Oncogene (2014) 33, 4558–4567 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE HOXB13 downregulates intracellular zinc and increases NF-kB signaling to promote prostate cancer metastasis

Y-R Kim1, I-J Kim1, TW Kang2, C Choi3, KK Kim4, MS Kim5, KI Nam1 and C Jung1

Characteristically, prostate cancer (PCa) cells exhibit marked decrease in intracellular zinc; however, the mechanism responsible is not clearly understood. HOXB13 is involved in PCa progression and is overexpressed in castration-resistant PCa. DNA microarray analysis of LNCaP Pca cells showed that ZnT zinc output transporters were strikingly upregulated among androgen-independent HOXB13 target . Furthermore, exogenous HOXB13 caused intracellular zinc concentrations to fall in PCa cells, stimulated NF-kB-mediated signaling by reducing inhibitor of NF-kB alpha (IkBa) and enhanced the nuclear translocation of RelA/p65. Human prostate tumors also exhibited strong inverse correlation between the expressions of HOXB13 and IkBa. Consequently, HOXB13 stimulated PCa cell invasion, and this was inhibited by the suppression of ZnT4. In addition, studies in a PC3 orthotopic mouse model of PCa metastasis showed that HOXB13 is a strong metastatic stimulator. Taken together, these results show that HOXB13 promotes PCa invasion and metastasis by decreasing intracellular zinc levels, thus stimulating NF-kB signals, and suggest that HOXB13 acts as a modulator of intracellular zinc levels that promotes the malignant characteristics of PCa.

Oncogene (2014) 33, 4558–4567; doi:10.1038/onc.2013.404; published online 7 October 2013 Keywords: HOXB13; metastasis; prostate cancer; zinc; ZnT4

INTRODUCTION available for those with advanced PCa. Nevertheless, most cases HOX genes typically function as DNA-binding transcription factors progress to recurrent CRPC. In order to improve treatment for and participate in embryogenesis and cellular differentiation. recurrent CRPC, we need to understand the mechanism that However, they also implicated in diverse pathogenic processes allows CRPC cells to develop and survive in a harsh androgen- in human cancers.1 Mice with Hoxb13 loss-of-function mutations deprived environment. A balance between the effects of exhibit overgrowth of structures derived from tail buds androgens and other growth-regulating is probably and malformation of the ventral prostate ducts.2 In fact, the required for homeostasis to prevent the abnormal cell growth expression of HOXB13 is restricted to prostate tissues and has and/or the continuous proliferation of cancer cells. The majority been reported to be highly correlated with androgen of CRPC cells overexpress AR, and AR overexpression has expression.3,4 Furthermore, HOXB13 has been shown to suppress been suggested to be sufficient to convert PCa growth from a androgen-mediated signals and to inhibit prostate cancer hormone-therapy-sensitive to a -resistant state.7–9 Furthermore, it (PCa) cell growth, which suggests that HOXB13 functions as a is believed that certain transcription factors are involved in this prostate-specific tumor suppressor under androgen-rich progression, and several studies have demonstrated that NF-kBis conditions. On the other hand, HOXB13 has also been reported constitutively activated in CRPC xenografts overexpressing to be overexpressed in castration-resistant PCa (CRPC), in which its AR.7,10,11 NF-kB contributes to development and progression of suppression drove PCa cells to cell death.5 These findings imply prostatic malignancies by regulating the expressions of genes that HOXB13 displays functional dichotomy and that its functions involved in proliferation, apoptosis and angiogenesis and by depend on cellular context. Cell growth-promoting activity of promoting tumor invasion and metastasis.12,13 Interestingly, NF-kB HOXB13 in an androgen-deficient environment has been shown upregulates AR and its transactivation activity, and inhibitors of to be mediated via the dramatic suppression of p21waf/cip1 NF-kB exhibit antitumor activity in CRPC xenografts,14 which followed by RB/ activation.5 However, because of the high suggests a link between the NF-kB and AR signaling pathways frequency of RB mutations in PCa, there seems to be HOXB13- that promotes the growth and survival of CRPC cells. mediated cell growth regulation independent of p21/RB/E2F In this study, we investigated the role of HOXB13 in signals. zinc-mediated regulation of NF-kB signaling to improve our PCa is the second leading cause of cancer mortality among understanding of the involvement of HOXB13 in the progression western men and was responsible for an estimated 29 720 deaths of PCa to CRPC. The normal human prostate accumulates higher in the United States in 2013.6 The most significant problem levels of zinc than any other soft tissue, and thus, zinc is presented by PCa is its tendency to recur; for example, PCa recurs considered an essential component of the healthy prostate. in 30% of men with early stage disease. On the other hand, However, zinc levels are markedly diminished in prostatic androgen withdrawal or blockade is the only effective therapy epithelial tumors and continue to decline during progression

