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

Research Article

Yke Jildouw Arnoldussen,1 Petra I. Lorenzo,1 Maria E. Pretorius,2,3 Ha˚kon Wæhre,2,3,5 Bjørn Risberg,2,3,4 Gunhild M. Mælandsmo,6 Ha˚vard E. Danielsen,2,3 and Fahri Saatcioglu1

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

Abstract A decrease in circulating androgen levels, by either surgical means Androgen ablation during the initial stages of prostate cancer (castration) or through androgen deprivation therapy, results in causes regression of the tumor due to an increase in apoptosis decreased cell proliferation, increased apoptosis, and atrophy in and reduced cellular proliferation. However, prostate cancer prostate epithelial cells (4). However, prostate tumors relapse and invariably progresses to an androgen-independent state for the disease progresses to an androgen-independent state (5). To poorly understood reasons. Previous studies showed that c-Jun date, the molecular mechanisms of transition from androgen dependence to androgen independence remain poorly understood, NH2 terminal kinase (JNK) is required for 12-O-tetradecanoylphorbol-13-acetate (TPA)– and thapsigargin (TG)–induced and little is known about the link between androgens and apoptosis in the androgen-responsive prostate cancer cell line apoptosis signaling pathways in prostate cancer. LNCaP. Androgens protect LNCaP cells from TPA-induced or One of the signaling pathways that has been implicated in TG-induced apoptosis via down-regulation of JNK activation. prostate carcinogenesis is the mitogen-activated protein kinase However, the molecular mechanisms of this inhibition are (MAPK) pathway. The MAPK cascades play an important role in not clear. Here, we systematically investigated the possible transducing environmental stimuli to changes in gene expression by regulation of mitogen-activated protein kinase phosphatases/ virtue of their ability to phosphorylate and regulate the activity of dual-specificity phosphatases during apoptosis of LNCaP various transcription factors and other molecules (6). The MAPK cells and found that Vaccinia H1–related protein (VHR/DUSP3) cascades are composed of three protein kinases that act in series by is up-regulated by androgens during inhibition of apoptosis activating one another through phosphorylation. A MAPK is in LNCaP cells, but not in androgen-independent DU145 activated by a MAPK kinase (MAPKK), which in turn is activated cells. Ectopic expression of wild-type VHR, but not a by a MAPKK kinase. In mammals, there are three well-characterized catalytically inactive mutant, interfered with TPA- and MAPKs: extracellular signal-regulated kinase (ERK), c-Jun NH2 TG-induced apoptosis. Consistently, small interfering RNA– terminal kinase (JNK)/stress-activated protein kinase, and p38 mediated knockdown of endogenous VHR increased apoptosis MAPK. The ERK pathway is typically stimulated by growth-related in response to TPA or TG in the presence of andro- signals and is mainly involved in growth, differentiation, and gens. Furthermore, COS7 cells stably expressing wild-type development. p38 MAPK and JNK are activated by growthfactors, VHR, but not a mutant, had a decrease in JNK phosphorylation. proinflammatory cytokines, and cellular stress, and their activation In vivo, VHR expression decreased in the androgen-dependent has been implicated in apoptosis, as well as in oncogenic trans- human prostate cancer xenograft CWR22 upon androgen formation, inflammation, development, and differentiation (7, 8). withdrawal and was inversely correlated to JNK phosphoryla- The MAPK pathways can be inactivated by tyrosine phospha- tion. Expression analysis in human prostate cancer specimens tases, serine/threonine phosphatases, and MAPK phosphatases showed that VHR is increased in prostate cancer compared with (MKP)/dual-specificity phosphatases (DUSP; ref. 9). These phos- normal prostate. These data show that VHR has a direct role in phatase families are implicated in the regulation of mitogenic and the inhibition of JNK-dependent apoptosis in LNCaP cells and other signaling pathways that are mediated by MAPKs. The MKPs, may therefore have a role in prostate cancer progression. in contrast to the other two phosphatase families, specifically [Cancer Res 2008;68(22):9255–64] dephosphorylate the MAPKs at phosphorylated threonine/serine and tyrosine residues located in their activation loop (9). To date, Introduction 10 typical MKPs have been identified in humans that share the common CDC25 homology domain but have different subcellular Androgens play an important role in regulating growth, localization, MAPK affinity, and structure, where determined differentiation, and cell deathresponses in thenormal and (9–11). In addition, there is a group of 19 atypical MKPs, of which cancerous prostate. The actions of androgens are mediated several are MAPK-specific (10). Atypical phosphatases generally through the androgen receptor (AR), a ligand-activated transcrip- consist of less than 250 amino acid residues and are characterized tion factor that belongs to the nuclear receptor superfamily (1–3). 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). Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). In HeLa cells, VHR regulates cell cycle progression and its Requests for reprints: Fahri Saatcioglu, University of Oslo, P.O. Box 1041, Blindern, knockdown leads to cell cycle arrest and cell senescence (16). Oslo, 0316, Norway. Phone: 47-22854569; Fax: 47-22857207; E-mail: [email protected]. O I2008 American Association for Cancer Research. We have previously shown that 12- -tetradecanoylphorbol- doi:10.1158/0008-5472.CAN-08-1224 13-acetate (TPA) and thapsigargin (TG) induce apoptosis in the www.aacrjournals.org 9255 Cancer Res 2008; 68: (22). November 15, 2008

