Translational Cancer Mechanisms and Therapy

Leukemia Inhibitory Factor Promotes Castration-resistant Prostate Cancer and NeuroendocrineDifferentiation by Activated ZBTB46Yen-Nien Liu1,2,3, Shaoxi Niu4,Wei-Yu Chen5,6, Qingfu Zhang7, Yulei Tao8,Wei-Hao Chen1,Kuo-Ching Jiang1, Xufeng Chen8, Huaiyin Shi9, Aijun Liu9, Jinhang Li9, Yanjing Li8,Yi-Chao Lee10,11, Xu Zhang4, and Jiaoti Huang8

Abstract

Purpose: The molecular targets for castration-resistantprostate cancer (CRPC) are unknown because the diseaseinevitably recurs, and therapeutic approaches for patients withCRPC remain less well understood. We sought to investigateregulatory mechanisms that result in increased therapeuticresistance, which is associated with neuroendocrine differen-tiation of prostate cancer and linked to dysregulation of theandrogen-responsive pathway.

Experimental Design: The underlying intracellular mech-anism that sustains the oncogenic network involved inneuroendocrine differentiation and therapeutic resistance ofprostate cancer was evaluated to investigate and identifyeffectors. Multiple sets of samples with prostate adenocarci-nomas and CRPC were assessed via IHC and other assays.

Results: We demonstrated that leukemia inhibitory factor(LIF)was inducedby androgendeprivation therapy (ADT) and

was upregulated by ZBTB46 in prostate cancer to promoteCRPCandneuroendocrine differentiation. LIFwas found tobeinduced in patients with prostate cancer after ADT and wasassociated with enriched nuclear ZBTB46 staining in high-grade prostate tumors. In prostate cancer cells, high ZBTB46output was responsible for the activation of LIF-STAT3 signal-ing and neuroendocrine-like features. The abundance of LIFwas mediated by ADT-induced ZBTB46 through a physicalinteraction with the regulatory sequence of LIF. Analysis ofserum from patients showed that cases of higher tumor gradeand metastatic prostate cancer exhibited higher LIF titers.

Conclusions: Our findings suggest that LIF is a potentserum biomarker for diagnosing advanced prostate cancerand that targeting the ZBTB46–LIF axis may thereforeinhibit CRPC development and neuroendocrine differentia-tion after ADT.

IntroductionTo date, prostate cancer therapies have attempted to suppress

androgen receptor (AR) activity, but despite initial responses,recurrent castration-resistant prostate cancer (CRPC) eventuallyensues (1). A subset of patientswith advancedCRPCmayprogressto an AR-suppressed phenotype and histologically exhibit strongneuroendocrine characteristics called castration-resistant neuro-endocrine prostate cancer (CRPC-NE; ref. 2). CRPC-NE has beendemonstrated to be resistant to androgen deprivation therapy

(ADT) and displays a high metastatic propensity with an averagesurvival of less than a year (3). Currently, there are no targetedtherapies available that can lead to more effective treatments forpatients with CRPC-NE (4). Chemotherapeutic strategies and anew generation of drugs that target AR signaling often involvemanagement to increase the efficacy of CRPC-NE treatment;however, side effects and the development of treatment resistanceremain problematic (5). Therefore, identifying consensus targetsnot involving AR signaling could lead to more curative therapiesfor CRPC-NE.

