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Molecular and Cellular Pathobiology

AR-Regulated TWEAK-FN14 Pathway Promotes ProstateCancer Bone Metastasis

JuanJuan Yin1, Yen-Nien Liu2, Heather Tillman1, Ben Barrett1, Stephen Hewitt3, Kris Ylaya3, Lei Fang1,Ross Lake1, Eva Corey4, Colm Morrissey4, Robert Vessella4, and Kathleen Kelly1

AbstractThe recurrence of prostate cancer metastases to bone after androgen deprivation therapy is a major clinical

challenge. We identified FN14 (TNFRSF12A), a TNF receptor family member, as a factor that promotes prostatecancer bonemetastasis. In experimental models, depletion of FN14 inhibited bonemetastasis, and FN14 could befunctionally reconstituted with IKKb-dependent, NFkB signaling activation. In human prostate cancer, upre-gulated FN14 expression was observed inmore than half of metastatic samples. In addition, FN14 expression wascorrelated inversely with androgen receptor (AR) signaling output in clinical samples. Consistent with this, ARbinding to the FN14 enhancer decreased expression. We show here that FN14 may be a survival factor in low ARoutput prostate cancer cells. Our results define one upstreammechanism, via FN14 signaling, through which theNFkB pathway contributes to prostate cancer metastasis and suggest FN14 as a candidate therapeutic andimaging target for castrate-resistant prostate cancers. Cancer Res; 74(16); 4306–17. �2014 AACR.

IntroductionMetastasis to the bone is one of the most clinically

important features of prostate cancer, although relativelylittle is known about specific pathways that promote tumorgrowth in the bone. Metastatic prostate cancer is treatedwith androgen deprivation therapy, which initially reducessymptoms and tumor cell growth, although recurrence ofcastration-resistant prostate cancer (CRPC) is almost uni-versal (1). Reconstitution of androgen receptor (AR)–medi-ated signaling is a central mechanism leading to CRPC (2, 3).Although some level of AR output, measured by expressionof AR target gene signatures, is almost always observed inprostate cancer, there is significant intertumoral heteroge-neity, especially associated with progression (4–6). Follow-ing the development of castration resistance, a decrease inthe level of an AR gene signature commonly expressed inprostate cancer indicates either reduced AR transcriptionalactivity or a shift in AR-dependent transcriptional specificity

(6, 7). AR output is influenced by factors such as PTEN levelsand the differentiation status of tumors (8–10). In the case ofPTEN loss or N-cadherin expression, compensatory non-ARpathways are thought to significantly contribute to tumorgrowth.

Histopathology of end-stage bone metastases acquired atautopsy or as a result of surgical resections for spinal cordcompressions or pathologic fractures (11–13) has shownthat bone metastases are heterogeneous, even within asingle patient. Importantly, although nuclear AR staining isusually prominent in most cells, non-neuroendocrine AR-negative tumor cells are clearly observed in both CRPC andtreatment-na€�ve metastases (11–14). These findings implythat AR-independent survival in the bone microenvironmentoccurs, and the mechanisms contributing to such survivalare of great interest. The heterogeneity of metastatic diseasesuggests that second-generation AR-directed therapiessuch as abiraterone and enzalutimide most likely will needto be complemented by therapies directed against non-ARpathways.

We describe here the characterization of Fn14 (TNFRSF12A)as a marker of clinical prostate cancer metastases and adeterminant of experimental metastatic capability. Fn14, aTNF receptor family member, is the cognate receptor forTWEAK, a TNF-like cytokine, produced most prominently byinfiltrating immune cells (15). Fn14 expression is low or absentin most normal tissues but can be activated by physiologicmediators such as mitogens, hormones, and cytokines inepithelial, mesenchymal, and neuronal cell types (16). Activa-tion of the TWEAK–Fn14 axis leads to context-dependentresponses, including a prominent role in normal tissue repairand inflammation (15, 16). TWEAK-stimulated Fn14 signalingoccurs through multiple pathways, including NFkB, MAPK,and CDC42/RAC (16, 17).

1Cell and Cancer Biology Branch, Center for Cancer Research, NationalCancer Institute, Bethesda, Maryland. 2Graduate Institute of Cancer Biol-ogy and Drug Discovery, College of Medical Science and Technology,TaipeiMedical University, Taipei, Taiwan. 3Laboratory of Pathology, Centerfor Cancer Research, National Cancer Institute, Bethesda, Maryland.4Department of Urology, University of Washington, Seattle, Washington.

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

J. Yin, Y.-N. Liu, and H. Tillman contributed equally to this article.

Corresponding Author: Kathleen Kelly, Cell and Cancer Biology Branch,Center for Cancer Research, National Cancer Institute, 37 Convent Drive,Room 1068, Bethesda, MD 20892. Phone: 301-435-4651; Fax: 301-435-4655; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3233

�2014 American Association for Cancer Research.