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Cancer Research androgen-responsive prostate cancer cell line LNCaP through a phenylindole (DAPI; Sigma-Aldrich). Fluorescence was observed using an mechanism that requires JNK activation (17). Furthermore, we have Axioplan2 imaging microscope (Zeiss), and pictures were taken withan shown that androgens protect LNCaP cells from TPA- and AxioCam HRc camera (Zeiss). At least three areas and a minimum of 300 TG-induced apoptosis, which is mediated by down-regulation of cells per area were counted, and the number of TUNEL-positive cells was expressed per 100 of the total number of cells. JNK activity (18). Inhibition of JNK activation by androgens was de novo Tissue microarray samples. Tissue microarrays (TMA) were prepared dependent on AR, androgen dose and time, and required from radical prostatectomy specimens from patients operated at the gene transcription. In the presence of androgens, an increase in Norwegian Radium Hospital between 1988 and 1996 and followed up after phosphatase activity was observed and the dephosphorylation rate surgery. Prostate-specific antigen (PSA) measurements were performed of JNK was faster than in vehicle-treated LNCaP cells (18). This before and after operation and at every subsequent clinical examination. suggested that phosphatases, at least in part, may mediate JNK- Follow-up period ranged from 2 to 176 mo (mean, 73.3 mo). Patients were dependent apoptosis. We therefore systematically investigated considered to have clinically evident recurrence of disease if any of the possible changes in the expression and regulation of MKPs during following were present: (a) evidence of local recurrence (confirmed by b TPA-induced and TG-induced apoptosis of LNCaP cells, as well as histologic biopsies or ultrasound) or ( ) evidence of distant metastasis during prostate cancer progression. (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 Materials and Methods of elevated PSA was set as the relapse date (Supplementary Fig. S1). Cell culture. The different cell lines were maintained, and treatments H&E-stained sections were made from eachselected primary tumor were done as described previously (18, 19). block (donor blocks) of paraffin-embedded material to define representative Xenografts. Transplantation, growth, and harvesting of tumors from tumor regions. With the use of the tissue array instrument (Beecher mice bearing CWR22 xenografts were as described previously (20). Instruments), two tissue cylinders (0.6 mm in diameter) were punched from Transfection and RNA interference. Transfection of LNCaP cells with regions of the donor block. Control samples of noncancer tissue from the pcDNA3-VHR, pcDNA3-VHR-C124S (generous gifts from John M. Denu; paraffin blocks were also taken. Gleason score used in the analysis was the ref. 14), HA-JNKK2-JNK1 (generous gift from Anning Lin; ref. 21), and pEGFP highest Gleason score in each of the prostatectomy series. were performed using FuGene 6, as per the manufacturer’s instructions Immunohistochemistry. Imunohistochemistry analysis was done as (Roche Diagnostics). For obtaining COS7 cells that stably express wild-type described previously (22). An anti-VHR rabbit polyclonal antibody VHR or its mutant, cells were transfected withpcDNA3-VHR or pcDNA3- (Cell Signaling) was used. Scoring was essentially identical to as previously VHR-C124S using FuGene 6. At 24 hafter transfection, thecells were plated reported (19). SPSS 15.0 software was used for statistical analysis. Mann- in 15-cm dishes, and after an additional 48 h, G418 (500 Ag/mL) was added Whitney test was applied to compare the expression level of VHR between to the medium. Outgrowth of single colonies were harvested and expanded normal and malignant tissues. For correlating VHR intensity to Gleason as separate clones. A control cell line was created under similar conditions score, linear logistic regression was used, and for univariate survival using a pcDNA3 vector without insert. Small interfering RNA (siRNA) was analysis, a log-rank test was performed together with Kaplan-Meier graphs. used to silence VHR. The siRNA duplex used for targeting human VHR was Statistics. Statistical analysis was performed using the Student’s t test, (sense strand): 5¶-GGCAGAAGAUGGACGUCAAdTT-3¶ (Dharmacon). A unless indicated otherwise. Values of P < 0.05 were considered as duplex targeting the luciferase gene was used as a negative control significant. (Qiagen). siRNA (200 nmol/L) was transfected into LNCaP cells using Oligofectamine, as per the manufacturer’s instructions (Invitrogen). Where Results indicated, R1881 was added 1 hbefore siRNA transfection. Expression and regulation of MKPs during apoptosis of Quantitative PCR. RNA extraction, cDNA synthesis, and quantitative PCR (Q-PCR) were as previously described (22). PCR primers used for VHR LNCaP cells. To determine if MKPs are involved in androgen- were forward 5¶-CGTCTGGCTCAGGACATC-3¶ and reverse 5¶-CATT- mediated inhibition of JNK-dependent apoptosis in LNCaP cells, we GAGCTGGCAGAGTGG-3¶. A standard curve made from serial dilutions of used Q-PCR to assess possible changes in the expression levels of cDNA was used to calculate the relative amount of VHR in each sample. the MKP family members in cells treated with TPA and TG in the These values were normalized to the relative amount of the internal presence or absence of the synthetic androgen R1881. As shown in standard TBP. Primers used for TBP were forward 5¶-GAATATAATCC- Table 1, MKP expression was differentially regulated in response to CAAGCGGTTTG-3¶ and reverse 5¶-ACTTCATCACAGCTCCCC-3¶.The these agents (detailed data for two MKPs, VH3 and MKP6, are experiments were performed in duplicate and repeated thrice. Primers presented in Supplementary Fig. S2). In addition, the possible used for other MKPs tested are available upon request. androgen regulation of most MKPs was checked (Table 1). In most Western analysis. Western analysis was carried out as previously cases, R1881 alone induced the expression of various MKPs and described (18). Antibodies used were against VHR, phJNK, totJNK, phERK, totERK (all from Cell Signaling), glyceraldehyde-3-phosphate dehydrogenase addition of TPA or TG increased this further. Among the MKPs VHR, MKP1, VH3 MKP6 (GAPDH; Santa Cruz), and a-tubulin (Sigma-Aldrich). ECL Western blotting tested, , and were most consistent in analysis system was used for the detection of the immunoreactive bands displaying significant changes in expression in response to both according to the manufacturer’s instructions (Amersham Pharmacia TPA and TG, as well as androgen treatment. BothMKP1 and VHR Biotech). effectively dephosphorylate JNK (14, 23), whereas VH3 is not Immunofluorescence microscopy and TUNEL assay. LNCaP cells specific for JNK (24), and MKP6 has not been studied in detail were transfected withtheconstructs described above. For JNK expression previously. We therefore chose to analyze VHR in further detail. studies, immunofluorescence microscopy was done as described previously VHR mRNA accumulation was significantly increased upon TPA (18). Ectopic expression of JNK was visualized by an anti-HA antibody + R1881 treatment compared withTPA alone at all time points (Sigma-Aldrich). For ectopic expression studies of VHR, cells were tested (Fig. 1A). There was also a modest up-regulation of VHR in incubated withanti-VHR rabbit antibody (generous gift from JohnM. VHR Denu) overnight at 4jC. Cells were then incubated with secondary antibody response to R1881 alone. Similarly, was significantly up- Alexa fluor 594–linked goat anti-rabbit IgG (Invitrogen) for 1 h. To detect regulated by TG + R1881 treatment compared withTG alone. apoptosis, an In situ Cell DeathDetection kit (TUNEL assay) was used However, at later time points, this effect was lost (Fig. 1B). These according to the manufacturer’s instructions (Roche Diagnostics) and as data indicate that VHR mRNA expression is increased during described previously (18). Cells were counterstained with4 ¶,6-diamidino-2- R1881-mediated inhibition of apoptosis in LNCaP cells.

