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Molecular and Cellular Pathobiology

Hepcidin Regulation in Prostate and Its Disruptionin Prostate CancerLia Tesfay1, Kathryn A. Clausen2, Jin Woo Kim3, Poornima Hegde4, Xiaohong Wang4,Lance D. Miller5, Zhiyong Deng1, Nicole Blanchette1, Tara Arvedson6, Cindy K. Miranti7,Jodie L. Babitt8, Herbert Y. Lin8, Donna M. Peehl9, Frank M. Torti10, and Suzy V. Torti1

Abstract

Hepcidin is a circulating peptide hormone made by the liverthat is a central regulator of systemic iron uptake and recycling.Here, we report that prostate epithelial cells also synthesizehepcidin, and that synthesis and secretion of hepcidin aremarkedly increased in prostate cancer cells and tissue. Prostatichepcidin functions as an autocrine hormone, decreasing cellsurface ferroportin, an iron exporter, increasing intracellulariron retention, and promoting prostate cancer cell survival.Synthesis of hepcidin in prostate cancer is controlled by a

unique intersection of pathways that involves BMP4/7, IL6,Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epi-genetic silencing of SOSTDC1 through methylation is increasedin prostate cancer and is associated with accelerated diseaseprogression in patients with prostate cancer. These resultsestablish a new connection between iron metabolism andprostate cancer, and suggest that prostatic dysregulation ofhepcidin contributes to prostate cancer growth and progres-sion. Cancer Res; 75(11); 2254–63. �2015 AACR.

IntroductionHepcidin is a peptide hormone secreted by the liver that is a

master regulator of systemic iron homeostasis (1). When iron issufficient, hepcidin is synthesized. Hepcidin binds to ferroportin,an iron efflux pump expressed in enterocytes andmacrophages ofthe reticuloendothelial system. Binding of hepcidin to ferroportintriggers ferroportin degradation, thereby blocking iron deliveryfrom enterocytes to the systemic circulation, as well as blockingdelivery of iron catabolized bymacrophages to the circulation (2).Thus, hepcidin reduces levels of systemic iron when iron isabundant. Conversely, when iron is insufficient, hepcidin syn-thesis is reduced, enabling increased iron uptake and recycling,

and restoring iron homeostasis. Inappropriate hepcidin synthesisis triggered under inflammatory conditions and can contribute tothe anemia of chronic disease by reducing iron uptake in patientswith chronic inflammation, infection, and cancer (3).

Threemajor drivers of hepatic hepcidin transcription have beendescribed. These are bone morphogenetic proteins (BMP), par-ticularly BMP6 (4), inflammatory cytokines, particularly IL6, andtransferrin-bound iron (Tf-Fe). These effectors cross-talk in aregulatory pathway that is incompletely understood (5, 6).

Hepcidin is elevated in the serumof patientswith cancer. This isgenerally considered an indirect consequence of the increasedlevels of cytokines present in patients with cancer and the stim-ulatory effect of these cytokines on hepatic hepcidin synthesis (5).However, hepcidin synthesis is not restricted to the liver (7, 8) andtumor tissue itself may also play a role in the increased hepcidinobserved in cancer (7). Furthermore, due to its ability to poten-tiate iron retention in cells, dysregulated hepcidinmay contributenot only to altered systemic iron regulation, but also exert localeffects on tumor growth and malignant potential.

Prostate cancer is the most common cancer in men, and thesecond leading cause of cancer-related mortality among men (9).Locally advanced prostate cancer is treated effectively by androgendeprivation therapies; however, treatments for recurrent meta-static prostate cancer, which is generally androgen insensitive, aresubstantially less effective (10). In this manuscript, we report thathepcidin is synthesized in prostate cancer cells and contributes toprostate cancer cell survival. Furthermore, we demonstrate thathepcidin synthesis in prostate cancer is controlled by a uniqueregulatory cascade that is distinct from the pathway controllinghepcidin synthesis in the liver, and that alterations in this pathwayare associated with biochemical recurrence in patients with pros-tate cancer. These data establish a new link between iron metab-olism and prostate cancer, identify an unanticipated role forhepcidin in prostate cancer, and suggest new targets for regulatingprostate cancer growth and metastases.

1Department of Molecular Biology and Biophysics, University of Con-necticut Health Center, Farmington, Connecticut. 2JK Associates,Conshohocken, Pennsylvania. 3Panagene Inc., Yuseong-gu, Daejeon,Republic of Korea. 4Department of Pathology, University of Connecti-cut Health Center, Farmington, Connecticut. 5Department of CancerBiology, Wake Forest School of Medicine, Winston Salem, NorthCarolina. 6Departmentof Inflammation,Amgen Inc., Seattle,Washing-ton. 7Laboratoryof IntegrinSignaling, Program inSkeletal Disease andTumor Microenvironment, Center for Cancer and Cell Biology, VanAndel Research Institute, Grand Rapids, Michigan. 8Program in Ane-mia Signaling Research, Division of Nephrology, Program in Mem-brane Biology, Center for Systems Biology, Massachusetts GeneralHospital, Harvard Medical School, Boston, Massachusetts. 9Depart-ment of Urology, Stanford University School of Medicine, Stanford,California. 10DepartmentofMedicine,UniversityofConnecticutHealthCenter, Farmington, Connecticut.