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The overexpression of Fn14 has been reported in varioussolid tumors, where higher Fn14 expression in some tumortypes has been shown to correlate with more advanced gradeand poorer prognosis (18, 19), although it has not beenpreviously addressed whether Fn14 is functionally requiredfor an aggressive phenotype in vivo. Fn14-targeted agentsbased upon antibody binding specificities are being developedand have shown promise in preclinical oncology studies as anew class of therapeutics (20, 21). The study described hereuniquely addresses the role of Fn14 in the development of bonemetastasis, identifies the p50/p65NFkBpathway as a sufficientand necessary downstream mediator of Fn14-dependent bonemetastasis, and establishes an association between Fn14expression and a low canonical AR gene signature phenotype.

Materials and MethodsshRNA-mediated gene silencing and expression rescue inDU145/RasB1 cellsAll genetically engineered cell lines were established using

lentiviral vectors following standard procedures (22). Fn14gene (TNFRSF12A) was silenced with two Fn14 shRNAs in thepLKO.1 lentiviral vector, designed by The RNAi Consortium(TRC; Open Biosystems) and targeting the followingsequences: 50-ATGAATGAATGATGAGTGGGC�30 (sh1) and,50-AGGATGGGCCAAAGCAGCCGG-30 (sh2). Expression-res-cued genes included Fn14 (Genecopeia), IKKbS177E,S181E andNIKDT3 in a pFUGW lentiviral vector with a FerH promoter(23). To achieve controlled expression of target genes, DU145/RasB1 cells stably expressing a Tet-On 3G transactivator werefirst established and further infected with a pFUGW lentiviralexpression clone containing either AR (NM_000044.3) ormutant IkBa super repressor (IkBaSR; IkBaS32A,S36A) underthe control of the TRE3G promoter with an IRES-mCherryreporter. AR or IkBaSR expression was induced with 1 mg/mLdoxycycline.

Chromatin immunoprecipitation assaysChromatin immunoprecipitation (ChIP) assays were per-

formed using the EZ-Magna ChIP A Kit (Millipore) with amodified protocol (24). ChIP reactions were set up using rabbitmonoclonal AR antibody [Clone ER179(2), Epitomics] at 4�Covernight. PCR primers are listed in Supplementary Table S1.

In vivo metastasis assayMetastatic activity was determined using 6- to 7-week-old

male athymic nude mice (Ncr nu/nu) as previously described(22).

Tissue microarray analysisTissue microarray analyses (TMA) containing primary pros-

tate cancer (UWTMA48) or metastases (UWTMA22) wereconstructed at the University of Washington (25). The NYUProstate Cancer Biochemical Recurrence array, CAP6 TMA,was acquired from the Prostate Cancer Biorepository Network(PCBN; http://www.prostatebiorepository.org).Immunohistochemical (IHC) staining conditions are

described in Supplementary Table S2. The intensity scoringmethod for Fn14, TWEAK and p65: 0, none; 1, weak; 2,

moderate/intermediate; 3, strong; and 4, strong and obscuringdetails. The percentages of tumor cells scored for Fn14 andTWEAK were 0, none; 1, up to 25%; 2, up to 50%; 3, up to 75%;and 4, up to 100%. Because a large number of cores contained alow percentage of nuclear p65 staining, an expanded scale wasused for scoring: 0, none; 1, up to 10%; 2, up to 25%; 3, up to 50%;4, up to 75%; and 5, up to 100%. This system yielded a 16 or20-point combined staining score. For Fn14 and TWEAK,combined scores were <2, negative/weak; 3–6, intermediate;and 8–16, strong. For nuclear p65, combined scores were 0,negative; 1–3, weak; 4–6, intermediate; and 8–20, strong.

Clinical outcome analysis using human tumor datasetsmRNA expression data under the GEO accession numbers

GSE21034 (26) and GSE35988 (27) were used. The Taylordataset represents 151 primary and 19metastatic tumors frompatients treated by radical prostatectomy at Memorial Sloan-Kettering Cancer Center. TheGrasso dataset represents a largecohort of heavily pretreated patients with lethal metastaticdisease comprising cancer-matched benign prostate tissues, 59localized prostate cancers, and 35 metastatic CRPCs. Theexpression data and resulting Z-scores were log2 normalized.

A previously described androgen-responsive gene signature(28) was used to determine the correlation of Fn14 expressionlevel andAR output. This gene setwas scored for each tumor bysumming the expression Z-scores within the human prostatecancer cohort. Tumors were median-stratified by Fn14 expres-sion and the mean score of androgen response signature wasdetermined in each group.

Statistical methodsIn vivo animal results, TMA analysis, and clinical outcome

analysis are expressed as plots showing the median and boxboundaries extending between 25th to 75th percentiles, withwhiskers down to theminimum and up to themaximum value.All in vitro data are expressed as mean � SE. Data wereanalyzed using Prism software (GraphPad Software, Inc.) anddifferences between individual groups were determined by theStudent t test or ANOVA followed by Bonferroni post test forcomparisons among three or more groups. P < 0.05 wasconsidered statistically significant. Log-rank test was used forsurvival curve analysis. The association of Fn14 and nuclearp65was analyzed using contingency tables and the Fisher exacttest.