Cancer Res 2008; 68: (22). November 15, 2008 9256 www.aacrjournals.org

To assess whether VHR protein was similarly regulated, whole performed. Whereas wild-type VHR expression inhibited apoptosis cell extracts were made from cells treated withTPA or TG in the induced by both TPA and TG, the mutant VHR had largely lost this presence or absence of R1881 and examined by Western blot ability (Fig. 2). For example, wild-type VHR inhibited TPA-induced analysis. VHR protein was increased in response to TPA + R1881 apoptosis by >95% at 24 hours, but inhibition by mutant VHR was and TG + R1881 compared withTPA or TG alone consistent with only 50% (Fig. 2B). Similarly, whereas wild-type VHR inhibited the mRNA expression data presented above (Fig. 1C and D). f90% of apoptosis induced by TG, inhibition by the mutant VHR Interestingly, although no significant regulation of VHR mRNA was only 35% (Fig. 2C). The effect of mutant VHR in decreasing expression was observed in long time points withTG treatment, apoptosis, albeit significantly less than wild-type VHR, could be due VHR protein levels were increased at these times, indicating that to the possibility that it can still bind and sequester JNK through the action of androgen on VHR expression involves both VHR JNK-interacting protein-1, thereby blocking its activation, as has mRNA and protein. These data indicate that VHR may have an been shown for two other MKPs (25). In parallel to the TUNEL essential role in the inhibition of JNK-dependent apoptosis in assay, DAPI staining and changes in nuclear morphology and LNCaP cells. nuclear fragmentation were also used to assess apoptosis and To investigate if the increase in VHR expression upon TPA or TG virtually identical results were obtained (data not shown). The treatment in the presence of R1881 is specific for androgen- same experiment was also performed in the presence of R1881, and responsive LNCaP cells, the androgen-independent cell line DU145 similar results were obtained, except for lower levels of total cell was used. Cells were treated as before, and VHR protein expression death (data not shown). In summary, these data show that VHR was investigated (Supplementary Fig. S3). VHR protein levels were inhibits apoptosis in LNCaP cells. slightly increased in response to R1881 compared with vehicle- Knockdown of VHR increases cell death induced by TPA and treated cells, but there was no increase in expression in cells TG in R1881-treated LNCaP cells. The data presented above treated with TPA or TG in the presence of R1881. These data suggested that VHR inhibits apoptosis induced by TPA and TG in indicate that induction of VHR expression is dependent on intact LNCaP cells. If this is the case, knockdown of VHR should have the androgen signaling. opposite effect, namely, increased apoptosis. To assess this, we Ectopic expression of VHR protects LNCaP cells from TPA- used a siRNA that efficiently knocks down VHR (16). LNCaP cells induced and TG-induced apoptosis. The data presented above were transfected withsiRNA directed against luciferase as a control suggested that up-regulation of VHR can down-regulate JNK or the siRNA for VHR. A significant reduction in both VHR mRNA activation and thereby prevent apoptosis in LNCaP cells. To assess and protein expression was observed in cells transfected withVHR this possibility, LNCaP cells were transiently transfected with siRNA compared withluciferase siRNA thatlasted at least up to expression vectors specifying wild-type VHR, the catalytically 96 hours, both in the presence or absence of R1881 (Supplementary inactive mutant, VHR-C124S, or withan expression vector for Fig. S4). green fluorescent protein (pEGFP) as a control. The cells were To investigate the effect of VHR knockdown on apoptosis, grown in the presence or absence of R1881, and apoptosis was TUNEL assays were performed on LNCaP cells transfected with induced with TPA or TG. At the indicated times, the cells were fixed siRNA targeting luciferase or VHR. Cells were treated withR1881 or and incubated withVHR antibody and TUNEL assay was vehicle for 40 hours before addition of TPA or TG for the indicated

