10). IGF-1 mediates growth and survival responses by binding to the tyrosine kinase IGF-1 receptor (IGF-1R). Once activated, IGF-1R phosphorylates insulin receptor substrate (IRS), which leads to activation of many signaling pathways, including Ras/Raf/MAPK or PI3k/Akt. Activation of PI3k/Akt by IGF-1 leads to Bad phosphorylation on Ser136 and the activation of forkhead-regulated transcription factor (11). Activation of Erk phosphorylates p90 ribosomal S6 kinase (p90Rsk) which, in turn, leads to phosphorylation of Bad on Ser112 (12). Hence, activation of PI3K and Erk pathways converge to inhibit apoptosis by phosphorylating (on serine 136 and 112, respectively) and inactivating the proapoptotic protein Bad.

Hsp27 is a stress-induced molecular chaperone that is highly and uniformly expressed in CRPC (13, 14) and other cancers (15) (16) (17) and is often associated with metastasis and poor prognosis (18, 19). Hsp27 expression is induced by hormone- or chemo-therapy and inhibits treatment-induced apoptosis through multiple mechanisms (8, 18, 20–22). Hsp27 has been shown to interact and inhibit components of both stress- and receptor-induced apoptotic pathways. Hsp27 prevents activation of caspases by sequestering cytochrome C in the cytoplasm (23). Cytochrome-c interacts with Apaf-1 and caspase-9 to form the “apoptosome” which activates caspase-3 and a cascade of downstream caspases, the so called “effectors” of cell death (24). Hsp27 interacts with and inhibits caspase-3 activation, an effect related to the ability of Hsp27 to stabilize actin microfilaments (25). Hsp27 binds to F-actin to prevent disruption of the cytoskeleton resulting from either heat shock and other stresses (26) and also inhibits mitochondrial release of Smac to confer resistance to dexamethasone in multiple myeloma cells (27). Hsp27 is also involved in regulation of AKT, inhibiting Bax activation to enhance cell survival (28). Hsp27 can inhibit apoptosis induced by etoposide or TNF-α by increasing IκBα ubiquitination and degradation, which increases the activity of the survival transcription factor nuclear factor-κB (NF-κB) (29).

While both increased IGF-1/IGF-1R signaling and Hsp27 expression are associated with CRPC progression (6, 7, 30, 31), the precise mechanisms by which the pro-survival effects of IGF-1/Hsp27 drive CRPC progression are incompletely defined. Here we define links between Hsp27 phosphorylation and IGF-1 signaling in CRPC, identifying p90Rsk as a downstream effector of IGF-1-activated Erk that directly phosphorylates Hsp27 as a key step in inhibition of Bad/14-3-3 complex formation and apoptosis.

Materials and methods

Plasmids

Hsp27 wild type (WT) was subcloned into lentivirus vector pHR-cytomegalovirus (CMV) as previously described (8). Hsp27 Triple mutant (TM) Alanine (Ala) was generated by direct mutagenesis replacing Ser15, Ser78, and Ser82 respectively with Alanine using QuikChange® mutagenesis kit according to manufacture instructions (Strategene). p90Rsk WT and DN plasmids were generously provided by Dr Blenis (Harvard University).

Antibodies and reagents

IGF-1 was purchased from Research Diagnostic Inc (NJ, USA). SB 203580, Ro 31-8220, PD 98056, Cyclohexamide from Calbiochem (San Diego, CA); total Hsp27/pHsp27 (Assay Designs, Michigan USA), pAkt/Akt, Bad, total Erk and poly(ADP ribose)polymerase (PARP) (Cell Signaling Technology, MA), p90Rsk/p-p90Rsk, pErk, p38 kinase, AR, IGF-1R, 14-3-3 antibodies, p90Rsk siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Purified recombinant non phosphorylated Hsp27 (expressed in bacteria) and active p90Rsk (expressed in Sf21 cells) were purchased from Assay Designs and Upstate Biotechnology, respectively. Secondary antibodies FITC and Rhodamine Red-x (Jackson ImmunoResearch Laboratories, Inc. West Grove, PA); and Flag-Agarose were purchased (Sigma Aldrich, St. Louis, MO).