1Graduate Institute of Cancer Biology and Drug Discovery, College of MedicalScience and Technology, Taipei Medical University, Taipei, Taiwan. 2TMUResearch Center of Cancer Translational Medicine, Taipei Medical University,Taipei, Taiwan. 3Ph.D. Program for Cancer Molecular Biology and Drug Discov-ery, College of Medical Science and Technology, Taipei Medical University,Taipei, Taiwan. 4Department of Urology, State Key Laboratory of KidneyDiseases, Chinese PLAGeneral Hospital, Chinese PLAMedical Academy, Beijing,China. 5Department of Pathology,Wan Fang Hospital, Taipei Medical University,Taipei, Taiwan. 6Department of Pathology, School of Medicine, College ofMedicine, Taipei Medical University, Taipei, Taiwan. 7Department of Pathology,The First Affiliated Hospital and College of BasicMedical Sciences, ChinaMedicalUniversity, Shenyang, China. 8Department of Pathology, Duke University Med-ical Center, Durham, North Carolina. 9Department of Pathology, The PLAGeneral Hospital, Beijing, China. 10PhD Program for Neural Regenerative Med-icine, College of Medical Science and Technology, Taipei Medical University,Taipei, Taiwan. 11Center for Neurotrauma andNeuroregeneration, Taipei MedicalUniversity, Taipei, Taiwan.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Y.-N. Liu and S. Niu contributed equally to this article.

CorrespondingAuthors:Yen-Nien Liu, Taipei Medical University. 250Wu-HsingStreet, Taipei 11031, Taiwan. Phone: 8862-2736-1661, ext. 7626; Fax: 8862-2655-8562; E-mail: liuy@tmu.edu.tw; Xu Zhang, Department of Urology, Chinese PLAGeneral Hospital, 28 Fuxing Road, Haidian District, Beijing 100853, China.Phone: 8610-6693-8008; Fax: 8610-6822-3575; E-mail:xzhang@tjh.tjmu.edu.cn; and Jiaoti Huang, Department of Pathology, DukeUniversity School of Medicine. Room 301M, Duke South, DUMC 3712, 40 DukeMedicine Circle, Durham, NC 27710. Phone: 919-668-3712; Fax: 919-681-0778;E-mail: jiaoti.huang@duke.edu

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doi: 10.1158/1078-0432.CCR-18-3239

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Severalmolecularmechanisms have been implicated in ligand-induced NE differentiation events, including activation ofPI3K (6), MAPK (7), and STAT pathways (8). Dysregulation ofSTAT signaling at the level of ligands, receptors, or effectors hasbeen shown to promote the initiation and progression of manymalignancies (9, 10). Leukemia inhibitory factor (LIF) activatesboth the Janus kinase (JAK)/STAT and RAS/MAPK pathways bymediating a specific LIF receptor (LIFR; ref. 11). LIF is overex-pressed in breast cancer and activates STAT3 signaling, leading tothe induction of proliferation, epithelial-to-mesenchymal transi-tion (EMT), and resistance to drug responses (12, 13). In theprostate microenvironment, activation of LIF–STAT signalingoccurs specifically through the phosphorylation of STAT3 and isinvolved in the appearance of metastasis and aggressive process-es (8, 14). Our understanding of the mechanisms by which LIF isstimulated in prostate cancer is incomplete; in particular, itsinvolvement in CRPC development and NE differentiation andits contribution to ADT-induced drug resistance are unclear.

In prostate cancer cells, prolonged exposure to IL6 has beenshown to reduce AR expression (15) and induce resistance toAR-targeted treatment (16). LIF is a member of the IL6 cytokinefamily and regulates embryonic stem cell self-renewal (17). LIF isa particularly important factor during critical stages of centralnervous system development (18) and can induce neuronalplasticity in pancreatic cancer (19). LIF stimulates the differenti-ation of neuroglial cells and induces their migration by activatingJAK/STAT3/AKT signaling (19). LIF acts as a key differentiationregulator of stem cells and as a neuropoietic cytokine (20, 21). LIFalso promotes the differentiation of an NE phenotypic switch inmurine corticotropic cells (22). LIF signaling is often aberrantlyactivated in later stages of advanced metastatic cancers (23);however, it is not known whether LIF contributes to the devel-opment of CRPC or NE differentiation in patients with prostatecancer following ADT. Herein, we addressed this question byfocusing on regulatory mechanisms between LIF and the progres-sion of CRPC and NE differentiation during the dysregulation ofAR signaling pathways.

