Repression of NR4A1 by a Chromatin Modifier Promotes Docetaxel

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FEBS Letters 587 (2013) 2542–2551

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Repression of NR4A1 by a chromatin modiﬁer promotes docetaxel resistance in PC-3 human prostate cancer cells ⇑ ⇑ Liang Yu a,b,1, Yan-sheng Su c,a,1, Jie Zhao d, He Wang a, , Wei Li d,

a Department of Urology, Xijing Hospital, Fourth Military Medical University, No. 127 Changle West Road, Xi’an 710032, China b Department of Urology, 3rd Hospital of PLA, No. 45 Dongfeng Road, Bao Ji 721004, China c Department of Urology, No.323 Hospital of PLA, Xi’an 710054, China d Department of Human Anatomy, Histology and Embryology, Fourth Military Medical University, No. 169 Changle West Road, Xi’an 710032, China

article info abstract

Article history: Epigenetic silencing mechanisms play an important role in chemoresistance of human cancer. Here Received 15 January 2013 we report the upregulated expression of metastasis-associated protein 1 (MTA1), a component of the Revised 19 June 2013 nucleosome remodeling deacetylation (NuRD) complex, in chemoresistant prostate cancer (PCa). Accepted 19 June 2013 MTA1 knockdown in PC-3 cells inhibited cell proliferation and enhanced docetaxel (DTX)-induced Available online 2 July 2013 cell death. Conversely, overexpression of MTA1 promotes DTX chemoresistance in PC-3 cells. MTA1 acted as a potent corepressor of the nuclear receptor NR4A1 transcription by interacting with Edited by Ned Mantei histone deacetylase 2 (HDAC2). These ﬁndings suggest that MTA1 may serve as a novel DTX-resistance promoter in PC-3 cells. Keywords: Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Metastasis-associated protein 1 (MTA1) Nucleosome remodeling deacetylation (NuRD) complex Prostate cancer (PCa) Chemoresistance

1. Introduction plays a central role in the regulation of divergent cellular pathways by modifying the acetylation status of crucial target genes [8,9].In Prostate cancer (PCa) causes substantial morbidity and mortal- PCa, MTA1 expression is higher in hormone-refractory metastatic ity worldwide and has become one of the leading male cancers in PCa compared to clinically localized disease and benign prostatic some Asian countries [1]. As prostate cancer progresses it becomes tissues [10]. MTA1 pro-angiogenic and pro-invasive functions cre- refractory to hormone manipulation. At this stage, chemotherapy ate a permissive environment for PCa tumor growth and likely sup- remains the main treatment option in the setting of castration- port metastasis by regulating the expression of E-cadherin [11]. resistant prostate cancer (CRPC), providing modest survival and Recent advances demonstrated that phosphatase and tensin homo- palliative benefits [2]. Among different chemotherapies, docetaxel log (PTEN), a master tumor suppressor gene during PCa pathogen- (DTX) chemotherapy offers both symptomatic and survival bene- esis, is also the transcriptional regulation target of MTA1. MTA1 fits in men with metastatic hormone-refractory PCa. However, transcriptionally represses PTEN expression by recruiting class II resistance to such chemotherapy will eventually develop in histone deacetylase along with the transcription factor Yin-Yang approximately half of the patients, and the disease then becomes 1 (YY1) onto the PTEN promoter [12]. In addition, many recent re- difficult to control [3–5]. The precise mechanisms of this phenom- ports in this field have demonstrated the functional importance of enon have not been clearly elucidated up to date. Preclinical and MTA1 in PCa progression and metastasis at both the in vitro and clinical evidence suggests that multiple mechanisms are certain in vivo levels [13–15]. Based on these findings, enhanced expres- to coexist to confer DTX chemoresistant phenotypes [6,7]. sion of endogenous MTA1 has been implicated in promoting PCa Metastasis-associated protein 1 (MTA1), a component of the tumorigenesis, and MTA1 can also be utilized both as a prognostic Mi-2/nucleosome remodeling deacetylation (NuRD) complex, marker and a therapy target in PCa [12]. NR4A orphan receptors, including NR4A1 (Nur77, TR3, NGFI-B), NR4A2 (Nurr1), and NR4A3 (Nor1), are members of the nuclear ⇑ Corresponding authors. Fax: +86 29 84774511 (W. Li), +86 29 84771238 receptor (NR) superfamily of transcription factors, and regulate (H. Wang). several key biological pathways in cancerous tissues [16]. Knock- E-mail addresses: [email protected] (W. Li), dusxijinghewang@fmmu. down of NR4A1 resulted in inhibition of cell growth, induction of edu.cn (H. Wang). 1 These authors contributed equally to this work. apoptosis, and decreased angiogenesis, suggesting that NR4A1 is