Animal manipulation

All animal experimentation was conducted in accordance with accepted standards of the UBC Committee on Animal Care. LNCaP and PC-3 xenograft tumors were grown in athymic nude mice at four sites as modified from a previously reported method (5). PSA levels were measured by tail vein sera samples weekly using an immunoassay kit (ClinPro, Union City, CA). Mice were castrated at 6 weeks post inoculation with LNCaP cells. Tumors were harvested from the same mouse pre-castration (intact), and at 35 days post-castration (CRPC). Tumors were excised, dissected and immediately frozen in liquid nitrogen.

Immunohistochemistry

A tissue microarray was constructed using a Beecher microarray from paraffin-embedded specimens with matching H&E slides. Each case is represented with 3 cancer cores in the tissue microarray analysis. Sections were deparafinized and rehydrated with xylene and ethanol, and transferred to the 0.02% triton for permeabilization. Slides in citrate buffer (pH = 6) were heated in the steamer for 30 minutes, washed for 5 minutes in PBS, incubated in 3% BSA transferred to 3% H2O2 and incubated overnight with anti-pHsp27 antibodies (cell signaling) as described (8).

Cell proliferation assay

PC-3 cells were plated in DMEM with 5% FBS. The next day medium was changed to serum-free with or without 50 ng/ml of IGF-1 and cell proliferation was performed (32)

Western Blot analysis

PC-3 cells were first serum starved in DMEM for overnight. IGF-1 was then added at different concentrations or at different times. Inhibitors were pre-incubated for 1 hour prior addition of IGF-1. After treatment, cells were lysed in RIPA buffer and submitted to western blot as previously described (32)

Kinase Assay

1 ug of recombinant Hsp27 was incubated at 30°C for 1 hour with the different concentrations of purified active form of p90Rsk in reaction buffer(50mM Hepes, pH 7.4, 10mM MgCl2, 1mM EGTA, 1 mM DTT, 10 mM β-mercaptoethanol, 0.5mM vanadate, 50uM ATP and 5uCi of 32P-γ-ATP). The p90Rsk inhibitor (Ro-31-8220), was added to show the specificity of the reaction.

Immunoprecipitation

Total proteins (500 ug) were pre-cleared with protein-G sepharose (Invitrogen-Life Technologies, Inc) for 1 h at 4°C and immunoprecipitated with 2 ug of anti-Bad overnight at 4°C. The immune complexes were recovered with protein-G sepharose for 2 h and then washed with RIPA buffer at least three times, centrifuged, and submitted to SDS-PAGE following by western blotting. For p90 Rsk pull-down, cells were lysed in RIPA buffer 48 hours after transfection and 750ug of protein were incubated with Flag-Agarose for 3 hours; the immune complexes were washed and submitted to western blotting.

Caspase 3 activity

PC-3 cells were treated once daily for 2 days with 10 nM siRNA or Scr control. After the second transfection, cells were incubated +/− IGF-1 for 48 hours. Caspase 3 assay was performed according to the manufacturer’s recommendation (Biomol Research Laboratories, Inc, PA, USA). Briefly, 50 μg of total proteins were assessed to cleave the Ac-DEVD-AMC substrate. The cleaved products yielded fluorogenic reaction and were read in a microplate reader using a filter with wavelength 360 nm excitation/460 emission

Immunofluorescence

PC-3 cells were grown on coverslips and fixed in Parafolmaldehyde for 10 min at room temperature and immunofluorescence was performed as described previously (32) using Hsp27 (1:500) and p90Rsk (1:100) antibodies.

Flow cytometry analysis

PC-3 cell lines were plated in 10-cm dishes and 24 hrs later cells were serum starved and treated with or without 50ng/ml of IGF-1 or 10μg/ml of cyclohexamide for 24 hours. Sub-G0 was analyzed as described previously in (32).

Statistical Analysis

All data were analyzed by student’s t test. Levels of statistical significance were set at P < 0.05 (two-sided), and all statistical calculations were done by use of the Statview 4.5 (Abacus Concepts, Inc., Berkeley, CA).

