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Cancer Research 68, 9255, November 15, 2008. doi: 10.1158/0008-5472.CAN-08-1224
© 2008 American Association for Cancer Research
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Endocrinology

The Mitogen-Activated Protein Kinase Phosphatase Vaccinia H1–Related Protein Inhibits Apoptosis in Prostate Cancer Cells and Is Overexpressed in Prostate Cancer

Yke Jildouw Arnoldussen1, Petra I. Lorenzo1, Maria E. Pretorius2,3, Håkon Wæhre2,3,5, Bjørn Risberg2,3,4, Gunhild M. Mælandsmo6, Håvard E. Danielsen2,3 and Fahri Saatcioglu1

1 Department of Molecular Biosciences and 2 Center for Cancer Biomedicine, University of Oslo; 3 Institute for Medical Informatics, Divisions of 4 Pathology and 5 Surgery, and 6 Department of Tumor Biology, Rikshospitalet University Hospital, Oslo, Norway

Requests for reprints: Fahri Saatcioglu, University of Oslo, P.O. Box 1041, Blindern, Oslo, 0316, Norway. Phone: 47-22854569; Fax: 47-22857207; E-mail: fahris{at}imbv.uio.no.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Androgen ablation during the initial stages of prostate cancer causes regression of the tumor due to an increase in apoptosis and reduced cellular proliferation. However, prostate cancer invariably progresses to an androgen-independent state for poorly understood reasons. Previous studies showed that c-Jun NH2 terminal kinase (JNK) is required for 12-O-tetradecanoylphorbol-13-acetate (TPA)– and thapsigargin (TG)–induced apoptosis in the androgen-responsive prostate cancer cell line LNCaP. Androgens protect LNCaP cells from TPA-induced or TG-induced apoptosis via down-regulation of JNK activation. However, the molecular mechanisms of this inhibition are not clear. Here, we systematically investigated the possible regulation of mitogen-activated protein kinase phosphatases/dual-specificity phosphatases during apoptosis of LNCaP cells and found that Vaccinia H1–related protein (VHR/DUSP3) is up-regulated by androgens during inhibition of apoptosis in LNCaP cells, but not in androgen-independent DU145 cells. Ectopic expression of wild-type VHR, but not a catalytically inactive mutant, interfered with TPA- and TG-induced apoptosis. Consistently, small interfering RNA–mediated knockdown of endogenous VHR increased apoptosis in response to TPA or TG in the presence of androgens. Furthermore, COS7 cells stably expressing wild-type VHR, but not a mutant, had a decrease in JNK phosphorylation. In vivo, VHR expression decreased in the androgen-dependent human prostate cancer xenograft CWR22 upon androgen withdrawal and was inversely correlated to JNK phosphorylation. Expression analysis in human prostate cancer specimens showed that VHR is increased in prostate cancer compared with normal prostate. These data show that VHR has a direct role in the inhibition of JNK-dependent apoptosis in LNCaP cells and may therefore have a role in prostate cancer progression. [Cancer Res 2008;68(22):9255–64]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Androgens play an important role in regulating growth, differentiation, and cell death responses in the normal and cancerous prostate. The actions of androgens are mediated through the androgen receptor (AR), a ligand-activated transcription factor that belongs to the nuclear receptor superfamily (1–3). A decrease in circulating androgen levels, by either surgical means (castration) or through androgen deprivation therapy, results in decreased cell proliferation, increased apoptosis, and atrophy in prostate epithelial cells (4). However, prostate tumors relapse and the disease progresses to an androgen-independent state (5). To date, the molecular mechanisms of transition from androgen dependence to androgen independence remain poorly understood, and little is known about the link between androgens and apoptosis signaling pathways in prostate cancer.

One of the signaling pathways that has been implicated in prostate carcinogenesis is the mitogen-activated protein kinase (MAPK) pathway. The MAPK cascades play an important role in transducing environmental stimuli to changes in gene expression by virtue of their ability to phosphorylate and regulate the activity of various transcription factors and other molecules (6). The MAPK cascades are composed of three protein kinases that act in series by activating one another through phosphorylation. A MAPK is activated by a MAPK kinase (MAPKK), which in turn is activated by a MAPKK kinase. In mammals, there are three well-characterized MAPKs: extracellular signal-regulated kinase (ERK), c-Jun NH2 terminal kinase (JNK)/stress-activated protein kinase, and p38 MAPK. The ERK pathway is typically stimulated by growth-related signals and is mainly involved in growth, differentiation, and development. p38 MAPK and JNK are activated by growth factors, proinflammatory cytokines, and cellular stress, and their activation has been implicated in apoptosis, as well as in oncogenic transformation, inflammation, development, and differentiation (7, 8).