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

Name TPA + R1881 TG + R1881 R1881

Significance Highest fold up-regulation Significance Highest fold up-regulation Highest fold up-regulation

DUSP1/MKP1 ++ 3.7 ++/À 1.8 1.4 DUSP3/VHR +++ 2.0 + 1.8 1.5 DUSP4/MKP2 + 1.8 ÀÀ 0.9 0.5 DUSP5/VH3 +++ 19.3 + 3.4 2.2 DUSP6/MKP3 À 0.8 + 1.4 0.6 DUSP7/MKPX À 1.0 + 1.8 0.8 DUSP8 + 6.4 À 1.4 1.3 DUSP9/MKP4 +/ÀÀ 1.8 +/À 1.7 0.6 DUSP10/MKP5 + 2.5 NC 1.3 1.7 DUSP14/MKP6 ++ 2.4 + 1.9 1.6 DUSP22/DUSP2 NC 1.0 ÀÀ 0.9 0.9

NOTE: LNCaP cells were treated withTPA or TG for 6, 12, 24, 36, and 48 hafter 40-hpretreatment of R1881, and mRNA expression levels were investigated by Q-PCR. TPA or TG in the presence of R1881 was compared with TPA or TG treatment alone. Abbreviations: + or À, one or two time points have a significant up-regulation or down-regulation, respectively; ++ or ÀÀ, three or four time points have a significant up-regulation or down- regulation, respectively; +++ or ÀÀÀ, five time points have a significant up-regulation or down-regulation, respectively; NC, no significant changes. In addition, the highest fold up-regulation is shown for LNCaP cells treated with TPA + R1881 or TG + R1881 compared with TPA or TG alone. LNCaP cells were also treated with vehicle or R1881 alone for 88 h and the fold up-regulation for R1881 treatment versus vehicle is as indicated.

www.aacrjournals.org 9257 Cancer Res 2008; 68: (22). November 15, 2008

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. a-Tubulin was used as loading control. times. As expected, there was an increase in apoptosis in response further suggests that JNK inhibition by VHR is linked to inhibition to TPA compared withvehicle-treatedcells (Fig. 3 A). However, the of apoptosis in LNCaP cells. extent of apoptosis was decreased in cells treated withR1881 plus To further assess the role of JNK in inhibition of apoptosis under luciferase siRNA, and this decrease was significantly reversed in the conditions where VHR is up-regulated, LNCaP cells were tran- presence of VHR siRNA (Fig. 3A). Quantification indicated that the siently transfected withJNKK2-JNK1 (MKK7-JNK1), whichencodes inhibitory action of R1881 on TPA-induced apoptosis was partially a hybrid JNK molecule that is constitutively active (21). Transfected reversed when VHR levels were decreased, indicating that R1881 cells were either left untreated or treated with R1881, and apoptosis inhibition of apoptosis in LNCaP cells, at least in part, is mediated was assessed using the TUNEL assay. LNCaP cells that express by VHR (Fig. 3B). JNKK2-JNK1 underwent significant apoptosis (33%) when treated Essentially, the same results were obtained with TG (Fig. 3C with vehicle, confirming the direct role of active JNK in apoptosis and D). In fact, at 36 hours of TG treatment, knockdown of VHR in LNCaP cells (Fig. 4C and D). Furthermore, the apoptosis- almost completely reversed the inhibitory effect of R1881 on TG- inducing effect of JNKK2-JNK1 was almost completely lost upon mediated apoptosis. At 48 hours of TG treatment, there was still a androgen treatment (Fig. 4C and D). This suggests that VHR and significant effect of VHR knockdown, but the reversal of the R1881 possibly other MKPs that are up-regulated by R1881 can reverse effect was not complete. apoptosis induced by JNK. In parallel to the TUNEL assays, DAPI staining was performed as Androgen withdrawal decreases VHR expression in human an independent measure of apoptosis under these conditions, and prostate cancer xenografts. As androgens have a protective effect similar results were obtained (Fig. 3 and Supplementary Fig. S5A on prostate cancer and R1881 increases VHR expression in LNCaP and B). Coupled to the results presented above with ectopic cells, we investigated the expression of VHR, as well as phJNK, in expression of VHR, these data show that VHR has a direct role in the human androgen-dependent prostate cancer xenograft CWR22. the inhibition of apoptosis in LNCaP cells. These tumors regress markedly after castration due to a decrease Stable expression of VHR leads to a decrease in JNK in growthand an increase in apoptosis (20, 26). Western blot phosphorylation. To investigate if VHR has a role in specific analysis of whole cell extracts made from CWR22 tumors collected dephosphorylation of JNK activated by TPA or TG, COS7 cell lines from mice at different times after castration showed that VHR that stably express either an empty vector or expression vectors expression had an initial increase at 1 week, which significantly specifying wild-type VHR or VHR-C124S mutant were generated. decreased by 2 weeks and continued to decline at 4 weeks (Fig. 5A). The cells were treated with TPA, TG, or vehicle for the indicated There was no difference in VHR expression in noncastrated mice at times, and the phosphorylation status of JNK and ERK was the same time points (Supplementary Fig. S6). This indicates that investigated by Western blot analysis (Fig. 4A). Quantification of VHR is regulated by androgens in vivo, and its expression is these blots indicated a significant decrease in JNK phosphorylation inversely correlated to apoptosis and tumor regression. In contrast, for all the treatments in cells expressing wild-type VHR but not in phJNK levels significantly increased upon castration reaching cells expressing mutant VHR or empty vector (Fig. 4B). Further- 3.5-fold higher levels at 4 weeks after castration compared with more, VHR did not affect ERK signaling as a significant decrease in t = 0, whereas totJNK levels did not significantly change (Fig. 5A). phERK was only observed in cells expressing empty vector (Fig. 4B). In noncastrated mice, there were no differences in phJNK levels at These results indicate that under the conditions in which it blocks the same time points (Supplementary Fig. S6). These data show apoptosis, VHR specifically inactivates JNK but not ERK, which that VHR and phJNK expressions are inversely correlated to each