Results

Phospho-Hsp27 is highly expressed in human CRPC tumors

To characterize levels of both total and phosphorylated Hsp27 (p-Hsp27) levels with CRPC, we analyzed staining patterns in human prostate cancer tissues before and after androgen ablation therapy. Figure 1A illustrates that total and p-Hsp27 levels are low in untreated tumors but the staining intensity increases after androgen ablation, becoming uniformly strong in CRPC and statistically higher compared to untreated patients (Fig. 1B). The increased levels of p-Hsp27 correlated with total Hsp27. We next characterized changes in total and phosphorylated Hsp27 levels in the human LNCaP xenograft model which mimics progression to CRPC after castration. Western blot analysis of treatment naïve and CRPC LNCaP tumors showed that p-Hsp27 levels increased after castration with no significant difference in total Hsp27 levels (Fig. 1C). This increase in p-Hsp27 levels correlated with increased levels of phosphorylated (but not total) Erk/p90Rsk and Akt protein levels (Fig. 1C). Increased p-Hsp27 levels also correlated with increased serum PSA levels, suggesting correlation between Hsp27 phosphorylation and androgen receptor (AR) -regulated PSA expression as reported previously (32). Next we evaluated p-Hsp27 levels in androgen-responsive LNCaP cells, castrate-resistant C4-2 cells, and AR-negative PC-3 cells. Western blot analysis indicates that while total Hsp27 levels are slightly higher in PC-3 xenografts, p-Hsp27 levels are highly up-regulated in PC-3 tumors compared to treatment naïve LNCaP tumors (Fig. 1D). Hsp27 expression and phosphorylation levels are also higher in C4-2 compared to LNCaP cells (data not shown). Collectively, these data indicate that increased p-Hsp27 levels correlate with CRPC.

Figure 1. Total and p-Hsp27 levels in prostate cancer tumors.

Figure 1

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A, Immunohistochemistry analysis for Hsp27 and p-Hsp27 in human prostate cancer tumors. Specimens from untreated tumors (0NHT), post-treated with 3 months neoadjuvant hormone therapy (3M NHT), 8M NHT, or CRPC were stained with p-Hsp27 or total Hsp27 in back-to-back sections. B, Mean Hsp27 phosphorylation (left panel) and Hsp27 expression (right panel) immunoreactivity in prostate cancer TMA before and after androgen ablation. Specimens were graded from 0 to +3 intensity representing the range from no staining to heavy staining by visual scoring and automated quantitative image analysis by pro-plus image software. Data from 110 patient samples (330 cores) were used to calculate mean and standard deviation. All comparison of stain intensity was made at x200 magnification. C, Phosphorylation levels of Hsp27, Erk, and p90Rsk in LNCaP xenografts. Proteins were extracted from LNCaP xenografts before castration (intact) or after progression to castration resistance as indicated by rising PSA levels. Western blots were performed using total (T)-Hsp27/p-Hsp27, T-Erk/p-Erk, T-p90Rsk/p-p90Rsk, T-Akt/p-Akt or vinculin as a loading control. D, Hsp27, Erk, and p90Rsk phosphorylation increase in AR negative PC-3 xenografts. Protein extracts from intact LNCaP and PC3 tumors were submitted to western blot using T-Hsp27/p-Hsp27, T-Erk/p-Erk, T-p90Rsk/p-p90Rsk, T-Akt/p-Akt or vinculin as a loading control.

IGF-1 increases PC-3 cell proliferation

We selected PC-3 cells to study the effect of IGF-1 on cell proliferation and survival because PC-3 cells are IGF-1R positive, AR negative and express high basal levels of p-Hsp27 (Fig. 2A). IGF-1 increased proliferative (Fig. 2B left panel) and decreased apoptotic (Fig. 2B right panel) rates in PC-3 cells grown in serum free media. Next we investigated the effect of IGF-1 on PC-3 cell survival using the apoptotic inducer cycloheximide (CHX). As expected, CHX treatment increased cleaved PARP (Fig. 2C left panel) and sub-G0 population (Fig. 2C right panel) as indicators of apoptosis, which were both reduced in the presence of IGF-1. Together, these results indicate that IGF-1 is cytoprotective in PC-3 cells.