The MAPK pathways can be inactivated by tyrosine phosphatases, serine/threonine phosphatases, and MAPK phosphatases (MKP)/dual-specificity phosphatases (DUSP; ref. 9). These phosphatase families are implicated in the regulation of mitogenic and other signaling pathways that are mediated by MAPKs. The MKPs, in contrast to the other two phosphatase families, specifically dephosphorylate the MAPKs at phosphorylated threonine/serine and tyrosine residues located in their activation loop (9). To date, 10 typical MKPs have been identified in humans that share the common CDC25 homology domain but have different subcellular localization, MAPK affinity, and structure, where determined (9–11). In addition, there is a group of 19 atypical MKPs, of which several are MAPK-specific (10). Atypical phosphatases generally consist of less than 250 amino acid residues and are characterized by the lack of a CDC25 homology domain. Vaccinia H1–related protein (VHR) is an atypical MKP (12), which has been shown to dephosphorylate both ERK and JNK in different cell lines (13–15). In HeLa cells, VHR regulates cell cycle progression and its knockdown leads to cell cycle arrest and cell senescence (16).

We have previously shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and thapsigargin (TG) induce apoptosis in the androgen-responsive prostate cancer cell line LNCaP through a mechanism that requires JNK activation (17). Furthermore, we have shown that androgens protect LNCaP cells from TPA- and TG-induced apoptosis, which is mediated by down-regulation of JNK activity (18). Inhibition of JNK activation by androgens was dependent on AR, androgen dose and time, and required de novo gene transcription. In the presence of androgens, an increase in phosphatase activity was observed and the dephosphorylation rate of JNK was faster than in vehicle-treated LNCaP cells (18). This suggested that phosphatases, at least in part, may mediate JNK-dependent apoptosis. We therefore systematically investigated possible changes in the expression and regulation of MKPs during TPA-induced and TG-induced apoptosis of LNCaP cells, as well as during prostate cancer progression.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture. The different cell lines were maintained, and treatments were done as described previously (18, 19).

Xenografts. Transplantation, growth, and harvesting of tumors from mice bearing CWR22 xenografts were as described previously (20).

Transfection and RNA interference. Transfection of LNCaP cells with pcDNA3-VHR, pcDNA3-VHR-C124S (generous gifts from John M. Denu; ref. 14), HA-JNKK2-JNK1 (generous gift from Anning Lin; ref. 21), and pEGFP were performed using FuGene 6, as per the manufacturer's instructions (Roche Diagnostics). For obtaining COS7 cells that stably express wild-type VHR or its mutant, cells were transfected with pcDNA3-VHR or pcDNA3-VHR-C124S using FuGene 6. At 24 h after transfection, the cells were plated in 15-cm dishes, and after an additional 48 h, G418 (500 µg/mL) was added to the medium. Outgrowth of single colonies were harvested and expanded as separate clones. A control cell line was created under similar conditions using a pcDNA3 vector without insert. Small interfering RNA (siRNA) was used to silence VHR. The siRNA duplex used for targeting human VHR was (sense strand): 5'-GGCAGAAGAUGGACGUCAAdTT-3' (Dharmacon). A duplex targeting the luciferase gene was used as a negative control (Qiagen). siRNA (200 nmol/L) was transfected into LNCaP cells using Oligofectamine, as per the manufacturer's instructions (Invitrogen). Where indicated, R1881 was added 1 h before siRNA transfection.

Quantitative PCR. RNA extraction, cDNA synthesis, and quantitative PCR (Q-PCR) were as previously described (22). PCR primers used for VHR were forward 5'-CGTCTGGCTCAGGACATC-3' and reverse 5'-CATTGAGCTGGCAGAGTGG-3'. A standard curve made from serial dilutions of cDNA was used to calculate the relative amount of VHR in each sample. These values were normalized to the relative amount of the internal standard TBP. Primers used for TBP were forward 5'-GAATATAATCCCAAGCGGTTTG-3' and reverse 5'-ACTTCATCACAGCTCCCC-3'. The experiments were performed in duplicate and repeated thrice. Primers used for other MKPs tested are available upon request.

Western analysis. Western analysis was carried out as previously described (18). Antibodies used were against VHR, phJNK, totJNK, phERK, totERK (all from Cell Signaling), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz), and {alpha}-tubulin (Sigma-Aldrich). ECL Western blotting analysis system was used for the detection of the immunoreactive bands according to the manufacturer's instructions (Amersham Pharmacia Biotech).