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

Corresponding Author: Suzy V. Torti, Department of Molecular Biology andBiophysics, University of Connecticut Health Center, 263 Farmington Avenue,Farmington, CT 06030-3305. Phone: 860-679-6503; Fax: 860-679-3408;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-2465

�2015 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst April 9, 2015; DOI: 10.1158/0008-5472.CAN-14-2465

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Materials and MethodsCell culture

Nonmalignant prostate epithelial cells (PEC; ref. 11) werecultured in keratinocyte –SFM medium (GIBCO) supplemen-ted with 50 mg/L bovine pituitary extract and 5 mg/L humanrecombinant EGF (GIBCO). Cells were used at passages 4–8.Nonmalignant PrEC cells (Lonza) were cultured in LonzaPrEGM media with supplements (Lonza PrEGM Bullet-kit).DU145 cells were cultured in EMEM medium (ATCC) contain-ing 10% FBS (BenchMark). LNCaP cells were cultured in RPMI-1640 (GIBCO) containing 10% FBS. PC3 cells were cultured inF-12K medium (GIBCO) containing 10% FBS. All cells weremaintained at 37�C in a humidified incubator at 5% CO2.DU145 and PC3 cell lines used in these studies were obtainedfrom the ATCC and reauthenticated using STR profiling (ATCC:DU145 July 15, 2014 and PC3 November 18, 2014). LNCaPcells were purchased from the ATCC for this study and werepassaged three to six times.

Western blot analysesFor ferroportin analysis, nonreduced samples were used; other

sampleswere reduced.Western blotswere probedwith antibodiesto Phospho-Smad-1-5-8 (Cell Signaling Technology), total Smad-1 (Cell Signaling Technology), Smad-5, Phospho-STAT3 (CellSignaling Technology), STAT3 (Cell Signaling Technology), hep-cidin (Fitzgerald), ferroportin (Novus Biologicals), GAPDH (Fitz-gerald), or b-actin (Abcam).

Real-time qPCRqRT-PCR was performed essentially as described (12). Addi-

tional details, including primer sequences, are provided in Sup-plementary Methods.

Neutralizing antibody and recombinant protein treatmentsCells were treated with 1 or 3 mg/mL anti-BMP4, anti-BMP6,

anti-BMP7, or anti-IL6 neutralizing antibodies (R&DSystems).Ofnote, 3 mg/mL Isotope-matched anti-IgG was used as a control(R&D Systems). Human recombinant BMP6 (R&D Systems),BMP4, and BMP7 (GIBCO) were used at 50 to 200 ng/mLfor 24 hours. Cells were treated with human recombinant IL6(Peprotech) for 24 hours.

ELISA analysis for secreted hepcidin and IL6Hepcidin was measured in conditioned RPMI-1640 media

using an ELISA from Bachem or USCN Life Science Inc.; bothassays gave similar results in direct comparisons. IL6 was mea-sured using an ELISA from R&D Systems.

Labile iron pool assayCells were cultured in 96-well plates overnight. The labile iron

pool was measured after treatment with 800 nmol/L hepcidin(Peptide International) or 200 mmol/L ferric ammonium citrate(Sigma) for 4 hours (see Supplementary Methods).

ImmunofluorescenceCells were plated on an 8-chamber slide (BD Falcon) for 24

hours before treatment with 800 nmol/L hepcidin (Peptide Inter-national) for 24 hours. Cells were fixed with 4% paraformalde-hyde for 15 minutes and blocked with 5% BSA at 4�C overnight.Anti-human ferroportin 38C8 (Amgen) was applied for one hour

followed by rhodamine-red conjugated secondary antibody(Jackson ImmunoResearch).

Cell viabilityViability was measured using anMTS (Promega) or clonogenic

assay. See Supplementary Methods for details.

Statistical analysisStatistics are reported as the mean � SD. Unless otherwise

noted, significant differences between control and treatmentgroups were determined using the two-tailed unpaired Studentt tests.

Demethylation studies of SOSTDC1DU145 cells were cultured in 2 mmol/L 5-Aza-20-deoxycytidine

(Sigma) or vehicle (DMSO) control for 96 hours with mediachanges every 24 hours. RNA was isolated using TRIzol reagent(Invitrogen), and a Micro-to-Midi total RNA purification system(Invitrogen). GenomicDNAwas isolated using TRIzol andGentraPuregene reagent (Qiagen).