1Department of Anatomy, Chonnam National University Medical School, Gwangju, Korea; 2Department of Urology, Chonnam National University Medical School, Gwangju, Korea; 3Department of Pathology, Chonnam National University Medical School, Gwangju, Korea; 4Department of Pharmacology, Chonnam National University Medical School, Gwangju, Korea and 5Department of Statistics, College of Natural Sciences, Chonnam National University, Gwangju, Korea. Correspondence: Dr C Jung, Department of Anatomy, Chonnam National University Medical School, 5 Hak-Dong, Dong-Gu, Gwangju 501-190, Korea. E-mail: [email protected] Received 25 February 2013; revised 30 July 2013; accepted 23 August 2013; published online 7 October 2013 The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4559 towards androgen-independent growth.15,16 In this present study, to profile androgen-independent genes targeted by HOXB13, -1.5 0.0 1.5 we performed complementary DNA microarray analysis in LNCaP cells grown under androgen-deprived conditions. In particular, B1 B2 B3 one of these target genes, ZnT4 zinc output transporter, was G1 G2 G3 analyzed in detail in order to identify any association between LOC221442 HOXB13 and zinc-mediated biomolecular cascades. This study was POPDC3 undertaken to determine how prostate cells adapt to survive via BCHE tumorigenesis and to progress to CRPC cells. SLITRK3 SLC30A10 EPHA5 SNORD116-18 RESULTS GABRA2 ZnT4 (zinc output transporter) is a target of HOXB13 UGT2B7 C12or f5 In our previous studies, we found that HOXB13 was overexpressed UGT2B15 in most CRPCs and that its overexpression provided a positive UGT2B15 growth signal to PCa cells under an androgen-deficient condition.5 LRRII1 In order to explore in detail the roles of HOXB13 in CRPC, we CEP170 first profiled HOXB13-targeted genes in an androgen-deficient SLC30A4 EPHA7 environment. LNCaP PCa cells were grown under androgen- SEMA3A deprived conditions for 3 days before treatment with either ACPP adenoviral HOXB13 or control adenovirus. To avoid the xenotoxic EPHA4 effect of HOXB13 observed in our previous study,17 a low dose of ATP10D virus (0.2 multiplicity of infection) was administered to cells NI PAL1 (Supplementary Figure S1A). LNCaP cells expressed only a tenth of the HOXB13 RNA expressed by normal prostate cells,17 and the above mentioned viral dose increased HOXB13 RNA expression to approximately the normal level (Supplementary Figure S1B). Purified RNA was subjected to microarray analysis, and hierarchical Ad controlAd-HOXB13 clustering analysis showed androgen-independent HOXB13 HOXB13 target genes over a twofold increase (Figure 1a). Supplementary ZnT10 Table S1 provides a detailed description of the genes targeted. Notably, SLC30A10 (ZnT10) and SLC30A4 (ZnT4) are zinc output ZnT4 transporters and were highly induced by HOXB13. In addition, HOXB13VehicledHD several ephrin receptors (that is, EPHA7, A5 and A4) were also Zip1 HOXB13 upregulated by HOXB13. Highly homologous HOXA13 and b-actin 18,19 HOXD13 are known to directly target EphAs. Further RT–PCR dHD-HOXB13 analysis confirmed the positive regulations of ZnT10 and ZnT4 by 100 Ad-GFP HOXB13, although HOXB13 did not affect Zip1 zinc input 90 ZnT4 80 Ad-HOXB13 transporter (Figure 1b top panel). Densitometry was used to 70 quantify the levels (Figure 1b bottom panel) and, although 60 β-actin

-actin 50

ZnT10 was more significantly upregulated by HOXB13 than  5 ZnT4 (7.8- versus 2.7-fold), levels of ZnT10 were very low in 4 most PCa cells, including LNCaP (Supplementary Figure S1C). gene/ 3 ZnT4 protein was also upregulated by HOXB13 transfection in 2 1 western blot analysis (Figure 1c). Moreover, we demonstrated 0 that ZnT4 expression was not altered by DNA-binding homeo- ZnT4 domain-deleted HOXB13-mutant overexpression, suggesting ZnT10 that HOXB13-mediated ZnT4 regulation is not a non- HOXB13 specific phenomenon. Based on these results, we decided to Figure 1. ZnT4, zinc output transporter, is a target of HOXB13. focus on ZnT4 as a representative HOXB13-targeted zinc output (a) Hierarchical clustering analysis of genes differentially expressed transporter. in LNCaP cells under hormone-deprived conditions. To investigate HOXB13 target genes, DNA microarray analysis was performed under a physiological condition in which HOXB13 (B1BB3) was HOXB13 diminished intracellular zinc levels and stimulated NF-kB minimally overexpressed in LNCaP as compared with controls signaling in PCa cells (G1BG3). This figure shows upregulated and downregulated In order to determine whether HOXB13 affects intracellular zinc transcripts in red and green, respectively. This overview of the relative expression levels of HOXB13-regulated genes shows target levels, we employed HOXB13-inducible LNCaP cells (S4) and genes whose expressions varied at least twofold from the mean HOXB13-suppressed LNCaP cells (1-3 and 4-1). Wild-type LNCaP 17 abundances in control. (b) The expression of zinc transporter mRNA cells express HOXB13 endogenously. Cells were pre-incubated in LNCaP cells (top panel). The expression of HOXB13 was controlled under androgen-deprived conditions for 3 days, and HOXB13 by adenovirus. The shown are the means of three overexpression was induced by treating HOXB13-inducible independent real time RT–PCR analyses and are presented as S4-LNCaP cells with 200 nM doxycycline (Dox) in the presence of means±s.d. (bottom panel). (c) ZnT4 protein expression using TNF-a. Zinc concentrations were measured using 25-Br-PAP as western blot. HEK293 cells were transfected by pFLAG-HOXB13, previously described.20 Dox-induced HOXB13 reduced the level of pFLAG or Homedoamin-deleted pFLAG-HOXB13. intracellular zinc compared with the untreated (P ¼ 0.0032) (Figure 2a), whereas tet-on only control cells were not affected scrambled controls (by one-way analysis of variance P ¼ 0.0001) by Dox (Figure 2b). On the other hand, the suppression of HOXB13 (Figure 2c). In HOXB13-suppressed LNCaP cells, ICP (inductively resulted in an accumulation of intracellular zinc as compared with coupled plasma) and atomic emission spectrometry produced