Immunofluorescence microscopy and TUNEL assay. LNCaP cells were transfected with the constructs described above. For JNK expression studies, immunofluorescence microscopy was done as described previously (18). Ectopic expression of JNK was visualized by an anti-HA antibody (Sigma-Aldrich). For ectopic expression studies of VHR, cells were incubated with anti-VHR rabbit antibody (generous gift from John M. Denu) overnight at 4°C. Cells were then incubated with secondary antibody Alexa fluor 594–linked goat anti-rabbit IgG (Invitrogen) for 1 h. To detect apoptosis, an In situ Cell Death Detection kit (TUNEL assay) was used according to the manufacturer's instructions (Roche Diagnostics) and as described previously (18). Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Fluorescence was observed using an Axioplan2 imaging microscope (Zeiss), and pictures were taken with an AxioCam HRc camera (Zeiss). At least three areas and a minimum of 300 cells per area were counted, and the number of TUNEL-positive cells was expressed per 100 of the total number of cells.

Tissue microarray samples. Tissue microarrays (TMA) were prepared from radical prostatectomy specimens from patients operated at the Norwegian Radium Hospital between 1988 and 1996 and followed up after surgery. Prostate-specific antigen (PSA) measurements were performed before and after operation and at every subsequent clinical examination. Follow-up period ranged from 2 to 176 mo (mean, 73.3 mo). Patients were considered to have clinically evident recurrence of disease if any of the following were present: (a) evidence of local recurrence (confirmed by histologic biopsies or ultrasound) or (b) evidence of distant metastasis (detected by skeletal scintigraphy and/or magnetic resonance imaging). If a patient who suffered from relapse had postoperative serum PSA of >4 ng/mL before the date of either local recurrence or metastasis, the date of elevated PSA was set as the relapse date (Supplementary Fig. S1).

H&E-stained sections were made from each selected primary tumor block (donor blocks) of paraffin-embedded material to define representative tumor regions. With the use of the tissue array instrument (Beecher Instruments), two tissue cylinders (0.6 mm in diameter) were punched from regions of the donor block. Control samples of noncancer tissue from the paraffin blocks were also taken. Gleason score used in the analysis was the highest Gleason score in each of the prostatectomy series.

Immunohistochemistry. Imunohistochemistry analysis was done as described previously (22). An anti-VHR rabbit polyclonal antibody (Cell Signaling) was used. Scoring was essentially identical to as previously reported (19). SPSS 15.0 software was used for statistical analysis. Mann-Whitney test was applied to compare the expression level of VHR between normal and malignant tissues. For correlating VHR intensity to Gleason score, linear logistic regression was used, and for univariate survival analysis, a log-rank test was performed together with Kaplan-Meier graphs.

Statistics. Statistical analysis was performed using the Student's t test, unless indicated otherwise. Values of P < 0.05 were considered as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Expression and regulation of MKPs during apoptosis of LNCaP cells. To determine if MKPs are involved in androgen-mediated inhibition of JNK-dependent apoptosis in LNCaP cells, we used Q-PCR to assess possible changes in the expression levels of the MKP family members in cells treated with TPA and TG in the presence or absence of the synthetic androgen R1881. As shown in Table 1 , MKP expression was differentially regulated in response to these agents (detailed data for two MKPs, VH3 and MKP6, are presented in Supplementary Fig. S2). In addition, the possible androgen regulation of most MKPs was checked (Table 1). In most cases, R1881 alone induced the expression of various MKPs and addition of TPA or TG increased this further. Among the MKPs tested, VHR, MKP1, VH3, and MKP6 were most consistent in displaying significant changes in expression in response to both TPA and TG, as well as androgen treatment. Both MKP1 and VHR effectively dephosphorylate JNK (14, 23), whereas VH3 is not specific for JNK (24), and MKP6 has not been studied in detail previously. We therefore chose to analyze VHR in further detail.


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  		Table 1. Overview of MKP expression in response to apoptosis inducers TPA and TG in the presence of R1881 compared with the expression in cells treated with TPA or TG alone

 
VHR mRNA accumulation was significantly increased upon TPA + R1881 treatment compared with TPA alone at all time points tested (Fig. 1A ). There was also a modest up-regulation of VHR in response to R1881 alone. Similarly, VHR was significantly up-regulated by TG + R1881 treatment compared with TG alone. However, at later time points, this effect was lost (Fig. 1B). These data indicate that VHR mRNA expression is increased during R1881-mediated inhibition of apoptosis in LNCaP cells.


Figure 1
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  		Figure 1. mRNA and protein expression levels of VHR in LNCaP cells treated with TPA or TG in the presence or absence of R1881. LNCaP cells were pretreated with vehicle or R1881 for 40 h before adding TPA or TG for the indicated times. Controls were treated with vehicle or R1881 for 48 h after the 40-h pretreatment with R1881. The mRNA expression levels of VHR were investigated by Q-PCR. TPA (A) and TG (B) treatments. Columns, mean of three independent experiments performed in duplicate; bars, SE. *, P < 0.05 indicate significant difference between R1881 + TPA–treated or R1881 + TG–treated cells compared with cells treated with TPA or TG alone. C and D, representative Western blots of VHR expression in TPA and TG experiments, respectively. {alpha}-Tubulin was used as loading control.

