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Cancer Research 67, 11402, December 1, 2007. doi: 10.1158/0008-5472.CAN-07-1792
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

The p38 MAPK Pathway Mediates Aryl Propionic Acid–Induced Messenger RNA Stability of p75NTR in Prostate Cancer Cells

Emily J. Quann1, Fatima Khwaja1 and Daniel Djakiew1,2

1 Department of Biochemistry and Molecular & Cellular Biology and 2 Vincent T. Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia

Requests for reprints: Daniel Djakiew, Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, 3900 Reservoir Road Northwest, Washington, DC 20057-1436. Phone: 202-687-1203; Fax: 202-687-1823; E-mail: djakiewd{at}georgetown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The p75NTR acts as a tumor suppressor in the prostate, but its expression is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines such as PC-3, DU-145, and LNCaP. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75NTR expression in PC-3 and DU-145 cells leading to p75NTR-mediated decreased survival. Here, we investigate the mechanism by which these drugs induce p75NTR expression. We show that the observed increase in p75NTR protein due to R-flurbiprofen and ibuprofen treatment was accompanied by an increase in p75NTR mRNA, and this increase in mRNA was the result of increased mRNA stability and not by an up-regulation of transcription. In addition, we show that treatment with R-flurbiprofen or ibuprofen led to sustained activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Furthermore, inhibition of the p38 MAPK pathway with the p38 MAPK–specific inhibitor SB202190 or by small interfering RNA (siRNA) knockdown of p38 MAPK protein prevented induction of p75NTR by R-flurbiprofen and ibuprofen. We also observed that siRNA knockdown of MAPK-activated protein kinase (MK)-2 and MK3, the kinases downstream of p38 MAPK that are responsible for the mRNA stabilizing effects of the p38 MAPK pathway, also prevented an induction of p75NTR by R-flurbiprofen and ibuprofen. Finally, we identify the RNA stabilizing protein HuR and the posttranscriptional regulator eukaryotic translation initiation factor 4E as two possible mechanisms by which the p38 MAPK pathway may increase p75NTR expression. Collectively, the data suggest that R-flurbiprofen and ibuprofen induce p75NTR expression by increased mRNA stability that is mediated through the p38 MAPK pathway. [Cancer Res 2007;67(23):11402–10]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The p75NTR (neurotrophin receptor) is a member of the tumor necrosis factor (TNF) receptor superfamily that share a conserved intracellular death domain capable of inducing apoptosis and suppressing growth (1, 2). It binds neurotrophin ligands with similar affinity; however, unlike other members of the TNF receptor superfamily, it is able to induce apoptosis and suppress growth in the unbound state (3–8). p75NTR has been shown to act as a tumor suppressor in the prostate (5, 7, 9). However, whereas normal prostate epithelial cells express high levels of p75NTR, expression is lost as prostate cancer progresses (10). In addition, although the gene remains intact, expression is very low in metastatic prostate cancer cell lines PC-3, DU-145, and LNCaP (10). Exogenous reexpression of p75NTR in PC-3 cells led to increased apoptosis, reduced proliferation, and a decreased ability of these cells to form tumors in mice, thus indicating potential for treatments that result in the reexpression of p75NTR in prostate tumor cells (5, 7–9, 11).

Nonsteroidal anti-inflammatory drugs are typically used to relieve inflammation by inhibiting cyclooxygenase (COX) activity; however, many of these drugs seem to possess anticancer activity that is independent of their COX inhibitory activity (12). Flurbiprofen and ibuprofen are members of the aryl propionic acid class of nonsteroidal anti-inflammatory drugs, also referred to as profens, and have shown anticancer activity in the prostate. For example, treatment with the enantiomer R-flurbiprofen, which lacks COX inhibitory activity, was able to slow progression of prostate cancer in the transgenic adenocarcinoma of the mouse prostate mouse (13). In addition, long-term ibuprofen use has been associated with decreased prostate cancer risk, and treatment of prostate cancer cells with ibuprofen resulted in decreased survival (14–16). Recently, we showed that treatment of PC-3 and DU-145 prostate cancer cells with R-flurbiprofen or ibuprofen resulted in a strong induction of p75NTR, which led to p75NTR-mediated apoptosis and decreased survival (17). The results of this study were significant because they showed that p75NTR expression is inducible, and that drugs which induce p75NTR in prostate cancer cells have therapeutic potential.

The p38 mitogen-activated protein kinase (MAPK) pathway is activated in response to cell stresses and external stimuli such as heat, UV light, osmotic shock, and inflammatory cytokines (18). It mediates various cellular processes including apoptosis, senescence, inflammation, and tumorigenesis through decreased p38 MAPK activity, which has been associated with tumor progression (18–20). In addition, p38 MAPK has been strongly implicated as a regulator of mRNA stability (18, 21, 22). p38 MAPK is activated through phosphorylation by upstream kinases MAPK kinase kinase (MKK)-3 and MKK6 and, on activation, phosphorylates a number of downstream targets (18). Among these targets are the kinases MAPK-activated protein kinase (MK)-2 and MK3, which have been shown to be responsible for mediating the effects of the p38 MAPK pathway on mRNA stability (21, 23).