Cancer Res 2008; 68: (22). November 15, 2008 9258 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. VHR in Prostate Cancer other and differentially correlated to apoptosis. At 4 weeks after examined gene expression profiles during prostate cancer progres- castration, there was a shift in the apparent molecular weight of sion and found that VHR mRNA expression was significantly VHR that could indicate a posttranslational modification, e.g., increased in metastatic prostate cancer compared withprostate phosphorylation changes as suggested previously (27); this requires carcinoma tissues of different grades (Fig. 5C; ref. 29). further investigation. Altogether, these results show that VHR To determine if similar changes in expression are present at the expression is regulated by androgens, is inversely related to phJNK protein level, we examined VHR expression by immunohistochem- expression, and, thus, is associated with apoptosis of prostate ical analysis on TMAs that contained normal (n = 14) and cancer cells in vivo. malignant prostate tissue (n = 74). The TMAs included samples VHR expression is up-regulated in human prostate cancer representing various stages of prostate cancer progression and samples. As for other cancer types, prostate tumors are resistant to normal prostate glands. VHR protein was expressed in normal apoptosis (1, 28). Based on the data presented above, we prostate tissue, solely in epithelial cells with predominantly nuclear hypothesized that VHR may play a role in the inhibition of cell but also some cytosolic localization (Fig. 5D). Furthermore, its deathin prostate cancer tissue and, therefore, may be differentially expression was significantly increased in cancer tissue compared expressed during prostate cancer progression. Comparison of gene withnormal cells (Fig. 5 D; P < 0.001, Mann-Whitney test). Intensity expression profiles from a diverse array of normal tissues showed scoring among different tumor grades (Gleason score) indicated no that VHR mRNA expression is significantly higher in normal differences (data not shown; P = 0.393, linear logistic regression). prostate compared with21 othertissues (Fig. 5 B). We also Similarly, survival was not associated withVHR expression

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. www.aacrjournals.org 9259 Cancer Res 2008; 68: (22). November 15, 2008

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.

(data not shown). Together, these data show that VHR expression is R1881 there was a greater effect of TPA on MKP expression than of increased at boththemRNA and protein levels in prostate cancer TG, which could be due to the different pathways that these compared withnormal prostate withno significant correlation compounds affect. One of the MKPs that was regulated, VHR, was withwell-differentiated or poorly differentiated tumors at the of special interest to us because it was previously shown to protein level. effectively dephosphorylate JNK (14), which is required for apoptosis in prostate cancer cells (17, 18). Discussion VHR was significantly up-regulated at bothmRNA and protein MAPK signaling pathways have been implicated in diverse levels that coincided with the inhibition of apoptosis by R1881 in cellular processes, suchas differentiation and apoptosis. To LNCaP cells. R1881 alone increased VHR expression in LNCaP cells regulate these cellular responses, the activity of MAPKs must be but not in androgen-independent DU145 cells. Consistently, after strictly regulated; this can be achieved by regulating either their an initial increase, VHR levels decreased in androgen-dependent activation or inhibition, i.e., their phosphorylation or dephosphor- CWR22 xenografts after androgen withdrawal in vivo. The effect of ylation, respectively. Whereas there has been keen focus on androgens on the inhibition of JNK activation has been studied pathways that activate the MAPKs, until recently, there has been previously by bicalutamide treatment or siRNA-mediated knock- relatively little research on the phosphatases that inactivate them. down of AR in LNCaP cells, which showed that AR-dependent To assess the possible role of MKPs in prostate carcinogenesis, we transcriptional activity is required for this process (18). Bioinfor- carried out a comprehensive expression analysis of the MKP family matics analysis of the VHR gene and 2-kb upstream flanking region during apoptosis of prostate cancer cells. indicated that there are at least two putative androgen response The MKPs tested were regulated differently in prostate cancer elements that may mediate the effects of androgens on VHR cells in response to the different compounds. In the presence of expression by direct interactions withAR (data not shown).

Cancer Res 2008; 68: (22). November 15, 2008 9260 www.aacrjournals.org

Androgen treatment of LNCaP cells was previously suggested In response to different treatments, there were inconsistencies to be required for phorbol 12-myristate 13-acetate (PMA)– between the VHR mRNA and protein expression levels. For induced apoptosis and the synthesis of protein kinase Cy (PKCy; example, TPA increased VHR mRNA expression of f2.5-fold by 4 ref. 30). However, this study used low passage LNCaP cells (2–8), hours, but the effect was smaller at the protein level. In response to had shorter R1881 and PMA treatment times, and the effect of TG, there was also an increase in VHR mRNA levels, but this did R1881 on PKCy expression was lost already after 12 hours. In not exceed 1.5-fold at the mRNA level and was even smaller at the our experimental system, longer times of treatment withboth protein level. Of note is the 12-hour time point where the basal R1881 and TPA are required to see the effects on apoptosis, level of VHR mRNA expression was significantly lower than at which could affect PKCy expression differently witha different other time points. However, this did not translate into changes in outcome on apoptosis. In another study, androgens facilitated VHR protein levels. At present, the molecular mechanisms that are TPA-induced apoptosis, wherein the nuclear factor-nB signaling responsible for these observations are not clear. Because TPA and pathway was interrupted and JNK was activated (31). However, TG are known to affect multiple signaling pathways, it is possible once again, the treatment times and conditions with DHT and that distinctly different components of these pathways converge TPA were different from our experimental conditions. Further upon VHR expression, depending on the compound. Furthermore, work is required to determine the mechanistic basis of these in the AR-positive cell line CWR22Rv1, there was no effect of TPA differences. or TG on VHR expression in the presence or absence of R1881 (data