DNA methylation assayDNA samples were modified in preparation for sequencing as

described previously (13). Amplified PCR products were sub-cloned using the TOPO TA Cloning Kit (Invitrogen) and approx-imately 20 individual colonies for each sample picked. PlasmidDNA was directly amplified (Templiphi amplification kit; GEHealthcare) and sequenced using BigDye Terminator v1.1 CycleSequencing Kits (Applied Biosystems). Data were compared withthe UCSC genome reference sequence to assess the methylationstatus of each CpG site (BiQ Analyzer software). Clones with aminimum of 95% bisulfite conversion rate were included insubsequent analyses.

Published methylation data and analysisDNA methylation data from 92 primary prostate cancer

samples and 86 adjacent normal prostate tissues were down-loaded from Gene Expression Omnibus (GSE26126; ref. 14)and analyzed using the Bioconductor lumi package asdescribed (15). The b (methylation) value, a continuous var-iable between 0 and 1 (with b values approaching 1 indicatingcomplete methylation and 0 indicating no methylation), wascalculated as described (16). Association between phenotypeand DNA methylation was assessed using a generalized linearmodel and PROC GENMOD or PROC LIFETEST in SAS soft-ware (SAS Institute Inc; ref. 15).

Luciferase reporter assaysDU145 cells were transfected with 0.1 mg TCF/LEF firefly

luciferase (Qiagen) or hepcidin luciferase construct (17) andconstitutively active Renilla luciferase. Twenty-four hours post-transfection, cells were treated with 20 mmol/L lithium chloride(Calbiochem), 50 mmol/L endo-IWR (R&D Systems), 50 ng/mLWnt3a (R&D Systems), or 100 ng/mL rSOSTDC1/USAG1 (R&DSystems). Luciferase activity was measured using the Dual-Lucif-erase Assay System (Promega).

IHCIHC analysis was performed on formalin-fixed, paraffin-

embedded sections using a mouse monoclonal anti-humanhepcidin antibody (19D12 Amgen) or anti-SOSTDC1 rabbit

Hepcidin in Prostate Cancer

www.aacrjournals.org Cancer Res; 75(11) June 1, 2015 2255




antiserum (18). Additional details are provided in SupplementaryMethods.

ResultsHepcidin is expressed and regulates ferroportin inprostate cells

We first tested whether hepcidin was expressed in a represen-tative selection of cultured prostate cancer cells as well as non-malignant PECs. We examined cancer cells whose proliferation isinsensitive (DU145, PC3) and sensitive (LNCaP) to androgen.Hepcidinwas secretedbyprostate cells andwasmarkedly elevatedin all prostate cancer cells relative to benign prostate cells (Fig.1A). Hepcidin transcripts were similarly elevated in prostatecancer cells (Supplementary Fig. S1A). Amounts of hepcidinsecreted by DU145 cells were comparable with amounts secretedby HepG2 cells (a cell line frequently used to model hepatocytesecretion of hepcidin): 66 � 2 pg/mL/106 cells/24 hours cells inHepG2 cells versus 59 � 8 pg/mL/106 cells/24 hours in DU145cells.

Hepcidin increases intracellular iron by binding to and trig-gering the degradation of the iron efflux pump, ferroportin (2).We testedwhether this pathwaywas conserved in prostate cells. Asshown in Fig. 1B, the increase in hepcidin in prostate cancer cells

was associated with a decrease in ferroportin in these cells whencompared with nonmalignant prostate cells. Furthermore, theaddition of recombinant hepcidin to nonmalignant PECs expres-sing high ferroportin substantially reduced ferroportin (Fig. 1Cand D), and increased intracellular iron (Fig. 1E). To directlyassess whether hepcidin secreted by prostate cells functions in anautocrine fashion to regulate ferroportin, we treated prostatecancer cells with anti-hepcidin antibody. As shown in Fig. 1F,antibody-mediated blockade of hepcidin increased ferroportin.Thus, prostate cells synthesize functional hepcidin and its targetferroportin, and the ferroportin/hepcidin regulatory axis is afunctional autocrine pathway in prostate cells.

Hepcidin expression is increased in human prostate cancersamples

To assess whether hepcidin was similarly increased in prostatetissue from patients with cancer, we queried a database of geneexpression in tissue isolated from human subjects (19). Hepcidinexpressionwas observed in normal prostate, andwas significantlyupregulated in prostate cancer (Fig. 2A). Hepcidin was furtherupregulated inmetastatic disease (Fig. 2A). Ferroportin, the targetof hepcidin, was downregulated in prostate cancer, and furtherdownregulated in metastatic prostate cancer (Fig. 2A). Next, we

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Figure 1.Hepcidin is upregulated in prostatecancer cells and regulates ferroportinand intracellular iron in prostate cells. A,hepcidin in conditioned medium fromnormal PEC and prostate cancer celllines. B, Western blot analysis of pro-hepcidin and ferroportin in normalprostate cells and prostate cancer celllines. Shown is a single exposure of oneblot; dashed line indicates where anirrelevant lane was cropped out. C, PECcells were treated with hepcidin ingrowth medium for 4 hours and levelsof ferroportin assessed byWestern blotanalysis. D, immunofluorescence offerroportin in untreated PEC and PECtreated for 24 hours with 800 nmol/Lhepcidin; ferroportin (red) and nuclei(blue). E, labile iron pool in PEC treatedwith iron (ferric ammonium citrate) orhepcidin. F, Western blot analysis offerroportin in PC3 cells treated withanti-hepcidin antibody or IgG controlfor 24 hours. Mean and SD of triplicatedeterminations; data arerepresentative of one of threeindependent experiments.