Our earlier study demonstrated that ADT stimulated the abun-dance of a prostatic tumor promoter (ZBTB46) and promotedEMT through the transcriptional regulation of SNAI1 (24). Here-in, we demonstrated that the abundance of ZBTB46 was involvedin LIF induction by directly and physically interacting with LIF.It is not clear whether LIF is particularly associated withCRPC development and NE differentiation following additionalAR-targeted therapy.We identified consensusmolecular pathwaysin prostate cancer cells modulated by the LIF-induced CRPCand NE-like phenotype via activated ZBTB46. We demonstratedthat silencing of LIF by LIF-knockdown or treatment with anLIF inhibitor could suppress tumor growth and NE markerexpression. Our results thus have potential implications forthe development of effective treatment strategies for advancedprostate cancer.

Materials and MethodsCell culture

The prostate adenocarcinoma cell line LNCaP, castration-resistant adenocarcinoma cell line C4-2, and AR-suppressed pros-tate cancer cell line PC3 (25) were obtained from the ATCC andcultured in RPMI1640 medium (Invitrogen) supplemented with10% FBS (Invitrogen). The RasB1 cell line (an aggressive cellline expressing a constitutively active Ras in DU145 cells andisolated from a bone metastasis) was provided by Dr. KathleenKelly (NCI/NIH, Bethesda, MD) and maintained as describedpreviously (24, 26–28). The small-cell neuroendocrine carcinoma(SCNC) cell line NCI-H660 was purchased from the ATCC andcultured in RPMI1640medium supplementedwith 0.005mg/mLinsulin (Sigma-Aldrich), 0.01mg/mL transferrin (Sigma-Aldrich),30 nmol/L sodium selenite (Sigma-Aldrich), 10 nmol/L hydro-cortisone (Sigma-Aldrich), 10 nmol/L ß-estradiol (Sigma-Aldrich), 4 mmol/L L-glutamine (Invitrogen), and 5% FBS.Dihydrotestosterone (DHT; Sigma-Aldrich) and LIF protein (R&DSystems) were used to treat cells at 10 nmol/L and 200 ng/mL,respectively, for 24 hours in a 10% charcoal-stripped serum(CSS)-containing medium. The AR antagonist enzalutamide(MDV3100; Selleck) and the first-in-class steroidal LIF inhibitorEC330 (MedChemExpress) were used to treat cells at concentra-tions of 10 mmol/L and 35 nmol/L, respectively, for 24 hours in10% FBS-containing medium.

IHC stainingWe collected 18 cases of clinical tissue samples from patients

with prostate cancer who were treated before and after ADT fromTaipei Medical University-Wan Fang Hospital (Taipei, Taiwan).Tissue microarray (TMA) sections, including 16 normal prostaticepithelial samples, 100 primary prostate adenocarcinomas, and 8SCNCs were collected from Duke University School of Medicine(Durham,NC). Tissue samples were obtained and used accordingto protocols approved by the Taipei Medical University-JointInstitutional Review Board (approval no. N201711067) and theDuke University School of Medicine-Institutional Review Board(protocol ID: Pro00070193) in accordance with Declarationof Helsinki and U.S. Common Rule. IHC staining of ZBTB46,LIF, Chromogranin A (CgA/CHGA), neuron-specific enolase(NSE/ENO2), Ki67, phosphorylated (p)-STAT3, and cleaved cas-pase-3 was performed using the antibodies listed in Supplemen-tary Table S4. Thepathologic diagnoses andGleason score of thesecases were microscopically reconfirmed by two pathologists

Translational Relevance

We show the underlying mechanisms that contribute toandrogen deprivation therapy (ADT) resistance and reveal thepivotal mechanisms responsible for the development of cas-tration-resistant prostate cancer (CRPC). We investigated theoncogenic role of leukemia inhibitory factor (LIF), whichpromotes therapeutic resistance and neuroendocrine differen-tiation in ADT-treated prostate cancer. This research isimportant because it potentially integrates the impact ofcommon hormonal therapy resistance observed in patientswith CRPC. This property is supported by results from clinicalsamples exhibiting a direct link between increased LIF expres-sion in prostate tissue samples with ADT resistance andmetastasis. Importantly, we elucidate the effect of a first-in-class steroidal LIF inhibitor in reducing the tumor growthrate and neuroendocrine marker expression by inhibitingthe LIF–STAT3 pathway. Our findings propose a novel linkbetween neuroendocrine differentiation and ADT-inducedLIF expression and provide new biomarkers and therapeutictargets for advanced prostate cancer.