Figure 2. IGF-1 enhances PC-3 cell survival and Hsp27 phosphorylation.

Figure 2

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A, Hsp27 is highly phosphorylated in PC-3 cells. 30 ug of total proteins from LNCaP and PC-3 cells were submitted to western blot using AR, IGF-1R, Hsp27, p-Hsp27 antibodies. GAPDH was used as loading control. B, IGF-1 increases PC-3 cell growth. PC-3 cells were serum starved or treated with 50ng/ml of IGF-1 and cell growth rates were determined by MTS assay and compared with control (day of treatment defined as 100%) (left panel). Cells were treated with 50ng/ml for 4 d, and proportion of cells in sub-G0, G0-G1, S, G2-M was determined by propidium iodide staining (right panel). C, IGF-1 protects PC-3 from apoptosis induced by cyclohexamide. PC-3 were treated with or without CHX for 24 hours in the presence or absence of IGF-1, and PARP cleavage was measured by western blot; vinculin was used as loading control (upper panel). SubG0 was determined by propidium iodide staining (lower panel). D, IGF-1 phosphorylates Hsp27 in a dose-dependent manner. PC-3 cells were serum starved overnight and treated with indicated concentrations of IGF-1 for 10 min. Proteins were submitted to Western blots using specific p-Hsp27, p-Erk, and p-Akt to assess phosphorylation status, and total antibodies were used as control (left panel). IGF-1 phosphorylates Hsp27 in time-dependent manner. PC-3 cells were serum starved overnight and treated with 50ng/ml of IGF-1 at indicated times. Western blots were performed using phospho-specific antibodies against Hsp27, p38 kinase, Akt, Erk, p90Rsk to assess phosphorylation status, and total antibodies were used as control (middle panel). IGF-1 induces Bad/14-3-3 complex. PC-3 cells were serum starved overnight and treated with 50ng/ml of IGF-1 at indicated times. 750ug of total proteins were immunoprecipitated with 2 ug of Bad antibody, and western blot performed using 14-3-3 or Bad antibodies (right panel).

IGF-1 induces Hsp27 phosphorylation

To identify relationships between Hsp27 in IGF-1 signaling and cell survival in prostate cancer, we next determined if IGF-1 phospho-activates Hsp27. A dose-dependent and time-course analysis of p-Hsp27 after IGF-1 treatment was studied in parallel with activation of IGF-1R signaling pathway. PC-3 cells were serum starved overnight and treated with increasing concentrations of IGF-1. IGF-1 potently induced dose- and time-dependent increases in Hsp27, Akt, and Erk phosphorylation (Fig. 2D left panel). Interestingly, p-Hsp27 increased within 5 minutes after IGF-1 exposure (Fig. 2D middle panel) and as expected, IGF-1 stimulated the time-dependent phosphorylation of p38 kinase, Akt, Erk and p90Rsk. Moreover, IGF-1 induces Bad/14-3-3 interaction in a time-dependent manner (Fig. 2D right panel) as a consequence of Bad phosphorylation by both Akt and Erk pathways. These results confirm that IGF-1 phospho-activates Hsp27 and enhances Akt and Erk signaling in PC-3 prostate cancer cells.

IGF-1 leads to Hsp27 phosphorylation via the MAPK pathway

To identify upstream effectors of IGF-1-induced Hsp27 phosphorylation, we pretreated PC-3 cells prior to IGF-1 stimulation with selected specific inhibitors, SB 203580, LY 294002, PD 98059 targeting p38 kinase, Akt, and Erk, respectively. The phosphorylation status of Hsp27, p38 kinase, Akt, and Erk were analyzed by western blotting. While neither SB203580 nor LY294002 altered IGF-1-induced Hsp27 phosphorylation (Fig. 3A, B), the Erk inhibitor PD 98059 abolished IGF-1-induced Hsp27 phosphorylation (Fig. 3C). We next tested whether IGF-1-induced Hsp27 phosphorylation occurs directly via Erk kinase or via its downstream effector, p90Rsk. PC-3 cells treated with the Ro-31-8220 inhibitor completely abolished IGF-1-induced Hsp27 phosphorylation (Fig. 3D) suggesting that IGF-1-mediated phosphorylation of Hsp27 occurs via the MAPK pathway downstream of Erk kinase.