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. www.aacrjournals.org 9261 Cancer Res 2008; 68: (22). November 15, 2008

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 valuesat 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. not shown). This could be due to the androgen-insensitive features implicated, which may be differentially regulated by VHR and other of these cells, due to, at least in part, a lack of the ligand-binding MKPs. However, ERK and p38 MAPK inhibitors had no effect on domain of AR in these cells (32). androgen-induced inhibition of JNK phosphorylation. Consistently, Under conditions wherein VHR is up-regulated, there was a there was no increase in the activity of these two pathways in significant decrease in apoptosis induced by the constitutively response to androgen treatment, indicating that they are not active JNKK2-JNK1 fusion protein (Fig. 4C and D). This suggests involved in this process (18). Other arms of the MAPK pathways that the inhibitory effect of androgen on apoptosis is at the level of and their regulation by VHR and other MKPs may play a role in this JNK and/or downstream from it. The ability of VHR to specifically regard. Third, the phosphatidylinositol 3-kinase (PI3K)/protein dephosphorylate JNK (Fig. 4A and B) and the correlation of this to kinase B pathway, which is critical for cell survival in prostate the inhibition of apoptosis in LNCaP cells (Figs. 2 and 3) suggests cancer cells (18, 33), may be involved. We have, thus far, found no that JNK is the target of the androgen effect. However, the lack of increase in Akt activation in response to R1881 in LNCaP cells (data full activity of VHR in reversing apoptosis suggested that other not shown). Consistently, PI3K inhibition did not have an effect on signaling pathways may also be involved. First, VHR may work in the androgen-dependent block of JNK activation, suggesting that concert with other MKPs, as indicated by the data presented in the PI3K pathway is not involved. Fourth, sustained ROS Table 1. Second, it is possible that other MAPK pathways are accumulation can trigger JNK activation through the inactivation

Cancer Res 2008; 68: (22). November 15, 2008 9262 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. VHR in Prostate Cancer of MKPs (34), suggesting that inhibition of ROS accumulation could data that we present here involving VHR, suggest that MKPs may be responsible for the inhibition of JNK activation by androgens in have an important role in prostate cancer biology. LNCaP cells, perhaps through affecting MKP activity. However, ROS Consistent withits effects on prostate cancer cells in vitro, VHR levels in LNCaP cells increased in response to R1881, consistent expression is significantly increased in human prostate cancer with previously published data (18, 35). However, this was not compared with normal prostate. Whereas there was no correla- inhibited under conditions where JNK activation was blocked by tion between VHR expression and tumor grade, at the mRNA level R1881 when VHR levels were increased (data not shown). These VHR expression was increased in metastatic prostate cancer data suggest that regulation of VHR or other MKPs by ROS levels is compared withprostate carcinoma. Cancer and its metastases are not a mechanism for inhibition of apoptosis by androgens in known to have decreased potential to undergo apoptosis and are LNCaP cells. resistant to extracellular deathsignals (47). Therole of VHR in Some previous studies have investigated the possible role of inhibiting apoptosis in prostate cancer cells in vitro that we have MKPs in carcinogenesis. A recent study on MKP8/DUSP26, which is shown here may, therefore, also apply to prostate cancer in situ. closely related to VHR, suggested that it acts as an oncogene in This is supported by the significant down-regulation of VHR anaplastic thyroid cancer (36). The MKP8 gene was found to be concomitant withphJNKexpression during androgen ablation– amplified withconsequently increased expression and promoted induced regression of human prostate cancer xenograft CWR22 cell survival through inhibition of apoptosis (36). Furthermore, (Fig. 5A), which is known to occur, at least in part, by an several studies showed that MKP1 is overexpressed and associated apoptotic pathway (26). withtumorigenicity in ovarian (37), breast (38), and pancreatic In summary, these data suggest that knockdown of VHR in cancer (39). In other studies, MKP1 promoted cell survival by prostate cancer may result in the activation of JNK, leading to attenuating stress-responsive MAPK-mediated apoptosis (23, 40). apoptosis, and may therefore have therapeutic utility in the clinic. MKP2/DUSP4 was also shown to be overexpressed in human breast This hypothesis is strengthened by the fact that prostate is the tissue cancer (38). Furthermore, a recent study showed that the where VHR is most highly expressed among 21 normal tissues tested expression of DUSP22/DUSP2, closely related to VHR, is increased (Fig. 5B). VHR inactivation may, therefore, be a unique approach to after estradiol treatment in breast cancer cells (41). DUSP22 was sensitize prostate cancer to cell deathin combination with found to negatively regulate estrogen receptor a activity and cell conventional cancer chemotherapeutic strategies. 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). Disclosure of Potential Conflicts of Interest There is limited data about the possible biological roles of MKPs No potential conflicts of interest were disclosed. 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 Acknowledgments apoptosis (43). Another study found that overexpression of Received 4/1/2008; revised 8/21/2008; accepted 8/21/2008. MKP5/DUSP10 reduced the invasion of a highly metastatic Grant support: Norwegian ResearchCouncil and Norwegian Cancer Society grants prostate cancer cell line by dephosphorylating p38 MAPK (44). (F. Saatcioglu). Furthermore, previous studies have shown that MKP1 is overex- 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 pressed in high-grade PIN compared with normal prostate and that with18 U.S.C. Section 1734 solely to indicate thisfact. the proportion of apoptosis is significantly lower in PIN lesions 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 expressing MKP1 (45). In addition, JNK1 enzymatic activity was Torlakovic and Vera Abeler for Gleason scoring of the specimens, and Alexander inversely related to MKP1 levels (45, 46). These data, along with the Kristian for help with the xenografts.