& 2014 Macmillan Publishers Limited Oncogene (2014) 4558 – 4567 The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4560 LNCaP-S4 LNCaP-Teton LNCaP-shHOXB13 PC3; NFB-luc 0.025 4 scrambled 1-3 0.020 3 0.0050 0.0050 4-1 0.015 2 RLU IC zinc IC zinc

IC zinc 0.010 0.0025 0.0025 1 0.005

0.0000 0.0000 0.000 0 TNF-α -++- - - + + Dox - + Dox - + HOXB13 - -

LNCaP-S4; NFB-luc LNCaP-S4; NFB-luc 125000 140000 LNCaP-shHOXB13; NFB-luc * ** 50000 120000 scrambled 100000 100000 40000 1-3 75000 80000 4-1 30000 RLU 50000 RLU 60000 40000 RLU 20000 25000 20000 10000 0 0 α Dox -+- - + + TNF- - - + + 0 Dox --++ zinc - ++-++ pyrithione - - + --+ Figure 2. HOXB13 diminishes intracellular zinc levels and stimulates NF-kB signaling in PCa cells. LNCaP-S4 cells were grown under andro- gen-deprived conditions for 3 days and, thus, forced to express HOXB13. LNCaP-shHOXB13 are HOXB13-suppressed cells. PC3 cells were transfected with Flag-HOXB13. Either LNCaP-S4 cells (a) or Tet-on control cells (b) were treated with Dox to induce HOXB13. HOXB13- suppressed cells (1-3, 4-1) were also used and compared with scrambled cells (c). Cells were analyzed for their intracellular zinc contents. After PC3 cells (d) or LNCaP-S4 cells (e) were grown for 24 h, TNF-a was added to stimulate NF-kB activity. LNCaP-S4 (f) or shHOXB13- LNCaP (g) cells were pre-incubated with zinc and/or pyrithione for 30 min and then incubated with TNF-a for 2 h. Cells were evaluated using luciferase assays. *,** in the diagram indicate significant differences between the two groups indicated, as determined using the two-tailed Student’s t-test (Po0.05). Both zinc and transfection experiments were performed in triplicate. Bars represent means±s.d.