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(W.-Y. Chen and Q. Zhang). The intensity was denoted as 0(negative), 1þ (weakly positive), 2þ (moderately positive),and 3þ (strongly positive). H-score values (range 0�300) werecalculated according to the following formula: [(% cells withan intensity of 1þ)þ 2� (% cells with an intensity of 2þ)þ 3�(% cells with an intensity of 3þ)].

Invasion assayFor the invasion assay, LNCaP and C4-2 cells stably

expressing the LIF expression vector or PC3 and RasB1 cellswith LIF knockdown were resuspended at a concentration of2 � 105 cells/mL in FBS-containing medium. Matrigel (BDBiosciences)-coated transwell dishes were prepared by adding200 mL of Matrigel diluted 10-fold in FBS-containing medium.Cells that had invaded the Matrigel-coated transwells after 10hours were fixed and stained with a 0.5% crystal violet fixativesolution for 15 minutes. Invaded cells on the underside of themembrane were quantified in five medium-power fields foreach replicate in triplicate.

Proliferation assayLNCaP and C4-2 cells stably expressing the LIF expression

vector or PC3 and RasB1 cells with LIF knockdown were seededat a density of 2� 103 cells/well in 96-well plates. LNCaP, C4-2,PC3, and NCI-H660 cells were treated with 0, 10, 20, 30, 35,40, 50, and 100 nmol/L EC330 for 24 hours and analyzed usinga Cell Proliferation Assay Kit (Promega catalog # G4000)according to the manufacturer's protocol. For the experiment,multiple wells were assessed at each time point and thenaveraged. The cells were stained with a 0.5% crystal violetfixative solution for 15 minutes, rinsed in distilled water, anddissolved in 50% ethanol containing 0.1 mol/L sodium citrate.The absorbance was quantified at a wavelength of OD 550 nmon a plate reader.

Colony-forming assayA colony-forming assay was performed using single-cell sus-

pensions of C4-2 and LNCaP cells stably expressing the LIFexpression vector or PC3 and RasB1 cells with LIF knockdown.Cells were seeded at a density of 500 cells/well in 6-well plates intriplicate and incubated for 14 days at 37�C in a humidifiedincubator. After incubation in a 0.5% crystal violet fixative solu-tion for 15 minutes, colonies of more than 50 mm in diameterwere counted and quantified for each replicate in triplicate.

Tumorigenicity assays in miceAnimal work was performed in accordance with a protocol

approved by the Taipei Medical University Animal Care andUse Committee (approval no. LAC-2017-0269, Taiwan). For thetumorigenicity assays, 5-week-old male nude mice (NLAC,Taiwan) were subcutaneously injected with 2.5 � 106 LIF shRNAvector-transfected PC3 cells in 50%Matrigel (BDBiosciences). ForEC330 treatment, mice were injected with PC3 or NCI-H660cells for 1 month or 18 days, respectively, and then treated with2.5 mg/kg EC330 or DMSO as the control by intraperitonealinjection once every two days. The tumor size was measuredweekly or twice a week, respectively, with calipers, and thetumor volumewas calculated using the following formula: tumorvolume ¼ (4/3) � (L/2) (W/2)2, where L is the length and W isthe width. The results are presented as the mean � SE for eachexperimental group.