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.06.029 L. Yu et al. / FEBS Letters 587 (2013) 2542–2551 2543 a pro-oncogenic factor in most cancers. The only exception to these 10% Fetal Bovine Serum (FBS) (Invitrogen). PC-3 resistant sub-lines observations was observed in LNCaP cells, where antisense-medi- (PC-3R) were generated as described elsewhere [24]. Specific ated knockdown of NR4A1 appeared to increase cell growth [17]. knockdown of MTA1 was achieved by transfecting PC-3 with corre- Consistent with the last line of evidence, microarray studies have sponding siRNA against MTA1 (sc-35981) or with a control siRNA identified that downregulation of NR4A1 was in favor of cancerous (sc-37007) (Santa Cruz Biotechnology, CA). Briefly, 2 105 PC-3 metastasis [18]. These data collectively are indicative of a unique cells were cultured in six-well culture plates and transfected with role of NR4A1 in PCa. siRNA against MTA1 (which consist of pools of three to five target- Accumulated evidence suggests that the androgen-refractory PCa specific 19–25 nt siRNAs) or the same amount of non-target proliferates faster than the androgen-sensitive cancer. Also, the control siRNA following the manufacturer’s protocol. 48 h after androgen-refractory PCa has been generally considered as chemore- transfection, cells were collected and subjected to other experi- sistant [19]. On the other hand, emerging data point to a potential ments. pCMV6-AC-GFP-tagged NR4A1 plasmid was purchased from involvement of deacetylases in the chemoresistant phenotypes of OriGene. PC-3 cells stably overexpressing exogenous MTA1 or PCa [20]. Considering the close relationship between MTA1 function NR4A1 were established according to a previous report [25]. and recruitment of histone deacetylation complexes during transcriptional regulation, we hypothesized that MTA1 may be involved 2.3. Cell viability measurement in the pathogenesis of PCa chemoresitance. A functional and mechanistic study comprising multiple analyses was therefore designed to Ten thousand cells/well were cultured in a 96-well plate. 24 h elucidate the potential links between this chromatin modifier and later, cells were treated with different concentrations of DTX (Sig- the aggressive nature of chemoresistant PCa. In addition, since ma–Aldrich) for different durations. Cells were then subjected to upregulation of MTA1 and downregulation of NR4A1 are both MTT assay for viability measurement. strongly associated with cancerous metastasis, and NR4A1 has been 2.4. Assessment of apoptosis shown to be transcriptionally regulated by histone deacetylase complexes [21], we then evaluated the potential functional association Apoptotic events were assessed by Annexin V–FITC/PI staining between MTA1 and NR4A1. Our systematic analysis will pave the followed by FACS as described [25]. An apoptosis ELISA kit (Roche way for a better understanding of the role of MTA1 in PCa. Diagnostics, Mannheim, Germany) was also used to quantitatively measure cytoplasmic histone-associated DNA fragments (mononu- 2. Materials and methods cleosomes and oligonucleosomes). Briefly, cells were incubated in a microplate well with 5 nM DTX for 24 h and were then pelleted 2.1. Patients by centrifugation. Cells were resuspended in lysis buffer. After lysis, an aliquot of the supernatant was transferred to a streptavi- Samples have been obtained after patients gave written in- din-coated well of a microplate. The nucleosomes in the formed consent. In detail, benign prostatic tissues from patients supernatant were incubated with two monoclonal antibodies, who underwent radical cystoprostatectomy due to bladder cancer anti-histone (biotin-labeled) and anti-DNA (peroxidase-conju- (n = 5) and biopses from CRPC patients (n = 30) were included. The gated), at room temperature for 2 h. The antibody-nucleosome chemoresistant PCa patients were defined as CRPC patients with complexes are bound to the microplate by the streptavidin. The fi- tumors clearly progressing on after 4 cycles of three-weekly first- nal spectrophotometric assay was developed using peroxidase sub- line DTX-based chemotherapy (patients were treated with DTX strate and the absorbance was measured in triplicate with a 2 chemotherapy 75 mg/m intravenously on day 1 repeated every microplate reader at 405 nM (Bio-Rad680). three weeks along with prednisolone 10 mg daily). Androgen sup- pression with LHRH analogues was continued throughout the 2.5. Promoter reporter assay duration of chemotherapy. General characteristics of PCa patients are summarized in Supplementary Table 1 (note: The Gleason Luciferase assays were performed using a human MTA1 pro- score system, which was developed by Dr. Donald Gleason in the moter reporter construct system (SwitchGear Genomics, Menlo 1960s, is used to help evaluate the prognosis of men with prostate Park, CA) according to the manufacturer’s instructions (nucleotides cancer. A Gleason score is given to prostate cancer based upon its 777 to +242; Genbank accession number: U35113). The human microscopic appearance. Cancers with a higher Gleason score are NR4A1 promoter region (Nucleotides 869 to +123; Genbank more aggressive and have a worse prognosis. This system consists accession number: U17590.1) was generated by PCR amplification of five grades, and is measured on a scale of 1–5. Pathologists usu- using human genomic DNA [26] and cloned into the pGL3 lucifer- ally assign a grade to the most common tumor pattern, and a sec- ase reporter vector (Promega, Madison, WI) with the Infusion 2.0 ond grade to the next most common tumor pattern. The two grades Dry-Down PCR cloning kit (Clontech). The primers used are listed are added together to get a Gleason Score. The Gleason Grade in Supplementary Table 2. Cells were transiently transfected with ranges from 1 to 5, with 5 having the worst prognosis. The Gleason 1 lg of plasmid DNA using Lipofectamine 2000 (Invitrogen). For Score ranges from 2 to 10, with 10 having the worst prognosis the assessment of NR4A1 promoter activity, cells were also [22,23]). After collection, tissues were either immediately fixed in co-transfected with pcDNA3.1-His-MTA1 or pcDNA3.1-His. The 10% neutral buffered formalin for histochemical examination or pRL-TK Renilla reporter plasmid (Promega) was co-transfected to stored at 80 °C for biochemical analysis. The use of the human normalize the transfection efficiency. Luciferase activity measure- tissue in this study was approved by the Human Research Commit- ments were performed using the dual-luciferase reporter assay tee of the Fourth Military Medical University (Permit number: system (Promega) according to the manufacturer’s protocol. The 10002). The protocol employed strictly conformed to the standards firefly luciferase activity was normalized according to the Renilla set by The 2008 Revised Declaration of Helsinki. luciferase activity and expressed as relative luciferase units (RLU) to reflect the promoter activity [27]. 2.2. Cell culture and treatment 2.6. Colony formation assay LNCaP and PC-3 cells were purchased from ATCC. Normal human prostate epithelial cells (PEC) were purchased from Clonetics. Approximately 400 cells (transformed with the PC-3-His-vector Cells were maintained in RPMI-1640 medium supplemented with or PC-3-His-MTA1)/60-mm plastic dish (Falcon; Becton Dickinson, 2544 L. Yu et al. / FEBS Letters 587 (2013) 2542–2551