Figure 3. IGF-1 phosphorylates Hsp27 downstream of MAPK.

Figure 3

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A, p38 kinase inhibition does not affect IGF-1-mediated Hsp27 phosphorylation. PC-3 cells were serum starved overnight and treated with indicated concentrations of SB 203580 for 1 hour prior 10 min IGF-1 stimulation. Proteins were extracted and Western blot were performed using antibodies for phospho- or total Hsp27 or p38 kinase. B, Akt inhibition does not affect IGF-1-mediated Hsp27 phosphorylation. PC-3 cells were serum starved overnight and treated with 20 uM of LY 203580 for 1 hour prior 5 min IGF-1 stimulation. Western blot were performed using p-Hsp27, and p-Akt or T-Hsp27 or T-Akt for control loading. C, Erk kinase inhibitor blocks IGF-1-mediated Hsp27 phosphorylation. PC-3 cells were serum starved overnight and treated with indicated concentration of PD 90059 for 1 hr prior 10 min IGF-1 stimulation. Proteins were extracted and Western blot performed using p-Hsp27, and p-Erk or T-Hsp27 for control loading. D, Effect of p90Rsk inhibition on IGF-1-mediated Hsp27 phosphorylation. PC-3 cells were serum starved overnight and treated with indicated concentrations of Ro31-8220 for 1 hr prior 10 min IGF-1 stimulation. Proteins were extracted and Western blot performed using p-Hsp27/T-Hsp27, and p-p90Rsk/p90Rsk.

p90Rsk phosphorylates Hsp27 in vitro and in vivo

To determine whether p90Rsk phosphorylates Hsp27, we performed an in vitro kinase assay using purified unphosphorylated Hsp27 and constitutively active p90Rsk. Figure 4A shows that in the absence of p90Rsk, Hsp27 is unphosphorylated and that only 50ng of p90Rsk was sufficient to phosphorylate 1ug of Hsp27 (1:50) (Fig. 4A, left panel). Moreover, the p90Rsk inhibitor Ro-31-8220 significantly suppressed the ability of p90Rsk to phosphorylate Hsp27 (Fig. 5A right panel) suggesting that p90Rsk specifically and directly phosphorylates Hsp27 in vitro. To corroborate this finding, we examined the ability of p90Rsk to phosphorylate Hsp27 in vivo. Overexpression of wild type (WT) p90Rsk increased Hsp27 phosphorylation which was amplified in the presence of IGF-1 stimulation compared to empty vector. However, p90Rsk dominant negative/kinase dead (DN) overexpression decreases endogenous Hsp27 and p90Rsk phosphorylation in the absence or presence of IGF-1 stimulation (Fig. 4B left panel). To further support these results, p90Rsk siRNA decreased p-Hsp27 levels that directly correlated with p90Rsk knockdown (Fig. 4B, right panel). Interestingly, p90Rsk interacts with Hsp27 (Fig. 4C left panel), and this interaction is dependent on p90Rsk activation since the DN of p90Rsk abolished p90Rsk/Hsp27 interaction (Fig. 4C, right panel). Hsp27 and p90Rsk co-localize in the cytoplasm in PC-3 cells (Fig. 4D, left panel). Moreover, immunohistochemical analysis of human prostate cancer specimens indicates that Hsp27 and p90Rsk are present in the same subset of the cells after androgen ablation (Fig. 4D, right panel). Collectively, these results indicate that p90Rsk directly interacts with and phosphorylates Hsp27 in vitro and in vivo and this interaction is p90Rsk dependent.

Figure 4. p90Rsk phosphorylates and interacts with Hsp27.

