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NDRG2 acts as a negative regulator downstream of androgen receptor and inhibits the growth of androgen-dependent and castration-resistant prostate cancer

NDRG2 acts as a negative regulator downstream of androgen receptor and inhibits the growth of... RESEARCH PAPER Cancer Biology & Therapy 16:2, 287--296; February 2015; © 2015 Taylor & Francis Group, LLC NDRG2 acts as a negative regulator downstream of androgen receptor and inhibits the growth of androgen-dependent and castration-resistant prostate cancer 1,2,3,y 3,y 3,y 3 3 3 1 1 4 1 Chuigong Yu , Guojun Wu , Ruixiao Li , Lei Gao , Fan Yang , Yi Zhao , Jian Zhang , Rui Zhang , Jing Zhang , Libo Yao , 3, 1, * * Jianlin Yuan , and Xia Li 1 2 State Key Laboratory of Cancer Biology; Department of Biochemistry and Molecular Biology; the Fourth Military Medical University; Xi’an, China; Department of Urology; General 3 4 Hospital of Armed Police Forces; Beijing, China; Department of Urology; Xijing Hospital; The Fourth Military Medical University; Xi’an, China; Experiment Teaching Center; the Fourth Military Medical University; Xi’an, China These authors equally contributed to this work. Keywords: AR, c-Myc, castration resistance, lentivirus, NDRG2 Abbreviations: AR, Androgen Receptor; CRPC, Castration-Resistant Prostate Cancer; DHT, Dihydrotestosterone; NDRG2, N-Myc Downstream Regulated Gene 2; PC, Prostate Cancer. Castration resistance is a major issue during castration therapy for prostate cancer and thus more effective treatment are needed for castration-resistant prostate cancer (CRPC). NDRG2 (N-Myc downstream regulated gene 2), a recently identified tumor suppressor, was previously shown to inhibit the proliferation and invasion of prostate cancer, but whether NDRG2 is involved in CRPC remains to be known. Because androgen receptor (AR) axis plays an important role in castration resistance, we evaluate the role of NDRG2 in AR signaling and CRPC. Immunohistochemistry examination of prostate cancer tissues demonstrated that the expression of NDRG2 is negatively correlated with that of AR and c- Myc. Furthermore, AR negatively regulates NDRG2, as well as alters levels of c-Myc and prostate specific antigen (PSA). Forced expression of NDRG2 significantly inhibits the in vitro growth of androgen-dependent and castration-resistant prostate cancer cells; this was accompanied by alterations in PSA, but not by those of AR and c-Myc. Finally, by mimicking castration therapy in a xenograft mouse model, we showed that lentivirus-mediated NDRG2 overexpression efficiently overcomes castration resistance. Thus, by acting as a negative regulator downstream of AR, NDRG2 may emerge as a potential therapy molecule for CRPC. Introduction Androgen receptor (AR) is a ligand-dependent transcription factor that plays important roles in both normal prostate develop- Prostate cancer (PC) is the most frequently diagnosed cancer ment and prostate cancer. When activated by endogenous andro- among men. With the development of diagnostic techniques, the gen, testosterone and dihydrotestosterone (DHT), AR regulates 1,2 detection rate of PC has increased significantly in recent years. the growth, survival and differentiation of prostate cells by trans- If the cancer is detected early and is confined within the prostatic activation of a subset of target genes. Several mechanisms under- capsule, it can be cured by surgery or radiotherapy (including lying castration resistance have been documented, including (a) brachytherapy). But the prognosis is often poor if metastasis has AR mutations or amplification, (b) alterations in the balance already occurred at the time of diagnosis. Currently, the mainstay between AR and its transcriptional co-regulators and (c) growth of therapy for metastatic prostate cancer is castration, which can factors/kinases signal pathways that activate AR activity at post- 7-11 be accomplished by either orchectomy or androgen-antagonistic castration androgen levels. It is noteworthy that CRPC cells agents. However, although castration is initially effective and retain AR and AR-regulated gene expression in the absence of or prolongs the period free of disease progression, PC eventually with low levels of circulating androgens. In addition, tumors becomes castration resistant, resulting in disease relapse and less that are refractory to conventional androgen deprivation therapy 5 13,14 than 25 months of median survival. are susceptible to more potent androgen pathway inhibitors. *Correspondence to: Jianlin Yuan; Email: [email protected]; Xia Li; Email: [email protected] Submitted: 08/28/2014; Revised: 11/03/2014; Accepted: 12/18/2014 http://dx.doi.org/10.1080/15384047.2014.1002348 www.taylorandfrancis.com Cancer Biology & Therapy 287 These studies strongly indicate that AR signaling continues to suggest that there might be some regulatory relationship between play a significant role in CRPC. Thus, more comprehensive NDRG2, c-Myc and AR. understanding of the regulation of the AR axis and the develop- ment of novel AR signaling-targeting therapies is imperative in NDRG2 is regulated by AR and is involved in the AR this field. signaling pathway The human N-Myc downstream regulated gene 2 (NDRG2), To explore the involvement of NDRG2 in AR signaling and which was first cloned in our laboratory in 1999 (GenBank acces- AR-mediated castration resistance, LNCaP and 22Rv1 cell lines sion no.AF 159092), belongs to the NDRG gene family, con- were chosen in this study. LNCaP is an androgen-dependent sisting of NDRG1–4. The NDRGs share roughly 60% amino prostate cancer cell line that express mutant full-length AR; while acid identity and are highly conserved among species. Previous 22Rv1 is a model of CRPC that expresses full-length mutated studies from our group and others have demonstrated that AR (110KDa) and constitutively active AR splice variants 17 18 31–34 NDRG2 is involved in cell differentiation, proliferation, apo- (80KDa). We first asked whether NDRG2 can be regulated 19,20 21 ptosis and the stress response. NDRG2 in particular has by AR signaling. DHT or bicalutamide were applied to stimulate been proposed as a tumor suppressor because mounting evi- or inhibit AR signaling. As expected, cell growth curves, EdU dence demonstrates that NDRG2 is deregulated in various carci- proliferation assay and FCM analysis confirmed that DHT stim- 15 22 nomas, including glioblastoma, breast cancer, hepatocellular ulated, but bicalutamide inhibited, the cell growth of LNCaP 23 24 cancer and colorectal cancer, while upregulating NDRG2 and 22Rv1 (Fig. 2A and Supplementary Fig. 1). The levels of 21,25,26 could reverse the malignant phenotype of cancer cells. AR, c-Myc, NDRG2 and PSA were assessed by Western blotting. Our most recent studies showed that NDRG2 expression levels As shown in Figure 2B, DHT upregulated, while bicalutamide were lower in PC tissues than in their benign counterparts, and downregulated, the expression of AR compared with the corre- forced expression of NDRG2 inhibited the proliferation and sponding controls in both LNCaP and 22Rv1 cells. Moreover, 27,28 invasion of PC cells. Particularly, NDRG2 inhibited prolif- the immunofluorescence assay showed that DHT stimulation led eration of PC cell line PC3 by affecting several cell cycle regula- to an increase in AR nucleic localization, while bicalutamide tors (cyclin-D1, CDK4 and p21), supporting that NDRG2 exerted an opposite effect (Supplementary Fig. 2). As expected, plays a tumor suppressive role in PC. We previously confirmed the upregulation of AR expression and its nuclear localization that NDRG2 is repressed by c-Myc. Of note, it was also shown caused by DHT was accompanied by an increase in its known that c-Myc is a known downstream target of AR and confers downstream axis, c-Myc and PSA. Meanwhile, a decrease in 29,30 androgen-independent PC cell growth. These findings NDRG2 was observed in both cell lines upon