Lincoln Park, NJ) were cultured at 37 °C in a humidified atmo- group, the mean optical density of each unit area was automati- sphere of 5% CO2 and 95% air. At the end of 48 h of incubation, cally calculated by the computer. Quantification of Ki67 staining DTX (5 nM) was added to the cultures. After 3 days treatment, was carried out as described elsewhere [30]. Briefly, immunoposi- the DTX-containing media was replaced with fresh media and all tivity and immunonegativity were determined using a linear dis- cultures were incubated for an additional 9 days until colonies criminant analysis based on hue, saturation, and intensity of 500 were large enough to be clearly discerned. Colonies were stained immunostained cells and 200 cells of a negative control slide, for 5 min with a solution containing 0.5% crystal violet and 25% respectively. The positivity for Ki67 was defined as the total num- methanol, followed by three rinses with tap water to remove ex- ber of pixels from immunopositive areas divided by the total num- cess dye. The colonies, defined as groups of >50 cells, were finally ber of pixels from all nuclear areas detected in a given specimen. scored using Colony Counter software (Syngene, Frederick, MD). The average number of colonies was plotted (Mean ± S.D., n = 3). 2.11. Chromatin immunoprecipitation

2.7. In vivo nude mouse tumor xenograft model The ChIP assay was performed according to previous work [29]. The sequence of primers and the antibodies used are listed in Sup- 1 106 cultured PC-3-His-vector or PC-3-His-MTA1 trans- plementary Tables 2 and 3, respectively. formed cells in 200 ll PBS were injected subcutaneously in 8-week-old nude mice (n = 6 mice per group). Equivalent volumes 2.12. Statistical analysis of PBS were injected as an experimental control. At the 4th week after cell inoculation, mice were subjected to intraperitoneal injec- Data from in vitro experiments were compared using Student’s t tions of DTX in 10% DMSO, at a dose of 25 mg/kg B.W. every 24 h test. The significance of differences between the growth curves in for 3 weeks. Control mice received 10% DMSO alone. Tumor vol- the xenograft model for tumor volume changes was assessed by ume changes were recorded weekly by two perpendicular diame- analysis of variance (ANOVA). Statistical analyses were performed ter measurements as described [28]. Animal work was approved using SPSS 15.0 software with P<0.05 being considered as statisti- by the Ethics Committee for Animal Experiments of the Fourth Mil- cally significant. itary Medical University (Permit number: 10001). 3. Results 2.8. RT-PCR and quantitative RT-PCR (qRT-PCR) 3.1. Upregulation of MTA1 expression in chemotherapy-resistant PCa RT-PCR and qRT-PCR were carried out as described [29]. In brief, total RNA was extracted using RNeasy Mini Kit (QIAGEN Inc., In the first attempt to study the potential involvement of MTA1 Valencia, CA, USA) and first-strand cDNA was synthesized with in the chemoresistance of PCa, we examined the expression level of Superscript II (Rnase H- Reverse Transcriptase; Invitrogen). PCR MTA1 mRNA in human PCa specimens. RT-PCR analysis revealed was set up according to Promega’s reverse transcription system that the MTA1 transcripts were detectable in a panel of human protocol. Details of primers used are listed in Supplemental Table 2. prostate benign and tumor tissues (Fig. 1A). Subsequent qRT-PCR Amplification of 18S RNA served as internal control. PCR products analysis demonstrated that MTA1 mRNA was significantly elevated were quantified by SYBR green intercalation using the MiniOpti- in chemoresistant PCa samples (n = 15) compared to their chemo- con™ system (Bio-Rad Laboratories, CA, USA). The relative abun- sensitive counterparts (n = 15) and benign tissues (n =5)(Fig. 1B). dance of each target transcript was quantified using the Immunohistochemical analysis demonstrated a significant in- comparative 44Ct method. crease of MTA1 expression in the nuclei of chemoresistant PCa cells (Fig. 1C). Further quantitative analysis confirmed the upregulation 2.9. Western blotting of MTA1 expression in chemoresistant PCa samples (Supplemen- tary Table. 