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Cancer Res; 75(11) June 1, 2015 Cancer Research2256




used IHC to examine hepcidin levels in prostate tissue containingmalignant as well as benign glands. As shown in Fig. 2B andSupplementary Fig. S2, hepcidin immunoreactivity was observedin malignant glands, localized along the cell membranes withaccentuation towards the glandular lumina. Faint cytoplasmicstaining was also noted. Stromal cells were negative for hepcidin.Thus, PECs and not adjacent stroma are the major source ofhepcidin in prostate cancer.

Hepcidin secretion controls labile iron and affects survival ofprostate cancer cells

We next assessed the consequences of endogenous hepcidinsynthesis in prostate cancer cells. Because hepcidin reduces ironexport by triggering the degradation of ferroportin, the increasein hepcidin observed in prostate cancer cells should be associ-ated with an increase in metabolically available iron (the labileiron pool, LIP). As shown in Fig. 2C, DU145 prostate cancer cellsdo indeed exhibit a larger labile iron pool than benign prostatecells. We tested whether the hepcidin-mediated increase in labileiron promotes cell survival by incubating cells with anti-hepci-din antibody. To avoid confounding effects of exogenous iron,these experiments were performed in serum-starved cells. As

seen in Fig. 2D, blockade of hepcidin reduced viability ofprostate cancer cells. We confirmed this result by performinga clonogenic assay on cells treated with two different anti-hepcidin antibodies compared with cells treated with controlIgG. As seen in Fig. 2E, treatment with anti-hepcidin antibodiesresulted in a statistically significant decrease in cell number.Consistent with these results, treatment with deferoxamine(DFO), an iron chelator, also reduced prostate cancer cellviability (Supplementary Fig. S3). Thus, elevated hepcidin con-tributes to prostate cancer cell survival.

IL6 and BMP4/7 regulate hepcidin synthesis in prostate cellsthrough an autocrine loop

We next focused on mechanisms that regulate hepcidinsynthesis in prostate cells. We first examined pathways knownto regulate hepcidin in hepatocytes: IL6 and BMPs. We treatedDU145 prostate cancer cells with IL6, an inflammatory cyto-kine known to be upregulated in prostate cancer (20). IL6increased hepcidin transcripts (Supplementary Fig. S1B),phosphorylation of STAT3 (Fig. 3A), and secretion of hepcidin(Fig. 3B), suggesting that IL6 triggers hepcidin synthesisthrough STAT3. To test whether IL6 induces hepcidin through

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Figure 2.Hepcidin is upregulated and ferroportinis downregulated in prostate cancerpatient samples. A, transcript levelsof ferroportin and hepcidin innormal prostate (N, green,n ¼ 29), primary tumor (T, blue,n¼ 131), and metastatic tumor (M, pink,n ¼ 19). Data from ref. 19. B, IHC ofhepcidin in prostate tissue. Top,prostate cancer [Gleason score(3þ5) ¼ 8]. Middle, benign prostaticglands and stroma. Bottom, control IgGstaining. CaP, prostate cancer; NP,normal prostate glands. C, labile ironpool in normal PEC and DU145 prostatecancer cells. D,MTS assayofDU145 cellstreated with either IgG control oranti-hepcidin antibody. E, clonogenicassay of DU145 using 3 mg/mLanti-hepcidin antibody from Amgen(#1) or Abcam (#2). Mean and SD oftriplicate determinations; data arerepresentative of one of threeindependent experiments. P valuesreported in D represent differencesbetween anti-hepcidin–treated(1 mg/mL and 3mg/mL) and IgG control;difference between untreated and IgGcontrol was not significant (P > 0.05).






an autocrine loop, we incubated DU145 cells with anti-IL6antibody. As shown in Fig. 3C, anti-IL6 antibody reducedhepcidin secretion, indicating that endogenous synthesis ofIL6 contributes to the increase in hepcidin observed in prostatecancer cells.