 
To assess whether VHR protein was similarly regulated, whole cell extracts were made from cells treated with TPA or TG in the presence or absence of R1881 and examined by Western blot analysis. VHR protein was increased in response to TPA + R1881 and TG + R1881 compared with TPA or TG alone consistent with the mRNA expression data presented above (Fig. 1C and D). Interestingly, although no significant regulation of VHR mRNA expression was observed in long time points with TG treatment, VHR protein levels were increased at these times, indicating that the action of androgen on VHR expression involves both VHR mRNA and protein. These data indicate that VHR may have an essential role in the inhibition of JNK-dependent apoptosis in LNCaP cells.

To investigate if the increase in VHR expression upon TPA or TG treatment in the presence of R1881 is specific for androgen-responsive LNCaP cells, the androgen-independent cell line DU145 was used. Cells were treated as before, and VHR protein expression was investigated (Supplementary Fig. S3). VHR protein levels were slightly increased in response to R1881 compared with vehicle-treated cells, but there was no increase in expression in cells treated with TPA or TG in the presence of R1881. These data indicate that induction of VHR expression is dependent on intact androgen signaling.

Ectopic expression of VHR protects LNCaP cells from TPA-induced and TG-induced apoptosis. The data presented above suggested that up-regulation of VHR can down-regulate JNK activation and thereby prevent apoptosis in LNCaP cells. To assess this possibility, LNCaP cells were transiently transfected with expression vectors specifying wild-type VHR, the catalytically inactive mutant, VHR-C124S, or with an expression vector for green fluorescent protein (pEGFP) as a control. The cells were grown in the presence or absence of R1881, and apoptosis was induced with TPA or TG. At the indicated times, the cells were fixed and incubated with VHR antibody and TUNEL assay was performed. Whereas wild-type VHR expression inhibited apoptosis induced by both TPA and TG, the mutant VHR had largely lost this ability (Fig. 2 ). For example, wild-type VHR inhibited TPA-induced apoptosis by >95% at 24 hours, but inhibition by mutant VHR was only 50% (Fig. 2B). Similarly, whereas wild-type VHR inhibited ~90% of apoptosis induced by TG, inhibition by the mutant VHR was only 35% (Fig. 2C). The effect of mutant VHR in decreasing apoptosis, albeit significantly less than wild-type VHR, could be due to the possibility that it can still bind and sequester JNK through JNK-interacting protein-1, thereby blocking its activation, as has been shown for two other MKPs (25). In parallel to the TUNEL assay, DAPI staining and changes in nuclear morphology and nuclear fragmentation were also used to assess apoptosis and virtually identical results were obtained (data not shown). The same experiment was also performed in the presence of R1881, and similar results were obtained, except for lower levels of total cell death (data not shown). In summary, these data show that VHR inhibits apoptosis in LNCaP cells.


Figure 2
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  		Figure 2. Ectopic expression of VHR in LNCaP cells undergoing apoptosis induced by TPA or TG. LNCaP cells were cultured on coverslips and were transiently transfected with pEGFP (GFP), pcDNA3-VHR (WT), or pcDNA-VHR-C124S (Mut). The cells were treated with TPA or TG for the indicated time points. After fixation, cells were incubated with VHR antibody, TUNEL assay was performed, and cells were counterstained with DAPI. Arrows indicate apoptotic cells. A, DAPI staining, GFP, VHR immunofluorescence, and TUNEL staining are shown for cells transfected with GFP-VHR, WT-VHR, and Mut-VHR that were treated with TPA for 24 h or TG for 48 h. For two independent experiments performed in duplicate, at least three areas and a minimum of 300 cells per area were counted for each coverslip. Columns, mean of the percentage of cell death is shown for 12-h and 24-h TPA treatments (B) and 36-h and 48-h TG treatments (C); bars, SE. *, P < 0.05 indicates significant difference from nontransfected cells.

 
Knockdown of VHR increases cell death induced by TPA and TG in R1881-treated LNCaP cells. The data presented above suggested that VHR inhibits apoptosis induced by TPA and TG in LNCaP cells. If this is the case, knockdown of VHR should have the opposite effect, namely, increased apoptosis. To assess this, we used a siRNA that efficiently knocks down VHR (16). LNCaP cells were transfected with siRNA directed against luciferase as a control or the siRNA for VHR. A significant reduction in both VHR mRNA and protein expression was observed in cells transfected with VHR siRNA compared with luciferase siRNA that lasted at least up to 96 hours, both in the presence or absence of R1881 (Supplementary Fig. S4).