In this study, we investigated the mechanism by which the profens, R-flurbiprofen and ibuprofen, induce p75NTR expression in PC-3, DU-145, and LNCaP prostate cancer cells. We showed that induction of p75NTR protein expression corresponded to increased p75NTR mRNA levels that were due to increased mRNA stability and not to an up-regulation of transcription. In addition, we showed that R-flurbiprofen and ibuprofen treatment led to sustained activation of the p38 MAPK pathway and that this pathway was necessary for induction of p75NTR expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, culture conditions, and reagents. PC-3 and DU-145 cells, the only two prostate tumor cell lines included in the NIH Developmental Therapeutics Program anticancer drug discovery program, and the androgen receptor–responsive LNCaP human prostate cancer cells were purchased from the tissue culture core facility of the Georgetown University Lombardi Comprehensive Cancer Center and maintained as previously described (17).

Ibuprofen (Sigma Chemical Co.) and R-flurbiprofen (Myriad Pharmaceuticals, Inc.) were dissolved in DMSO and used at final concentrations of 0, 0.25, 0.5, 1.0, or 2.0 mmol/L. Actinomycin D (Tocris Bioscience) and SB202190 (Calbiochem) were dissolved in DMSO and used at final concentrations of 5 µg/mL and 20 µmol/L, respectively.

Reverse transcription-PCR. RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription-PCR (RT-PCR) was done with the SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase (Invitrogen) using equal amounts of RNA according to the following program: 47°C for 30 min; 94°C for 2 min; 30 cycles of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min; and 72°C for 5 min. Primers for p75NTR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were described previously (17).

Immunoblot analysis. Lysates were prepared as described previously (11). Cytoplasmic and nuclear fractions were prepared with a nuclear extract kit (Active Motif). SDS-PAGE and immunoblot analysis were done as described previously (17). The following primary antibodies were used: mouse monoclonal anti-p75NTR (Millipore; 1:2,000); rabbit polyclonal antibodies to phosphorylated p38 MAPK, p38 MAPK, p38{alpha}, phosphorylated MK2, MK2, phosphorylated eukaryotic translation initiation factor 4E (eIF4E), and eIF4E (Cell Signaling Technology; 1:1,000); rabbit polyclonal anti-HuR (Millipore; 1:1,000); mouse monoclonal anti-p38β (Zymed Laboratories) and anti-MK3 (Affinity BioReagents; 2 µg/mL); mouse monoclonal anti-Hu protein (Abcam, Inc.; 1:1,000); mouse monoclonal anti–{alpha}-tubulin and rabbit polyclonal anti–lamin B1 (Abcam; 1:3,000); and mouse monoclonal anti–β-actin (Sigma; 1:5,000). Goat anti-mouse and goat anti-rabbit horseradish peroxidase–conjugated secondary antibodies (Bio-Rad Laboratories) were used at 1:3,000. Membranes used for detection of phosphorylated p38 MAPK, MK2, or eIF4E were subsequently stripped and reprobed for total p38 MAPK, MK2, or eIF4E, respectively.

Small interfering RNA transfection. Cells were transfected for 72 h with nontargeting small interfering RNA (siRNA) or siRNA specific for p38{alpha} (J-003512-20), p38β (J-003972-11), MK2 (J-003516-11), MK3 (J-005014-07), or HuR (J-003773-08; Dharmacon RNA Technologies) at final concentrations of 100 nmol/L according to the manufacturer's protocol. Transfection reagent DharmaFECT 1 was used for DU-145 cells and DharmaFECT 2 was used for PC-3 and LNCaP cells (Dharmacon).

Luciferase assay. PC-3, DU-145, and A875 cells were cotransfected with either the pGL3-2.1 kb p75NTR promoter-luciferase construct (gift from Emil Bogenmann, Children's Hospital, Los Angeles, CA) or empty vector and the Renilla luciferase reporter vector phRL-TK (Promega) using GeneJammer transfection reagent (Stratagene) for 48 h. Following transfection, PC-3 and DU-145 cells were treated with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control for 24 h, whereas A875 cells were treated with isotonic medium (320 mosm/L) or hypoosmotic medium (160 mosm/L) prepared by a 1:1 dilution of complete medium with water. Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega) and normalized to the empty vector and Renilla luciferase activity.