ResultsFn14 is required for experimental prostate cancer bonemetastasis

We previously described the development of a metastaticprostate cancer xenograft model, based upon the activation ofRas effector pathways in the DU145 cell line (22). Activation ofthe Ras pathway is commonly observed associated with pros-tate cancer progression (26, 29). To enrich for bone metastaticactivity, DU145 RasG37 tumor cells were isolated from threeindependent bone metastases and expanded in culture. Inoc-ulation of the bone-derived clones (DU145/RasB), comparedwith the parental RasG37 transformed cells, demonstratedhigher metastatic capacity as determined by more rapid

Fn14 and Prostate Cancer Bone Metastases

www.aacrjournals.org Cancer Res; 74(16) August 15, 2014 4307




development of metastasis and formation of more numerousand larger metastatic lesions (30). To determine whetherchanges in secreted factors or surface receptors correlatedwith bonemetastatic capacity, supernatants from the culturedparental and bone-derived clones were assayed using a com-mercial antibody array designed to detect proteins within thisfunctional class. Two of the most robust differences observedin all three bone-derived clones compared with the non-enriched parental culture were increased levels of the cytokine,TWEAK, and its cognate receptor, Fn14 (Fig. 1A). Western blotanalyses performed on cell extracts confirmed high cell-asso-ciated Fn14 in bone-derived clones (Fig. 1B). To analyze thefunctional significance in bone metastasis development of theFn14–TWEAK pathway, Fn14 was depleted in DU145/RasB1

cells using two different lentivirus-encoded Fn14 shRNAs (Fig.1C). We investigated Fn14 in the development of metastasisbecause it is anticipated to act cell autonomously. In contrast,TWEAK, a secreted product of the microenvironment, poten-tially can be contributed by a nontumor source. Depletion ofFn14 had no effect on the in vitro growth rate (SupplementaryFig. S1A). Following inoculation of Fn14-depleted cells, micedeveloped significantly less bone (Fig. 1D) and brainmetastasis(Supplementary Fig. S1C) comparedwith controls asmeasuredby bioluminescent imaging at week 4 and by the percentageof mice with bone lesions as determined by histology and/orX-ray at morbidity (Table 1). Mice inoculated with Fn14-depleted cells maintained a normal body weight (Supplemen-tary Fig. S1B) and survived longer (Fig. 1E). Morbidity in these

P G37 B1 B2 B3

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Figure 1. Fn14 pathway activity isnecessary for experimental bonemetastasis. A, TWEAK and Fn14protein levels as determined bythe protein array analysis ofconditioned media from parentalDU145 (P), DU145/RasG37 (G37),and three bone metastatic clones(B1, B2, and B3). B, Western blotanalysis of whole-cell lysatesmeasuring Fn14 in parentalDU145(P), G37, and three bonemetastatic clones (DU145/RasB1,B2, and B3). C, Western blotanalysis for Fn14 inDU145/RasB1cells infectedwith lentivirus vectoralone (EV) or lentivirus encodingsilencing shRNA directedtoward Fn14 (Fn14shRNA1or Fn14shRNA2). D,bioluminescence signal of bonemetastasis per mouse for micebearing tumor cells described in Cat week 4. EV: n¼ 9; shRNA1 andshRNA2: n ¼ 10 per group;�, P < 0.05 versus control. E,survival curve of tumor-bearingmice in the three groups from D.�, P < 0.05 versus EV. F, theincidence of bone metastasis inmice bearing PC3/EV tumors andmice bearing PC3/Fn14 shRNA1or two tumors. ��, P < 0.01 versusEV.

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mice mainly was due to brain metastasis. The bone metastasisinhibitory effect was rescued by reexpression of Fn14 (Table 1),demonstrating specificity of the phenotype for Fn14 depletion.To address the generality of this finding, we depleted Fn14 inPC3 prostate cancer cells, originally isolated from a patientbone metastasis. PC3 cells express high levels of Fn14, andalthough depletion did not affect growth in vitro (Supplemen-tary Fig. S1D), it significantly inhibited bone metastasis asmeasured by bone metastasis development (Figure 1F), tumorburden, and survival (Supplementary Fig. S1E and S1F). Takentogether, these data support a functional role for Fn14 in bonemetastatic capability.