We next assessedwhether synthesis of hepcidin in prostate cellsis also dependent on BMPs. We focused on BMP6 and the relatedBMPs 2, 4, and 7, because BMP6 plays a major role in iron-mediated transcriptional induction of hepcidin in the liver (4).BMPs 4, 6, and 7 were all functional in triggering SMAD signalingin prostate cells (Supplementary Fig. S4). However, only BMP4and to a greater extent BMP7 induced hepcidin secretion inDU145 cells (Fig. 3D). BMP6, the major driver of hepcidinsynthesis in the liver, had relatively little effect on hepcidintranscripts (Supplementary Fig. S1C) or secretion (Fig. 3D) inprostate cells.

To test the role of autocrine BMP signaling in hepcidin induc-tion, we treated DU145 cells with antibodies to BMP4, BMP6, orBMP7. As shown in Fig. 3E, depletion of BMP7 (and to a lesserdegree BMP4) by neutralizing antibodies inhibited hepcidinsecretion in these cells. Consistent with the inability of BMP6 toinduce hepcidin in DU145 cells, antibody to BMP6was unable toblock a decrease in hepcidin transcripts (Supplementary Fig. S1D)or hepcidin secretion (Fig. 3E). A preferential ability of BMP7 toinduce hepcidin was also seen in PC3 and LNCaP prostate cancercells (Supplementary Fig. S5).

Hepcidin synthesis in prostate cells is controlled by the BMPantagonist SOSTDC1

BMP antagonists are a major mechanism through which theactivity of BMPs is regulated (21). In particular, the BMP antag-onist Sclerostin domain containing 1 protein (SOSTDC1) directlybinds to and inhibits the activity of BMPs 2, 4, and 7 (22, 23).Because SOSTDC1 is downregulated in cancer (18, 24–26), wetested whether downregulation of this antagonist contributed tothe increased hepcidin expression seen in prostate cancer cells.

We first verified that SOSTDC1 attenuates BMP4/7 signaling inprostate cancer cells by treating cells with either BMP4 or BMP7 inthe presence or absence of recombinant SOSTDC1 and assessingSMAD signaling. As shown in Fig. 4A, SOSTDC1 attenuatesBMP4/7-mediated SMADphosphorylation in these cells.We thentested whether SOSTDC1 would diminish hepcidin synthesis. Asshown in Fig. 4B, treatment of cells with recombinant SOSTDC1reduced hepcidin secretion 3- to 5-fold, as well as reducinghepcidin transcripts (Supplementary Fig. S1E).

Hepcidin is regulated by Wnt signaling in prostate cellsAn unusual feature of SOSTDC1 is its ability to interfere with

canonical Wnt signaling as well as block BMP signaling (27). Thepotent effect of SOSTDC1 on hepcidin synthesis led us to querywhether SOSTDC1 exerted its inhibitory effect on hepcidin syn-thesis by simultaneously targeting both BMP and Wnt pathways.We first tested the involvement of the Wnt pathway in hepcidin

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Figure 3.IL6 and BMP4/7 mediate induction ofhepcidin in prostate cancer cells. A,phosphorylated and total STAT3 inDU145 cells treated with IL6. B,extracellular hepcidin followingtreatment of DU145 cells with 100 or200 ng/mL IL6. C, extracellularhepcidin in DU145 cells treated withanti-IL6 antibody or IgG controlantibody. D, extracellular hepcidin inDU145 cells treated with 100 or200 ng/mL BMP4, BMP6, or BMP7. E,extracellular hepcidin in DU145 cellstreated with 3 mg/mL anti-BMP4, anti-BMP6, anti-BMP7, anti-IL6 antibody,or IgG control antibody. Mean and SDof triplicate determinations; data arerepresentative of one of threeindependent experiments.

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synthesis. In the canonical pathway, Wnt ligands prevent thedegradation of b-catenin by inhibiting a destruction complexcomposed of APC, Axin, GSK3b and casein kinase; stabilizedb-catenin is then able to interact with TCF/LEF transcriptionfactors to trigger the transcription of downstream genes (28). Wetreated DU145 prostate cancer cells with LiCl, an inhibitor ofGSK3b and activator of theWnt pathway (Supplementary Fig. S6;ref. 28), and measured effects on hepcidin transcripts. As shownin Fig. 4C, LiCl treatment increased hepcidin transcripts, an effectthat was attenuated by SOSTDC1. Hepcidin transcripts were alsoincreased in cells treated withWnt3a (Fig. 4C). Conversely, endo-IWR, a small-molecule stabilizer of Axin that acts as a Wntpathway antagonist (29), markedly inhibited hepcidin synthesis(Fig. 4C).