References 9. Owens DM, Keyse SM. Differential regulation of MAP 16. Rahmouni S, Cerignoli F, Alonso A, et al. Loss of the kinase signalling by dual-specificity protein phospha- VHR dual-specific phosphatase causes cell-cycle arrest 1. Dehm SM, Tindall DJ. Molecular regulation of tases. Oncogene 2007;26:3203–13. and senescence. Nat Cell Biol 2006;8:524–31. androgen action in prostate cancer. J Cell Biochem 10. Alonso A, Sasin J, Bottini N, et al. Protein tyrosine 17. Engedal N, Korkmaz CG, Saatcioglu F. C-Jun N- 2006;99:333–44. phosphatases in the human genome. Cell 2004;117: terminal kinase is required for phorbol ester- and 2. Heinlein CA, Chang C. Androgen receptor in prostate 699–711. thapsigargin-induced apoptosis in the androgen respon- cancer. Endocr Rev 2004;25:276–308. 11. Farooq A, Zhou MM. Structure and regulation of sive prostate cancer cell line LNCaP. Oncogene 2002;21: 3. Kaarbo M, Klokk TI, Saatcioglu F. Androgen signaling MAPK phosphatases. Cell Signal 2004;16:769–79. 1017–27. and its interactions with other signaling pathways in 12. Ishibashi T, Bottaro DP, Chan A, Miki T, Aaronson SA. 18. Lorenzo PI, Saatcioglu F. Inhibition of apoptosis in prostate cancer. Bioessays 2007;29:1227–38. Expression cloning of a human dual-specificity phos- prostate cancer cells by androgens is mediated through 4. Mercader M, Sengupta S, Bodner BK, et al. Early phatase. Proc Natl Acad Sci U S A 1992;89:12170–4. downregulation of c-Jun N-terminal kinase activation. effects of pharmacological androgen deprivation in 13. Alonso A, Saxena M, Williams S, Mustelin T. Neoplasia 2008;10:418–28. human prostate cancer. BJU Int 2007;99:60–7. Inhibitory role for dual specificity phosphatase VHR in 19. Xi Z, Klokk TI, Korkmaz K, et al. Kallikrein 4 is a 5. Feldman BJ, Feldman D. The development of andro- T cell antigen receptor and CD28-induced Erk and Jnk predominantly nuclear protein and is overexpressed in gen-independent prostate cancer. Nat Rev Cancer 2001; activation. J Biol Chem 2001;276:4766–71. prostate cancer. Cancer Res 2004;64:2365–70. 1:34–45. 14. Todd JL, Rigas JD, Rafty LA, Denu JM. Dual- 20. Nagabhushan M, Miller CM, Pretlow TP, et al. 6. Chang L, Karin M. Mammalian MAP kinase signalling specificity protein tyrosine phosphatase VHR down- CWR22: the first human prostate cancer xenograft cascades. Nature 2001;410:37–40. regulates c-Jun N-terminal kinase (JNK). Oncogene withstrongly androgen-dependent and relapsed 7. Engelberg D. Stress-activated protein kinases-tumor 2002;21:2573–83. strains both in vivo and in soft agar. Cancer Res suppressors or tumor initiators? Semin Cancer Biol 15. Todd JL, Tanner KG, Denu JM. Extracellular regulated 1996;56:3042–6. 2004;14:271–82. kinases (ERK) 1 and ERK2 are authentic substrates for 21. Zheng C, Xiang J, Hunter T, Lin A. The JNKK2–1 8. Wada T, Penninger JM. Mitogen-activated protein the dual-specificity protein-tyrosine phosphatase VHR. fusion protein acts as a constitutively active c-Jun kinase kinases in apoptosis regulation. Oncogene 2004;23: A novel role in down-regulating the ERK pathway. J Biol that stimulates c-Jun transcription activity. J Biol Chem 2838–49. Chem 1999;274:13271–80. 1999;274:28966–71. www.aacrjournals.org 9263 Cancer Res 2008; 68: (22). November 15, 2008