Fn14 is expressed in a high percentage of clinicalprostate cancer metastasesTo analyze the prevalence of Fn14 expression in patient

samples relative to prostate cancer progression, we per-formed IHC using antibodies directed against Fn14 in tissuearrays containing a large number of bone and soft-tissuemetastases as well as normal prostate, benign prostatichyperplasia (BPH), and primary prostate cancer specimens(Fig. 2A). Representative images in bone metastases of eitherweak or strong staining, composed of membranous andcytoplasmic labeling, are shown in Supplementary Fig.S2A. A semi-quantitative scoring system of 0 to 16, reflectingintensity of staining and percentage of positive tumor cells,was used. The mean total score (Fig. 2B) as well as individualintensity and extent (Supplementary Fig. S2B) scores ofmetastatic samples were significantly higher than samplesin the other categories. The distribution of scores is shownin Fig. 2C, illustrating Fn14 expression in 10% of normalprostate epithelium, 25% of BPH, 40% of primary prostatecancer, and 75% of metastatic samples. The distribution ofscores between bone and soft-tissue metastases was similar.We next investigated the correlation of Fn14 expression inprimary prostate specimens with tumor progression, mea-sured as increasing PSA levels with time after prostatectomy,usually an indication of recurrence outside the prostate (Fig.2D). Patients with Fn14-positive specimens (22.5% in thisarray) had significantly shorter time to biochemical recur-rence, consistent with published data from an independentcohort of patients (18). These data from patient populationsshow that Fn14 expression occurs in a significant proportionof clinical bone metastases and support the correlation in

primary prostate cancer of Fn14 expression with increasedaggressiveness.

An important question concerns the availability of theTWEAK ligand. TWEAK staining in tissue sections wasobserved across various cellular components, including thecytoplasm, membrane, and extracellular matrix. A high pro-portion of epithelial cells was stained inmost sections, whereasthe staining of stromal and immune components was variable(see Supplementary Fig. S2C for examples of staining). Semi-quantitative analyses demonstrated that relatively similarlevels of TWEAK ligand are constitutively present in normaland pathologic prostate as well as metastatic tissues (Fig. 2E).

The canonical NFkB pathway reconstitutes Fn14-depleted prostate cancer metastasis

The identification of signaling pathways that contribute tothe establishment of prostate cancer bone metastasis is oftherapeutic interest. Fn14 signaling activates canonical andnoncanonical NFkB pathways, mediated by p65/RELA andp52/RELB complexes, respectively (16). Depletion of Fn14 inDU145/RasB1 cells inhibited TWEAK-stimulated p65 and p52nuclear translocation by more than 50% (Fig. 3A). To deter-mine whether NFkB pathways are necessary for the develop-ment of DU145/RasB1 bone metastases, we made use of adoxycycline-inducible, degradation-resistant IkBaSR, whichprevents the translocation of p65 into the nucleus and indi-rectly inhibits synthesis of the p52 precursor, p100 (31). Priordoxycycline induction of IkBkSR led to inhibition of bothNFkBpathways following TWEAK treatment in vitro (Fig. 3B), where-as Fn14-dependent MAPK pathways were not affected (Sup-plementary Fig. S3A). Figure 3C shows the experimental designfor investigating the influence of NFkB signaling duringmetas-tasis development. IkBaSRwas induced either from the time oftumor cell inoculation into the arterial circulation (day 0) orafter micrometastases had been established (day 14). Expres-sion of IkBaSR beginning at either day 0 or 14 led to signif-icantly less metastases after 4 weeks as compared with controlmice (Fig. 3D), although IkBaSR had a relatively less dramaticeffect on in vitro cell growth (Supplementary Fig. S3B). Theinhibition of metastasis by IkBaSR also was reflected by themaintenance of body weight (Supplementary Fig. S3C) andincreased survival (Fig. 3E). These results indicate that block-ing NFkB even after the establishment of micrometastasesinhibits clinically observable metastasis development.

Table 1. The role of the canonical and noncanonical NFkB pathway in bone metastasis development

DU145/RasB1EV

DU145/RasB1Fn14KDEV

DU145/RasB1Fn14KDFn14

DU145/RasB1Fn14KDIKKBS177E/S181E

DU145/RasB1Fn14KDNIKDT3

Bone metastasis 14/18 (78%) 5/19 (26.3%) 12/19 (63.2%) 17/18 (94.4%) 5/16(31.2%)Vs. Fn14KD ��, P < 0.01 �, P < 0.05 �, P < 0.05 NS

NOTE: Shown are the number and percentage of mice that developed bonemetastasis. Fn14-depleted DU145/RasB1 (Fn14KD) weresuper-infected with lentiviruses expressing the indicated genes. Mice were euthanized at morbidity, between 6 to 9 weeks after tumorcell inoculation.Abbreviation: NS, nonsignificant.