Sequence inspection revealed apotential TCF/LEF transcriptionfactor binding site at –55/-48 nt of the human hepcidin promoter(Fig. 4D), suggesting that activation of TCF/LEF transcriptionfactors by Wnt signaling could directly drive hepcidin transcrip-tion. To test this possibility, we performed transient transfectionswith an hepcidin-luciferase reporter construct in DU145 cells. Asshown in Fig. 4D, treatment of transfectants with either LiCl orWnt3a increased hepcidin promoter-driven luciferase activity, aneffect that was antagonized by SOSTDC1 (Fig. 4D). Conversely,IWR-mediated inhibition of luciferase activitywas blocked in cells

cotreated with IWR and LiCl or Wnt3a (Fig. 4D). Interestingly, asimilar effect was not observed in HepG2 hepatocellular carci-noma cells (data not shown), suggesting that Wnt-mediatedhepcidin induction may be tissue restricted. Furthermore, com-bining SOSTDC1and anti-IL6 antibody to simultaneously inhibitWnt, BMP4/7, and IL6 signaling almost completely preventedhepcidin synthesis (Fig. 4E), suggesting that in conjunction, thesepathways drivemost or all of hepcidin synthesis in prostate cancercells.

SOSTDC1expression is suppressedbypromotermethylation inprostate cancer cells

We asked whether the increase in hepcidin in prostate cancercould be related to decreased levels of SOSTDC1. We first usedqRT-PCR to measure SOSTDC1 expression in prostate cells. Asshown in Fig. 5A and B, SOSTDC1 mRNA and secretion wasdramatically suppressed in DU145 prostate cancer cells whencompared with normal PECs. We tested whether this was due toDNA methylation, an important mechanism of gene silencing incancer (30). DNA methylation often occurs at CpG sites in theproximal promoter, and inhibits the access of transcription factorsto the promoter. We identified a candidate CpG region approx-imately 100 nt from the transcriptional start site of the SOSTDC1gene. As shown in Fig. 5C, this region was heavily methylated in

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Figure 4.SOSTDC1 antagonizes BMP and Wnt-mediated induction of hepcidin.A, phosphorylation of Smad-1-5-8 inDU145 cells treated with BMP4 orBMP7 in the presence or absence ofSOSTDC1 for 24 hours. B, extracellularhepcidin in DU145 cells treatedwith 50or 100 ng/mL rSOSTDC1 for 24 hours.C, hepcidin transcript levels in DU145cells treated with 20 mmol/L LiCl, 20mmol/L LiCl plus 100 ng/mL SOSTDC1,50 ng/mL Wnt3a, 50 ng/mL Wnt3aplus 100 ng/mL SOSTDC1, or50 mmol/L endo-IWR for 24 hours.D, hepcidin promoter-drivenluciferase activity in DU145 cellstreated as in C. Inset is a cartoon of thelocation of BMP and STAT3 (44) sitesand a candidate TCF/LEF site in thehuman hepcidin promoter. E, hepcidinsecretion in DU145 cells treated with1 mg/mL anti-IL6 antibody, 1 mg/mLcontrol IgG, 100 ng/mL recombinantSOSTDC1, or the combination of anti-IL6 and SOSTDC1 for 24 hours. Meanand SD of triplicate determinations;data are representative of one of threeindependent experiments.






prostate cancer cells. Treatment with 5-aza-dC demethylatedthis region, and dramatically increased expression of SOSTDC1(Fig. 5D).

Methylation of SOSTDC1 in prostate tumors is associated withincreased probability of cancer recurrence

To test whether the increasedmethylation of SOSTDC1 that weobserved in prostate cancer cells was also observed in prostatetumors, we queried a publicly available database of DNA meth-ylation profiles from prostate tumors and adjacent prostate tissue(14). This database also contained information on time to pros-tate cancer recurrence, where recurrence was scored by measuringprostate-specific antigen (PSA), a biochemical marker of prostatecancer. As shown in Fig. 6A, the SOSTDC1 promoter was moreextensively methylated in prostate tumors than in normal pros-tate. As predicted from this result, IHC revealed that SOSTDC1expression was reduced in prostate cancer in comparison withbenign prostatic glands in the same patient sample. The reductionwas particularly noticeable in areas of high grade (Fig. 6B andSupplementary Fig. S7).

To determine the prognostic impact of SOSTDC1methylation,we assessed the association of SOSTDC1 methylation with bio-

chemical recurrence of prostate cancer. As shown in Fig. 6C,methylation of SOSTDC1 was associated with increased risk ofprostate cancer recurrence. A model of hepcidin regulation inprostate cancer is shown in Fig. 7.

DiscussionOur data demonstrate that normal prostate cells synthesize

hepcidin, and that hepcidin synthesis is markedly upregulated inprostate cancer cells (Fig. 1) and tissue (Fig. 2).Hepcidin acts in anautocrine fashion and exerts important effects on both normaland malignant prostate cells. It reduces levels of the iron effluxpump, ferroportin (Fig. 1), increases metabolically available iron(Figs. 1 and 2), and promotes survival (Fig. 2). Because prostatecells represent not only a source, but also a local target of hepcidinactivity (Fig. 1), this autocrine regulatory axis may contribute tonormal prostate biology. In addition, although hepcidin hasprimarily been studied for its role in regulation of systemic ironuptake and recycling, these results demonstrate that local synthe-sis of hepcidin by peripheral tissues also have important effects.