4). We then generated DTX-resistant sublines (PC-3R). Western blotting was carried out as described previously [29]. PC-3R cells demonstrated partial but increasing resistance to DTX Briefly, protein samples were prepared in ice-cold RIPA buffer treatment over the different doses, when compared to the PC-3 (Tris–HCl 50 mM, NaCl 150 mM, Triton X-100 1% vol/vol, sodium cells (Supplementary Fig. 1). We then assessed MTA1 expression deoxycholate 1% wt/vol, and SDS 0.1% wt/vol pH 7.5) supple- levels in different human prostatic cell lines. PEC and LNCaP cells mented with complete proteinase-inhibitor cocktail tablets (Roche showed the lowest levels of MTA1, whereas the higher expression Diagnostic, Mannheim, Germany). 20 lg of protein sample were levels were found in PC-3 and PC-3R cells, with the highest level separated by SDS/PAGE and transferred to a nitrocellulose observed in PC-3R cells (Fig. 1D and E). Moreover, to investigate membrane (Millipore, Bedford, MA, USA). Membranes were then the transcriptional activity of the MTA1 gene in different cell types, incubated with different primary antibodies as indicated in Sup- human MTA1 promoter reporter constructs were transfected into plemental Table 3, with b-actin serving as internal control. Final different PCa cells. Luciferase activity was measured 48 h after signals were detected using an ECL kit (Amersham Biosciences, transfection. PC-3R cells showed the highest promoter activity Buckinghamshire, UK) according to the manufacturer’s (12.68 ± 2.12 RLU) when compared to other cells (Fig. 1F). These instructions. data are indicative of an association of elevated MTA1 expression with chemoresistance in PCa. 2.10. Immunohistochemistry 3.2. Endogenous MTA1 protects PC-3 cells from DTX-induced death The streptavidin–biotin complex (SABC) immunohistochemical method was conducted as previously described [29]. Primary anti- We next explored the role of MTA1 in the DTX-induced cytotox- bodies used are listed in Supplemental Table 3. Immunostaining of icity in PC-3 cells, because these cells represent a more resistant MTA1 in human PCa samples was evaluated using a Q500 com- phenotype. We transiently knocked down MTA1 in PC-3 cells using puter-assisted image analysis system (Leica, USA). In total, 50 unit siRNA treatment (Fig. 2A and B). The viability of these cells in re- areas in five sections in each age group were measured using a 40 sponse to DTX treatment was then analyzed by MTT assay. Abla- objective. The optical densities of the MTA1 immunoreactive tion of MTA1 in PC-3 cells resulted in a 50% drop in cell viability deposits were determined. At the end of the analysis for each at 22 h post-treatment, whereas the control group only lost about L. Yu et al. / FEBS Letters 587 (2013) 2542–2551 2545

A B P < 0.05 ) 1/18S A expression A MTA ( MTA1 mRNA

18S MTA1 1 2 3

C D P < 0.05 )

P < 0.05 TA1/18S T RNA expression RNA M ( mR MTA1

E F PEC LNCaP

-MTA1 moter activity RLU) (RLU) ( o ( omoter activity pro

-Actin pro MTA1 MTA1

PCPC-3 3 PC-3RPC 3R (RLU) (RLU) promoter activity promoter activity 1 1 1 MTA MTA1

Fig. 1. Upregulation of MTA1 in chemoresistant prostate cancer (PCa). (A) Expression of MTA1 in human prostate tissues. Total RNA was isolated from benign, chemosensitive and chemoresistant human prostate tissues and subjected to reverse transcription-PCR (RT-PCR) as described in Section 2. (B) Relative expression levels of MTA1 in human prostate tissues were quantiﬁed using quantitative RT-PCR (qRT-PCR). (C) Immunohistochemical staining of MTA1 in human prostate tissues. NC, negative control experiments using preabsorbed anti-MTA1 antibody. (D) Relative expression levels of MTA1 in normal human prostate epithelial cells (PEC), LNCaP, PC-3 and PC-3R cells were quantiﬁed using qRT-PCR. (E) The level of MTA1 protein in PEC, LNCaP, PC-3 and PC-3R cells was detected by Western blotting. b-Actin served as a loading control. (F) The MTA1 promoter activity was determined using a commercially available human MTA1 promoter reporter construct system according to the manufacturer’s instruction.