similar results, showing that suppression of HOXB13 increased HOXB13-mediated NF-kB activation was accomplished via the intracellular zinc levels (Supplementary Figure S2A). To further upregulation of ZnT4 expression elucidate the role of HOXB13 in the zinc-mediated signaling In order to confirm that the promoting role of HOXB13 on NF-kB pathway, we used androgen-independent PC3 PCa cells, which signaling is mediated via the regulation of intracellular zinc, we have been reported for the effects of zinc on NF-kB signaling.21,22 suppressed ZnT4 by using short interfering RNA in S4-LNCaP cells. In these cells, HOXB13 transfection resulted in a slight decrease Figure 3a shows that HOXB13-mediated promotion of NF-kB in intracellular zinc, whereas homeodomain deletion mutant activity was dose-dependently inhibited by si-ZnT4 (by linear of HOXB13 did not affect the intracellular zinc content regression analysis r2 ¼ 0.986 and P ¼ 0.010). Furthermore, si-ZnT4 (Supplementary Figure S2B). Physiological levels of intracellular at 100 mM almost negated the effect of HOXB13, whereas si- zinc are known to suppress NF-kB-mediated signaling activity in control RNA did not influence the effect of HOXB13. Figure 3b response to TNF-a stimulation.21,22 Thus, in order to determine describes the specificity of si-ZnT4 as determined using RT–PCR whether HOXB13 can alter NF-kB activity, PC3 cells were (top panel) and western blot (bottom panel). The si-ZnT4- transiently transfected with HOXB13. Cells were transfected mediated suppression of NF-kB activity was also verified in wild- with NFkB-luc and pFLAG-HOXB13 with or without 10 ng/ml type LNCaP cells (Figure 3c). To determine whether the observed TNF-a. HOXB13 stimulated NF-kB signaling in a dose-dependent si-ZnT4-mediated suppression of NF-kB activity was because of manner in both the absence and presence of TNF-a (by linear intracellular zinc reduction, zinc concentrations were measured in regression analysis r2 ¼ 0.962 and P ¼ 0.001 without TNF-a; HOXB13-induced LNCaP-S4 cells in the presence of si-ZnT4 r2 ¼ 0.937 and P ¼ 0.001 with TNF-a) (Figure 2d). This phenom- (Figure 3d). It was found that, whereas si-ZnT4 leads to an enon was also exhibited by LNCaP-S4 cells (Figure 2e). Although accumulation of zinc (*Po0.0001), HOXB13 reduced intracellular not affected by the physiological level (0.5–1.5 mg/ml) of extra zinc, zinc levels (**Po0.0001). On the other hand, in the presence of NF-kB activity in S4 cells was significantly reduced by treatment both HOXB13 and si-ZnT4, zinc levels were slightly increased with zinc ionophore, pyrithione (1 mM) (Supplementary Figures S2C (***P ¼ 0.0003). These results suggest that zinc inhibits NF-kB and D), as was demonstrated by another group.22 However, activity and that HOXB13 counteracts this by downregulating excessive intracellular accumulation of zinc by pyrithione was intracellular zinc concentrations. relieved by HOXB13 overexpression (*P ¼ 0.014) (Supplementary Figure S2E). In HOXB13-manipulated LNCaP cells, in the presence of TNF-a HOXB13 stimulated NF-kB activity (*P ¼ 0.0008) and NF-kB activation by HOXB13 was mediated by the downregulation partly restored NF-kB activity in the presence of surplus zinc of IkB (0.5 mg/ml) and pyrithione (1 mM) (**P ¼ 0.005) (Figure 2f). In NF-kB proteins (p65, p50, p52, c-REL and RELB) are present in the shHOXH13-LNCaP cells, the suppression of HOXB13 significantly cytoplasm in association with inhibitory proteins known as decreased NF-kB activity in the presence of TNF-a (by one-way inhibitors of NF-kB(IkBs). After activation, IkB proteins become analysis of variance P ¼ 0.0001) (Figure 2g). These data suggest phosphorylated and degraded, which result in the nuclear that the HOXB13-induced overexpression of ZnT4 facilitates translocations of NF-kB proteins. In the nucleus, NF-kBs bind to intracellular zinc outflow and further stimulates TNF-a-induced their cognate DNA-binding sites to regulate the transcriptions of NF-kB activity. many functional genes.23 To investigate the mechanism by which

Oncogene (2014) 4558 – 4567 & 2014 Macmillan Publishers Limited The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4561 ? LNCaP-S4; NFB-luc 200000

150000

100000 RLU

50000

0 Dox - - + + + + + TNF-α - + + + + + + si-Znt4 - - - 20 50 100 - si-control ------100

LNCaP; NFkB-luc LNCaP-S4 80000 0.008 * 60000 0.006 *** 40000 0.004 ** RLU IC zinc 20000 0.002

0.000 0 TNF-α -+++ + + Dox - -++ si-ZnT4 - +-+ si-Znt4 -2050- 100 - si-control ---- - 100 Figure 3. HOXB13-mediated NF-kB activation is accomplished through the upregulation of ZnT4 expression. (a) LNCaP-S4 cells were treated with various doses of ZnT4 short interfering RNA under the control of Dox and TNF-a. Luciferase assays were performed 24 h post transfection. Transfection experiments were performed in triplicate. Bars represents means±s.d. (b) ZnT4 mRNA expression in LNCaP-S4 cells (top panel) or ZnT4 protein expression in HEK293 cells (bottom panel) by si-ZnT4 or were determined. (c) Wild-type LNCaP cells were used to confirm the ZnT4-mediated suppression of NF-kB activity in the presence of HOXB13. (d) Intracellular zinc concentrations were analyzed in LNCaP-S4 cells in the presence of si-ZnT4 and/or HOXB13. Asterisks indicate Po0.05.