Immunofluorescence stainingA CRPC TMA section was obtained from Duke University

School of Medicine using LIF (Abcam cat. # ab135629,) and CgA(Abcam cat. # ab15160) antibodies at 1:100 and 1:150 dilutions,respectively. Tissue sections were washed with PBS containing0.1% Tween-20, incubated with Alexa-488- and/or Alexa-568-conjugated immunoglobulin G (IgG) in 2% BSA for 30 min atroom temperature, and finally washed and mounted usingFluoro-gel II anti-fade reagent with DAPI (Electron MicroscopySciences cat. # 17984-24). Fluorescent signal images were cap-tured using an inverted and/or upright fluorescence Axioplanmicroscope (Zeiss).

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assays were

performed using an EZ magna ChIP A kit (Millipore catalog no.17-10086) with a modified protocol (29). For each sample,107 cells in 10-cm dishes were treated with DHT (10 nmol/L) orMDV3100 (10 mmol/L) in 10% CSS- or FBS-containing mediumas indicated for 10 hours. Nuclear extract preparation, immuno-precipitation, and DNA-purification steps were performedaccording to the protocol provided by Millipore. Quantitative(q)PCRwas performed in triplicate with 1mL of eluted chromatin.Enrichment is presented as a percentage of the total input nor-malized to IgG. ChIP antibodies and PCR primers are listed inSupplementary Table S5.

Promoter reporter assayFor promoter reporter assays, cells in 12-well plates (5 � 104

cells/well) were transiently cotransfected with 1 mg of thepromoter reporter and 1 mg of the cDNA expression vectoror 100 nmol/L siRNA followed by DHT (10 nmol/L) orMDV3100 (10 mmol/L) treatment for 24 hours in 10% CSS- orFBS-containing medium, respectively. Reporter activity was ana-lyzed as the relative median fluorescence intensity (MFI) valuesfor the GFP andwasmeasured by FACS using FACSDiva softwareand normalized to the value of the vehicle.

ELISASera from healthy donors (20 samples), patients with benign

prostatic hyperplasia (BPH; 20 samples), patients with prostatecancer (20 samples), and patients with prostate cancer with bonemetastasis (20 samples) were collected from the Chinese PLAGeneral Hospital (Beijing, China). All human sera were collectedin June and July of 2018. The eight BPH patients and 18 patientswith prostate cancerwhohadbeen tested for LIF serum levelswereselected for LIF, CgA, and NSE examination by IHC staining.Written informed consent was obtained from all patients, and thestudy was approved by the Ethics Committee and InstitutionalReview Board of the Chinese PLA General Hospital in accordancewithDeclarationofHelsinki. LIF titration in serumwasperformedwith the LegendMaxHumanLIF ELISAKit (BioLegend catalog no.443507) according to the manufacturer's instructions.

Statistical analysisAll data are presented as the mean � SEM. Statistical calcula-

tions were performed with GraphPad Prism analytic tools. Differ-ences between individual groups were determined by Studentt test or one-way ANOVA followed by Bonferroni post hoc test forcomparisons among three or more groups. A log-rank test wasused for the survival curve analysis. The method for determining

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cutoffs was predecided for half of the patients, and P <0.05 wereconsidered statistically significant.