34% viability 24 h after the same DTX treatment (Fig. 2C). In addi- Akt signaling was inhibited (Fig. 2E). We also used another siRNAs tion, ELISA methods revealed that MTA1 knockdown led to signif- from Sigma (Cat. SASI_Hs01_00239682 and SASI_Hs01_00239683) icant increase of apoptosis after cells had been incubated with DTX to exclude potential off-targets effects in the knockdown experi- (5 nM) for 24 h (Fig. 2D). This elevated apoptotic rate was further ment. About 70% of endogenous MTA1 was efficiently knocked confirmed by the upregulated expression of proteolytic PARP frag- down by this siRNA (Supplementary Fig. 3A and B). ELISA methods ment in the MTA1-siRNA treated cells, in which the pro-survival showed that MTA1 knockdown led to a significant increase of 2546 L. Yu et al. / FEBS Letters 587 (2013) 2542–2551

A B ) expression /18S

-MTA1 MTA1/ ( mRNA mRNA * -Actin MTA1

C D P < 0.05 nM) is bility

P > 0.05 Apoptosi % Cell viab Cell % (OD @ 405 * Ctrl siRNA + - + - MTA1 siRNA - + - + DMSO + + - - Time of DTX treatment (h) DTX - - + +

E DTX (5nM): 24 h

-MTA1MTA1

-pAkt

-Akt

-cleaved PARP

-Actin

Fig. 2. Silencing of MTA1 expression enhances the DTX-induced apoptosis in PC-3 cells. (A) When compared to the control group, the relative expression level of MTA1 mRNA after siRNA treatment was 0.2172 ± 0.0761, ⁄P < 0.05 as demonstrated by qRT-PCR analysis. (B) The knockdown efﬁciency was also conﬁrmed by Western blotting at the translational level. (C) 48 h later after siRNA treatment, cells were treated with DTX (5 nM) for different durations and cell viability was recorded using MTT assay. Results are expressed as mean ± S.D.; n = 3 independent experiments, ⁄P < 0.05. (D) At the end of 24-h DTX treatment, cells were harvested and an apoptosis ELISA kit (Roche Diagnostics, Mannheim, Germany) was used to quantitatively measure cytoplasmic histone-associated DNA fragments. Results are expressed as mean ± S.D. (E) At the end of 24-h DTX treatment, cells were harvested and immunoblotting analysis of a pro-proliferation signal (pAkt) and apoptotic indicator (cleaved PARP) were carried out using b-actin as a loading control.

apoptotic rate after cells had been incubated with DTX (5nM) for detected after DTX treatment in PC-3 cells overexpressing His- 24 h (Supplementary Fig. 3C). Collectively, these data indicate that tagged MTA1 (Fig. 3C). In the tumor xenograft study, we implanted endogenous MTA1 may protect PC-3 cells from chemotherapeutic subcutaneously cells overexpressing His-tagged MTA1 or His- insult. vector at the back of nude mice. Starting at the 4th week after cell inoculation, mice were treated with DTX (25 mg/kg B.W. every 3.3. Overexpression of MTA1 enhances DTX resistance in PC-3 cells day) administered intraperitoneally for another 4 weeks. We analyzed tumor size on a weekly basis for 8 weeks and the tumor To further assess whether enhanced MTA1 expression is weight on week 8 after tumor cell injections. Data showed a signif- causative of or a result of chemotherapy-resistance in PCa, we icant difference in tumor size from week 6 afterwards (⁄P < 0.05) overexpressed His-tagged MTA1 in PC-3 cells (Fig. 3A) and then (Fig. 3D). Consistently, significant changes in tumor weight were incubated these cells with 5 nM DTX for 24 h. DTX-induced apop- observed at week 8 (Fig. 3E). The higher proliferation rate and tosis was significantly attenuated in the cells overexpressing lower apoptotic status of prostate tumors derived from cells over- His-tagged MTA1 when compared to that in control cells expressing His-tagged MTA1 were confirmed using immunohisto- (Fig. 3B). Conversely, higher colony formation efficiency was chemical staining of Ki67 and immunoblotting analysis of cleaved L. Yu et al. / FEBS Letters 587 (2013) 2542–2551 2547

A B P < 0.05 @ 405 nM) @ 405 poptosis

-His Ap (OD @ -MTA1

vector + - + - -Actin His-MTA1 - + - + DMSO + + - - DTX - - + + C D ) 3

P < 0.05 (mm olonies

*** Number of co Tumor volume Tumor vector + - + - Weeks after cell inoculation His-MTA1 - + - + DMSO + + - - DTX - - + +