AR activation by prompt us to question whether NDRG2, as a newly identified DHT (Fig. 2B). In contrast, bicalutamide-mediated downregula- c-Myc target gene, is also involved in AR-mediated castration tion of AR was accompanied by an increase in NDRG2 as well as resistance. the opposite changes in AR axis, c-Myc and PSA (Fig. 2B), with the alteration of secreted PSA being confirmed by the ELISA results (Fig. 2C). Taken together with the published evidences Results showing that c-Myc is a known downstream target of AR and 29,30 confers androgen-independent PC cell growth while The expression of NDRG2, c-Myc and AR are correlated NDRG2 can be repressed by c-Myc, these data suggest that in PC tissues NDRG2 may be regulated by AR through c-Myc and involved We previously showed that the expression level of NDRG2 is in the AR signaling pathway. negatively correlated with those of c-Myc in prostate cancer tis- sues, consistent with our previous finding that NDRG2 is Forced expression of NDRG2 inhibits downstream AR repressed by c-Myc in transcriptional level. Considering that c- signaling but not affects AR abundance and activity 29,30 Myc is a known target gene of AR, we hypothesized that Next, we asked whether NDRG2 affects AR and its down- NDRG2 may be involved in AR signaling. Therefore, we first stream signaling. To this end, we utilized lentivirus system to examined PC samples to see if there is any correlation among the overexpress NDRG2 in LNCaP and 22Rv1 and then examined expression of NDRG2, c-Myc and AR. For this purpose, the changes in AR’s expression and activity as well as its down- 107 PC tissue sections from PC patients were analyzed with stream axis. A high efficiency of lentivirus infection in LNCaP immunohistochemistry. The results showed that the rates of posi- and 22Rv1 cells was demonstrated by the fact that almost 100% tive NDRG2, c-Myc and AR expression in PC specimens were of Lenti-Cherry-infected cells express cherry proteins (Supple- 29.91% (32/107), 33.64% (36/107) and 71.96% (77/107), mentary Fig. 3). The Western blot results showed that Lenti- respectively. All three proteins were expressed both in the cyto- NDRG2-infected LNCaP and 22Rv1 cells express higher levels plasm and in the nuclei of PC cells. NDRG2 protein was mainly of NDRG2 protein than the controls (Fig. 3A). Of note, expressed in the cytoplasm, but the c-Myc and AR proteins were NDRG2 overexpression neither has any influence on the expres- expressed mainly in the nuclei (Fig. 1A). Moreover, the expres- sion levels of AR and c-Myc (Fig. 3A) nor changes the nuclear sion levels of NDRG2 were negatively correlated with those of localization pattern of AR (Fig. 3B) in both LNCaP and 22Rv1 c-Myc and those of AR (Fig. 1B), while the expression levels of cells. This indicates that NDRG2 does not affect the quantity c-Myc and AR were positively correlated (Fig. 1B). These data and nuclear translocation of AR. However, Western blot showed 288 Cancer Biology & Therapy Volume 16 Issue 2 that NDRG2 overexpression led to a downregulation of PSA expression in both LNCaP and 22Rv1 cells (Fig. 3A). This was further confirmed by ELISA, show- ing that the overexpression of NDRG2 decreased the levels of secreted PSA (Fig. 3C). Taken together, these results imply that NDRG2 can be repressed by AR/ c-Myc as a target of c-Myc, and importantly, NDRG2 negatively regulates AR downstream signaling pathway without affecting the quantity or activity of AR itself. Forced expression of NDRG2 inhibits growth of CRPC cell lines To further evaluate the role of NDRG2 in CRPC, we examined the effect of Lenti-NDRG2 on the malig- nant behavior as well as molecular changes in LNCaP and 22Rv1 cells. Androgen- independent growth and cas- tration resistance in LNCaP cells were also mimicked by IL-4 (50 ng/mL) treatment of these cells because IL-4 has been reported to sensitize the AR and stimulate cell growth through the activa- tion of pro-survival signaling pathways. As shown in Figure 4A, Lenti-NDRG2 infection sig- nificantly inhibits the growth of LNCaP cells when cul- tured either in routine medium or in the presence Figure 1. The expression levels of NDRG2 were negatively correlated with those of c-Myc and AR in prostate of IL-4 (Fig. 4A left), as well cancer tissues.(A) Immunohistochemical staining of the sections from human prostate cancer tissues with anti- NDRG2 (a, d), anti-AR (b, e) and anti-c-Myc (c, f) antibodies (Scale bar: 20 mm). The sections in a, b, c and those in as the growth of 22Rv1 cells d, e, f originated from the same paraffin blocks, respectively. Gleason score: a, b, c: 2 C 2 D 4; d, e, f: 3 C 4 D 7. (B) (Fig. 4A right). EdU staining Statistical analysis of the expression levels among NDRG2, c-Myc and AR. The number of specimens is shown using showed that Lenti-NDRG2 ‘scale’. Disparities across groups were analyzed using the Pearson correlation coefficient, and the graph was gener- significantly promotes prolif- ated using SPSS13.0 software (a: r D－0.252, P < 0.01; b: r D－0.277, P < 0.01; c: r D 0.610, P < 0.01). eration when compared with Lenti-LacZ (Fig. 4B). This is further confirmed by FCM analysis, showing that Lenti-NDRG2 NDRG2 infection can downregulate the expression of G1 phase infection leads to more G0/G1 accumulation (Supplementary regulators (cyclinD1, cyclinE, CDK2, CDK4) and upregulate Fig. 4). Consistently, Western blot demonstrated that Lenti- the G1 phase inhibitor p27 in both cell lines (Fig. 3A). In www.taylorandfrancis.com Cancer Biology & Therapy 289 addition, NDRG2 overexpression causes cell apoptosis relative to Lenti-LacZ (Fig. 4C). Together, these data indicated that NDRG2 overexpression retards cell growth of not only androgen-dependent prostate cancer but also CRPC by inhibit- ing cell proliferation and promoting cell apoptosis. NDRG2 retards the in vivo growth of CRPC Finally, we wanted to know whether ectopically overexpressed NDRG2 could inhibit the in vivo tumor growth of CRPC and thus potentially overcome castration resistance. To this end, we injected Lenti- LacZ- or Lenti-NDRG2-infected 22Rv1 cells into nude mice to establish a xenograft model. The mice were then given castration therapy by orchectomy, and tumor growth was monitored. Encouragingly, the mice transplanted with 22Rv1 cells developed xenograft outgrowth and tumor growth curves showed that castration alone had no effect on tumor growth (Lenti-LacZ C Cas- tration vs. Lenti-LacZ, F  0.658, P > 0.05) (Fig. 5A), reinforcing the castration resistance of 22Rv1 cells. However, Lenti- NDRG2 significantly suppressed the growth of 22Rv1-inoculated tumors follow- ing castration (Fig. 5A, B). As a result, the weights of the tumors in the Lenti-NDRG2 C Castration group were significantly ligh- ter than those of the Lenti-LacZ C Castra- tion group (Fig. 5B, C). The lower tumor/ body mass ratio in the Lenti-NDRG2 C Castration group indicated that the physical condition of the mice was much better than that of the mice in the other 2 groups (Fig. 5D). Taken together, forced overex- pression of NDRG2 can strikingly inhibit the in vivo tumor growth of CRPC, suggest- ing its potential usage as a novel therapeutic agent. Discussion Figure 2. AR activation or inhibition leads to alterations in AR, NDRG2, c-Myc and PSA in It has been well established that AR is a LNCaP and 22Rv1 cells. LNCaP and 22Rv1 cells were treated with vehicle controls (Ethanol or 7–11 key molecule for castration resistance. DMSO), DHT (10 nM), or bicalutamide (10 mM). (A) Cell growth curves were drawn based on the AR can promote ligand-independent pros- MTT assay (n D 6). (B) Western blot analysis of AR, c-Myc, NDRG2 and PSA protein levels with the corresponding antibodies. a-Tubulin protein levels were used as a loading control. Represen- tate cancer progression by directly regulat- tative blots are shown, and the relative expression levels of the proteins normalized to a-tubulin ing the transcription of c-Myc, which were derived from 4 independent