Figure 4

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A, p90Rsk phosphorylates Hsp27 in vitro. 1 ug of purified Hsp27 was incubated with indicated concentrations of active p90Rsk in kinase buffer in the presence of 32P-γ-ATP for 30 min at 30ºC. Kinase reactions were terminated using Laemeli buffer and resolved in 10% SDS-PAGE. Gels were stained with Coomassie blue, dried and exposed for autography (Left panel). 1 ug of purified Hsp27 was incubated with 250 ng of purified p90Rsk in the presence of absence of indicated concentrations of Ro31-8220. Kinase reactions were terminated using Laemeli buffer and resolved in 10% SDS-PAGE. Gels were stained with Coomassie blue, dried and exposed for autography (right panel). B, p90Rsk phosphorylates Hsp27 in vivo. PC-3 cells were transfected with p90Rsk WT-Flag or DN-Flag cDNA and empty vector for 48 hr, then serum starved overnight and stimulated with IGF-1 for 10 min. Proteins were submitted to Western blot using p-Hsp27/T-Hsp27, p-p90Rsk/T-p90Rsk (left panel). In the right panel, PC-3 cells were transfected with 20nM of siRNA p90Rsk2 for 48 hr, then, then serum starved overnight and stimulated with IGF-1 for 10 min. Proteins were submitted to Western blot using p-Hsp27/T-Hsp27, p-p90Rsk/T-p90Rsk. C, Hsp27 is associated with p90Rsk. 750ug of total proteins were immunoprecipitated with 2ug of Hsp27, p90Rsk or control IgG overnight. Immunoprecipitated material was submitted to western blot using Hsp27 and p90Rsk antibodies (left panel). p90Rsk WT or DN cDNA were transfected into PC-e3 cells; 72 hrs later proteins were extracted and 750ug of total proteins were pulled down with Flag-Agarose and precipitated materials were recovered by centrifugation. Western blot were performed using T-Hsp27, p-p90Rsk and T-p90Rsk as control loading (right panel). D, p90Rsk co-localizes with Hsp27 in PC-3 cells. PC-3 cells were fixed with paraformaldehyde and double stained with Hsp27 (1:500) and p90Rsk (1:100) antibodies (left panel). p90Rsk and Hsp27 stained the same subsets of the cells in NHT prostate cancer tumors. 3M NHT tumors were stained back to back with Hsp27 and p90Rsk showing that both proteins share the same subset of the cells (right panel).

Figure 5. Hsp27 phosphorylation and expression levels correlate with targets of MAPK and Akt pathways, including Bad/14-3-3 complex formation.

Figure 5

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A, Hsp27 knockdown decreases levels of downstream targets of MAPK and Akt pathways. PC-3 cells were treated with 10nM Scr or siRNA Hsp27 twice and 48 hours after transfection, cells were serum starved overnight and then stimulated with IGF-1 for 10 min. Proteins were extracted and used either for direct Western blot using T-Hsp27/p-Hsp27, T-Akt/p-Akt, T-Erk/p-Erk, T-pRsk/p-p90Rsk, T-Bad/p-Bad112-136 or Bad immunoprecipitation followed by Western blot for 14-3-3. B, Hsp27 over-expression increases levels of downstream targets of MAPK and Akt pathways. PC-3 cells over-expressing Hsp27-WT or empty vector were serum starved overnight and stimulated with IGF-1 for 10 min. Proteins were extracted and used either for direct Western blot using T-Hsp27/p-Hsp27, T-Akt/p-Akt, T-Erk/p-Erk, T-pRsk/p-p90Rsk, T-Bad/p-Bad112-136 or Bad immunoprecipitation followed by Western blot for 14-3-3. C, Hsp27 phospho-mutants suppresses downstream targets of MAPK and Akt pathways. PC-3 cells over-expressing Hsp27-TM or empty vector were serum starved overnight and stimulated with IGF-1 for 10 min. Proteins were extracted and used either for Western blot using T-Hsp27/p-Hsp27, T-Akt/p-Akt, T-Erk/p-Erk, T-pRsk/p-p90Rsk, T-Bad/p-Bad112-136 or Bad immunoprecipitation followed by Western blot for 14-3-3.