Immunoprecipitation RT-PCR. Equal numbers of treated cells were collected, washed in cold PBS, and incubated on ice for 30 min in 1 mL of lysis buffer (1% Igepal Ca-630, 0.5% sodium deoxycholate, 0.1% SDS in PBS) containing 10 µg/mL Protease Inhibitor Cocktail (Sigma) and 100 units of RNaseOUT (Invitrogen) per sample. Samples were then centrifuged at 10,000 x g for 15 min at 4°C. Ten micrograms of HuR antibody (Millipore) were added to the supernatant of each sample, with the exception of the negative controls, and samples were rocked at 4°C for 30 min. Fifty microliters of protein A/G Plus-agarose beads (Santa Cruz Biotechnologies) were added to each sample, and samples were rocked at 4°C for 30 min. Samples were centrifuged at 2,000 x g for 2 min at 4°C, the supernatant was discarded, and the beads were washed eight times with 1 mL of lysis buffer. RNA was isolated from the beads using TRIzol reagent (Invitrogen) according to the manufacture's protocol. RT-PCR was done as described above for determination of p75NTR mRNA level using equal volumes of RNA and 32 cycles of PCR.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
R-Flurbiprofen and ibuprofen increase p75NTR mRNA level. To determine the mechanism by which the profens, R-flurbiprofen and ibuprofen, induce p75NTR expression in PC-3 and DU-145 human prostate cancer cell lines, we first compared the levels of p75NTR protein and mRNA following treatment with various concentrations of each profen (Fig. 1A ). There was a strong correlation between p75NTR protein and mRNA, indicating that increased mRNA levels are responsible for the induction of p75NTR protein by R-flurbiprofen and ibuprofen. A strong increase in p75NTR protein and mRNA levels was observed in PC-3 cells following treatment with 0.5 mmol/L R-flurbiprofen and 1.0 mmol/L ibuprofen, and in DU-145 cells with 1.0 mmol/L R-flurbiprofen and 2.0 mmol/L ibuprofen. Therefore, in subsequent experiments, we used 2.0 mmol/L R-flurbiprofen and ibuprofen because at this concentration, each of these profens induced high levels of p75NTR in both cell types. The level of p75NTR mRNA was also determined at various time points following profen treatment, and an induction was first observed within 2 to 4 h of treatment (Fig. 1B). We also compared levels of p75NTR protein and mRNA in the androgen-responsive LNCaP cell line following treatment with several concentrations of ibuprofen, and again observed a strong correlation between p75NTR protein and mRNA (Supplementary Fig. S1A). In addition, we observed a similar time course for induction of p75NTR mRNA in LNCaP cells treated with ibuprofen (Supplementary Fig. S1B).


Figure 1
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  		Figure 1. A, p75NTR mRNA and protein levels following R-flurbiprofen and ibuprofen treatment. PC-3 and DU-145 cells were treated with 0 to 2 mmol/L R-flurbiprofen or ibuprofen for 24 h. Cell lysates were collected and equal amounts of protein were subjected to SDS-PAGE and immunoblot analysis with an antibody to p75NTR or β-actin (β-act) for the loading control. Alternatively, RNA was isolated from PC-3 and DU-145 cells after 24 h of R-flurbiprofen or ibuprofen treatment, and p75NTR and GAPDH mRNA levels were determined by RT-PCR. B, time course induction of p75NTR mRNA levels. PC-3 and DU-145 cells were treated with 2 mmol/L R-flurbiprofen or ibuprofen for 0, 2, 4, 8, or 24 h. RNA was isolated at each time point and levels of p75NTR and GAPDH were determined by RT-PCR.

 
R-Flurbiprofen and ibuprofen increase p75NTR mRNA stability. Because the amount of transcript in a cell is a function of the rate of transcription and the rate of degradation, the observed increase in p75NTR mRNA in PC-3 and DU-145 cells following R-flurbiprofen and ibuprofen treatment must be due to either an up-regulation of transcriptional activity or a decrease in the rate of degradation of the p75NTR transcript, or both. Thus, we examined transcriptional activity and mRNA stability following treatment with profens. PC-3 and DU-145 cells were transfected with a p75NTR promoter-luciferase construct followed by treatment with R-flurbiprofen or ibuprofen, and promoter activity was compared with DMSO control–treated cells (Fig. 2A ). Treatment with profens did not result in a significant change in p75NTR promoter activity, suggesting that the increase in mRNA was not due to transcriptional up-regulation of the p75NTR gene. Previously, p75NTR promoter activity was shown to be up-regulated in the A875 melanoma cell line on exposure to hypoosmotic medium (24). Therefore, to show that the p75NTR promoter-luciferase construct was functional, we transfected A875 cells with the construct and compared luciferase activity in cells exposed to isotonic versus hypoosmotic medium. Consistent with previously published data, treatment with hypoosmotic medium resulted in 17-fold increased p75NTR promoter activity (Fig. 2A; ref. 24). Therefore, increased levels of p75NTR mRNA and protein in PC-3 and DU-145 cells treated with R-flurbiprofen or ibuprofen were not a consequence of changed transcriptional activity.