experiments (n D 4). (C) ELISA analysis of PSA secretion. The 29 confers androgen independence. As a tar- histograms were drawn based on the average values (n D 3). In A, B, and D, data indicate the get gene of c-Myc, NDRG2 has been mean § SD *: P < 0.05; **: P < 0.01. widely accepted as a tumor suppressor in 290 Cancer Biology & Therapy Volume 16 Issue 2 many types of can- 15,21,25-27 cers, and our pre- vious study reported a negative correlation betw- een the expression of NDRG2 and that of c-Myc in prostate cancer. These findings prompted us to investigate whether NDRG2 is involved in AR signaling and plays an important role in CRPC. Initial immunohistochemi- cal staining of PC patient samples indicated that the expression of the NDRG2 protein was negatively cor- related with 2 molcules known to be intimately involved in CRPC, AR and c-Myc. Thus, we sought to determine the potential regulatory rela- tionship between them. We first examined whether NDRG2 is regu- lated by AR signaling. By utilizing an AR activator (DHT) and an AR inhibi- tor (bicalutamide), we con- firmed that the abundance and activity of AR, as well as PSA production, was oppositely regulated by DHT and bicalutamide, which is consistent with 36,37 the previous results. Accordingly, we demon- strated that the expression level of c-Myc was posi- tively regulated, while that of NDRG2 was negatively regulated, by alterations in AR. A previous study in Figure 3. Overexpression of NDRG2 causes downregulation of PSA but not affects AR quantity and activity in LNCaP rats showed that androgen and 22Rv1 cells. (A) Expression pattern of NDRG2 and the designated proteins upon NDRG2 overexpression. Cells infected with lentivirus encoding NDRG2 and LacZ were lysed, and Western blots were performed using the desig- deprivation by castration nated antibodies. a-Tubulin protein levels were used as a loading control. Representative blots are shown, and the plus adrenalectomy signifi- relative expression levels of the different proteins normalized to a-Tubulin were derived from 4 independent experi- cantly increased NDRG2 ments (n D 4). (B) Immunofluorescence detection of AR subcellular localization pattern with anti-AR antibody in expression, but DHT Lenti-NDRG2-infected and control cells (Scale bar: 20 mm). (C) ELISA analysis of PSA secretion in Lenti-NDRG2- decreased its expression in infected and control cells. The histograms were drawn based on the average values (n D 3). In A and C, data indicate the mean § SD *: P < 0.05; **: P < 0.01; ***: P < 0.001. the kidney cortical collect- ing duct. Together, these data clearly revealed that NDRG2 is a downstream molecule of NDRG2 and a downstream target of AR. However, we cannot AR. As for the mechanism, our results and current knowledge exclude the possibility that AR may regulate NDRG2 directly or favor the notion that c-Myc acts as an intermediate between AR independently of c-Myc, given that AR is a well-characterized and NDRG2, as c-Myc is a known upstream regulator of nuclear transcription factor. Additionally, we explored the www.taylorandfrancis.com Cancer Biology & Therapy 291 Figure 4. Lentivirus-mediated overexpression of NDRG2 inhibited the proliferation and induced apoptosis of LNCaP and 22Rv1 cells in vitro. LNCaP and 22Rv1 cells were infected with lentivirus encoding NDRG2 and LacZ and cultured in the presence or absence of IL-4 (50 ng/mL). (A) The growth curves were drawn based on the MTT assay (n D 6). (B) Cell proliferation was measured by EdU staining, with the average percentage of proliferative cells shown in the histograms (n D 3). (C) Cell apoptosis detection with FCM, with the average percentage of apoptotic cells shown on the histograms (n D 3). The EdU stain and FCM assays were performed 5 d after culture. Data indicate the mean § SD *: P < 0.05; **: P < 0.01. possibility that NDRG2 may affect AR/AR signaling. By modu- PSA is a widely accepted marker for the diagnosis and progno- lating NDRG2 with a lentivirus expression system, we found sis of prostate cancer. Our finding that NDRG2 attenuates the that overexpression of NDRG2 did not influence the expression AR-PSA axis strongly implies that overexpression of NDRG2 level and subcellular localization of AR, but could downregulate may reverse the malignant phenotype of prostate cancer. Indeed, the expression and production of PSA, a well-known downstream this has been confirmed by our in vitro data, showing that ectopi- target of AR. We thus assume that, as a target of AR/c-Myc, cally overexpressed NDRG2 inhibited the growth of both andro- NDRG2 plays an important inhibitory role in AR(/c-Myc)/ gen-dependent prostate cancer cell line LNCaP and CRPC cell NDRG2/PSA signaling pathway, but the exact mechanism still line 22Rv1. Importantly, we demonstrated that NDRG2 can awaits further investigation. also inhibit the IL-4-induced, androgen-independent growth of 292 Cancer Biology & Therapy Volume 16 Issue 2 Figure 5. Lentivirus-mediated overexpression of NDRG2 suppressed the in vivo growth of 22Rv1 xenografts following castration. Nude mice were injected subcutaneously with 22Rv1 cells stably expressing LacZ or NDRG2 and underwent castration therapy by orchectomy. (A) Tumor growth was monitored by assessing tumor volume (mm ) every week. (B) The tumor nodules were collected at the end of the experiment. (C) The tumor weight (g) was recorded. (D) Ratios of tumor weight/mouse weight were recorded. The curves and histograms were drawn based on the average values (n D 6). Data indicate the mean § SD *: P < 0.05; **: P < 0.01. LNCaP cells through G1 phase arrest and induction of apoptosis. NDRG2, by acting downstream of AR and inhibiting the AR sig- IL-4 has been shown to be significantly elevated and directly cor- nal pathway, is able to efficiently inhibit CRPC cell growth in related with elevated PSA in CRPC. By activating AR signaling vitro and in vivo. These studies not only offer new insight into 35,40 33 through Akt/NF-kB and CBP/p300, IL-4 can not only the mechanism of castration resistance in prostate cancer but also increase the sensitivity of AR-positive cancer cells to androgen provide a potential and powerful candidate for the treatment of CRPC in the future. but also promote androgen-independent growth. Thus, by act- ing as an intermediate negative regulator between AR and PSA, NDRG2 is able to exert a broad inhibitory role on androgen- dependent prostate cancer and CRPC. To pursue the potential use of NDRG2 as a novel therapeutic Materials and Methods candidate, we established 22Rv1 xenograft nude mouse models and then evaluated the in vivo effect of NDRG2 on castration Collection of clinical prostate cancer samples resistance. The results clearly demonstrated that lentivirus-medi- A total of 107 PC samples were collected between 2008 and ated overexpression of NDRG2 could inhibit the growth of 2010 from patients with prostatic tumors. The donor age range 22Rv1 xenografts following castration therapy, whereas single was 50–89 years, with a mean age of 65.7 § 8.9 y Before treat- orchectomy did not achieve any therapeutic effect. Thus, with ment, the tissues were gathered by transurethral resection or nee- improvements in delivery techniques, NDRG2 may serve as a dle biopsy in the Department of Urologic Surgery, Xijing new promising therapeutic agent or at least a hypurgia for castra- Hospital, FMMU (Xi’an, CHN). tion therapy in CRPC. As for whether NDRG2 can broadly The collection and use of human prostatic tissues were cooperate or synergize with other AR-signaling-targeted thera- approved by the Fourth Military Medical University medical pies, it still awaits systematic investigation in the future. ethics committee. All patients provided written informed con- In summary, integrating the PC patient tissue examination sent. Each tumor was scored for pathological stage based on the and in vitro and in vivo experiments, we demonstrate that Gleason system, which was introduced in 