We observed that hepcidin was elevated in metastatic as com-pared with localized prostate cancer (Fig. 2A). Metastatic prostate

Control

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Figure 5.Expression of SOSTDC1 is controlledby promoter methylation in prostatecancer cells. A, transcript levels ofSOSTDC1 in DU145 and normal PECcells. B, extracellular SOSTDC1 inDU145 and PEC cells. C, methylation ofthe SOSTDC1 promoter at thedesignated positions in DU145 cellstreated with DMSO or 5-Aza-20-deoxycytidine. D, SOSTDC1 transcriptsin DU145 cells treatedwith 2 mmol/L 5-Aza-20-deoxycytidine or DMSO inthree independent experiments. Meanand SD of triplicate determinations;data in A and B are representative ofone of three independentexperiments.

Tesfay et al.





cancer eventually evolves into disease that no longer responds toandrogen deprivation therapy. Although we did not study theregulation of hepcidin by androgen, we found that hepcidinsynthesis was elevated in both androgen receptor positive andandrogen receptor-negative cell lines (Fig. 1), suggesting thathepcidin may not be a direct target of androgen signaling. Thus,elevated levels of prostatic hepcidinmay persist despite androgendeprivation therapy, possibly contributing to prostate cancerprogression by enhancing prostate cancer cell survival (Fig. 2Eand D). Future studies will be required to directly test thishypothesis.

The regulation of hepcidin synthesis in prostate cancer exhibitsseveral distinctive features. First, it utilizes different BMPs than areused to control systemic hepcidin synthesis in the liver, withBMP4/7 rather than BMP6 exhibiting a prominent role (Fig. 3).BMPs and their receptors are expressed in normal prostate tissueas well as in prostate cancer (31, 32), and both BMP6 (33) andBMP7 (34) are elevated in prostate cancer. Because BMP6 andBMP7 activated SMAD signaling in prostate cells (SupplementaryFig. S5), defects in signaling are unlikely to account for thedifference in hepcidin response to these BMPs.

A second unanticipated feature of hepcidin regulation in theprostate is the involvement of Wnt signaling (Fig. 4), which hasnot previously been implicated in hepcidin regulation. Transfec-tion of prostate cancer cells with a luciferase reporter gene driven

by 2.7 kb of the hepcidin promoter suggests that hepcidin is adirect downstream target of Wnt (Fig. 4). Constitutive Wnt sig-naling has been implicated in many cancers, including prostatecancer (35). Thus, aberrant activation of this pathway mayincrease hepcidin expression in other cancers as well.

The third component of hepcidin regulation in prostate canceris IL6, which also regulates expression of hepatic hepcidin inresponse to inflammation. In the prostate, autocrine secretion ofIL6makes a significant contribution to hepcidin secretion (Figs. 3and 4). IL6 is synthesized by prostate cancer cells (36) and affectstheir differentiation, therapy resistance, and proliferation (37).Higher levels of IL6 have been associated with tumor burden,bone metastasis, and poor survival of patients with prostatecancer (38). Intriguingly, high levels of circulating hepcidin arealso significantly correlated with the presence of metastases inpatients with prostate cancer (39). Thus upregulation of hepcidinmay be onemechanism throughwhich IL6 contributes to prostatecancer progression.

A fourth and new member of the regulatory network control-ling hepcidin synthesis in prostate cancer is SOSTDC1, a BMPantagonist notable for its ability to antagonizeWnt aswell as BMPsignaling (40). BMP antagonists are secreted proteins that controlBMP activity by binding directly to BMPs and preventing themfromproductively engaging their receptors (21). SOSTDC1 antag-onizes BMPs 2, 4, and 7; downregulation of SOSTDC1 has been

P < 0.0001A B

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Figure 6.SOSTDC1 promoter is hypermethylated inprostate tumors. A, methylation of SOSTDC1in benign and malignant human prostatetissue (data from ref. 14). B, SOSTDC1expression in prostate tissue, analyzed byIHC. Benign prostatic glands at left are moreintensely stained than the high grade[Gleason score (5þ4) ¼ 9] prostaticcarcinoma to the right. C, probability ofbiochemical recurrence in prostate cancerpatients with SOSTDC1 methylation in tumortissue that is above or below the mean.






observed in kidney (18), breast (24), and gastric (25, 26) cancers,implicating SOSTDC1 as a potential tumor suppressor.

We observed that SOSTDC1 plays a central role in regulation ofhepcidin in prostate cells through its dual inhibition of BMP andWnt signaling (Fig. 4). Our results demonstrate that SOSTDC1 isdownregulated in prostate cancer cells (Fig. 5) and prostate cancertissue (Fig. 6).Corroborating this result,weobserved that SOSTDC1is silenced inprostate cancer cell linesbypromotermethylation (Fig.6). Silencing of SOSTDC1 has substantial clinical impact, sincemethylation of the SOSTDC1 promoter in patient samples wasassociated with more rapid disease progression (Fig. 6).