HOXB13 stimulates NF-kB signaling, we western blotted HOXB13- score of 2–3 or 0–1, respectively, the expressions of HOXB13 and regulated LNCaP cells. As shown in Figure 4a, whole-cell lysates IkBa were found to be inversely correlated (w2 ¼ 4.887, Po0.05) from LNCaP-S4 cells demonstrated the inhibition of IkBa (Figure 5B). By logistic regression analysis, the probability of high expression but no effects to p65 expression. The regulation of IkBa expression was 12 times greater when samples showed low IkBa expression appears to be mediated via the upregulation of HOXB13 expression (log p/1 À p ¼ (3.871–2.485) Â HOXB13, odds IkB kinase a (IKKa). The nuclear fractions of LNCaP-S4 cells showed ratio ¼ 0.083). These expressional scores for HOXB13 and IkBa are more p65 nuclear translocation than no Dox-treated cells. In listed in Supplementary Table S2. shHOXB13-LNCaP cells, HOXB13 suppression increased IkBa levels in whole-cell lysates and inhibited the nuclear translocation of p65 (Figure 4b). These observations imply that HOXB13 facilitates IkBa HOXB13 increased the invasive potential of PCa cells degradation by upregulation of IKKa to activate NF-kB signaling. In order to determine whether HOXB13 affects the biological roles of NF-kB signaling, particularly with respect to cancer cell invasion, HOXB13-regulated cells were placed in transwell chambers in the HOXB13 expression was inversely correlated with IkBa expression absence of androgen with or without TNF-a. Significantly fewer in prostate tumors invasive cells were observed among HOXB13-suppressed cells As HOXB13 regulated NF-kB activity by downregulating IkBa in than among scrambled cells (Figures 6a and b). The inhibition of PCa cells, we evaluated the tissue expressions of HOXB13 and IkBa ZnT4 with short interfering RNA dramatically reduced the invasive in 9 CRPC and 12 androgen-dependent PCas (ADPC). However, as effect of HOXB13 in HOXB13-inducible S4-LNCaP cells (Figure 6c). PCa were heterogeneous, we only evaluated The downstream targets of NF-kB are major proangiogenic and (AR)-positive PCa cells. Figure 5A shows expressions of HOXB13 prometastatic molecules and include VEGF, interleukin-6, inter- and IkBa, and that HOXB13-overexpressing tumors did not leukin-8 and matrix metalloproteinases 9 (MMP-9). We investi- express IkBa (a–d). This was especially the case in CRPCs, as only gated whether HOXB13 regulates the expressions of genes HOXB13-overexpressing CRPCs did not express IkBa. A few downstream of NF-kB using western blotting. As shown in HOXB13-overexpressing ADPCs exhibited IkBa expression (e–h), Figure 6d, the expressions of MMP-9 and VEGF were inhibited whereas some tumor cells and cells from benign prostatic by HOXB13 suppression in shHOXB13-LNCaP cells. The p21 hyperplasia exhibited low HOXB13 and high IkBa expressions expression was used as a known HOXB13 target as described (i–l). When high and low expressions were arbitrarily awarded previously.5 To confirm that alterations in MMP-9 expression by

& 2014 Macmillan Publishers Limited Oncogene (2014) 4558 – 4567 The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4562 metastases to para-aortic lymph nodes (P ¼ 0.032, Fisher’s exact test). No evidence of gross and microscopic metastases was identified in any other organs.