22. Klokk TI, Kilander A, Xi Z, et al. Kallikrein 4 is a 32. Tepper CG, Boucher DL, Ryan PE, et al. Character- 41. Sekine Y, Ikeda O, Hayakawa Y, et al. DUSP22/LMW- proliferative factor that is overexpressed in prostate ization of a novel androgen receptor mutation in a DSP2 regulates estrogen receptor-a-mediated signaling cancer. Cancer Res 2007;67:5221–30. relapsed CWR22 prostate cancer xenograft and cell line. through dephosphorylation of Ser-118. Oncogene 2007; 23. Zhou JY, Liu Y, Wu GS. The role of mitogen-activated Cancer Res 2002;62:6606–14. 26:6038–49. protein kinase phosphatase-1 in oxidative damage- 33. Li L, Ittmann MM, Ayala G, et al. The emerging role 42. Henkens R, Delvenne P, Arafa M, et al. Cervix induced cell death. Cancer Res 2006;66:4888–94. of the PI3-K-Akt pathway in prostate cancer progres- carcinoma is associated withan up-regulation and 24. Mandl M, Slack DN, Keyse SM. Specific inactivation sion. Prostate Cancer Prostatic Dis 2005;8:108–18. nuclear localization of the dual-specificity protein and nuclear anchoring of extracellular signal-regulated 34. Kamata H, Honda S, Maeda S, Chang L, Hirata H, phosphatase VHR. BMC Cancer 2008;8:147. kinase 2 by the inducible dual-specificity protein Karin M. Reactive oxygen species promote TNFa- 43. SrikanthS, Franklin CC, Duke RC, Kraft RS. Human phosphatase DUSP5. Mol Cell Biol 2005;25:1830–45. induced deathand sustained JNK activation by DU145 prostate cancer cells overexpressing mitogen- 25. Willoughby EA, Perkins GR, Collins MK, Whitmarsh inhibiting MAP kinase phosphatases. Cell 2005;120: activated protein kinase phosphatase-1 are resistant to AJ. The JNK-interacting protein-1 scaffold protein 649–61. Fas ligand-induced mitochondrial perturbations and targets MAPK phosphatase-7 to dephosphorylate JNK. 35. Pinthus JH, Bryskin I, Trachtenberg J, et al. Androgen cellular apoptosis. Mol Cell Biochem 1999;199:169–78. J Biol Chem 2003;278:10731–6. induces adaptation to oxidative stress in prostate 44. Chen L, He HY, Li HM, et al. ERK1/2 and p38 26. Smitherman AB, Gregory CW, Mohler JL. Apoptosis cancer: implications for treatment withradiation pathways are required for P2Y receptor-mediated levels increase after castration in the CWR22 human therapy. Neoplasia 2007;9:68–80. prostate cancer invasion. Cancer Lett 2004;215:239–47. prostate cancer xenograft. Prostate 2003;57:24–31. 36. Yu W, Imoto I, Inoue J, Onda M, Emi M, Inazawa J. A 45. Magi-Galluzzi C, Mishra R, Fiorentino M, et al. 27. Alonso A, Rahmouni S, Williams S, et al. Tyrosine novel amplification target, DUSP26, promotes anaplastic Mitogen-activated protein kinase phosphatase 1 is phosphorylation of VHR phosphatase by ZAP-70. Nature thyroid cancer cell growth by inhibiting p38 MAPK overexpressed in prostate cancers and is inversely Immunol 2003;4:44–8. activity. Oncogene 2007;26:1178–87. related to apoptosis. Lab Invest 1997;76:37–51. 28. Lorenzo PI, Arnoldussen YJ, Saatcioglu F. Molecular 37. Denkert C, Schmitt WD, Berger S, et al. Expression of 46. Magi-Galluzzi C, Montironi R, Cangi MG, Wishnow mechanisms of apoptosis in prostate cancer. Crit Rev mitogen-activated protein kinase phosphatase-1 (MKP- K, Loda M. Mitogen-activated protein kinases and Oncogen 2007;13:1–38. 1) in primary human ovarian carcinoma. Int J Cancer apoptosis in PIN. Virchows Arch 1998;432:407–13. 29. Rhodes DR, Yu J, Shanker K, et al. ONCOMINE: a 2002;102:507–13. 47. Gupta GP, Massague J. Cancer metastasis: building a cancer microarray database and integrated data-mining 38. Wang HY, Cheng Z, Malbon CC. Overexpression of framework. Cell 2006;127:679–95. platform. Neoplasia 2004;6:1–6. mitogen-activated protein kinase phosphatases MKP1, 48. Su AI, Cooke MP, Ching KA, et al. Large-scale analysis 30. Gavrielides MV, Gonzalez-Guerrico AM, Riobo NA, MKP2 in human breast cancer. Cancer Lett 2003;191: of the human and mouse transcriptomes. Proc Natl Kazanietz MG. Androgens regulate protein kinase Cy 229–37. Acad Sci U S A 2002;99:4465–70. transcription and modulate its apoptotic function in 39. Liao Q, Guo J, Kleeff J, et al. Down-regulation of the 49. WelshJB, Sapinoso LM, Su AI, et al. Analysis of gene prostate cancer cells. Cancer Res 2006;66:11792–801. dual-specificity phosphatase MKP-1 suppresses tumor- expression identifies candidate markers and pharmaco- 31. Altuwaijri S, Lin HK, Chuang KH, et al. Interruption igenicity of pancreatic cancer cells. Gastroenterology logical targets in prostate cancer. Cancer Res 2001;61: of nuclear factor nB signaling by the androgen receptor 2003;124:1830–45. 5974–8. facilitates 12-O-tetradecanoylphorbolacetate-induced 40. Wang Z, Xu J, Zhou JY, Liu Y, Wu GS. Mitogen- 50. Tomlins SA, Mehra R, Rhodes DR, et al. Integrative apoptosis in androgen-sensitive prostate cancer LNCaP Activated Protein Kinase Phosphatase-1 Is Required for molecular concept modeling of prostate cancer pro- cells. Cancer Res 2003;63:7106–12. Cisplatin Resistance. Cancer Res 2006;66:8870–7. gression. Nat Genet 2007;39:41–51.

Cancer Res 2008; 68: (22). November 15, 2008 9264 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. The Mitogen-Activated Protein Kinase Phosphatase Vaccinia H1 −Related Protein Inhibits Apoptosis in Prostate Cancer Cells and Is Overexpressed in Prostate Cancer

Yke Jildouw Arnoldussen, Petra I. Lorenzo, Maria E. Pretorius, et al.

Cancer Res 2008;68:9255-9264.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/68/22/9255

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2008/11/07/68.22.9255.DC1

Cited articles This article cites 50 articles, 16 of which you can access for free at: http://cancerres.aacrjournals.org/content/68/22/9255.full#ref-list-1

Citing articles This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/68/22/9255.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/68/22/9255. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.