F His-vector (1) His-MTA1 (2) His-vector+DTX (3) His-MTA1+DTX (4) E

-His C -Actin Ki67 IHC

P < 0.05

cells P < 0.05005 ht (g) % of Ki67 of % 1 2 3 4 Tumor weigh Tumor

G -HisHis -cleaved caspase 3 -Actin 1 2 3 4

Fig. 3. Ectopic overexpression of MTA1 protects PC-3 cells from chemotherapeutic insult. (A) Establishment of PC-3 cells overexpressing MTA1 was confirmed by immunoblotting analysis using anti-His and anti-MTA1 antibodies. b-Actin was used as a loading control. (B) PC-3-His-MTA1 or PC-3-His-vector cells were treated with 5 nM DTX for 24 h and then were subjected to ELISA assay to evaluate the apoptotic status. Results are expressed as mean ± S.D. Each experiment was performed in triplicate (n =3) (⁄P < 0.05). (C) PC-3-His-MTA1 or PC-3-His-vector cells were cultured at 37 °C for 48 h. DTX (5 nM) was then added to cultures. After 3 days of treatment, the DTX-containing media was replaced with fresh media and all cultures were incubated for an additional 9 days until colonies were large enough to be clearly discerned. A colony formation assay was performed as described in Section 2. (D) In vivo effects of MTA1 overexpression on tumorigenicity and sensitivity to DTX was assessed using a murine xenograft model as described in Section 2. Tumor volume changes were recorded weekly by two perpendicular diameter measurements. Data are expressed as mean ± S.D. (⁄P < 0.05). (E) At the end of the 8-week xenograft experiments, representative images for tumor sizes from different groups were also collected and tumors were weighed. MTA1 overexpression in the xenograft experiments was verified by immunoblotting analysis using anti-His antibody. (F) At the end of 8-week xenograft experiments, tumor tissues were collected, and the expressions of Ki67 and cleaved caspase-3 were evaluated in different groups using immunohistochemistry and immunoblotting, respectively. Quantification of Ki67 staining was carried out as described in Section 2 (lower panel in Fig. 3F). Data are expressed as means ± S.D. ⁄P < 0.05. caspase-3, respectively (Fig. 3F–G). These data suggest that upreg- nificant reduction of NR4A1 expression in PC-3R cells with ele- ulation of MTA1 expression could influence the proliferation and vated levels of MTA1 (data not shown), raising the possibility clonogenicty in PC-3 cells upon cytotoxic stress. that MTA1 might be an upstream regulator of the NR4A1 signaling. To verify this, we treated cells overexpressing His-tagged MTA1 or 3.4. Augmentation of MTA1 expression by DTX treatment represses the His-vector with 5nM DTX. The transcriptional level of NR4A1 in expression of NR4A1 in PC-3 cells cells expressing His-vector was notably increased during the first 2 h of DTX incubation. In contrast, we did not find a significant in- In an effort to search for the potential mechanism underlying crease of NR4A1 expression in cells overexpressing His-tagged the role of MTA1 in chemoresistance in PC-3 cells, we found a sig- MTA1 during the study period (Fig. 4A). This expression profile 2548 L. Yu et al. / FEBS Letters 587 (2013) 2542–2551 was confirmed at the translational level by immunoblotting chromatin, and its gradual de-recruitment by increasing doses of (Fig. 4B). In line with this observation, the relative expression level TSA. MTA1 recruitment to the NR4A1 promoter at 4 h of DTX treat- of NR4A1 mRNA in cells depleted of endogenous MTA1 remained ment was more robust that at the 1 h of DTX treatment, well con- continuously high in response to DTX treatment (Supplementary sistent with the NR4A1 expression level in DTX treated PC-3 cells Fig. 2). MTA1 efficiently repressed the NR4A1 transcriptional activ- expressing His-vector (Fig. 4B and E). Collectively, our results indi- ities in DTX treated PC-3 cells as measured by promoter reporter cated that MTA1 represses NR4A1 expression in an HDAC-depen- assay (Fig. 4C). To confirm that the NR4A1 gene chromatin is a di- dent manner in DTX treated PC-3 cells (Fig. 4F). rect target of MTA1, we performed ChIP assays, using primers encompassing different fragments of the NR4A1 promoter region. 3.5. Ectopic overexpression of NR4A1 enhances chemosensitivity to MTA1 was recruited to the 318 to 31 bp region of the NR4A1 DTX in PC-3R cells promoter after 4 h of DTX treatment (Fig. 4D). Because MTA1 en- gages HDAC complexes to repress transcription, we performed a Previous study showed that inhibition of NR4A1 expression in- double ChIP in DTX treated PC-3 cells to further demonstrate the creases viability of PCa cells upon cytotoxic stress [17]. In our physical interaction of the MTA1/nucleosome remodeling and his- study, we overexpressed GFP-tagged NR4A1 and its control vector tone deacetylase complex with the NR4A1 chromatin. We found in PC-3R cells (Fig. 5A), and then incubated the cells with DTX simultaneous co-association of MTA1 and HDAC2 with the NR4A1 (20 nM) for 24 h. Annexin V/PI double staining and FACS analysis

A B

* Time of DTX (5 nM) treatment * 0 1 2 4 12 (h)

pression -His 8S) x x * -NR4A1 -MTA1 -Actin PC-3: pcDNA3.1-His -Actin

-NR4A1 mRNA e

(NR4A1/18 PC-3: pcDNA3.1-His-MTA1 -Actin NR4A1 0 1 2 4 12 Time of DTX (5 nM) treatment (h)

C * D * PC-3: DTX (5 nM) activity v vel) treatment, 4h P1 P2 P3 P1: -2521 to -2272 bp -Input promoter P2: -1591 to -1347 bp (Relative le