To investigate the effect of VHR knockdown on apoptosis, TUNEL assays were performed on LNCaP cells transfected with siRNA targeting luciferase or VHR. Cells were treated with R1881 or vehicle for 40 hours before addition of TPA or TG for the indicated times. As expected, there was an increase in apoptosis in response to TPA compared with vehicle-treated cells (Fig. 3A ). However, the extent of apoptosis was decreased in cells treated with R1881 plus luciferase siRNA, and this decrease was significantly reversed in the presence of VHR siRNA (Fig. 3A). Quantification indicated that the inhibitory action of R1881 on TPA-induced apoptosis was partially reversed when VHR levels were decreased, indicating that R1881 inhibition of apoptosis in LNCaP cells, at least in part, is mediated by VHR (Fig. 3B).


Figure 3
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  		Figure 3. Knockdown of VHR increases cell death which is inhibited by R1881. A and C, LNCaP cells were plated on coverslips and transfected with either luciferase (Luc) siRNA or VHR siRNA. Cells were then pretreated with vehicle or R1881 for 40 h before adding TPA or TG for the indicated times. TUNEL assay was performed, and the cells were counterstained with DAPI. Representative pictures from 24-h TPA treatment and 48-h TG treatment. Insets indicate magnified areas and arrowheads point to cells undergoing apoptosis. B and D, quantification of cell death observed in TPA and TG treatments, respectively. A minimum of 400 cells were counted in at least four different areas per coverslip. Columns, average of three independent experiments performed in duplicate; bars, SE. *, P < 0.05 indicates significant difference from luciferase siRNA-transfected cells.

 
Essentially, the same results were obtained with TG (Fig. 3C and D). In fact, at 36 hours of TG treatment, knockdown of VHR almost completely reversed the inhibitory effect of R1881 on TG-mediated apoptosis. At 48 hours of TG treatment, there was still a significant effect of VHR knockdown, but the reversal of the R1881 effect was not complete.

In parallel to the TUNEL assays, DAPI staining was performed as an independent measure of apoptosis under these conditions, and similar results were obtained (Fig. 3 and Supplementary Fig. S5A and B). Coupled to the results presented above with ectopic expression of VHR, these data show that VHR has a direct role in the inhibition of apoptosis in LNCaP cells.

Stable expression of VHR leads to a decrease in JNK phosphorylation. To investigate if VHR has a role in specific dephosphorylation of JNK activated by TPA or TG, COS7 cell lines that stably express either an empty vector or expression vectors specifying wild-type VHR or VHR-C124S mutant were generated. The cells were treated with TPA, TG, or vehicle for the indicated times, and the phosphorylation status of JNK and ERK was investigated by Western blot analysis (Fig. 4A ). Quantification of these blots indicated a significant decrease in JNK phosphorylation for all the treatments in cells expressing wild-type VHR but not in cells expressing mutant VHR or empty vector (Fig. 4B). Furthermore, VHR did not affect ERK signaling as a significant decrease in phERK was only observed in cells expressing empty vector (Fig. 4B). These results indicate that under the conditions in which it blocks apoptosis, VHR specifically inactivates JNK but not ERK, which further suggests that JNK inhibition by VHR is linked to inhibition of apoptosis in LNCaP cells.


Figure 4
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  		Figure 4. Stable expression of VHR results in decreased JNK phosphorylation. A, COS7 cells stably transfected with empty vector or vectors specifying expression of VHR (WT) or VHR-C124S (Mut) were treated with vehicle, TPA, or TG for the indicated time points. Whole cell protein extracts were made and used for Western blot analysis. Representative Western blots of phJNK, phERK, and VHR expression in TPA-treated and TG-treated cells. B, quantitative presentation of data obtained in A. Average of three independent experiments. *, P < 0.05 indicates significant difference between cells expressing WT or Mut VHR or as indicated otherwise. C, R1881 reverses apoptosis induced by activated JNK. LNCaP cells were transiently transfected with JNKK2-JNK1 and either treated with vehicle or R1881 for 64 and 88 h. Transfected cells were visualized by immunofluorescence microscopy using the HA epitope on JNKK2-JNK1, and TUNEL assay was performed. Representative images from 88-h treatment. D, quantification of cell death observed in transfected cells from C. Three independent experiments in duplicate were performed. Result from one representative experiment. Apoptosis induced in the presence of JNKK2-JNK1 (33%) was set to 100. *, P < 0.05 indicates significant difference.

 
To further assess the role of JNK in inhibition of apoptosis under conditions where VHR is up-regulated, LNCaP cells were transiently transfected with JNKK2-JNK1 (MKK7-JNK1), which encodes a hybrid JNK molecule that is constitutively active (21). Transfected cells were either left untreated or treated with R1881, and apoptosis was assessed using the TUNEL assay. LNCaP cells that express JNKK2-JNK1 underwent significant apoptosis (33%) when treated with vehicle, confirming the direct role of active JNK in apoptosis in LNCaP cells (Fig. 4C and D). Furthermore, the apoptosis-inducing effect of JNKK2-JNK1 was almost completely lost upon androgen treatment (Fig. 4C and D). This suggests that VHR and possibly other MKPs that are up-regulated by R1881 can reverse apoptosis induced by JNK.