ResultsThe LIF–STAT3 pathway induces neuroendocrinedifferentiation

To better understand the transcriptome profile consistent withCRPC-NE, we checked the levels of mRNAs of AR, LIF, andneuroendocrine markers [chromogranin A (CgA/CHGA) andneuron-specific enolase (NSE/ENO2)] expression in a panel ofthe cell lines. We found that the AR-suppressed PC3 and RasB1cells and SCNC cell lineNCI-H660 expressed lower AR and higherLIF and neuroendocrine markers than did the prostate adenocar-cinoma cell lines LNCaP and C4-2 (Supplementary Fig. S1A–S1D). We sought to determine whether activation of the LIF-STAT3 pathway could modulate the neuroendocrine differentia-tion of prostate cancer. We investigated the effect of LIF on theneuroendocrine differentiation of adenocarcinoma cells andobserved an induction of p-STAT3, LIFR, and neuroendocrinemarkers in LNCaP and C4-2 cells treated with increased LIFprotein (Fig. 1A; Supplementary Fig. S1E). To determine whetherLIF overexpression could induce neuroendocrine differentiation,we generated LNCaP and C4-2 cells that stably express LIFcomplementary (c)DNA and found that forced expression of LIFsignificantly induced the mRNA and protein levels of LIFR andneuroendocrine markers (Supplementary Fig. S1F; Fig. 1B). Incontrast, decreases in mRNA levels of the LIFR and neuroendo-crine markers were observed in PC3 and NCI-H660 cells with LIFknockdown (Fig. 1C). Western blotting confirmed that the reduc-tionof p-STAT3, LIFR, andneuroendocrinemarkerswaspositivelyassociated with LIF knockdown in PC3 and NCI-H660 cells(Fig. 1D). Moreover, the mRNA and protein levels of theLIFR and neuroendocrine markers were decreased in PC3 andNCI-H660 cells when the cells were treated with EC330, an LIFinhibitor (Supplementary Fig. S1G; Fig. 1E). These resultssupport that an abundance of LIF and activation of the LIF–STAT3pathway induces neuroendocrine differentiation of prostate can-cer cells.

Inhibition of AR signaling induces LIF and is associated withneuroendocrine differentiation

To assess whether the abundance of LIF was mediated by ADT,we analyzed LIF expression with signatures that reflect AR signal-ing components in the Taylor and TCGA prostate cancer datasetsby gene set enrichment analyses (GSEA). We found that tissuesexpressing low levels of LIFwere significantly associatedwith genesignatures of upregulated androgen responsiveness (refs. 30, 31;Supplementary Fig. S2A), whereas high levels of LIF were asso-ciated with a downregulated androgen-responsive signature(ref. 32; Fig. 1F). We further analyzed LIF expression by z-scoreanalysis, which was grouped based on relative AR activitiesdefined by the levels of gene signatures regulated by andro-gens (30, 31).We found that higher LIF expression was associatedwith the downregulation of androgen-responsive genes (Supple-mentary Fig. S2B), whereas tissues that expressed low levels ofLIF had gene signatures associated with androgen upregulation(Supplementary Fig. S2C). We validated the expression of LIF inAR-positive LNCaP and C4-2 cells relative to the AR signalingresponse. We found that cells treated with the AR ligandDHT hadlower levels of LIF, LIFR, p-STAT3, and NE markers than did cells

with androgen withdrawal (after culturing in CSS-containingmedium; Fig. 1G). In contrast, higher expression levels of LIF,LIFR, p-STAT3, and neuroendocrine markers were found inDHT-treated cells exposed to the AR antagonist MDV3100(Fig. 1G). We further performed LIF knockdown in LNCaP andC4-2 cells and treated the cells with androgen withdrawal. Theresults showed that LIF knockdown reduced LIF, LIFR, p-STAT3,and neuroendocrine markers regardless of the treatment withCSS-containing medium (Fig. 1H; Supplementary Fig. S2D andS2E), confirming that the abundance of LIF was involved inneuroendocrine differentiation after ADT. We next analyzedsamples consisting of tissue specimens from 18 patients withprostate cancer before and after undergoing ADT, collected fromTaipei Medical University-Wan Fang Hospital (Taipei, Taiwan).We found that the cytoplasmic LIF was increased in prostatetumors from patients who had received ADT compared with thecorresponding levels in the same patients before ADT treatment(Fig. 1I and J). These results suggest that ADT or inhibition ofAR signaling induces LIF expression, which is associated withneuroendocrine differentiation of prostate cancer.