Figure 2
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  		Figure 2. A, p75NTR promoter activity following R-flurbiprofen and ibuprofen treatment. PC-3 and DU-145 cells were transfected for 48 h with a p75NTR promoter-luciferase construct and then treated with 2 mmol/L R-flurbiprofen (FLU), 2 mmol/L ibuprofen (IBU), or DMSO vehicle control (C) for 24 h. Relative luciferase activity was determined after normalization to Renilla and graphed as fold induction relative to DMSO control–treated cells. As a positive control, A875 cells were transfected for 48 h with the p75NTR promoter-luciferase construct and then exposed to isotonic (Iso) or hypoosmotic (Hypo) medium for 24 h. Fold induction of luciferase activity was determined for hypoosmotic treated cells relative to cells in isotonic medium. Bars, SE. ***, P < 0.0001 (ANOVA). B and C, increase in p75NTR mRNA stability following R-flurbiprofen or ibuprofen treatment. PC-3 and DU-145 cells were treated with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control for 8 h followed by the addition of 5 µg/mL actinomycin D (ActD). RNA was isolated at 0, 2, 4, 8, and 12 h following the addition of actinomycin D, and p75NTR mRNA levels were determined by RT-PCR. The intensity of the bands for p75NTR was analyzed by densitometry and graphed as a percentage of the 0-h time point.

 
The stability of the p75NTR transcript was determined with the transcriptional inhibitor actinomycin D. PC-3 and DU-145 cells were treated with R-flurbiprofen, ibuprofen, or DMSO for 8 h followed by the addition of actinomycin D. p75NTR mRNA level was determined by RT-PCR at several time points following actinomycin D addition (Fig. 2B and C). The majority of p75NTR mRNA from DMSO control–treated PC-3 and DU-145 cells was degraded within 2 and 4 h, respectively. In contrast, p75NTR mRNA was still present at high levels in profen-treated cells 12 h following the addition of actinomycin D. Therefore, profen treatment strongly enhanced p75NTR mRNA stability but did not influence transcription, suggesting that increased mRNA stability is responsible for the induction of p75NTR expression by R-flurbiprofen and ibuprofen (Fig. 2). We also observed an increase in p75NTR mRNA stability in LNCaP cells treated with ibuprofen relative to DMSO control–treated cells (Supplementary Fig. S1C and D).

R-Flurbiprofen and ibuprofen activate the p38 MAPK pathway. The p38 MAPK pathway has been strongly implicated in the stabilization of several mRNAs through the downstream kinases MK2 and MK3, which are activated upon phosphorylation by p38 MAPK (21, 23). p38 MAPK is also activated by phosphorylation. Therefore, we determined the phosphorylation status of p38 MAPK and MK2 at several time points in PC-3 and DU-145 cells following treatment with R-flurbiprofen or ibuprofen and in LNCaP cells following treatment with ibuprofen (Fig. 3A and Supplementary Fig. S2A). In all cases, profen treatment led to sustained activation of the p38 MAPK pathway, which could be observed even 8 h after treatment of each cell line.


Figure 3
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  		Figure 3. A, activation of the p38 MAPK pathway by R-flurbiprofen and ibuprofen. PC-3 and DU-145 cells were treated with 2 mmol/L R-flurbiprofen or 2 mmol/L ibuprofen for 0, 5 min, 1 h, 4 h, or 8 h. Cell lysates were collected and equal amounts of protein were subjected to SDS-PAGE and immunoblot analysis with antibodies to phosphorylated p38 MAPK (p-p38) or phosphorylated MK2 (p-MK2). Blots for phosphorylated p38 MAPK and phosphorylated MK2 were stripped and reprobed for total p38 MAPK and total MK2, respectively. β-actin was used as the loading control. B, the p38 MAPK inhibitor SB202190 prevents induction of p75NTR by R-flurbiprofen and ibuprofen. PC-3 and DU-145 cells were pretreated with or without 20 µmol/L SB202190 (SB) for 1 h and then treated with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control (CON) for 24 h. Cell lysates were collected and equal amounts of protein were subjected to SDS-PAGE and immunoblot analysis with an antibody to p75NTR or β-actin for the loading control.

 
The p38 MAPK pathway is involved in the induction of p75NTR by R-flurbiprofen and ibuprofen. Increased p75NTR mRNA stability seems to be the mechanism by which R-flurbiprofen and ibuprofen induce p75NTR expression, and these drugs activate the p38 MAPK pathway, which is known to be involved in mRNA stabilization. Pretreatment of PC-3 and DU-145 cells with the p38 MAPK inhibitor SB202190 followed by treatment with R-flurbiprofen or ibuprofen prevented an induction of p75NTR, indicating that p38 MAPK is involved in profen-mediated induction of p75NTR (Fig. 3B). To further confirm the role of the p38 MAPK pathway in profen-mediated induction of p75NTR, we also used p38 MAPK siRNA to specifically knock down p38 MAPK protein. There are two ubiquitously expressed isoforms of p38 MAPK, p38{alpha} and p38β, which are capable of phosphorylating MK2 and MK3 (18, 23, 25). PC-3 and DU-145 cells were transfected with nontargeting siRNA, p38{alpha} siRNA, p38β siRNA, or siRNAs for p38{alpha} and p38β together. The efficacy and specificity of the siRNAs were determined by Western blot (Fig. 4A ). Transfection of cells with p38{alpha} siRNA or p38{alpha} and p38β siRNAs together before profen treatment prevented an induction of p75NTR relative to untransfected cells or cells transfected with nontargeting siRNA (Fig. 4B). Transfection with p38β siRNA alone was less effective in preventing induction of p75NTR than transfection with p38{alpha} siRNA. Because the p38β isoform seems to be expressed at much lower levels than p38{alpha}, it is not surprising that knockdown of p38β alone only modestly affected induction of p75NTR because p38{alpha} was still present. Expression of p38{alpha} was also much greater than that of p38β in LNCaP cells (not shown), and consistently, induction of p75NTR by ibuprofen was prevented in LNCaP cells transfected with p38{alpha} siRNA (Supplementary Fig. S2C).