To determine whether either NFkB pathway individually issufficient for replacing the Fn14-dependent signal leading tobone metastasis, we used a rescue approach in Fn14-depletedcells. A constitutively active form of IKKb, IKKbS177E,S181E, wasused to reconstitute the p50/p65 pathway, and a truncatedform of NIK lacking the TRAF3 binding site (NIKDT3) was usedto rescue the p52/RELB pathway (Supplementary Fig. S3D;ref. 23). As shown in Fig. 3F, IKKbS177E,S181E expressing Fn14-depleted cells demonstrated elevated levels of nuclearp65 with or without TWEAK treatment, whereas control cellsdemonstrated nondetectable nuclear p65. Similarly, NIKDT3-expressing Fn14-depleted cells showed increased p52 nucleartranslocation independent of TWEAK treatment (Fig.3F). Table 1 summarizes the bone metastatic capacity in eachgroup, demonstrating that NFkB activation is sufficient toreconstitute Fn14-dependent metastasis. The bone metastasisphenotype was reconstituted in Fn14-depleted cells by

IKKbS177E,S181E expression, whereas NIKDT3 expression aloneappeared to be insufficient, suggesting a greater contributionby the p65 pathway. Taken together, these data show thatNFkB activation is necessary and sufficient for the develop-ment of Fn14-dependent bone metastasis.

Nuclear NFkB p65 is observed in clinical prostate cancermetastases

Todetermine the prevalence of the canonical NFkBpathwayin prostate cancer primary tumors and metastases, tissuearrays previously characterized for Fn14 labeling were stainedwith an anti-total p65 antibody. Labeling was observed both inthe nuclear and cytoplasmic compartments. Nuclear labelingof prostate epithelial cells marked activation of the NFkBpathway. Representative images of total p65 labeling in normalprostate and benign and malignant prostate pathologies areshown in Fig. 4A. A semi-quantitative scoring system of 0

B

D

C

Normal BPH PCa Bone metastasisA

E

Figure 2. Expression of Fn14 innormal and pathologic prostatetissues. A, representativehistologic images depictingnormal prostate, BPH, prostatecancer (PCa), and bonemetastasisstained for Fn14. B, plotssummarizing the Fn14 scores indifferent groups of clinical samplesquantified for UWTMA22 andUWTMA48. The number of patienttissue samples is indicated.�, P < 0.05; ��, P < 0.001.C, percentage of tissue sampleswith negative/weak (0–2, openbar), intermediate (3–6, gray bar),or high (�8, black bar) Fn14expression from B. The shadedsections of the bars in themetastasis group represent soft-tissue metastasis, whereas thesolid sections represent bonemetastasis. D, Kaplan–Meier curveof biochemical recurrence rate(BCR) in Fn14-positive (�3) andFn14-negative (�2) patientpopulations from CAP6 TMA.�, P < 0.05 with log-rank test.E, average scores for TWEAKstaining in epithelium inUWTMA22andUWTMA48.n¼27 (normal), 28(BPH), 51 (PCa), and 75(metastasis).

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(negative) to 20 (extensive, strong labeling), reflecting a com-bination of staining intensity and the percentage of positivecells, was used to examine nuclear total-p65 expression acrosscores. Cores were marked as negative if less than 1% of theprostate epithelial cells were positive for nuclear NFkB p65staining.Primary prostate tumors and metastases had a statistically

significant increase in the combined values of intensity andpercentage positivity compared with normal prostate or BPHsamples (Fig. 4B & C). The relatively low overall scores are dueto the majority of samples demonstrating a low overall fre-quency of nuclear staining, consistent with another large-scalestudy (32). This may be the result of transient p65 nuclearlocalization secondary to robust negative feedback mechan-

isms, which have been described for the p50/p65 pathway (33).Interestingly, the categorization of staining intensities forsamples with 1% to 10% positive cells, which fall into theoverall weak/intermediate categories, demonstrated a higherpercentage of intense staining in metastatic samples (Fig. 4D),suggesting relatively increased signaling in advanced diseasesamples. Notably, we also observed a significant associationbetween Fn14 and nuclear p65 staining in metastatic prostatecancer specimens (Fig. 4E), suggesting that Fn14 signalingleads to NFkB activation in clinical disease.

Fn14 expression is inversely correlated with AR statusOne clinically important property of tumor cells is AR

transcriptional output, which can be heterogeneous within

A

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Figure 3. The NFkB pathwaymediates the effects of TWEAK–Fn14. A, Western blot analysis ofnuclear p52 and p65 in DU145/RasB1 and DU145/RasB1Fn14shRNA cells treated withTWEAK for various times. B,Western blot analysis of DU145/RasB1 cells expressing Tet-on TAand TRE3G-IkBaSR with andwithout prior doxycyclineinduction showing IkBa, p65(RelA), p52, p100, and Fn14expression relative to time afterTWEAK treatment. IkBa, Fn14,and the p52 precursor, p100, weredetected in the cytoplasmicfraction; p65 and p52 weredetected in the nuclear fraction.C, schematic of in vivoexperimental design. Mice wereinoculated on day 0 with DU145/RasB1 cells expressing Tet-on TAand TRE3G-IkBaSR. Mice werefed with doxycycline diets startingon day 0 or day 14 to induceIkBaSR expression and imagedweekly to monitor tumor burden.n ¼ 8 to 9 per group. D, BLIanalysis of bone and brainmetastasis per mouse for tumor-bearing mice described in C.Numbers represent mice thatdeveloped metastasis over totalmice in each group at week 4.�, P < 0.05 versus control. E, thesurvival rate of tumor-bearingmicefrom D. F, DU145/RasB1 cellsdepleted of Fn14 were super-infectedwith lentiviruses encodingFn14, IKKB177E,S181E, or NIKDT3.The DU145/RasB1 EV cells areshown for comparison. Westernblot analysis shows nuclear p65and p52 translocation with (þ) orwithout TWEAK (�) treatment for60 minutes. Dox, doxycycline.