LIF promotes a malignant phenotype in prostate cancerTo investigate the functional relevance of LIF-mediated induc-

tion of the malignant phenotype in nonmetastatic cell lines,LNCaP and C4-2 cells were overexpressed with LIF. We foundthat cellswith ectopic LIF expressionhad induced cell invasivenessthrough Matrigel (Fig. 2A) and exhibited higher cell proliferationand colony formation than did cells carrying the empty vector(Fig. 2B and C; Supplementary Fig. S3A). In contrast, when RasB1and PC3 metastatic cell lines were exposed to LIF knockdown,cell invasiveness decreased (Fig. 2D). We also found that LIFknockdown significantly reduced the proliferation rate of RasB1and PC3 cells (Fig. 2E; Supplementary Fig. S3B) and reducedcolony formation in RasB1 and PC3 cells (Fig. 2F). LIF expressionwas confirmed by Western blotting in cells harboring LIF altera-tions (Fig. 2G). These results were supported by further in vivoexperiments. Mice that were administered subcutaneousinjections of PC3 cells with LIF knockdown had significantlyreduced tumor formation and tumor weights compared withthose in mice injected with PC3 cells carrying the empty vector(Fig. 2H–J). These data suggest that LIF acts as a tumor promoterthat induces tumor formation in prostate cancer cells, thus pro-moting a variety of invasive properties and the growth rate ofprostate cancer cells.

LIF induces prostate cancer resistance to AR-targetedtherapeutics

To examine the biological effect of LIF after ADT, we culturedAR-positive cells with CSS-containing medium to mimic anandrogen-deprivation condition. Notably, overexpression of LIFin C4-2 and LNCaP cells significantly promoted cell proliferationand colony formation regardless of androgenwithdrawal or beingcombined with MDV3100 treatment (Fig. 3A–C; SupplementaryFig. S3C–S3E), suggesting that an abundance of LIF could inducetumor growth irrespective of AR signaling inhibition in AR-positive cells. Moreover, we treated cells with an LIF inhibitorand found that AR-negative PC3 and NCI-H660 cells had greaterdrug sensitivity than AR-positive LNCaP and C4-2 cells (Fig. 3D).We also found a significant reduction of colony formation in PC3andNCI-H660 cells or inC4-2 andLNCaP cells overexpressing LIFfollowing treatmentwith an LIF inhibitor (Fig. 3E; Supplementary

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Fig. S3F and S3G). We further studied the role of the LIF inhibitorin suppressing the tumor growth of AR-negative PC3 andNCI-H660 cells in vivo. We found that in mice administeredsubcutaneous injections of PC3 and NCI-H660 cells, treatmentwith the LIF inhibitor produced significant decreases in the tumorgrowth rate and tumor weights (Fig. 3F–H; Supplementary

Fig. S4A–S4C). IHC analyses showed that LIF inhibitor–treatedtumors had reduced p-STAT3, NE (CgA and NSE), and prolifer-ation (Ki67) markers and had gained an apoptotic marker(cleaved caspase-3; Fig. 3I; Supplementary Fig. S4D–S4F). Thesedata suggest that targeting LIF might suppress CRPC-NE progres-sion and efficiently inhibit tumor growth in prostate cancer cells.

Figure 1.

ADT induces LIF abundance and neuroendocrine differentiation of prostate cancer cells. A,Western blotting for LIFR, p-STAT3, STAT3, CgA, and NSE in LNCaPand C4-2 cells treated with LIF protein at various concentrations for 24 hours. B, Protein levels of LIF, LIFR, p-STAT3, STAT3, CgA, and NSE in LNCaP and C4-2cells following stable expression of an LIF cDNA vector. C,mRNA levels of LIF, LIFR, STAT3, CHGA, and ENO2 in PC3 and NCI-H660 cells following stableexpression of an LIF shRNA vector. D,Western blotting for LIF, LIFR, p-STAT3, STAT3, CgA, and NSE in PC3 and NCI-H660 cells stably expressing the LIF shRNAvector. E,Western blotting for LIFR, p-STAT3, STAT3, CgA, and NSE in PC3 and NCI-H660 cells treated with EC330 (35 nmol/L) for 24 hours. F, GSEA of theTCGA prostate cancer dataset showing that higher LIF expression was associated with an androgen-inactivated gene signature. NES, normalized enrichmentscore; FDR, false discovery rate. G, Effects of DHT and MDV3100 (MDV), relative to that of the vehicle (methanol or DMSO), on the expression of LIF, LIFR,p-STAT3, STAT3, CgA, and NSE proteins in LNCaP and C4-2 cells cultured with 10% CSS-containing medium. H, LIF, LIFR, p-STAT3, STAT3, CgA, and NSEprotein levels in control (Luc) and LIF-knockdown LNCaP or C4-2 cells and those treated with androgen withdrawal (CSS) for 1 week. IHC staining (I) and analysis(J) of cytoplasmic LIF in prostate cancer tissue sections from patients before and after ADT treatment. The 18 samples were obtained from Taipei MedicalUniversity-Wan Fang Hospital. Scale bars, 100 mm. Statistical analysis by the two-tailed Student t test. Data from the quantification of mRNA are presented asthe mean� SEM, n¼ 3 (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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LIF associates with ZBTB46 expression and contributes toCRPC-NE progression