Figure 4
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  		Figure 4. A, validation assay for siRNA knockdown of p38 MAPK. PC-3 and DU-145 cells were transfected for 72 h with nontargeting siRNA or siRNA targeting p38{alpha}, p38β, or siRNAs for p38{alpha} and p38β together. Cell lysates were collected and efficacy of siRNA knockdown was determined by SDS-PAGE and immunoblot analysis with antibodies to p38{alpha} and p38β. β-actin was used as the loading control. B, knockdown of p38 MAPK prevents induction of p75NTR by R-flurbiprofen and ibuprofen. PC-3 and DU-145 cells were transfected with nontargeting siRNA, siRNA for p38{alpha}, siRNA for p38β, or siRNAs for p38{alpha} and p38β together for 72 h. Following transfection, cells were treated with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control for 24 h. Cell lysates were collected and expression of p75NTR was determined by SDS-PAGE and immunoblot analysis. β-actin was used as the loading control.

 
Because the MK2 and MK3 kinases are downstream of p38 MAPK and have both been shown to mediate mRNA stabilization, we used siRNA for MK2 and MK3 to determine if they are involved in the induction of p75NTR by R-flurbiprofen or ibuprofen. PC-3 and DU-145 cells were transfected with nontargeting siRNA, MK2 siRNA, MK3 siRNA, or siRNAs for MK2 and MK3 in combination. The efficacy and specificity of these siRNAs were tested in PC-3 and DU-145 cells by Western blot (Fig. 5A ). Transfection with MK2 siRNA or MK3 siRNA separately before profen treatment resulted in decreased induction of p75NTR (Fig. 5B). However, transfection with MK2 siRNA and MK3 siRNA together, resulting in the simultaneous knockdown of both proteins, was more effective in preventing induction of p75NTR by R-flurbiprofen or ibuprofen than knockdown of MK2 or MK3 separately (Fig. 5B). This is consistent with previous observations that MK2 and MK3 are activated in parallel by p38 MAPK, have similar substrates, and are both strongly expressed in PC-3 and DU-145 cells (Fig. 5A; refs. 23, 25). Likewise, in LNCaP cells, simultaneous siRNA knockdown of MK2 and MK3 was successful in preventing induction of p75NTR expression by ibuprofen (Supplementary Fig. S2C).


Figure 5
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  		Figure 5. A, validation assay for siRNA knockdown of MK2 and MK3. PC-3 and DU-145 cells were transfected for 72 h with nontargeting siRNA, siRNA for MK2, siRNA for MK3, or siRNAs for MK2 and MK3 together. Cell lysates were collected and efficacy of siRNA knockdown was determined by SDS-PAGE and immunoblot analysis with antibodies to MK2 and MK3. β-actin was used as the loading control. B, knockdown of MK2 and MK3 prevents induction of p75NTR by R-flurbiprofen and ibuprofen. PC-3 and DU-145 cells were transfected with nontargeting siRNA, siRNA for MK2, siRNA for MK3, or siRNAs for MK2 and MK3 together for 72 h. Following transfection, cells were treated with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control for 24 h. Cell lysates were collected and expression of p75NTR was determined by SDS-PAGE and immunoblot analysis. β-actin was used as the loading control.