Hsp27 Stimulates Bad Binding to 14-3-3

Akt and Erk activation leads to phosphorylation of Bad at Ser112 and Ser136 which increases 14-3-3 binding and sequesters Bad in the cytoplasm to suppress its pro-apoptotic activity (33). Since Hsp27 is a cytoprotective chaperone and is phosphorylated by p90Rsk, and Hsp27 knockdown increases apoptosis rates, we hypothesized that Hsp27 levels may modulate cell survival via the modulation of Bad phosphorylation and formation the Bad/14-3-3 complex. We first characterized effects of Hsp27 on levels of IGF-1R downstream targets after IGF-1 stimulation and found that Hsp27 knockdown abolished IGF-1-induced phosphorylation of Hsp27, Akt, Erk, Bad on Ser112 and Ser136 without affecting their total expression levels (Fig. 5A). In contrast, Hsp27-WT over-expression enhances the phosphorylation of Akt, Erk, and p90Rsk compared to empty vector (Fig. 5B). An Hsp27-triple mutant (Serine 15, 78 and 82 were mutated with Alanine) also decreased levels of phospho-Akt, Erk, and p90Rsk, consistent with its dominant-negative effect and loss of Hsp27 function (Fig. 5C). Next, we investigated the effect of Hsp27 on Bad phosphorylation as a downstream effector of Akt and MAPK pathways. Over-expression of Hsp27-WT increases phosphorylation of Bad and Bad/14-3-3 complex formation, while Hsp27 downregulation at expression (siRNA) or phosphorylation (TM) levels decreased levels of Bad phosphorylation on Ser 112 and 136 and Bad/14-3-3 complex, even in the presence of IGF-1. We also found that silencing Hsp27 using OGX-427 (data not shown) or siRNA in PC-3 cells treated with or without IGF-1 increased sub G0 fraction (Fig. 6A) and caspase 3 activity (Fig. 6B). These results show that Hsp27 silencing increases apoptotic rates and this effect was not reversed by IGF-1. Collectively, these results indicate that levels of total and p-Hsp27 are important determinants of Bad/14-3-3 complex stability and represent one mechanism regulating cancer cell survival.

Figure 6. Hsp27 knockdown induces cell apoptosis.

Figure 6

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A, Effect of Hsp27 knockdown on cell cycle population. PC-3 cells were treated with 10 nM Hsp27 siRNA or Scr control and then incubated +/− IGF-1 for 48 hours. Cell cycle populations were analyzed using propodium iodide staining (left panel). B, Effect of Hsp27 knockdown on Caspase 3 activity. PC-3 cells were treated with 10 nM Hsp27 siRNA or Scr control and then incubated +/− IGF-1 for 48 hours. Total proteins (50ug) from each condition were lysed in RIPA buffer and monitored for their ability to cleave the fluorogenic substrate Ac-DEVD-AMC. Fluorescence generated by the cleavage was quantified by using a spectrofluorometer.

Discussion

Survival proteins and signaling pathways up-regulated following androgen ablation that function to inhibit cell death are of special importance to treatment resistance and progression of castrate resistant prostate cancer (CRPC). Increased activity in the IGF-1R signaling pathway and altered expression of IGF-1 axis components are strongly implicated in prostate carcinogenesis and CRPC progression (30, 34), and contributes to nearly 50% of cell growth and proliferation in some models (35). Castrate-resistant progression of human prostate cancer xenografts is associated with increased expression of IGF-1R and IGF-I signaling pathways (30, 36). In prostate cancer and other cancer models, IGF-I is mitogenic and anti-apoptotic, and IGF-1R blockade inhibits castration-induced prostate cancer xenograft growth (37). The PI3/Akt and Erk signaling pathways are the principle cell survival pathways activated by the IGF-1 axis (38), and include the phosphorylation and subsequently deactivation of the pro-apoptotic Bad protein (39). Hsp27 is phospho-activated during cell stress to form oligomers that prevent protein aggregation and/or regulate activity/degradation of certain client proteins (40). Hsp27 phosphorylation is catalysed by MAPKAP kinase-2, a target of the p38 MAP kinase (41, 42). Higher levels of Hsp27 are commonly detected in many cancers (18), where it plays a role in thermotolerance, cytoprotection, steroid hormone response and molecular chaperoning (43). Hsp27 is also involved in regulation of Akt (44), and enhances NF-κB activity by increasing degradation of I-κBα (29). Hsp27 also chaperones and shuttles stat3 and AR into the nucleus, enhancing transcription of several Stat3- and AR- regulated survival genes (8). In prostate cancer, stress-induced increases in Hsp27 after hormone therapy inhibit castration-induced cell death, accelerate CRPC progression, and render cells more resistant to chemotherapy (8, 19).