DISCUSSION We previously found that HOXB13 was highly overexpressed in CRPCs than in ADPCs.5 This study demonstrated that HOXB13 is a positive growth regulator of PCa cells under androgen-deprived conditions because of the inhibition of p21waf/cip1expression and the promotion of RB/E2F signaling. Furthermore, in breast cancer, Ma et al.24 found that HOXB13 was overexpressed in -positive hormone refractory tumors. Since the tumor suppressor retinoblastoma (RB) gene is inactivated in up to 60% of clinical prostate tumors,25 the RB/E2F signaling pathway may not be an only target of HOXB13 for the promotion of androgen- independent PCa growth. Therefore, in this study we used DNA microarray to profile androgen-independent target genes of HOXB13 in LNCaP cells. Further investigation demonstrated that HOXB13 positively targeted zinc efflux transporter ZnTs, but not influx transporter Zip1, and, consequently, regulated the intracellular concentration of zinc. Furthermore, ZnT upregulation by HOXB13 was found to deplete intracellular zinc levels in PCa cells. Kolenko and colleagues22,26,27 reported that intracellular zinc inhibits the invasive ability of PC3 PCa cells by regulating NF-kB signaling, and the present study shows intracellular zinc level reductions by HOXB13 and zinc-mediated NF-kB inhibition are accomplished via the regulation of IKKa. Figure 4. NF-kB activation by HOXB13 is mediated by IkB. Healthy human prostates contain higher zinc levels than any (a) HOXB13 expression was induced in LNCaP-S4 cells with Dox, other soft body tissue,28 although little is known of the functional and corresponding lysates were collected and western blotted (left roles of zinc. Furthermore, zinc concentrations are decreased in panel, whole lysates; right panel, nuclear extracts). (b) HOXB13- PCa as compared with those in nonmalignant cells.16 However, suppressed cells (1-3 and 4-1) and scrambled control cells were whereas naturally occurring malignant prostate cells lose the evaluated for pNF-kB protein expression (left panel, whole lysate; ability to accumulate zinc, some malignant cell lines, such as right panel, nuclear extracts). LNCaP and PC3, exhibit zinc accumulation29 and require specialized plasma membrane transporters to mediate zinc uptake.30 It is still not clear why in situ PCa cells depressed zinc HOXB13 are functionally relevant to cancer cell migration and levels, although it has been proposed that the transformation of invasion, the conditioned media of cells were subjected to a normal zinc-accumulating citrate producing normal cells to gelatin zymography assay (Figure 6e). In HOXB13-supressed and citrate-oxidizing malignant cells that lose the ability to -overexpressed cells, proforms of MMP-9 were expressionally accumulate zinc during the tumorigenic process is responsible.15 down- and upregulated, respectively. These results suggest that In addition, a genetic alteration in the expression of zinc HOXB13 promotes the invasive potential of LNCaP cells by transporter is associated with this metabolic transformation. Zinc stimulating the expression of NF-kB-targeted genes, especially input transporter Zip1 has been widely investigated to determine under hormone-deprived conditions. how Zip1 expression is suppressed in PCa.31,32 The present study shows that zinc output transporter is involved in the minimization of intracellular zinc that stimulates PCa growth and progression. HOXB13 stimulated the lymph node metastasis of PCa cells We found that the overexpression of HOXB13 in CRPC upregulates In our preliminary study, the tumor incidence of successful tumor ZnT4 and, thus, depletes zinc levels. ZnT proteins are members of implantation in mouse prostate using HOXB13-overexpressing the cation diffusion facilitator family and either transport zinc out LNCaP-S4 cells was low (1 in 10 tumors). Therefore, we used an of cells or sequester zinc in intracellular compartments.33 orthotopic mouse model of PC3 PCa to observe the effect of Although the expression of zinc input transporters, such as Zip1, HOXB13 on metastasis. First, we constructed HOXB13-stable is suppressed during PCa development, HOXB13 seemingly transfectants in PC3 cells, in which FLAG-tagged HOXB13 was facilitates the depletion of intracellular zinc by upregulating zinc overexpressed (Figure 7A). After the intraprostatic inoculation of output transporters such as ZnT4, which suggests that zinc influx PC3-HOXB13 or PC3-mock cells, tumors were grown for up to does not wholly account low zinc levels in tumor tissues. Unlike 8 weeks when the animals were killed and their major organs other zinc transporters, the tissue expression of ZnT4 is restricted were collected. At the time of killing, the average weights of all to mammary glands, mast cells, the prostate, intestine and primary tumors were approximately the same, regardless of cell others.34–37 It is also noteworthy that the expression of ZnT4 types (data not shown). The incidence of lymph node metastases was markedly reduced in prostate tumors as compared with was studied in serial sections of prostate and selected lymph normal prostate tissues, whereas the expression of influx nodes with hematoxylin and eosin and immunohistochemical transporters were relatively unchanged.38 In addition, ZnT4 staining. Local tumors were developed in 7 of 11 mice carrying mRNA was found to be overexpressed in tumor specimens PC3-mock cells and 8 of 12 mice carrying PC3-HOXB13 cells. obtained by radical prostatectomy as compared with normal Metastases in para-aortic lymph nodes were observed in 75.0% tissues.36 (6/8) of mice bearing PC3-HOXB13 tumors and in only 14.3% (1/7) Rel/NF-kB transcription factors are composed of several homo- of those bearing PC3-mock tumors (Figures 7B and C), which and heterodimers of p50, p52, p65 (RelA), RelB and c-Rel. suggests that HOXB13 significantly promoted the incidence of Furthermore, NF-kB dimers are normally retained in the cytoplasm

Oncogene (2014) 4558 – 4567 & 2014 Macmillan Publishers Limited The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4563

HOXB13

IkBa *

HOXB13

IkBa **

0 1 2 3 Expression score

HOXB13 overexpressed HOXB13 underexpressed Figure 5. HOXB13 expression correlates inversely with IkBa in prostate tumors. (A) Immunostaining was performed in various prostate tissues using serial sections. AR was used as an internal control. Scale bar represents 100 mm. Insets were shown as  40 magnification. (B) Bar graph showing the HOXB13 scores of HOXB13-overexpressed and -underexpressed PCas. Expressions of HOXB13 and IkBa in tumors were scored (0, absent; 1, weak; 2, intermediate; and 3, strong). Asterisks indicate Po0.05. in inactive forms by binding between them and IkB inhibitory p65 expression.40,41 NF-kB contributes to development and proteins, such as IkBa,IkBb,IkBg,IkBe, Bcl3, p100 and p105. The progression of prostatic malignancies by regulating the most common Rel/NF-kB dimer in mammals contains p50-RelA expressions of genes involved in proliferation, apoptosis and (p65) subunits bound to IkB and is called NF-kB. However, angiogenesis, as well as those involved in tumor invasion and stimulation by NF-kB inducers, such as TNF-a, leads to phosphor- metastasis.12,13 Accordingly, NF-kB targeted proteins, such as ylation and degradation of IkB by IKK, and the NF-kB so released is BCL-2, cyclin D1, MMP-9 and VEGF, are overexpressed in high-grade translocated to the nucleus where it induces the expressions of PCa tissues as compared with low-grade and benign tissues.40 numerous regulatory genes.39 Furthermore, NF-kB is constitutively Moreover, the suppression of NF-kB in human PCa cells inhibits activated in androgen-insensitive PCa cells and in tumor samples their tumorigenic and metastatic properties in vivo by suppressing with high Gleason grades, which also show high levels of nuclear angiogenesis and invasion via the downregulations of VEGF,