 
p75NTR mRNA is a target of the RNA stabilizing protein HuR. The HuR is an RNA binding protein that enhances the stability of target mRNAs (26, 27). It has been shown that activation of the p38 MAPK pathway results in the translocation of HuR from the nucleus to the cytoplasm where it binds to the 3'-untranslated regions (UTR) of various mRNAs containing a core AUUUA sequence (20, 28–31). The p75NTR transcript contains two AUUUA sites in the 3'-UTR, suggesting HuR binding as a possible mechanism by which R-flurbiprofen and ibuprofen may stabilize p75NTR mRNA. R-Flurbiprofen– and ibuprofen-treated cells exhibited modestly increased levels of cytoplasmic HuR (Fig. 6A ). In addition, siRNA knockdown of HuR protein before profen treatment of PC-3 and DU-145 cells partially prevented induction of p75NTR relative to untransfected or nontargeting siRNA–transfected cells, suggesting that HuR is involved in the induction of p75NTR by R-flurbiprofen and ibuprofen (Fig. 6B). Similarly, transfection of LNCaP cells with HuR siRNA before ibuprofen treatment was effective in preventing increased p75NTR expression (Supplementary Fig. S2C). HuR belongs to the embryonic lethal abnormal vision family of proteins, which also includes HuB, HuC, and HuD, all RNA stabilizing proteins that are typically expressed in neurons (32). Whereas HuR expression was detected in PC-3 and DU-145 cells (Fig. 6A), expression of the other family members could not be detected (not shown), indicating that they most likely do not participate in profen-mediated induction of p75NTR. A direct interaction between the HuR protein and the p75NTR transcript was shown by an immunoprecipitation RT-PCR assay. Lysates of profen-treated PC-3 and DU-145 cells were immunoprecipitated with, or without, an HuR antibody and RNA was isolated from the immunoprecipitated beads, followed by RT-PCR for p75NTR mRNA. PC-3 and DU-145 cells treated with R-flurbiprofen or ibuprofen showed a direct interaction between HuR and p75NTR mRNA, whereas DMSO control–treated cells did not (Fig. 6C). In addition, p75NTR mRNA was not detected in samples from cells treated with ibuprofen but not incubated with the HuR antibody, indicating that the p75NTR mRNA detected in the treated samples was not nonspecific (Fig. 6C).


Figure 6
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  		Figure 6. A, translocation of HuR from the nucleus to the cytoplasm following treatment with R-flurbiprofen or ibuprofen. PC-3 and DU-145 cells were treated for 2 h with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control. Nuclear (N) and cytoplasmic (C) fractions were subjected to SDS-PAGE and immunoblot analysis with antibodies to HuR, lamin B1, and {alpha}-tubulin. B, knockdown of HuR prevents induction of p75NTR by R-flurbiprofen and ibuprofen. PC-3 and DU-145 cells were transfected for 72 h with nontargeting siRNA or siRNA targeting HuR. For the test for siRNA knockdown, cell lysates were collected and efficacy of siRNA knockdown was determined by SDS-PAGE and immunoblot analysis with an antibody to HuR or β-actin for the loading control. For examination of p75NTR expression, cells were treated with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control for 24 h following siRNA transfection. Cell lysates were collected and expression of p75NTR was determined by SDS-PAGE and immunoblot analysis. β-actin was used as the loading control. C, HuR binds to the p75NTR transcript following treatment with R-flurbiprofen or ibuprofen. PC-3 and DU-145 cells were treated for 12 h with 2 mmol/L R-flurbiprofen, 2 mmol/L ibuprofen, or DMSO vehicle control. Cell lysates from equal numbers of cells were incubated with an antibody to HuR, with the exception of the negative controls, and then incubated with protein A/G Plus-agarose beads. Beads were washed, RNA was isolated from the beads, and p75NTR mRNA levels were determined by RT-PCR. D, R-flurbiprofen and ibuprofen increase phosphorylation of eIF4E through the p38 MAPK pathway. PC-3 and DU-145 cells were transfected for 72 h with nontargeting siRNA or p38{alpha} siRNA. Following transfection, cells were treated for 30 min with DMSO, 2 mmol/L R-flurbiprofen, or 2 mmol/L ibuprofen. Cell lysates were collected and phosphorylated eIF4E was detected by SDS-PAGE and immunoblot analysis. Blots for phosphorylated eIF4E were stripped and reprobed for total eIF4E. β-actin was used as the loading control.