Androgen withdrawal decreases VHR expression in human prostate cancer xenografts. As androgens have a protective effect on prostate cancer and R1881 increases VHR expression in LNCaP cells, we investigated the expression of VHR, as well as phJNK, in the human androgen-dependent prostate cancer xenograft CWR22. These tumors regress markedly after castration due to a decrease in growth and an increase in apoptosis (20, 26). Western blot analysis of whole cell extracts made from CWR22 tumors collected from mice at different times after castration showed that VHR expression had an initial increase at 1 week, which significantly decreased by 2 weeks and continued to decline at 4 weeks (Fig. 5A ). There was no difference in VHR expression in noncastrated mice at the same time points (Supplementary Fig. S6). This indicates that VHR is regulated by androgens in vivo, and its expression is inversely correlated to apoptosis and tumor regression. In contrast, phJNK levels significantly increased upon castration reaching 3.5-fold higher levels at 4 weeks after castration compared with t = 0, whereas totJNK levels did not significantly change (Fig. 5A). In noncastrated mice, there were no differences in phJNK levels at the same time points (Supplementary Fig. S6). These data show that VHR and phJNK expressions are inversely correlated to each other and differentially correlated to apoptosis. At 4 weeks after castration, there was a shift in the apparent molecular weight of VHR that could indicate a posttranslational modification, e.g., phosphorylation changes as suggested previously (27); this requires further investigation. Altogether, these results show that VHR expression is regulated by androgens, is inversely related to phJNK expression, and, thus, is associated with apoptosis of prostate cancer cells in vivo.


Figure 5
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  		Figure 5. VHR expression analysis in human prostate cancer xenograft CWR22, as well as in normal and malignant human prostate tissue samples. A, CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. PSA levels dramatically dropped upon castration, indicating the success of the process and regression of the tumor (Supplementary Fig. S7). Whole cell protein extracts were made and used for Western blot analysis. Representative blots for VHR, phJNK, totJNK, and GAPDH. VHR and phJNK levels were normalized to GAPDH and totJNK levels, respectively. For GAPDH and totJNK, the values at t = 0 are set to 1. B, large-scale analysis of the human transcriptome by gene expression profiling of samples of a diverse array of tissues, organs, and cell lines. Data were obtained from the Oncomine database (29). Prostate tumors were profiled to identify overexpressed genes. Samples of different human tissue samples used in this analysis were obtained from commercial resources and research collaborations, as described by Su and colleagues (48, 49). Twenty-one tissues (n = 68), other than prostate (n = 3), were analyzed for VHR expression. C, from 44 individuals with different histories of prostate disease as described by Tomlins and colleagues (50), 101 laser capture microdissected cell populations from a progression of prostate cancer cell types (PIN, low-grade prostate cancer, high-grade prostate cancer, metastatic prostate cancer) were analyzed with a microarray chip. Prostate carcinoma, n = 30; metastatic prostate cancer, n = 19. *, P < 0.001 indicates significant difference. D, TMAs with normal and tumor glands from human prostate were stained with the VHR antiserum, as described in Materials and Methods. VHR expression was predominantly observed in the nuclei of the prostate epithelium, with significantly stronger staining in tumor glands. Representative images are shown. The intensity of staining (score, 0–3) in the TMAs was scored by a pathologist and is shown in the table for normal and cancerous prostate glands.

 
VHR expression is up-regulated in human prostate cancer samples. As for other cancer types, prostate tumors are resistant to apoptosis (1, 28). Based on the data presented above, we hypothesized that VHR may play a role in the inhibition of cell death in prostate cancer tissue and, therefore, may be differentially expressed during prostate cancer progression. Comparison of gene expression profiles from a diverse array of normal tissues showed that VHR mRNA expression is significantly higher in normal prostate compared with 21 other tissues (Fig. 5B). We also examined gene expression profiles during prostate cancer progression and found that VHR mRNA expression was significantly increased in metastatic prostate cancer compared with prostate carcinoma tissues of different grades (Fig. 5C; ref. 29).