& 2014 Macmillan Publishers Limited Oncogene (2014) 4558 – 4567 The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4564

shHOXB13-LNCaP LNCaP-S4 75 200 scrambled 1-3 4-1 150 50 100 25 50 Invaded cell number 0 Invaded cell number 0 (-) TNF-α (+) TNF-α Dox -+ - + si-ZnT4 - - + +

Figure 6. HOXB13 increases the invasive potential of PCa cells. (a) Cell invasion was measured using the matrigel transwell. HOXB13- suppressed cells (1-3 and 4-1) and scrambled cells were grown under androgen-deprived conditions for 2 days. Media containing fibronectin was added to bottom chambers. After 24 h, cells were stained and images were captured. (b) Cells were counted in randomly selected five fields at  200 magnification; results are presented as a bar graph. (c) Dox-induced HOXB13 S4 cells were plated and then transfected with si-ZnT4 (50 nM) or si-control, and transfected cells were applied to invasion assays as described above. Assays were performed in triplicate; bars represent means±s.d. (d) shHOXB13-LNCaP cells were grown under androgen-deprived conditions for 2 days. Cells were then extracted and lysates were evaluated using western blotting. (e) Conditioned media from LNCaP-shHOXB13 (left panel) or LNCaP-S4 (right panel) cells were subjected to gelatin zymography assays. Proforms of MMP-9 and MMP-2 are shown.

interleukin-8 and MMP-9,42 and NF-kB provides a link between Taken together, our results suggest that HOXB13 ultimately zinc and PCa growth and invasion.43 Physiological levels of zinc stimulates the invasive properties of PCa cells by downregulating (0.25–0.5 mg/ml) inhibit the activation of NF-kB in PCa cells, intracellular zinc levels, particularly under androgen-deprived probably by blocking IkB kinase.27,44 Zinc was also reported conditions. We propose a hypothetical model for the action to inhibit NF-kB activity in androgen-independent PC-3 and mechanism of HOXB13 during PCa invasion (Figure 8): (1) HOXB13 DU-145 PCa cells, to reduce the constitutive expression of the upregulates zinc efflux channels, including ZnT4, under androgen- anti-apoptotic protein c-IAP2, to activate JNK and to sensitize PCa deprived conditions. (2) ZnT4 facilitates zinc outflow from cells. cells to apoptosis induced by cytotoxic agents.22 The present (3) Reduced levels of intracellular zinc stimulate the expression study confirms that physiological levels of zinc in combination of IKKa, which further phosphorylates and degrades IkBa. with zinc ionophore pyrithione significantly inhibit TNF-a- (4) Released stimulatory NF-kBs, such as p65 and p50, translocate mediated NF-kB signaling in PC3 and LNCaP PCa cells. to the nucleus and activate the expression of genes involved in Moreover, HOXB13-manipulated LNCaP cells also upregulated tumor cell invasion and metastasis. NF-kB signaling in the presence and absence of TNF-a, although was counteracted by the suppression of ZnT4, suggesting that HOXB13 activates NF-kB signaling by depleting intracellular zinc MATERIALS AND METHODS level. The promotion of the invasive properties of LNCaP cells Cell culture, transfections and luciferase assays was also inhibited by the suppression of ZnT4, and we found LNCaP, PC3 and DU-145 cells were cultured as previously described. that zinc-mediated suppression of NF-kB is accomplished via the HOXB13-inducible S4 and Tet-on control cells were previously described.5 downregulation of IKKa and the promotion of IkBa stabilization. Cell transfections and luciferase assays are described in Supplementary Furthermore, attempts to link HOXB13 and IkBa in tumor Materials and methods. tissues revealed that expressions of HOXB13 and IkBa are inversely correlated. Our in vivo study also shows that HOXB13 RNA preparation and gene expression profiling is strong metastatic promoter. These findings suggest that the Total RNA was isolated using RNeasy Mini Kit columns as described by the overexpression of HOXB13 in malignant PCa is a reason why in situ manufacturer (Qiagen, Valencia, CA, USA). Gene chip analysis was malignant prostate cells do not accumulate zinc, which would performed on a contractual basis by DNA Link (Seoul, Korea) using enhance their invasive characteristics. Considering that NF-kB Affymetrix GeneChip Human Gene 1.0 ST oligonucleotide arrays. To determine whether genes were differentially expressed in two groups, the activity is highly activated in CRPC, in which HOXB13 is paired t-test was performed on robust multichip average expression overexpressed, the reduction in intracellular zinc by HOXB13 values, and genes with P-values o0.05 were extracted. Then by comparing may facilitate CRPC growth and metastasis by increasing NF-kB the signal values of control and test groups, genes showing 42.0-fold signals. expression differences were selected (Supplementary Table S1).