P3: -318 to -31 bp -IgG NR4A1

-IP DTX (5 nM): - + + + pcDNA3.1-His: - - + - pcDNA3.1-His-MTA1: - - - +

E F IP: Input IgG MTA1 HDAC2 + - + - + - P3

2nd IP: HDAC2 + 1st IP: IgG MTA1

- - - 20 40 TSA (ng/ml)

1 1 4 4 4 Time of DTX (5 nM) treatment (h)

Fig. 4. MTA1 regulates NR4A1 expression. PC-3 cells overexpressing exogenous MTA1 or transfected with control vector were subjected to DTX (5 nM) treatment and expression levels of NR4A1 were then evaluated using qRT-PCR (A) and Western blotting (B) at different time-points. (C) The NR4A1 promoter reporter construct together with pcDNA3.1-His-MTA1 or pcDNA3.1-His were cotransfected into PC-3 cells followed by luciferase assays. Data are presented from at least three independent experiments and expressed as means ± S.D. ⁄P < 0.05. (D) ChIP analysis showing recruitment of MTA1 onto a specific region of the NR4A1 promoter. (E) Double ChIP analysis of MTA1/HDAC2 complex on NR4A1 promoter in PC-3 cells. (F) A working model depicts that MTA1 is a negative regulator of NR4A1 transcription. L. Yu et al. / FEBS Letters 587 (2013) 2542–2551 2549 revealed that NR4A1 overexpression markedly increased the per- the nuclear (Fig. 1C) [8]. In line with the human tissue data, MTA1 centage of early apoptotic cells induced by DTX (Fig. 5B). Therefore, expression level was higher in PC-3 cells than that in LNCaP and it is possible that NR4A1 may be terminally responsible for the PEC cells. This is understandable, since androgen-sensitive LNCaP pathogenesis of chemoresistance caused by MTA1 upregulation cells are susceptible to apoptosis but androgen-insensitive PC-3 in PC-3 cells. cells are relatively resistant to chemotherapy [31]. MTA1 expression and transcriptional activities were even more robust in exper- 4. Discussion imentally produced chemoresistant PC-3R cells, further validating the potential involvement of MTA1 expression in the pathogenesis Upregulation of MTA1 has been associated with increased risk of PCa chemoresistance. of breast, liver, and more recently with highly progressive prostate Emerging evidence points to a frequent participation of deacet- cancer [9]. However, whereas deregulation of MTA1 may influence ylases in tumor chemoresistance. For example, loss of class III beta- prostate cancer progression, few studies have examined MTA1 pro- tubulin induced by histone deacetylation is associated with tein expression in prostate cancer tumor tissue [13–15]. Hofer and chemosensitivity to paclitaxel in malignant melanoma cells [32]. colleagues had addressed this important point, and they found that The interaction between HA/CD44-stimulated p300 (acetyltrans- metastatic PCa demonstrated significantly higher mean MTA1 pro- ferase) and resveratrol-activated deacetylase plays pivotal roles tein expression intensity compared with clinically localized PCa or in regulating the balance between multidrug resistance and sensi- benign prostate tissue [10]. Additionally, our results, showing that tivity in breast tumor cells [33]. More recently, upregulation of MTA1 upregulation is also involved in the differential response to SIRT1, a transcriptional coregulator belonging to the family of type DNA damage agents and is positively associated with chemoresis- III histone deacetylases, has been shown to play a key role in pro- tant PCa and higher Gleason score, can also explain why the evolu- moting chemoresistance in androgen-refractory PC-3 and DU145 tion of the disease could be favored by the elevation of MTA1 cells [17]. Although the mechanisms whereby MTA1 expression expression. Mechanistically, MTA1 functions as an essential tran- is up-regulated in chemoresistant PC-3 cells remain to be defined, scriptional coregulator. This may explain why most of the positive several regulators have been so far reported to determine the level signals of upregulated MTA1 expression were immunolocalized in of MTA1 expression. For example, E3 ubiquitin ligase COP1 regulates the stability and functions of MTA1 [34]. Emerging data suggest that E3 ubiquitin ligases are frequently overexpressed in human cancers, which correlates well with increased chemoresis- A tance and poor clinical prognosis [35]. Similarly, it was previously reported that miR-661 inhibits MTA1 expression and negatively regulates its function [36]. There is increasing evidence that drug-induced dysregulation of microRNA (miRNA) function may modulate the sensitivity of cancer cells to chemotherapeutic agents, and may be involved in the acquisition of cancer cell resis- -MTA1 tance to chemotherapy [37]. Thus, deregulation of E3 ubiquitin ligase or miRNA may at least in part explain the elevated expression -Anti-GFP of MTA1 in response to DTX treatment. MTA1 acts as a component of the NuRD complex and plays an