1984 by Gleason. All www.taylorandfrancis.com Cancer Biology & Therapy 293 of the samples had been reviewed and given a final diagnosis by 3 MTT assay different clinical pathologists. Cells were seeded in 96-well plates (2000 cells/well for LNCaP, 1000 cells/well for 22Rv1) in sextuplicate and cultured the medium with or without the indicated reagents for up to 6 d Immunohistochemistry detection The viable cells were determined using the 3-(4,5-dimethylthia- Immunohistochemistry staining was performed with a mouse zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and anti-human NDRG2 antibody (Santa Cruz Biotechnology, CA), reading the absorbance at 490 nm. Cell growth curves were rabbit anti-human c-Myc and rabbit anti-human AR antibodies drawn based on the average absorption values. (Cell Signaling, MA), using an immunohistochemistry kit (Bos- ter Company, Wuhan, CHN) according to the manufacturer’s EdU staining instructions. The intensity were quantified using a scoring system Cells were seeded in 96-well plates in triplicate and cultured in from 0 to 12 (0 D no signal, 1–4 D weak signal, 5–8 D interme- reagent-free medium or medium containing the indicated diate signal, and 9–12 D strong signal). Each sample was exam- reagents for 5 d EdU staining was performed according to the ined separately and scored by 2 pathologists. The photos were manufacturer’s instruction (Ribo Company, Guangzhou, CHN). collected through light microscopy and using SPOT Advanced Briefly, EdU was added into the culture media and the cells were Software (Olympus, Nagano, JPN). fixed and examined via fluorescence microscope. Six visual fields (400£) were chosen randomly to count and calculate the propor- Cell lines and reagents tion of proliferative cells, whose nuclei were stained red. LNCaP and 22Rv1 cells (from ATCC) were maintained in RPMI 1640 (Gibco BRL, NY, USA) containing 10% fetal Flow cytometry assays (FCM) bovine serum (Gibco BRL, NY, USA) at 37 C in a humidified Cells were plated in 6-well culture dishes (1 £ 10 cells/well) atmosphere with 5% CO . When reaching approximately 80% in triplicate and cultured as in EdU staining. Five days later, cells confluence, the cells were collected or split through were harvested, fixed and stained with 50 mg/ml propidium trypsinization. iodide in the presence of RNAseA (10 mg/ml) and Triton X-100 DHT (Sigma-Aldrich, MO, USA) was dissolved in ethanol (0.1%). The cell cycle distribution was measured with a flow (ready-to-use concentration: 10 nM). Bicalutamide (Sigma- cytometer (BD Company, NJ, USA). Data are presented as the Aldrich, MO, USA) was dissolved in DMSO (ready-to-use mean § SD. The experiments were repeated at least 3 times. concentration: 10 mM). IL-4 (Sigma-Aldrich, MO, USA) was dissolved in PBS (ready-to-use concentration: 50 ng/mL). All the Immunofluorescence assay for AR dissolved regents were stored at ¡20 C. Cells were seeded in 48-well plates (1 £ 10 cells per well) in triplicate and cultured as in EdU staining. After permeabilization Lentivirus generation and infection (0.5% Triton X-100), fixation (4% formaldehyde) and blocking Recombinant lentiviral vectors were constructed with the (10% normal goat serum and 0.5% Triton X-100), rabbit anti- TM ViraPower Lentiviral System (Invitrogen, NY, USA) as human AR monoclonal antibody (Cell Signaling Technology, described previously. Lenti-LacZ was used as a negative con- MA, USA) was added to the cells overnight at 4 C. Cells were trol, while Lenti-Cherry was used to optimize the infection effi- then stained with FITC-conjugated goat-anti-rabbit polyclonal ciency. For cell infection, LNCaP and 22Rv1 cells were seeded antibody at 37 C for 2 h in the dark. The fluorescence staining into 25 cm plastic culture flasks and grown to approximately intensity was then examined using immunofluorescence micros- 40% confluence, following which the lentivirus-containing copy and photographed. The experiments were repeated at least supernatants were added into the cell culture medium in the pres- 3 times. ence of 1 mg/ml Polybrene (Sigma-Aldrich, USA). Twenty-four hours after infection, the cells were selected with blasticidin PSA ELISA (LNCaP, 4 mg/mL; 22Rv1, 6 mg/mL) for approximately 3 Cells were seeded in 6-well plates (5 £ 10 cells per well) in weeks, so that the uninfected cells were eliminated entirely. triplicate and cultured as in EdU staining. The conditioned media in different groups was assayed for PSA levels using com- Western blot analysis mercial sandwich ELISA kits (Cusabio, Wuhan, CHN) accord- Western blotting was performed as described previously. ing to the manufacturer’s instructions. Each sample was tested in The primary antibodies are as following: mouse anti-human duplicate and quantified using an Emax precision microplate NDRG2 (1:500 dilution, Santa Cruz Biotechnology, CA, USA), reader (Molecular Devices Corp, CA, USA) at 450 nm. The lev- rabbit anti-human PSA, c-Myc and AR (1:1000 dilution, Cell els of PSA in different groups were normalized relative to the cor- Signaling Technology, MA, USA), rabbit anti-human cyclinD1, responding control group. Data are presented as the mean § SD. cyclinE, CDK2, CDK4, and p27 (1:1000 dilution, Santa Cruz The experiments were repeated at least 3 times. Biotechnology, CA, USA). Detection of a–tubulin served as a loading control. The images were scanned and quantified using Tumor growth assays in vivo Kodak Digital Science ID software (Kodak, NY, USA). The Male BALB/c nude mice were purchased from Shanghai experiments were repeated 4 times. Experimental Animal Center (Shanghai, CHN). All of the 294 Cancer Biology & Therapy Volume 16 Issue 2 protocols were approved by the Animal Care and Use Committee analyzed using the Pearson correlation coefficient. Analysis of at the Fourth Military Medical University and were performed in variance (ANOVA) and Student-Newman-Keuls (SNK) tests accordance with the animal care rules set forth by the university were computed to determine whether there were differences (Permit number 10001). LNCaP and 22Rv1 cells that were among the different groups of in vitro and in vivo assays. A P infected with Lenti-LacZ or Lenti-NDRG2 were harvested, value of less than 0.05 was considered statistically significant, and resuspended in sterile PBS and injected subcutaneously into the histograms were prepared using Origin 6.0 (Microcal Software, left flanks of the 6-week-old nude mice (1 £ 10 cells/mouse; 6 Inc., Northampton, MA). mice/group). In the groups of Lenti-LacZ C castration and Lenti-NDRG2 C castration, the mice were given orchectomy surgery to mimic castration therapy just after cell injection. The Disclosure of Potential Conflicts of Interest mice were monitored every day; tumor volumes were calculated every week based on caliper measurements of the length and No potential conflicts of interest were disclosed. width of the lesions using the formula: V (mm ) D length £ width /2. After 10 weeks, the mice were sacrificed by cervical dis- location under anesthesia (130 mg ketamine/8.8 mg xylazine/kg Funding body weight), and tumor specimens were taken, photographed, measured and weighed. Data are presented as the mean § SD. This study was supported by grants from the Key Project of Chinese National Programs for Fundamental Research and Statistical analysis Development (Project No. 2009CB521704) and the National Statistical analyses were performed using SPSS 13.0 software. 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Regulation NDRG2 upregulation inhibits the invasion of human ligand-independent prostate cancer progression of heat shock protein 70-1 expression by androgen hepatocellular carcinoma via suppressing ERK1/2 sig- through c-Myc upregulation. PloS One 2013; 8: receptor and its signaling in human prostate cancer naling pathway. PloS One 2013; 8:e61079; e63563; PMID:23704919; http://dx.doi.org/10.1371/ cells. Int J Oncol 2010; 36:459-67; PMID:20043082; PMID:23630579; http://dx.doi.org/10.1371/journal. journal.pone.0063563 http://dx.doi.org/10.3892/ijo_00000538 pone.0061079 31. Dozmorov MG, Hurst RE, Culkin DJ, Kropp BP, 37. Lu S, Wang A, Lu S, Dong Z. A novel synthetic com- Frank MB, Osban J, Penning TM, Lin HK. Unique pound that interrupts androgen receptor signaling in 296 Cancer Biology & Therapy Volume 16 Issue 2 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