Oncogene (2014) 4558 – 4567 & 2014 Macmillan Publishers Limited The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4565

100

80 *

60

40 Occurrence of

metastasis (%) 20

0 mock HOXB13

Figure 7. Metastasis of PC3-HOXB13 tumors to para-aortic lymph nodes. (A) Stably transfected PC3 cells were immunoblotted for FLAG. (B) Para-aortic lymph nodes were sectioned to detect metastasis, and numbers of metastases were counted. The frequency of metastasis in mice lymph nodes was 75.0% (six of eight tumor-bearing mice) in the PC3-HOXB13 group and 14.3% (one of seven tumor-bearing mice) in the PC3-mock group. (C) Selected local (a–c) and metastatic (d–f) PC3-HOXB13 tumors are shown. The boxes in ‘a’ and ‘d’ are magnified in ‘b’ and ‘e’, respectively. Serial sections ‘b’ and ‘e’ were immunostained for FLAG (c, f).

Immunoblotting and immunohistochemistry reader and SOFTmax PRO software (Molecular Devices, Sunnyvale, CA, HOXB13 was detected using rabbit polyclonal antibodies as previously USA). described.5 Antibodies to NF-kB/p65, BCL-2 and b-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to VEGF Analysis of cell invasiveness and MMP-9 were obtained from R&D Systems (Minneapolis, MN, USA). Anti-IkBa and IKKa were obtained from Cell signaling Technology Transwell chambers (Corning Inc, Corning Life Sciences, Tewksbury, MA, (Danvers, MA, USA), and antibodies against p21waf/cip1 were from USA) were coated with a thin layer of matrigel (BD Biosciences, San Jose, Upstate Biotechnology (Lake Placid, NY, USA). Anti-ZnT4 antibodies were CA, USA), which was diluted in serum-free media. One hundred microlitre from Abcam (Cambridge, MA, USA). All antibodies were detected as of gel suspension was added to the upper chamber and incubated for 1 h described in Supplementary Materials and methods. For immunohisto- at room temperature. The matrigel solution was then removed, and the chemical analysis of HOXB13 and IkBa, human primary PCas were obtained upper chamber was washed once with 100 ml of serum-free media and placed in the bottom chamber containing 400 ml of media containing from patients at the Chonnam National University Hospital between 1997 5 and 2005. Specimens from 21 patients comprised 9 CRPCs and 10 ADPCs. fibronectin (20 mg/ml). LNCaP-shHOXB13 cells (2.4 Â 10 cells) were then Immunohistochemistry was performed as previously described.5 seeded in serum-free RPMI media (120 ml) into the upper chamber. For LNCaP-S4 cells, cultured cells were first transfected with si-ZnT4 (Santa Cruz Biotechnology) or si-control for 24 h. Cells transfected with short RNA extraction and RT–PCR analysis interfering RNA were loaded into the top chamber with the addition of 45 200 nM Dox to induce HOXB13 expression and were incubated for 24 h in a RNA was extracted from cells as previously described. RT–PCR was CO incubator. Cells on upper sides of inserts were removed with cotton performed in order to verify expressions of HOXB13, ZnT4 and Zip1. 2 46 buds, and invading cells were stained with the Diff Quick (Sysmex Corp, Quantitative RT–PCR was also performed as previously described. Primer Kobe, Japan) and analyzed using microscope (Leica Microsystems, Wetzlar, sets are listed in Supplementary Materials and methods. Germany) by counting cells in five random fields per transwell. Experiments were performed in triplicate. Zinc assays Zinc concentrations in cells were measured using 2-(5-bromo-2-pyridy- Gelatin zymography lazo)-5-(N-propyl-N-sulfopropylamino) phenol disodium salt dehydrate MMP activities of HOXB13-altered cells were determined using gelatin (5-Br-PAPS, Dojindo Laboratory, Kumamoto).20 Briefly, cells were cultured zymography. Equal numbers of cells were cultured in serum-free media for in RPMI containing 5% CDT-FBS (charcoal dextran-treated fetal bovine 24 h and media were collected. The conditioned media obtained were serum) for 2 days, washed with phosphate-buffered saline, pelleted, mixed with substrate gel sample buffer and loaded into 10% SDS–PAGE treated with 10% trichloroacetic acid on ice for 15 min and centrifuged at containing 2.65 mg/ml gelatin (Sigma-Aldrich, Seelze, Germany) under 4 1C for 10 min. Resulting supernatants were incubated with 0.08 nM nonreducing conditions. After electrophoresis, the gel was washed in 2.5% 5-Br-PAPS and 29 mM salicyladoxime for 10 min at room temperature. Triton X-100, rinsed and incubated overnight at 37 1C in developing buffer Absorbances of mixtures were measured at 570 nm using a microplate (50 mmol/l Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM CaCl2 and 0.01% sodium

& 2014 Macmillan Publishers Limited Oncogene (2014) 4558 – 4567 The role of HOXB13 in prostate cancer metastasis Y-R Kim et al 4566 ACKNOWLEDGEMENTS HOXB13 This work was supported by the Korea Science and Engineering Foundation through the Medical Research Center for Gene Regulation (2012-0009445), by the Basic Zinc Zinc Science Research Program of the National Research Foundation of Korea (NRF) grant influx efflux funded by the Korea Government (2010-0003838), and by a grant from the Chonnam National University Research Institute of Medical Sciences ((2010-CURIMS-DR011). cytoplasm

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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