 
R-Flurbiprofen and ibuprofen increase phosphorylation of eIF4E. Although HuR seems to bind to the p75NTR transcript upon profen treatment, the inhibition of p75NTR induction in the presence of a substantial HuR knockdown was modest. This result suggests that there are additional mechanisms involved in the induction of p75NTR by the p38 MAPK pathway. eIF4E is a downstream, indirect target of p38 MAPK and is involved in several mechanisms of posttranscriptional regulation including translation initiation through 5'-cap binding, mRNA nuclear export, and mRNA stability (33, 34). We examined eIF4E phosphorylation following profen treatment in untransfected, nontargeting siRNA–transfected, and p38{alpha} siRNA–transfected PC-3 and DU-145 cells. R-Flurbiprofen and ibuprofen treatment resulted in increased phosphorylated eIF4E levels in both cell types, and the increase was inhibited to varying degrees in the p38{alpha} siRNA–transfected cells (Fig. 6D). These results identify eIF4E phosphorylation as another mechanism through which the p38 MAPK pathway may regulate p75NTR expression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The p75NTR exhibits tumor suppressor activity in prostate cancer cells, which is mediated through an intracellular death domain capable of inducing apoptosis and suppressing growth (2, 5, 7, 9). However, p75NTR expression is progressively lost in organ confined prostate cancer and is minimal in established metastatic prostate cancer cell lines (10). Significantly, our recent observations that the profens, R-flurbiprofen and ibuprofen, induced reexpression of p75NTR protein levels causal of p75NTR-dependent decreased survival of prostate cancer cells has stimulated an investigation of the associated signal transduction cascade that mediates profen effects on prostate cancer cells (17). Increased p75NTR protein levels in R-flurbiprofen– or ibuprofen-treated cells were accompanied by increased mRNA levels. This indicated that profen treatment influenced either the rate of mRNA degradation or the rate at which the gene was transcribed. Profen-treated cells showed no significant change in luciferase activity of the p75NTR promoter construct relative to untreated cells. However, the transcriptional inhibitor actinomycin D revealed a strong increase in p75NTR mRNA stability in cells treated with R-flurbiprofen or ibuprofen. Previously, our investigation of the mechanism by which several prostate cancer cell lines lose expression of p75NTR showed that although p75NTR is actively transcribed at levels comparable to the high-expressing A875 melanoma cell line, the prostate cancer cells contain <1% of the p75NTR mRNA found in A875 cells (10). Furthermore, transfection of prostate cancer cells with full-length p75NTR cDNA led to very modest p75NTR protein expression, whereas transfection with p75NTR cDNA lacking most of the 3'-UTR resulted in high protein expression. This suggested that although p75NTR is transcribed at a high level, prostate cancer cells have very little mRNA or protein due to increased mRNA instability that is mediated through the 3'-UTR. Interestingly, the p75NTR 3'-UTR is ~2 kb long, which is about four times the length of the average human 3'-UTR (35). It has been suggested that increased 3'-UTR length provides increased potential for posttranscriptional regulation through the 3'-UTR (35). Therefore, it seems likely that R-flurbiprofen and ibuprofen are able to induce p75NTR expression by modulating the stability of the transcript and that mRNA stability is an important determinant in regulating the expression level of p75NTR protein.

Because induction of p75NTR by R-flurbiprofen and ibuprofen treatment seemed to be largely due to increased mRNA stability, we examined activity of the p38 MAPK pathway, which is considered an important regulator of mRNA stability (18, 21, 22). p38 MAPK is activated by phosphorylation, and R-flurbiprofen and ibuprofen caused increased p38 MAPK phosphorylation within 5 min of treatment. Increased activation was still noticeable 8 h following treatment. Because pretreatment with the p38 MAPK selective inhibitor SB202190 or siRNA knockdown of p38 MAPK before profen treatment prevented an induction of p75NTR, it seems that the p38 MAPK pathway was involved in regulating profen-mediated induction of p75NTR. The data suggest that activation of the p38 MAPK pathway is the primary mechanism responsible for increased p75NTR expression. However, inhibition of p38 MAPK did not always result in complete inhibition of p75NTR induction, and this may be due to participation of additional pathways. R-Flurbiprofen and ibuprofen induced activation of the kinase MK2, which is directly downstream of p38 MAPK. MK2 and the closely related MK3 are known to be responsible for mediating the mRNA stabilizing effects of the p38 MAPK pathway (22, 23). They are activated with similar kinetics, are able to compensate for one another, have similar substrates, and lead to stabilization of the same group of transcripts in response to lipopolysaccharide (LPS; refs. 23, 25). Not surprisingly, siRNA knockdown of MK2 and MK3 together was more effective in preventing induction of p75NTR by R-flurbiprofen or ibuprofen than knockdown of either MK2 or MK3 separately, indicating that p38 MAPK is able to induce p75NTR by acting through both MK2 and MK3.

Activation of the p38 MAPK pathway seems to be responsible for R-flurbiprofen– and ibuprofen-mediated induction of p75NTR in prostate cancer cells. Significantly, there is an accumulating body of evidence linking p38 MAPK to tumor suppression. For example, inactivation of p38 MAPK, inactivation of the p38 MAPK activating kinases MKK3 and MKK6, and overexpression of the p38 MAPK phosphatase Wip1 are associated with increased tumorigenesis (36, 37). In addition, Wip1 was found to be amplified in primary breast tumors (36, 37). The tumor suppressor p53 is a direct substrate of p38 MAPK and is therefore one way in which p38 MAPK exerts its tumor-suppressing effects (18, 21, 36). However, p53 is inactivated by mutation in roughly half of all cancers (38). This is also the case for PC-3 cells, which are p53-null, and for DU-145 cells, which express mutated p53 (39). Here, we identify induction of p75NTR expression as a novel mechanism by which p38 MAPK may achieve tumor suppressor activity. This may be applicable to other cancer types in addition to prostate cancer. For example, we previously showed that ibuprofen up-regulates p75NTR in bladder cancer cells, which resulted in p75NTR-mediated apoptosis (40). In addition, basic fibroblast growth factor treatment resulted in the p38 MAPK–dependent death of Ewing's sarcoma family of tumor cells and was associated with induction of p75NTR that was prevented in the presence of a p38 MAPK selective inhibitor (41). Interestingly, p38 MAPK and p75NTR have many overlapping roles in addition to induction of apoptosis and tumor suppression. For example, both are involved in the regulation of inflammation as well as response to brain injury (18, 24, 42–45). Therefore, it is possible that p38 MAPK achieves these roles, in part, by increasing expression of p75NTR. Consistent with this hypothesis is the observation that LPS, which leads to the stabilization of several transcripts through the p38 MAPK/MK2 pathway, also induces p75NTR expression (23, 46, 47).