NDRG2 acts as a negative regulator downstream of androgen receptor and inhibits the growth of androgen-dependent and castration-resistant prostate cancer

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Abstract

RESEARCH PAPER Cancer Biology & Therapy 16:2, 287--296; February 2015; © 2015 Taylor & Francis Group, LLC NDRG2 acts as a negative regulator downstream of androgen receptor and inhibits the growth of androgen-dependent and castration-resistant prostate cancer 1,2,3,y 3,y 3,y 3 3 3 1 1 4 1 Chuigong Yu , Guojun Wu , Ruixiao Li , Lei Gao , Fan Yang , Yi Zhao , Jian Zhang , Rui Zhang , Jing Zhang , Libo Yao , 3, 1, * * Jianlin Yuan , and Xia Li 1 2 State Key Laboratory of Cancer Biology; Department of Biochemistry and Molecular Biology; the Fourth Military Medical University; Xi’an, China; Department of Urology; General 3 4 Hospital of Armed Police Forces; Beijing, China; Department of Urology; Xijing Hospital; The Fourth Military Medical University; Xi’an, China; Experiment Teaching Center; the Fourth Military Medical University; Xi’an, China These authors equally contributed to this work. Keywords: AR, c-Myc, castration resistance, lentivirus, NDRG2 Abbreviations: AR, Androgen Receptor; CRPC, Castration-Resistant Prostate Cancer; DHT, Dihydrotestosterone; NDRG2, N-Myc Downstream Regulated Gene 2; PC, Prostate Cancer. Castration resistance is a major issue during castration therapy for prostate cancer and thus more effective treatment are needed for castration-resistant prostate cancer (CRPC). NDRG2 (N-Myc downstream regulated gene 2), a recently identified tumor suppressor, was previously shown to inhibit the proliferation and invasion of prostate cancer, but whether NDRG2 is involved in CRPC remains to be known. Because androgen receptor (AR) axis plays an important role in castration resistance, we evaluate the role of NDRG2 in AR signaling and CRPC. Immunohistochemistry examination of prostate cancer tissues demonstrated that the expression of NDRG2 is negatively correlated with that of AR and c- Myc. Furthermore, AR negatively regulates NDRG2, as well as alters levels of c-Myc and prostate specific antigen (PSA). Forced expression of NDRG2 significantly inhibits the in vitro growth of androgen-dependent and castration-resistant prostate cancer cells; this was accompanied by alterations in PSA, but not by those of AR and c-Myc. Finally, by mimicking castration therapy in a xenograft mouse model, we showed that lentivirus-mediated NDRG2 overexpression efficiently overcomes castration resistance. Thus, by acting as a negative regulator downstream of AR, NDRG2 may emerge as a potential therapy molecule for CRPC. Introduction Androgen receptor (AR) is a ligand-dependent transcription factor that plays important roles in both normal prostate develop- Prostate cancer (PC) is the most frequently diagnosed cancer ment and prostate cancer. When activated by endogenous andro- among men. With the development of diagnostic techniques, the gen, testosterone and dihydrotestosterone (DHT), AR regulates 1,2 detection rate of PC has increased significantly in recent years. the growth, survival and differentiation of prostate cells by trans- If the cancer is detected early and is confined within the prostatic activation of a subset of target genes. Several mechanisms under- capsule, it can be cured by surgery or radiotherapy (including lying castration resistance have been documented, including (a) brachytherapy). But the prognosis is often poor if metastasis has AR mutations or amplification, (b) alterations in the balance already occurred at the time of diagnosis. Currently, the mainstay between AR and its transcriptional co-regulators and (c) growth of therapy for metastatic prostate cancer is castration, which can factors/kinases signal pathways that activate AR activity at post- 7-11 be accomplished by either orchectomy or androgen-antagonistic castration androgen levels. It is noteworthy that CRPC cells agents. However, although castration is initially effective and retain AR and AR-regulated gene expression in the absence of or prolongs the period free of disease progression, PC eventually with low levels of circulating androgens. In addition, tumors becomes castration resistant, resulting in disease relapse and less that are refractory to conventional androgen deprivation therapy 5 13,14 than 25 months of median survival. are susceptible to more potent androgen pathway inhibitors. *Correspondence to: Jianlin Yuan; Email: [email protected]; Xia Li; Email: [email protected] Submitted: 08/28/2014; Revised: 11/03/2014; Accepted: 12/18/2014 http://dx.doi.org/10.1080/15384047.2014.1002348 www.taylorandfrancis.com Cancer Biology & Therapy 287 These studies strongly indicate that AR signaling continues to suggest that there might be some regulatory relationship between play a significant role in CRPC. Thus, more comprehensive NDRG2, c-Myc and AR. understanding of the regulation of the AR axis and the develop- ment of novel AR signaling-targeting therapies is imperative in NDRG2 is regulated by AR and is involved in the AR this field. signaling pathway The human N-Myc downstream regulated gene 2 (NDRG2), To explore the involvement of NDRG2 in AR signaling and which was first cloned in our laboratory in 1999 (GenBank acces- AR-mediated castration resistance, LNCaP and 22Rv1 cell lines sion no.AF 159092), belongs to the NDRG gene family, con- were chosen in this study. LNCaP is an androgen-dependent sisting of NDRG1–4. The NDRGs share roughly 60% amino prostate cancer cell line that express mutant full-length AR; while acid identity and are highly conserved among species. Previous 22Rv1 is a model of CRPC that expresses full-length mutated studies from our group and others have demonstrated that AR (110KDa) and constitutively active AR splice variants 17 18 31–34 NDRG2 is involved in cell differentiation, proliferation, apo- (80KDa). We first asked whether NDRG2 can be regulated 19,20 21 ptosis and the stress response. NDRG2 in particular has by AR signaling. DHT or bicalutamide were applied to stimulate been proposed as a tumor suppressor because mounting evi- or inhibit AR signaling. As expected, cell growth curves, EdU dence demonstrates that NDRG2 is deregulated in various carci- proliferation assay and FCM analysis confirmed that DHT stim- 15 22 nomas, including glioblastoma, breast cancer, hepatocellular ulated, but bicalutamide inhibited, the cell growth of LNCaP 23 24 cancer and colorectal cancer, while upregulating NDRG2 and 22Rv1 (Fig. 2A and Supplementary Fig. 1). The levels of 21,25,26 could reverse the malignant phenotype of cancer cells. AR, c-Myc, NDRG2 and PSA were assessed by Western blotting. Our most recent studies showed that NDRG2 expression levels As shown in Figure 2B, DHT upregulated, while bicalutamide were lower in PC tissues than in their benign counterparts, and downregulated, the expression of AR compared with the corre- forced expression of NDRG2 inhibited the proliferation and sponding controls in both LNCaP and 22Rv1 cells. Moreover, 27,28 invasion of PC cells. Particularly, NDRG2 inhibited prolif- the immunofluorescence assay showed that DHT stimulation led eration of PC cell line PC3 by affecting several cell cycle regula- to an increase in AR nucleic localization, while bicalutamide tors (cyclin-D1, CDK4 and p21), supporting that NDRG2 exerted an opposite effect (Supplementary Fig. 2). As expected, plays a tumor suppressive role in PC. We previously confirmed the upregulation of AR expression and its nuclear localization that NDRG2 is repressed by c-Myc. Of