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 genes. 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 protein 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 proteins 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 receptor 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/E2F 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 gene 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