Since Hsp27 is involved in many pathways regulating the response of a cell to stress and therapeutic stimuli, including MAPK and Akt pathways, and that increased Hsp27 and IGF-1 activity are both associated with CRPC progression (19, 30), we explored interactions between IGF-1 signaling and Hsp27 phospho-activation that could promote castrate-resistant disease. Here we report that both total and p-Hsp27 are highly expressed in and correlates with CRPC in human prostate tissues (Fig. 1A) and LNCaP xenografts post-castration (Fig. 1B). Increased p-Hsp27 levels are accompanied by Akt, Erk and p90Rsk phosphorylation, all involved in cell survival and proliferation. We previously reported that Hsp27 phosphorylation plays a key role on AR activation and stability (32), that increased Hsp27 levels accelerate castrate-resistant LNCaP xenograft growth, and that Hsp27 knockdown using antisense (OGX-427, OncoGenex Technologies) or siRNA delays progression post-castration. Collectively, these data functionally link phospho-activated Hsp27 to stress-induced prostate cancer cell survival and castrate-resistant progression (8) (32).

Depending on cell context and stimulus, Hsp27 phosphorylation is regulated by several protein kinases involved in cell survival and proliferation, including p38 kinase and Akt (44, 45). PKC α, δ and cAMP-dependent kinase have also been reported to phosphorylate Hsp27 (46) suggesting that Hsp27 regulates activity of many varied signaling pathways. We show here for the first time that Hsp27 is directly phosphorylated by p90Rsk kinase, a downstream effector of MAPK pathway, in an IGF-1 dependent manner. Several lines of evidence support a link between Hsp27 phosphorylation and p90Rsk in prostate cancer progression. First, both Hsp27 and p90Rsk are overexpressed in prostate cancer and both enhance AR transcription activity (32, 47). Using in vitro and in vivo assays, we show that p90Rsk directly phosphorylates Hsp27 and that a p90Rsk dominant negative inhibits Hsp27 phosphorylation. Immunoprecipitation and immunofluorescence studies indicate that Hsp27 is associated and co-localized with p90Rsk. Moreover, Hsp27 and p90Rsk are present in the same subsets of the cells in prostate cancer tumors after androgen ablation. Collectively, these studies indicate that p90Rsk interacts and phosphorylated Hsp27, a key functionally relevant mechanism linked to CRPC growth and survival.

p-Hsp27 is a downstream effector of several signaling pathways linked to cell survival and CRPC including MAPK/Erk, PI3K/Akt, and p38 kinase. In the LNCaP xenograft model, increased p-Hsp27 levels correlate with increased levels of phosphorylated Erk, p90Rsk, and Akt, suggesting that growth factor signaling pathways like IGF-1 and IL-6 are activated after castration. Enhanced IGF-1 signaling, mediated by increased IGF-1R (30), IGFBP-2 (48) and IGFBP-5 (7) levels is believed to be a functionally and clinically relevant driver of CRPC progression (49). Interactions between IGF-1 signaling and Hsp27 via activation of MAPK/p90Rsk and Hsp27 phospho-activation lead to Bad/14-3-3 complex stabilization, Bad sequestration in the cytoplasm, and inhibition of apoptosis. In summary, Hsp27 is a key interacting component of the IGF-1 signaling in the development of CRPC. Hsp27 is directly phosphorylated by p90Rsk to serve as a downstream effector of IGF-1 signaling, promoting cell survival pathway by chaperoning and stabilizing Bad/14-3-3 complexes. These data support the development of targeted inhibitors of Hsp27 in CRPC.

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