note, it was also shown caused by DHT was accompanied by an increase in its known that c-Myc is a known downstream target of AR and confers downstream axis, c-Myc and PSA. Meanwhile, a decrease in 29,30 androgen-independent PC cell growth. These findings NDRG2 was observed in both cell lines upon AR activation by prompt us to question whether NDRG2, as a newly identified DHT (Fig. 2B). In contrast, bicalutamide-mediated downregula- c-Myc target gene, is also involved in AR-mediated castration tion of AR was accompanied by an increase in NDRG2 as well as resistance. the opposite changes in AR axis, c-Myc and PSA (Fig. 2B), with the alteration of secreted PSA being confirmed by the ELISA results (Fig. 2C). Taken together with the published evidences Results showing that c-Myc is a known downstream target of AR and 29,30 confers androgen-independent PC cell growth while The expression of NDRG2, c-Myc and AR are correlated NDRG2 can be repressed by c-Myc, these data suggest that in PC tissues NDRG2 may be regulated by AR through c-Myc and involved We previously showed that the expression level of NDRG2 is in the AR signaling pathway. negatively correlated with those of c-Myc in prostate cancer tis- sues, consistent with our previous finding that NDRG2 is Forced expression of NDRG2 inhibits downstream AR repressed by c-Myc in transcriptional level. Considering that c- signaling but not affects AR abundance and activity 29,30 Myc is a known target gene of AR, we hypothesized that Next, we asked whether NDRG2 affects AR and its down- NDRG2 may be involved in AR signaling. Therefore, we first stream signaling. To this end, we utilized lentivirus system to examined PC samples to see if there is any correlation among the overexpress NDRG2 in LNCaP and 22Rv1 and then examined expression of NDRG2, c-Myc and AR. For this purpose, the changes in AR’s expression and activity as well as its down- 107 PC tissue sections from PC patients were analyzed with stream axis. A high efficiency of lentivirus infection in LNCaP immunohistochemistry. The results showed that the rates of posi- and 22Rv1 cells was demonstrated by the fact that almost 100% tive NDRG2, c-Myc and AR expression in PC specimens were of Lenti-Cherry-infected cells express cherry proteins (Supple- 29.91% (32/107), 33.64% (36/107) and 71.96% (77/107), mentary Fig. 3). The Western blot results showed that Lenti- respectively. All three proteins were expressed both in the cyto- NDRG2-infected LNCaP and 22Rv1 cells express higher levels plasm and in the nuclei of PC cells. NDRG2 protein was mainly of NDRG2 protein than the controls (Fig. 3A). Of note, expressed in the cytoplasm, but the c-Myc and AR proteins were NDRG2 overexpression neither has any influence on the expres- expressed mainly in the nuclei (Fig. 1A). Moreover, the expres- sion levels of AR and c-Myc (Fig. 3A) nor changes the nuclear sion levels of NDRG2 were negatively correlated with those of localization pattern of AR (Fig. 3B) in both LNCaP and 22Rv1 c-Myc and those of AR (Fig. 1B), while the expression levels of cells. This indicates that NDRG2 does not affect the quantity c-Myc and AR were positively correlated (Fig. 1B). These data and nuclear translocation of AR. However, Western blot showed 288 Cancer Biology & Therapy Volume 16 Issue 2 that NDRG2 overexpression led to a downregulation of PSA expression in both LNCaP and 22Rv1 cells (Fig. 3A). This was further confirmed by ELISA, show- ing that the overexpression of NDRG2 decreased the levels of secreted PSA (Fig. 3C). Taken together, these results imply that NDRG2 can be repressed by AR/ c-Myc as a target of c-Myc, and importantly, NDRG2 negatively regulates AR downstream signaling pathway without affecting the quantity or activity of AR itself. Forced expression of NDRG2 inhibits growth of CRPC cell lines To further evaluate the role of NDRG2 in CRPC, we examined the effect of Lenti-NDRG2 on the malig- nant behavior as well as molecular changes in LNCaP and 22Rv1 cells. Androgen- independent growth and cas- tration resistance in LNCaP cells were also mimicked by IL-4 (50 ng/mL) treatment of these cells because IL-4 has been reported to sensitize the AR and stimulate cell growth through the activa- tion of pro-survival signaling pathways. As shown in Figure 4A, Lenti-NDRG2 infection sig- nificantly inhibits the growth of LNCaP cells when cul- tured either in routine medium or in the presence Figure 1. The expression levels of NDRG2 were negatively correlated with those of c-Myc and AR in prostate of IL-4 (Fig. 4A left), as well cancer tissues.(A) Immunohistochemical staining of the sections from human prostate cancer tissues with anti- NDRG2 (a, d), anti-AR (b, e) and anti-c-Myc (c, f) antibodies (Scale bar: 20 mm). The sections in a, b, c and those in as the growth of 22Rv1 cells d, e, f originated from the same paraffin blocks, respectively. Gleason score: a, b, c: 2 C 2 D 4; d, e, f: 3 C 4 D 7. (B) (Fig. 4A right). EdU staining Statistical analysis of the expression levels among NDRG2, c-Myc and AR. The number of specimens is shown using showed that Lenti-NDRG2 ‘scale’. Disparities across groups were analyzed using the Pearson correlation coefficient, and the graph was gener- significantly promotes prolif- ated using SPSS13.0 software (a: r D－0.252, P < 0.01; b: r D－0.277, P < 0.01; c: r D 0.610, P < 0.01). eration when compared with Lenti-LacZ (Fig. 4B). This is further confirmed by FCM analysis, showing that Lenti-NDRG2 NDRG2 infection can downregulate the expression of G1 phase infection leads to more G0/G1 accumulation (Supplementary regulators (cyclinD1, cyclinE, CDK2, CDK4) and upregulate Fig. 4). Consistently, Western blot demonstrated that Lenti- the G1 phase inhibitor p27 in both cell lines (Fig. 3A). In www.taylorandfrancis.com Cancer Biology & Therapy 289 addition, NDRG2 overexpression causes cell apoptosis relative to Lenti-LacZ (Fig. 4C). Together, these data indicated that NDRG2 overexpression retards cell growth of not only androgen-dependent prostate cancer but also CRPC by inhibit- ing cell proliferation and promoting cell apoptosis. NDRG2 retards the in vivo growth of CRPC Finally, we wanted to know whether ectopically overexpressed NDRG2 could inhibit the in vivo tumor growth of CRPC and thus potentially overcome castration resistance. To this end, we injected Lenti- LacZ- or Lenti-NDRG2-infected 22Rv1 cells into nude mice to establish a xenograft model. The mice were then given castration therapy by orchectomy, and tumor growth was monitored. Encouragingly, the mice transplanted with 22Rv1 cells developed xenograft outgrowth and tumor growth curves showed that castration alone had no effect on tumor growth (Lenti-LacZ C Cas- tration vs. Lenti-LacZ, F  0.658, P > 0.05) (Fig. 5A), reinforcing the castration resistance of 22Rv1 cells. However, Lenti- NDRG2 significantly suppressed the growth of 22Rv1-inoculated tumors follow- ing castration (Fig. 5A, B). As a result, the weights of the tumors in the Lenti-NDRG2 C Castration group were significantly ligh- ter than those of the Lenti-LacZ C Castra- tion group (Fig. 5B, C). The lower tumor/ body mass ratio in the Lenti-NDRG2 C Castration group indicated that the physical condition of the mice was much better than that of the mice in the other 2 groups (Fig. 5D). Taken together, forced overex- pression of NDRG2 can strikingly inhibit the in vivo tumor growth of CRPC, suggest- ing its potential usage as a novel therapeutic agent. Discussion Figure 2. AR activation or inhibition leads to alterations in AR, NDRG2, c-Myc and PSA in It has been well established that AR is a LNCaP and 22Rv1 cells. LNCaP and 22Rv1 cells were treated with vehicle controls (Ethanol or 7–11 key molecule for castration resistance. DMSO), DHT (10 nM), or bicalutamide (10 mM). (A) Cell growth curves were drawn based on the AR can promote ligand-independent pros- MTT assay (n D 6). (B) Western blot analysis of AR, c-Myc, NDRG2 and PSA protein levels with the