One mechanism by which the p38 MAPK pathway has been shown to stabilize target transcripts involves the RNA binding protein HuR (22, 29–31). HuR is the only RNA binding protein repeatedly shown to stabilize transcripts containing the AUUUA sequence (22, 31). Its ability to stabilize target mRNAs is linked to its subcellular localization, and activation of p38 MAPK and MK2 has been shown to cause translocation of HuR from the nucleus to the cytoplasm, resulting in increased mRNA stability of a number p38 MAPK regulated genes (22, 29–31). The human p75NTR transcript contains AUUUA sites located in the 3'-UTR at positions 2,946 and 3,124, and these are conserved in rat and mouse, suggesting that they may be involved in the regulation of p75NTR expression. We showed that treatment with R-flurbiprofen or ibuprofen resulted in the binding of HuR to the p75NTR transcript, and a modest increase in the cytoplasmic level of HuR was observed following profen treatment. However, siRNA knockdown of HuR before profen treatment only partially prevented an induction of p75NTR. These data suggest that binding of HuR to the p75NTR transcript is not the sole mechanism responsible for increased p75NTR expression. This is not surprising given the elaborate picture of mRNA regulation that is emerging. Recent data suggest that posttrascriptional regulation of gene expression occurs through a plethora of RNA binding proteins that control the splicing, nuclear export, stability, localization, translation, and degradation of transcripts, often through specific sequential or structural elements located in the untranslated regions (48, 49). Therefore, given the exceptionally long 3'-UTR of the p75NTR transcript, it is probable that other RNA binding proteins regulate expression as well, and this area requires further investigation. To provide insight into additional mechanisms through which the p38 MAPK pathway may influence p75NTR mRNA stability, we examined phosphorylation of eIF4E. eIF4E is phosphorylated by kinases downstream of p38 MAPK (33). It is most well known as a 5'-cap binding protein involved in translation initiation. However, recent data suggest that it regulates gene expression at several levels (34). Interestingly, an increase in eIF4E activity is not associated with an increase in global translation, but rather translation of only a subset of transcripts (34). In addition, eIF4E has been shown to control the nuclear export of a different subset of transcripts (34). Finally, eIF4E is linked to control of mRNA stability in that removal of the 5'-cap is a key step in mRNA degradation, and competition between eIF4E and decapping enzymes has been shown (50). We show that profen treatment increases the level of phosphorylated eIF4E, and this seems to occur, at least partially, through the p38 MAPK pathway because the increase in phosphorylation is substantially inhibited in the presence of p38 MAPK siRNA. Therefore, these data identify modulation of eIF4E activity as another mechanism by which the p38 MAPK pathway may control posttranscriptional events in response to profen treatment.

In this study, we show for the first time that R-flurbiprofen and ibuprofen activate the p38 MAPK pathway in prostate cancer cells and that this pathway is necessary for induction of the p75NTR tumor suppressor by these drugs. This mechanism of induction occurs in both androgen-independent PC-3 and DU-145 cells as well as androgen-responsive LNCaP cells. Retrospective epidemiologic studies have shown that certain nonsteroidal anti-inflammatory drugs, including ibuprofen, reduce the incidence of prostate cancer (15, 16). Numerous recent studies have provided compelling evidence that inhibition of cancer cell growth by nonsteroidal anti-inflammatory drugs can occur through COX-independent mechanisms that have yet to be fully elucidated (12, 13, 17, 40). The enantiomer R-flurbiprofen lacks COX inhibitory activity, indicating that the activation of p38 MAPK and subsequent induction of p75NTR is a COX-independent mechanism by which these profens can achieve anticancer activity in the prostate (13). Because p38 MAPK is activated within 5 min of profen treatment, it is likely that R-flurbiprofen and ibuprofen are interacting with signaling components proximal to p38 MAPK. Further delineation of this mechanism may identify novel therapeutic targets and facilitate the discovery of more effective compounds that are able to induce p75NTR expression at lower concentrations.


   Acknowledgments
 
Grant support: NIH grant R01DK52626 and the Department of Defense Prostate Cancer Research Program grant PC060409.

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 Dr. Emil Bogenmann for providing the p75NTR promoter-luciferase construct and Dr. Albert J. Fornace for critical reading of the manuscript.


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

Received 5/15/07. Revised 9/ 7/07. Accepted 10/ 4/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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