To determine if similar changes in expression are present at the protein level, we examined VHR expression by immunohistochemical analysis on TMAs that contained normal (n = 14) and malignant prostate tissue (n = 74). The TMAs included samples representing various stages of prostate cancer progression and normal prostate glands. VHR protein was expressed in normal prostate tissue, solely in epithelial cells with predominantly nuclear but also some cytosolic localization (Fig. 5D). Furthermore, its expression was significantly increased in cancer tissue compared with normal cells (Fig. 5D; P < 0.001, Mann-Whitney test). Intensity scoring among different tumor grades (Gleason score) indicated no differences (data not shown; P = 0.393, linear logistic regression). Similarly, survival was not associated with VHR expression (data not shown). Together, these data show that VHR expression is increased at both the mRNA and protein levels in prostate cancer compared with normal prostate with no significant correlation with well-differentiated or poorly differentiated tumors at the protein level.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
MAPK signaling pathways have been implicated in diverse cellular processes, such as differentiation and apoptosis. To regulate these cellular responses, the activity of MAPKs must be strictly regulated; this can be achieved by regulating either their activation or inhibition, i.e., their phosphorylation or dephosphorylation, respectively. Whereas there has been keen focus on pathways that activate the MAPKs, until recently, there has been relatively little research on the phosphatases that inactivate them. To assess the possible role of MKPs in prostate carcinogenesis, we carried out a comprehensive expression analysis of the MKP family during apoptosis of prostate cancer cells.

The MKPs tested were regulated differently in prostate cancer cells in response to the different compounds. In the presence of R1881 there was a greater effect of TPA on MKP expression than of TG, which could be due to the different pathways that these compounds affect. One of the MKPs that was regulated, VHR, was of special interest to us because it was previously shown to effectively dephosphorylate JNK (14), which is required for apoptosis in prostate cancer cells (17, 18).

VHR was significantly up-regulated at both mRNA and protein levels that coincided with the inhibition of apoptosis by R1881 in LNCaP cells. R1881 alone increased VHR expression in LNCaP cells but not in androgen-independent DU145 cells. Consistently, after an initial increase, VHR levels decreased in androgen-dependent CWR22 xenografts after androgen withdrawal in vivo. The effect of androgens on the inhibition of JNK activation has been studied previously by bicalutamide treatment or siRNA-mediated knockdown of AR in LNCaP cells, which showed that AR-dependent transcriptional activity is required for this process (18). Bioinformatics analysis of the VHR gene and 2-kb upstream flanking region indicated that there are at least two putative androgen response elements that may mediate the effects of androgens on VHR expression by direct interactions with AR (data not shown).

Androgen treatment of LNCaP cells was previously suggested to be required for phorbol 12-myristate 13-acetate (PMA)–induced apoptosis and the synthesis of protein kinase C{delta} (PKC{delta}; ref. 30). However, this study used low passage LNCaP cells (2–8), had shorter R1881 and PMA treatment times, and the effect of R1881 on PKC{delta} expression was lost already after 12 hours. In our experimental system, longer times of treatment with both R1881 and TPA are required to see the effects on apoptosis, which could affect PKC{delta} expression differently with a different outcome on apoptosis. In another study, androgens facilitated TPA-induced apoptosis, wherein the nuclear factor-{kappa}B signaling pathway was interrupted and JNK was activated (31). However, once again, the treatment times and conditions with DHT and TPA were different from our experimental conditions. Further work is required to determine the mechanistic basis of these differences.

In response to different treatments, there were inconsistencies between the VHR mRNA and protein expression levels. For example, TPA increased VHR mRNA expression of ~2.5-fold by 4 hours, but the effect was smaller at the protein level. In response to TG, there was also an increase in VHR mRNA levels, but this did not exceed 1.5-fold at the mRNA level and was even smaller at the protein level. Of note is the 12-hour time point where the basal level of VHR mRNA expression was significantly lower than at other time points. However, this did not translate into changes in VHR protein levels. At present, the molecular mechanisms that are responsible for these observations are not clear. Because TPA and TG are known to affect multiple signaling pathways, it is possible that distinctly different components of these pathways converge upon VHR expression, depending on the compound. Furthermore, in the AR-positive cell line CWR22Rv1, there was no effect of TPA or TG on VHR expression in the presence or absence of R1881 (data not shown). This could be due to the androgen-insensitive features of these cells, due to, at least in part, a lack of the ligand-binding domain of AR in these cells (32).