corresponding antibodies. a-Tubulin protein levels were used as a loading control. Represen- tate cancer progression by directly regulat- tative blots are shown, and the relative expression levels of the proteins normalized to a-tubulin ing the transcription of c-Myc, which were derived from 4 independent experiments (n D 4). (C) ELISA analysis of PSA secretion. The 29 confers androgen independence. As a tar- histograms were drawn based on the average values (n D 3). In A, B, and D, data indicate the get gene of c-Myc, NDRG2 has been mean § SD *: P < 0.05; **: P < 0.01. widely accepted as a tumor suppressor in 290 Cancer Biology & Therapy Volume 16 Issue 2 many types of can- 15,21,25-27 cers, and our pre- vious study reported a negative correlation betw- een the expression of NDRG2 and that of c-Myc in prostate cancer. These findings prompted us to investigate whether NDRG2 is involved in AR signaling and plays an important role in CRPC. Initial immunohistochemi- cal staining of PC patient samples indicated that the expression of the NDRG2 protein was negatively cor- related with 2 molcules known to be intimately involved in CRPC, AR and c-Myc. Thus, we sought to determine the potential regulatory rela- tionship between them. We first examined whether NDRG2 is regu- lated by AR signaling. By utilizing an AR activator (DHT) and an AR inhibi- tor (bicalutamide), we con- firmed that the abundance and activity of AR, as well as PSA production, was oppositely regulated by DHT and bicalutamide, which is consistent with 36,37 the previous results. Accordingly, we demon- strated that the expression level of c-Myc was posi- tively regulated, while that of NDRG2 was negatively regulated, by alterations in AR. A previous study in Figure 3. Overexpression of NDRG2 causes downregulation of PSA but not affects AR quantity and activity in LNCaP rats showed that androgen and 22Rv1 cells. (A) Expression pattern of NDRG2 and the designated proteins upon NDRG2 overexpression. Cells infected with lentivirus encoding NDRG2 and LacZ were lysed, and Western blots were performed using the desig- deprivation by castration nated antibodies. a-Tubulin protein levels were used as a loading control. Representative blots are shown, and the plus adrenalectomy signifi- relative expression levels of the different proteins normalized to a-Tubulin were derived from 4 independent experi- cantly increased NDRG2 ments (n D 4). (B) Immunofluorescence detection of AR subcellular localization pattern with anti-AR antibody in expression, but DHT Lenti-NDRG2-infected and control cells (Scale bar: 20 mm). (C) ELISA analysis of PSA secretion in Lenti-NDRG2- decreased its expression in infected and control cells. The histograms were drawn based on the average values (n D 3). In A and C, data indicate the mean § SD *: P < 0.05; **: P < 0.01; ***: P < 0.001. the kidney cortical collect- ing duct. Together, these data clearly revealed that NDRG2 is a downstream molecule of NDRG2 and a downstream target of AR. However, we cannot AR. As for the mechanism, our results and current knowledge exclude the possibility that AR may regulate NDRG2 directly or favor the notion that c-Myc acts as an intermediate between AR independently of c-Myc, given that AR is a well-characterized and NDRG2, as c-Myc is a known upstream regulator of nuclear transcription factor. Additionally, we explored the www.taylorandfrancis.com Cancer Biology & Therapy 291 Figure 4. Lentivirus-mediated overexpression of NDRG2 inhibited the proliferation and induced apoptosis of LNCaP and 22Rv1 cells in vitro. LNCaP and 22Rv1 cells were infected with lentivirus encoding NDRG2 and LacZ and cultured in the presence or absence of IL-4 (50 ng/mL). (A) The growth curves were drawn based on the MTT assay (n D 6). (B) Cell proliferation was measured by EdU staining, with the average percentage of proliferative cells shown in the histograms (n D 3). (C) Cell apoptosis detection with FCM, with the average percentage of apoptotic cells shown on the histograms (n D 3). The EdU stain and FCM assays were performed 5 d after culture. Data indicate the mean § SD *: P < 0.05; **: P < 0.01. possibility that NDRG2 may affect AR/AR signaling. By modu- PSA is a widely accepted marker for the diagnosis and progno- lating NDRG2 with a lentivirus expression system, we found sis of prostate cancer. Our finding that NDRG2 attenuates the that overexpression of NDRG2 did not influence the expression AR-PSA axis strongly implies that overexpression of NDRG2 level and subcellular localization of AR, but could downregulate may reverse the malignant phenotype of prostate cancer. Indeed, the expression and production of PSA, a well-known downstream this has been confirmed by our in vitro data, showing that ectopi- target of AR. We thus assume that, as a target of AR/c-Myc, cally overexpressed NDRG2 inhibited the growth of both andro- NDRG2 plays an important inhibitory role in AR(/c-Myc)/ gen-dependent prostate cancer cell line LNCaP and CRPC cell NDRG2/PSA signaling pathway, but the exact mechanism still line 22Rv1. Importantly, we demonstrated that NDRG2 can awaits further investigation. also inhibit the IL-4-induced, androgen-independent growth of 292 Cancer Biology & Therapy Volume 16 Issue 2 Figure 5. Lentivirus-mediated overexpression of NDRG2 suppressed the in vivo growth of 22Rv1 xenografts following castration. Nude mice were injected subcutaneously with 22Rv1 cells stably expressing LacZ or NDRG2 and underwent castration therapy by orchectomy. (A) Tumor growth was monitored by assessing tumor volume (mm ) every week. (B) The tumor nodules were collected at the end of the experiment. (C) The tumor weight (g) was recorded. (D) Ratios of tumor weight/mouse weight were recorded. The curves and histograms were drawn based on the average values (n D 6). Data indicate the mean § SD *: P < 0.05; **: P < 0.01. LNCaP cells through G1 phase arrest and induction of apoptosis. NDRG2, by acting downstream of AR and inhibiting the AR sig- IL-4 has been shown to be significantly elevated and directly cor- nal pathway, is able to efficiently inhibit CRPC cell growth in related with elevated PSA in CRPC. By activating AR signaling vitro and in vivo. These studies not only offer new insight into 35,40 33 through Akt/NF-kB and CBP/p300, IL-4 can not only the mechanism of castration resistance in prostate cancer but also increase the sensitivity of AR-positive cancer cells to androgen provide a potential and powerful candidate for the treatment of CRPC in the future. but also promote androgen-independent growth. Thus, by act- ing as an intermediate negative regulator between AR and PSA, NDRG2 is able to exert a broad inhibitory role on androgen- dependent prostate cancer and CRPC. To pursue the potential use of NDRG2 as a novel therapeutic Materials and Methods candidate, we established 22Rv1 xenograft nude mouse models and then evaluated the in vivo effect of NDRG2 on castration Collection of clinical prostate cancer samples resistance. The results clearly demonstrated that lentivirus-medi- A total of 107 PC samples were collected between 2008 and ated overexpression of NDRG2 could inhibit the growth of 2010 from patients with prostatic tumors. The donor age range 22Rv1 xenografts following castration therapy, whereas single was 50–89 years, with a mean age of 65.7 § 8.9 y Before treat- orchectomy did not achieve any therapeutic effect. Thus, with ment, the tissues were gathered by transurethral resection or nee- improvements in delivery techniques, NDRG2 may serve as a dle biopsy in the Department of Urologic Surgery, Xijing new promising therapeutic agent or at least a hypurgia for castra- Hospital, FMMU (Xi’an, CHN). tion therapy in CRPC. As for whether NDRG2 can broadly The collection and use of human prostatic tissues were cooperate or synergize with other AR-signaling-targeted thera- approved by the Fourth Military Medical University medical pies, it still awaits systematic investigation