and between tumors (4, 14). We observed that Fn14 andTWEAK expression were significantly and highly expressed inAR-negative compared with AR-positive prostate cancer celllines (Fig. 5A), in agreement with published results (18). Todetermine whether AR signaling was able to influence Fn14 orTWEAK expression in prostate cancer cells, we overexpressedAR in PC3 and DU145 cells using a doxycycline-regulatedsystem. As shown in Fig. 5B, when AR is induced in DU145/RasB1 cells, Fn14 and TWEAK RNA levels were decreased afterDHT treatment, whereas the AR antagonist MDV3100 reducedthis effect. Consistent with this finding, Fn14 protein levelsalso were reduced by the presence of AR (Fig. 5C). In LAPC4cells, Fn14 protein levels were regulated by endogenous AR

in the anticipated pattern, demonstrating decreased andincreased levels with DHT and MDV3100 treatment, respec-tively (Fig. 5D).

To interrogate Fn14 and TWEAK expression in patientpopulations, we analyzed a microarray dataset fromMemorialSloan-Kettering Cancer Center composed of 131 primary and19 metastatic prostate cancer samples (26). Because TWEAKand Fn14 expression appeared to be regulated approximatelyin parallel in cell lines, we analyzed the relative expression ofthe two genes in individual patient samples. Interestingly, Fn14and TWEAK RNA levels in individual patients revealed asignificant correlation (R ¼ 0.59; P < 0.0001; Fig. 6A). TWEAKprotein levels tend to be constitutively present, whereas Fn14

ANormal BPH Pca Bone metastasis

CB

ED

Figure 4. NFkB activation inprostate cancer clinical samples.A, representative images and(scores) of tissue cores, includingnormal prostate tissue (0), BPH (0),primary prostate cancer (4;UWTMA 48), and bone metastasis(3; UWTMA22) stained for p65. Inthe bone metastasis core, thecytoplasmic staining is strong,whereas nuclear staining is <10%.B, plots summarizing the p65scores in different groups ofpatient samples. The numbers ofpatient tissue samples in eachgroup are: normal (29), BPH (28),primary prostate cancer (55), andmetastasis (115). ��, P < 0.01versus normal; ##, P < 0.01;###, P < 0.001 versus BPH. C,percentage of TMA tissue sampleswith negative, weak (1–3),intermediate (4–6), or high (>6)nuclear p65 staining from B. D,tissue samples from B with 1% to10% of cell nuclear p65 positivewere further analyzed for stainingintensity. Shown is the distributionof the selected samples for stainingintensity with weak (1),intermediate/moderate (2), andstrong (3) intensity. E, distributionof Fn14 (positive¼�3) and nuclearp65 (positive ¼ �1) expression inmetastatic samples from B(n ¼ 103). P < 0.05.

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protein levels increase with progression (Fig. 2), implying thatthere may be different posttranscriptional regulation ofTWEAK and Fn14. We then analyzed AR pathway activationrelative to Fn14 mean-stratified expression by using a previ-ously reported mRNA signature of AR target genes (28), whichdemonstrated an inverse relationship between Fn14 expres-sion and AR transcriptional output (Fig. 6B). This finding wasvalidated in an independent dataset (27) for a cohort of CRPCsamples (Supplementary Fig. S4A). The Fn14-high samplesdemonstrated more variability in the AR activity signaturethan the Fn14-low samples, suggesting more heterogeneitywithin Fn14-high prostate cancers.

AR downregulates the transcription of TWEAK and Fn14To analyze whether AR directly regulates the transcription

of Fn14 and TWEAK, potential androgen receptor responseelements (ARE) were mapped in the TWEAK and Fn14 pro-moter/enhancer regions (Fig. 6C). LNCaP cells, which expressendogenous AR, were used to perform ChIP assays with anti-AR antibodies, which confirmed AR binding to two AREs (A1and A3) inTWEAK and twoAREs (A5 and A7) in Fn14 upstreamregions (Fig. 6D). Similar ChIP results were obtained in doxy-cycline-induced AR-expressing DU145 cells (DU145-AR; Sup-plementary Fig. S4B). In LNCaP, binding sites for FOXA1, an ARcomplex cofactor, were detected at the same positions as AR