Under conditions wherein VHR is up-regulated, there was a significant decrease in apoptosis induced by the constitutively active JNKK2-JNK1 fusion protein (Fig. 4C and D). This suggests that the inhibitory effect of androgen on apoptosis is at the level of JNK and/or downstream from it. The ability of VHR to specifically dephosphorylate JNK (Fig. 4A and B) and the correlation of this to the inhibition of apoptosis in LNCaP cells (Figs. 2 and 3) suggests that JNK is the target of the androgen effect. However, the lack of full activity of VHR in reversing apoptosis suggested that other signaling pathways may also be involved. First, VHR may work in concert with other MKPs, as indicated by the data presented in Table 1. Second, it is possible that other MAPK pathways are implicated, which may be differentially regulated by VHR and other MKPs. However, ERK and p38 MAPK inhibitors had no effect on androgen-induced inhibition of JNK phosphorylation. Consistently, there was no increase in the activity of these two pathways in response to androgen treatment, indicating that they are not involved in this process (18). Other arms of the MAPK pathways and their regulation by VHR and other MKPs may play a role in this regard. Third, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway, which is critical for cell survival in prostate cancer cells (18, 33), may be involved. We have, thus far, found no increase in Akt activation in response to R1881 in LNCaP cells (data not shown). Consistently, PI3K inhibition did not have an effect on the androgen-dependent block of JNK activation, suggesting that the PI3K pathway is not involved. Fourth, sustained ROS accumulation can trigger JNK activation through the inactivation of MKPs (34), suggesting that inhibition of ROS accumulation could be responsible for the inhibition of JNK activation by androgens in LNCaP cells, perhaps through affecting MKP activity. However, ROS levels in LNCaP cells increased in response to R1881, consistent with previously published data (18, 35). However, this was not inhibited under conditions where JNK activation was blocked by R1881 when VHR levels were increased (data not shown). These data suggest that regulation of VHR or other MKPs by ROS levels is not a mechanism for inhibition of apoptosis by androgens in LNCaP cells.

Some previous studies have investigated the possible role of MKPs in carcinogenesis. A recent study on MKP8/DUSP26, which is closely related to VHR, suggested that it acts as an oncogene in anaplastic thyroid cancer (36). The MKP8 gene was found to be amplified with consequently increased expression and promoted cell survival through inhibition of apoptosis (36). Furthermore, several studies showed that MKP1 is overexpressed and associated with tumorigenicity in ovarian (37), breast (38), and pancreatic cancer (39). In other studies, MKP1 promoted cell survival by attenuating stress-responsive MAPK-mediated apoptosis (23, 40). MKP2/DUSP4 was also shown to be overexpressed in human breast cancer (38). Furthermore, a recent study showed that the expression of DUSP22/DUSP2, closely related to VHR, is increased after estradiol treatment in breast cancer cells (41). DUSP22 was found to negatively regulate estrogen receptor {alpha} activity and cell survival but positively regulated AR activity. In addition, VHR was recently found to be up-regulated in cervical carcinoma and in cervical cancer cell lines (42).

There is limited data about the possible biological roles of MKPs in prostate cancer. One study showed that overexpression of MKP1 in the androgen-independent prostate cancer cell line DU145 blocked activation of JNK that inhibited Fas ligand-induced apoptosis (43). Another study found that overexpression of MKP5/DUSP10 reduced the invasion of a highly metastatic prostate cancer cell line by dephosphorylating p38 MAPK (44). Furthermore, previous studies have shown that MKP1 is overexpressed in high-grade PIN compared with normal prostate and that the proportion of apoptosis is significantly lower in PIN lesions expressing MKP1 (45). In addition, JNK1 enzymatic activity was inversely related to MKP1 levels (45, 46). These data, along with the data that we present here involving VHR, suggest that MKPs may have an important role in prostate cancer biology.

Consistent with its effects on prostate cancer cells in vitro, VHR expression is significantly increased in human prostate cancer compared with normal prostate. Whereas there was no correlation between VHR expression and tumor grade, at the mRNA level VHR expression was increased in metastatic prostate cancer compared with prostate carcinoma. Cancer and its metastases are known to have decreased potential to undergo apoptosis and are resistant to extracellular death signals (47). The role of VHR in inhibiting apoptosis in prostate cancer cells in vitro that we have shown here may, therefore, also apply to prostate cancer in situ. This is supported by the significant down-regulation of VHR concomitant with phJNK expression during androgen ablation–induced regression of human prostate cancer xenograft CWR22 (Fig. 5A), which is known to occur, at least in part, by an apoptotic pathway (26).

In summary, these data suggest that knockdown of VHR in prostate cancer may result in the activation of JNK, leading to apoptosis, and may therefore have therapeutic utility in the clinic. This hypothesis is strengthened by the fact that prostate is the tissue where VHR is most highly expressed among 21 normal tissues tested (Fig. 5B). VHR inactivation may, therefore, be a unique approach to sensitize prostate cancer to cell death in combination with conventional cancer chemotherapeutic strategies.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Norwegian Research Council and Norwegian Cancer Society grants (F. Saatcioglu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank John M. Denu and Anning Lin for the generous gifts of plasmids, Thomas Pretlow for the CWR22 xenografts, John M. Denu for the VHR antiserum, Goran Torlakovic and Vera Abeler for Gleason scoring of the specimens, and Alexander Kristian for help with the xenografts.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 4/ 1/08. Revised 8/21/08. Accepted 8/21/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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