PC-3R essential role in chromatin remodeling and transcriptional regula- -Actin tion of genes [9]. It is therefore a logical hypothesis that this chromatin modifier may directly participate in PCa chemoresistance. Our results showed that siRNA mediated MTA1 knockdown in B PC-3 cells inhibited the ability to proliferate or to resist apoptosis, and thereafter resulted in significant cell death upon DTX treatment. In contrast, the overexpression of exogenous MTA1 sub- P <005<0.05 stantially promoted cell proliferation and protected cells from DTX-induced apoptosis. In a subcutaneous xenograft implantation mouse model to assess in vivo drug sensitivity, overexpression of MTA1 enhanced the chemoresistance of PCa cells and led to a significant increase of the tumor burden. These data support our emerging view that MTA1 probably serves as a key transcriptional regulator during the pathogenesis of chemoresistance in PC-3 cells.

of apoptosis Of note, the effect of MTA1 overexpression on PC-3 cell prolifera-

%o tion in the absence of DTX treatment appeared to be associated with the cell density initially seeded. When we used 400 cells (PC-3-His-vector or PC-3-His-MTA1)/60-mm plastic for the colony formation assay, we did not find a significant effect of overexpression of MTA1 on cell proliferation rate (Fig. 3C). However, we did find a significant increase in the colony formation rate in the absence of DTX treatment if we seeded cells at 1200 cells/60-mm plastic (Supplementary Fig. 4). Two possibilities may account for this observation. First, higher density cell culture may result in a hypoxic environment. Hypoxia induces MTA1 expression, and Fig. 5. Overexpression of exogenous NR4A1 increases the chemosensitivity of PC- MTA1 could enhance the stability and activity of hypoxia-inducible 3R cells. Overexpression of exogenous GFP-tagged NR4A1 in PC-3R cells was factor-1a (HIF-a), thus promoting tumor growth under hypoxic confirmed by immunoblotting for GFP. Expression of MTA1 was not affected (A). Cells were incubated with DTX (20 nM) for 24 h and the apoptotic status was then condition [38]. Moreover, the effect of MTA1 overexpression on determined using Annexin V/PI double staining followed by FACS analysis (B). the state of cell proliferation may be closely regulated by cellular 2550 L. Yu et al. / FEBS Letters 587 (2013) 2542–2551 interactions. In favor of the latter hypothesis, it has been reported elevation might confer a more aggressive clinical course for men that the hyaluronan-mediated motility receptor (HMMR) and E- with DTX treatment, and personalized interventions may be war- cadherin, two key components of extracellular matrix (ECM) mol- ranted. Moreover, since NR4A1 is a direct down-stream target of ecules regulating the motility, invasion, and metastatic potential of MTA1 and upregulation of MTA1/downregulation of NR4A1 is clo- cancer cells, are both tightly regulated by MTA1 [11,39]. The rele- sely associated with chemoresistance in PCa, testing patients for vance of the potential crosstalk between MTA1 overexpression and MTA1/NR4A1 expression ratios may provide more accurate prog- hypoxia/cellular contact in the regulation of cell proliferation in nostic information and could influence the recommended course the absence of genotoxic insult is yet to be further defined. of treatment. Many apoptosis-inducing drugs induce nuclear export of NR4A1 In summary, we showed here for the first time that MTA1 level and activate apoptosis in cancer cell lines [13]. It has been shown is significantly elevated in chemoresistant PCa samples, and that that antisense-mediated knockdown of NR4A1 increased LNCaP MTA1 promotes DTX resistance in PC-3 cells via its transcriptional cells growth in response to etoposide treatment, indicating a pro- regulation property. A major pathway in this response is the direct apoptotic effect of this NR in etoposide-induced PC-3 cell death control of NR4A1 transcription, which in turn governs the ability of [14]. However, the transcriptional or posttranscriptional mecha- MTA1 to reduce genotoxic stress-induced apoptosis in PC-3 cells. nisms responsible for controlling NR4A1 expression in response to DNA damage are poorly understood. Our findings extend these Acknowledgments understanding by identifying MTA1 as a potent corepressor of NR4A1 transcription [We confirmed the inhibitory effect of endog- To Hui Wang for her careful assistance during the preparation enous MTA1 on NR4A1 transcription in DTX-treated PC-3 cells by of our report, with thanks, and to the editor and reviewer’s excel- using another siRNA system from Sigma (Supplementary lent work and suggestions. This work was supported by the Natural Fig. 3D)]. Repression of NR4A1 transcription by MTA1-HDAC2 Science Foundation of China: 31271248. may represent a novel mechanism contributing to the pathogenesis of PCa chemoresistance. To be noted, we believe that HDAC2 Appendix A. Supplementary data indirectly binds to the NR4A1 promoter region via first binding to MTA1. There are two lines of evidence supporting this assumption: Supplementary data associated with this article can be found, in (1) In single step ChIP assay, the positive signals of HDAC2 were al- the online version, at http://dx.doi.org/10.1016/j.febslet.2013. ways weaker than those for MTA1; (2) In the response to DTX 06.029. treatment we could not obtain positive ChIP signals for HDAC2 in cells depleted of endogenous MTA1 (our unpublished data). Thus, References HDAC2 may exert its transcriptional regulation effect through interaction with MTA1 in a piggy-back manner (Fig. 4F). Previ- [1] Zhang, L., Yang, B.X., Zhang, H.T., Wang, J.G., Wang, H.L. and Zhao, X.J. 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