in the future. ethics committee. All patients provided written informed con- In summary, integrating the PC patient tissue examination sent. Each tumor was scored for pathological stage based on the and in vitro and in vivo experiments, we demonstrate that Gleason system, which was introduced in 1984 by Gleason. All www.taylorandfrancis.com Cancer Biology & Therapy 293 of the samples had been reviewed and given a final diagnosis by 3 MTT assay different clinical pathologists. Cells were seeded in 96-well plates (2000 cells/well for LNCaP, 1000 cells/well for 22Rv1) in sextuplicate and cultured the medium with or without the indicated reagents for up to 6 d Immunohistochemistry detection The viable cells were determined using the 3-(4,5-dimethylthia- Immunohistochemistry staining was performed with a mouse zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and anti-human NDRG2 antibody (Santa Cruz Biotechnology, CA), reading the absorbance at 490 nm. Cell growth curves were rabbit anti-human c-Myc and rabbit anti-human AR antibodies drawn based on the average absorption values. (Cell Signaling, MA), using an immunohistochemistry kit (Bos- ter Company, Wuhan, CHN) according to the manufacturer’s EdU staining instructions. The intensity were quantified using a scoring system Cells were seeded in 96-well plates in triplicate and cultured in from 0 to 12 (0 D no signal, 1–4 D weak signal, 5–8 D interme- reagent-free medium or medium containing the indicated diate signal, and 9–12 D strong signal). Each sample was exam- reagents for 5 d EdU staining was performed according to the ined separately and scored by 2 pathologists. The photos were manufacturer’s instruction (Ribo Company, Guangzhou, CHN). collected through light microscopy and using SPOT Advanced Briefly, EdU was added into the culture media and the cells were Software (Olympus, Nagano, JPN). fixed and examined via fluorescence microscope. Six visual fields (400£) were chosen randomly to count and calculate the propor- Cell lines and reagents tion of proliferative cells, whose nuclei were stained red. LNCaP and 22Rv1 cells (from ATCC) were maintained in RPMI 1640 (Gibco BRL, NY, USA) containing 10% fetal Flow cytometry assays (FCM) bovine serum (Gibco BRL, NY, USA) at 37 C in a humidified Cells were plated in 6-well culture dishes (1 £ 10 cells/well) atmosphere with 5% CO . When reaching approximately 80% in triplicate and cultured as in EdU staining. Five days later, cells confluence, the cells were collected or split through were harvested, fixed and stained with 50 mg/ml propidium trypsinization. iodide in the presence of RNAseA (10 mg/ml) and Triton X-100 DHT (Sigma-Aldrich, MO, USA) was dissolved in ethanol (0.1%). The cell cycle distribution was measured with a flow (ready-to-use concentration: 10 nM). Bicalutamide (Sigma- cytometer (BD Company, NJ, USA). Data are presented as the Aldrich, MO, USA) was dissolved in DMSO (ready-to-use mean § SD. The experiments were repeated at least 3 times. concentration: 10 mM). IL-4 (Sigma-Aldrich, MO, USA) was dissolved in PBS (ready-to-use concentration: 50 ng/mL). All the Immunofluorescence assay for AR dissolved regents were stored at ¡20 C. Cells were seeded in 48-well plates (1 £ 10 cells per well) in triplicate and cultured as in EdU staining. After permeabilization Lentivirus generation and infection (0.5% Triton X-100), fixation (4% formaldehyde) and blocking Recombinant lentiviral vectors were constructed with the (10% normal goat serum and 0.5% Triton X-100), rabbit anti- TM ViraPower Lentiviral System (Invitrogen, NY, USA) as human AR monoclonal antibody (Cell Signaling Technology, described previously. Lenti-LacZ was used as a negative con- MA, USA) was added to the cells overnight at 4 C. Cells were trol, while Lenti-Cherry was used to optimize the infection effi- then stained with FITC-conjugated goat-anti-rabbit polyclonal ciency. For cell infection, LNCaP and 22Rv1 cells were seeded antibody at 37 C for 2 h in the dark. The fluorescence staining into 25 cm plastic culture flasks and grown to approximately intensity was then examined using immunofluorescence micros- 40% confluence, following which the lentivirus-containing copy and photographed. The experiments were repeated at least supernatants were added into the cell culture medium in the pres- 3 times. ence of 1 mg/ml Polybrene (Sigma-Aldrich, USA). Twenty-four hours after infection, the cells were selected with blasticidin PSA ELISA (LNCaP, 4 mg/mL; 22Rv1, 6 mg/mL) for approximately 3 Cells were seeded in 6-well plates (5 £ 10 cells per well) in weeks, so that the uninfected cells were eliminated entirely. triplicate and cultured as in EdU staining. The conditioned media in different groups was assayed for PSA levels using com- Western blot analysis mercial sandwich ELISA kits (Cusabio, Wuhan, CHN) accord- Western blotting was performed as described previously. ing to the manufacturer’s instructions. Each sample was tested in The primary antibodies are as following: mouse anti-human duplicate and quantified using an Emax precision microplate NDRG2 (1:500 dilution, Santa Cruz Biotechnology, CA, USA), reader (Molecular Devices Corp, CA, USA) at 450 nm. The lev- rabbit anti-human PSA, c-Myc and AR (1:1000 dilution, Cell els of PSA in different groups were normalized relative to the cor- Signaling Technology, MA, USA), rabbit anti-human cyclinD1, responding control group. Data are presented as the mean § SD. cyclinE, CDK2, CDK4, and p27 (1:1000 dilution, Santa Cruz The experiments were repeated at least 3 times. Biotechnology, CA, USA). Detection of a–tubulin served as a loading control. The images were scanned and quantified using Tumor growth assays in vivo Kodak Digital Science ID software (Kodak, NY, USA). The Male BALB/c nude mice were purchased from Shanghai experiments were repeated 4 times. Experimental Animal Center (Shanghai, CHN). All of the 294 Cancer Biology & Therapy Volume 16 Issue 2 protocols were approved by the Animal Care and Use Committee analyzed using the Pearson correlation coefficient. Analysis of at the Fourth Military Medical University and were performed in variance (ANOVA) and Student-Newman-Keuls (SNK) tests accordance with the animal care rules set forth by the university were computed to determine whether there were differences (Permit number 10001). LNCaP and 22Rv1 cells that were among the different groups of in vitro and in vivo assays. A P infected with Lenti-LacZ or Lenti-NDRG2 were harvested, value of less than 0.05 was considered statistically significant, and resuspended in sterile PBS and injected subcutaneously into the histograms were prepared using Origin 6.0 (Microcal Software, left flanks of the 6-week-old nude mice (1 £ 10 cells/mouse; 6 Inc., Northampton, MA). mice/group). In the groups of Lenti-LacZ C castration and Lenti-NDRG2 C castration, the mice were given orchectomy surgery to mimic castration therapy just after cell injection. The Disclosure of Potential Conflicts of Interest mice were monitored every day; tumor volumes were calculated every week based on caliper measurements of the length and No potential conflicts of interest were disclosed. width of the lesions using the formula: V (mm ) D length £ width /2. After 10 weeks, the mice were sacrificed by cervical dis- location under anesthesia (130 mg ketamine/8.8 mg xylazine/kg Funding body weight), and tumor specimens were taken, photographed, measured and weighed. Data are presented as the mean § SD. This study was supported by grants from the Key Project of Chinese National Programs for Fundamental Research and Statistical analysis Development (Project No. 2009CB521704) and the National Statistical analyses were performed using SPSS 13.0 software. 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Journal

Cancer Biology & Therapy – Taylor & Francis

Published: Feb 1, 2015

Keywords: AR; c-Myc; castration resistance; lentivirus; NDRG2

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