These four key observations allowus to develop amodel for theregulation of prostate cancer cell growth through hepcidin-medi-ated control of iron efflux (Fig. 7).

Data presented here provide novel mechanistic insights intopathways governing hepcidin synthesis in cancer cells, and for thefirst time demonstrate a critical role for hepcidin in prostatecancer. We previously observed that expression of ferroportinwas associatedwith breast cancer progression and affected growthof breast tumor xenografts in mice (7). Here, we demonstratethat prostate cancer cells synthesize hepcidin, and that through itseffects on ferroportin, hepcidin is a principal regulator of intra-cellular iron and survival in these cells. We show that hepcidinsynthesis in prostate cancer cells is governed by a unique pathwayinvolvingWnt and SOSTDC1, signalingmolecules not previouslyimplicated in hepcidin regulation in any tissue. Furthermore, wedemonstrate for the first time that epigenetic mechanisms, inparticular methylation of SOSTC1, play an important role in thisprocess. Future studieswill assess the role ofWnt and SOSTDC1 inregulating hepcidin synthesis in other cancer types.

In addition to hepcidin and ferroportin, other proteins con-tribute to maintenance of intracellular iron, including ferritin,which stores intracellular iron, TfR1, which is involved in ironimport, STEAP family proteins, which are involved in intracellulariron trafficking, and the iron regulatory proteins IRP1 and IRP2,among others (41). Ultimately, the contribution of these willneed to be evaluated to create a comprehensive picture of ironmetabolism in prostate cancer.

Ourwork suggests that hepcidin itself, and/or thepathways thatregulate its synthesis, may be a potential therapeutic target orbiomarker of prostate cancer progression (39). The discovery ofthe ferroportin/hepcidin regulatory axis has not only increasedfundamental understanding of mechanisms involved in mainte-nance of iron balance, but also has engendered a vigorous searchfor pharmacologic agents that can target this axis (5). Hepcidinhas been aparticularly attractive target, anddrugs that can increaseor mimic hepcidin as well as drugs that can downregulate hepci-din are both under active investigation as potential therapies(5, 42, 43).Notably, inhibition of hepcidin significantly inhibitedprostate cancer cell survival (Fig. 2), suggesting that agents thatinhibit hepcidinmight find additional application as modulatorsof tumor growth. Furthermore, because prostate cancer cellsexhibit a mechanism of hepcidin regulation that is distinct fromthat regulating systemic hepcidin, it may be possible to differen-tially inhibit these twopathways. Inparticular, itmaybe fruitful toexplore whether perturbation of SOSTDC1, a critical link betweeniron metabolism and prostate cancer, may be used in attainingsuch selective intervention.

Disclosure of Potential Conflicts of InterestJ.L. Babitt and H.Y. Lin have ownership interest (including patents) in

Ferrumax Pharmaceuticals, Inc. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: L. Tesfay, K.A. Clausen, N. Blanchette, H.Y. Lin,D.M. Peehl, F.M. Torti, S.V. TortiDevelopment of methodology: L. Tesfay, J.W. Kim, T. ArvedsonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.W. Kim, P. Hegde, L.D. Miller, Z. Deng, J.L. BabittAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Tesfay, L.D. Miller, N. BlanchetteWriting, review, and/or revision of the manuscript: L. Tesfay, J.W. Kim,N. Blanchette, T. Arvedson, C.K. Miranti, J.L. Babitt, H.Y. Lin, D.M. Peehl,F.M. Torti, S.V. TortiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Wang, C.K. MirantiStudy supervision: F.M. Tort, S.V. Torti

AcknowledgmentsThe authors thank Drs. Nathaniel Dyment and David Rowe for assistance in

image acquisition, and current and previousmembers of the Torti laboratory forinput and constructive discussions.

Grant SupportF.M. Torti and S.V. Torti were supported in part by NIH R01 CA-171101 and

R01 CA188025. H.Y. Lin was supported by NIH grants RO1 DK-069533and RO1 DK-071837. J.L. Babitt was supported in part by NIH grant RO1-DK087727 and aHoward Goodman Fellowship Award from theMassachusettsGeneral Hospital.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 20, 2014; revised December 29, 2014; accepted February 19,2015; published OnlineFirst April 9, 2015.

SOSTDC1 methylation

SOSTDC1 expression

BMP4,7 signaling

Hepcidin

Ferroportin

Tumor iron retention

Poor outcome

Wnt IL6

Tumor cell survival

Figure 7.Working model of transcriptional control of hepcidin in the prostate.


Tesfay et al.




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2015;75:2254-2263. Published OnlineFirst April 9, 2015.Cancer Res Lia Tesfay, Kathryn A. Clausen, Jin Woo Kim, et al. CancerHepcidin Regulation in Prostate and Its Disruption in Prostate

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