DOX − + +DHT − − +

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Figure 5. AR downregulatesTWEAK and Fn14 expression.A, RT-PCR analysis for Fn14 andTWEAK in two AR-positiveprostate cancer cell lines, LNCaPand 22RV1, and two AR-negativeprostate cancer cell lines, PC3 andDU145. B, RT-PCR analysis forAR, TWEAK, and Fn14 in DU145/RasB1 cells expressing Tet-on TAand TREG3-AR with or withoutprior doxycycline induction for 2days and subsequently treatedwith DHT (5 nmol/L) or MDV3100(10 mmol/L) for 2 days. C, Westernblot analysis of whole-cell lysatesfor AR and Fn14 protein levelsfollowing doxycycline induction ofAR in the indicated cells. Cellswere treated or untreatedwith DHT(5 nmol/L) for 2 days. D, Westernblot analysis of Fn14 protein levelin LAPC4 treated with DHT(5 nmol/L) or MDV3100 (10 mmol/L)in charcoal-stripped serum for2 days.






(Fig. 6D). To determine whether AR activation was able todirectly regulate TWEAK and Fn14 transcription, the TWEAKupstream region between �4,006 and �2,183 and the Fn14upstream region between �2,434 and þ1 were separatelycloned into an RFP reporter construct and transfected intoLNCaP cells. The two reporters demonstrated approximatelysimilar activity patterns. AR transcriptional activation withDHT suppressed reporter activity, whereas suppression ofbasal AR transcription with the AR antagonist, MDV3100,

induced reporter activity (Fig. 6E). Mutations in the definedAREs, especially TWEAK A1 and Fn14 A7, decreased DHT-dependent suppression and MDV-dependent induction rela-tive to no treatment, demonstrating AR-mediated effects.Consistent with the results in LNCaP, AR-mediated suppres-sion of reporter activity also was observed upon doxycyclineinduction in DU145 cells (Supplementary Fig. S4C). Takentogether, these data suggest that AR binding to Fn14 andTWEAK upstream regions is transcriptionally suppressive.

A

C

D

E

ARE−3,000

−3,804

2,076

1,8061,506

−3,647

−3,248

−2,843

A1 A2 A3 A4

TNFSF12A(TWEAK) −4,006/−2,183

+1

A6 A7 A5 −1,000

−2,434/+1

−2,000

TNFRSF12A(Fn14/TWEAK R)

+1

B

Figure 6. AR downregulates Fn14and TWEAK transcription inprostate cancer cells. A,correlation analysis of TWEAK andFn14 expression levels in theMSKCC prostate cancer dataset.r ¼ 0.63; P < 0.001. B, AR activitysignature genes expressed as asummed Z-score for samplesseparated on the basis of Fn14expression above the median(Fn14 high) or below the median(Fn14 low) in the MSKCC dataset.��, P < 0.001. C, schematic ofpredicted AREs in the Fn14 andTWEAK promoter regions. D,qChIP analysis of AR and FOXA1binding to predicted AREs in theTWEAK and Fn14 promoters/enhancers illustrated in C. LNCaPcells were maintained in DHT-containing media. The bindingactivity of each protein to each siteis given as a percentage of totalinput then normalized to individualimmunoglobulin G (IgG)background. E, TWEAK and Fn14promoter reporter activity asmeasured by relative medianfluorescence intensity (MFI) inLNCaP cells. Cells were treatedwith vehicle (Veh) or DHT (5 nmol/L)or MDV3100 (10 mmol/L) for 72hours. Mutant TWEAK (mutant A1,M1; mutant A3, M3) and Fn14(mutant A5, M5; mutant A7, M7)promoter reporters were used ascontrols. �, P < 0.05 versus Veh;#, P < 0.05 versus DHT. Datarepresent the mean � SEM.

Yin et al.





Because higher Fn14 andTWEAKRNA levels in patient samplescorrelate with lower AR signature gene expression, Fn14 andTWEAK may represent genes expressed in association withoverall lower AR activity or altered AR specificity.

DiscussionAlthough bone metastasis is a primary reason for morbidity

and mortality in patients with progressive prostate cancer,relatively little is known about themechanisms responsible forestablishing and expanding prostate cancer bone metastases(34). Using experimental models in addition to molecular andhistologic analyses of clinical samples, we present evidencethat Fn14 is one determinant of prostate cancer bone meta-static capacity. Fn14 staining also has been observed in about50% of clinical breast cancer bone metastases, although thenumber of samples analyzed was small (35).Of particular interest, Fn14 expression suggests a mecha-

nistic link between prostate cancer bone metastasis andinflammation as the TWEAK–Fn14 axis promotes local inflam-mation (15). An inflammatory microenvironment in the bonecontributes to the so-called "vicious cycle" mediated by inter-acting stromal, immune, and tumor elements and resulting inbone remodeling and enhanced tumor cell survival and pro-liferation (36). From the data presented here, it seems thatFn14-expressing prostate cancer cells often produce autocrineTWEAK in addition to TWEAK secretion from infiltratinginflammatory cells. The bone metastasis model used in thisstudy identifies the Fn14 pathway as one upstream regulator ofNFkB signaling in prostate cancer, and shows that the canon-ical NFkB pathway is sufficient to replace Fn14 downstreamsignaling in this context. Fn14-mediated responses are usuallyproinflammatory as a result of NFkB induction of inflamma-tory proteins, including cytokines, chemokines, adhesionmolecules, and metalloproteases (37). Thus, Fn14-dependenttumor responses are anticipated to increase inflammatory cellinfiltration and activation. In addition, the Fn14–TWEAK axishas been shown to autonomously regulate tumor cells withrespect to survival, proliferation, migration, and progenitorexpansion (15, 16). Thus, both autonomous and nonautono-mous Fn14-mediated functions may contribute to prostatecancer bone metastasis.Our data are consistent with various preclinical models that

support a role for NFkB in prostate cancer metastasis. Analternative mechanism of NFkB activation, loss of the DAB2IPtumor suppressor, leading to experimental prostate cancersoft-tissue metastasis has been described (38). Of interest,coordinated RAS and NFkB activation occur in the DAB2IPmodel and in the model investigated here. DAB2IP loss reg-ulates both RAS and NFkB activation, whereas the DU145/Rasmodel expresses constitutive RAS activation in addition toFn14-regulated NFkB. It remains an open question as to howdirectly or indirectly RAS signaling is associated with Fn14 andTWEAK expression in prostate cancer. In another study, themanipulation of NFkB pathway components in human pros-tate cancer cell lines demonstrated that NFkB activation isnecessary and sufficient for tumor cell growth following intra-tibial injections (39). Finally, a role for the NFkB pathway has

been implicated in models of castration-resistant growth (40,41); castration resistance strongly correlates clinically with thedevelopment of metastasis (12, 13).

The increased presence of NFkB signaling in tumor tissuerelative to normal tissue and a positive correlation with Fn14expression was confirmed with IHC staining for nuclear p65(Fig. 4). The low frequency of positive cells may reflect thenormally transient nature of p50/p65 signaling, and are con-sistent with a recent study showing a significant association ofeven low numbers of nuclear p65 positive cells with higherGleason score, increased biochemical recurrence, and thedevelopment of metastases (32, 42, 43).

Fn14 expression is regulated by a variety of extracellularstimuli, including growth factors and hormones, although themolecular mechanisms of such regulation are mostly unknown(16). As shown here, Fn14 expression seems to increase inprostate cancer cells in which AR activity is suppressed, intrin-sically low, or shifted in specificity. We describe an AR-depen-dent, negative regulatory component influencing TWEAK andFn14 expression in prostate cancer cells. (Fig. 6). The ability toidentify prostate cancer cells with a modified AR response,which are anticipated to be less sensitive to androgen depri-vation therapy (ADT), is of potential utility considering theinter- and intratumoral heterogeneity of prostate cancer (4–6).Thus, an Fn14-targeted agent may permit the imaging and/ortherapeutic treatment ofmetastatic tumors, especially cells thatescape first-line ADT due to modified AR signaling.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: J. Yin, Y.-N. Liu, K. KellyDevelopment of methodology: J. Yin, Y.-N. Liu, H. Tillman, B. BarrettAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Yin, H. Tillman, B. Barrett, S. Hewitt, L. Fang, R. Lake,E. Corey, C. Morrissey, R. VessellaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Yin, Y.-N. Liu, H. Tillman, B. Barrett, S. Hewitt,L. Fang, E. CoreyWriting, review, and/or revision of the manuscript: J. Yin, Y.-N. Liu,H. Tillman, S. Hewitt, E. Corey, C. Morrissey, K. KellyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Yin, Y.-N. Liu, H. Tillman, B. Barrett,K. Ylaya, L. FangStudy supervision: J. Yin, K. Kelly

AcknowledgmentsThe authors thank Drs. Celestia Higano, Martine Roudier, Lawrence True,

Paul Lange, Bruce Montgomery and Peter Nelson.

Grant SupportThis research was supported by the Intramural Research Program of the NIH,

National Cancer Institute, Center for Cancer Research. Y.-N. Liu was supportedby the New Staff Research Project Grant, Taipei Medical University, Taiwan(TMU101-AE1-B20). The Prostate Cancer Donor Program is supported by thePacific Northwest Prostate Cancer SPORE (P50CA97186), the PO1 NIH grant(PO1CA085859), and the Richard M. Lucas Foundation.

The costs of publication of this article were defrayed in part 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 November 12, 2013; revised May 20, 2014; accepted May 31, 2014;published OnlineFirst June 26, 2014.






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2014;74:4306-4317. Published OnlineFirst June 26, 2014.Cancer Res JuanJuan Yin, Yen-Nien Liu, Heather Tillman, et al. Bone MetastasisAR-Regulated TWEAK-FN14 Pathway Promotes Prostate Cancer

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