We have previously shown that hormonal therapy for pros-tate cancer enhances ZBTB46 overexpression (24). ZBTB46 is alineage-specific transcription factor that is crucial for EMT andmetastasis of prostate cancer (24). We hypothesized that theunderlying mechanism by which LIF induces neuroendocrinedifferentiation after ADT is associated with the abundance ofZBTB46. We analyzed 16 normal prostatic epithelial samples,100 primary prostate adenocarcinomas, and 8 SCNCs from aprostate cancer TMA collected from the Department of Pathol-ogy at Duke University School of Medicine (Durham, NC).IHC analyses showed that cytoplasmic LIF was associated withincreased nuclear ZBTB46 and had greater abundances in high-grade tumors and SCNC samples (Fig. 4A and B). QuantitativecoIF staining of the CRPC TMA confirmed that in high-gradeprostate tumors, LIF expression was restricted to NE-like (CgA-positive) tumor cells (Fig. 4C). Moreover, the abundance of LIF

was positively associated with gene signatures of CRPC-NE-differentiation (3) and neuronal development, as confirmedby z-score analyses and GSEAs (Fig. 4D and E; SupplementaryFig. S5A and S5B). Furthermore, the mean expression correc-tion was validated in the Taylor and TCGA prostate cancerdatasets, showing that LIF was positively correlated withZBTB46 and the expression of neuroendocrine markers butwas inversely correlated with androgen-responsive genes(FKBP5, NKX3.1, KLK3, and SPDEF; Fig. 4F and G). In studyingthe clinical relevance of LIF, we found that prostate tumorswith either higher ZBTB46 or higher LIF expression wereassociated with metastasis (Supplementary Fig. S5C andS5D) and a high pathologic grade in the Gleason score(PathGGS; Supplementary Fig. S5E and S5F). Moreover,Kaplan–Meier analyses showed that patients with tumorsexhibiting higher LIF gene expression had a shorter survivalthan those with low LIF expression in the Taylor prostatecancer dataset (Fig. 4H). These results indeed supported an

Figure 2.

LIF induces malignant progression of prostate cancer cells. A, Invasion assay in stable cell lines containing either a control (empty vector; EV) or LIF-expressingvector. Selected images are shown on the right. B, Proliferation assay in stably LIF-expressing C4-2 cells. C, Colony formation by LNCaP and C4-2 cells stablytransfected with the EV or LIF-expressing vector. D, Invasion assay in RasB1 and PC3 cells by stable transfection of either a control (shLuc) or an LIF-targetedshRNA (shLIF) vector. Proliferation (E) and colony formation (F) of RasB1 or PC3 cells stably transfected with shLuc or shLIF. G, Confirmation of theoverexpression (top) or knockdown (bottom) efficiency by an LIF-expressing vector or LIF shRNA in stable cell lines. H–J, Growth (H), images (I), and weights(J) of tumor xenografts in male nude mice 4 weeks after subcutaneous inoculation with PC3 cells stably expressing shLuc or shLIF. n¼ 4mice per group. Datafrom the invasion, proliferation, and colony formation assays are presented as the mean� SEM from three independent experiments (�, P < 0.05; �� , P < 0.01;��� , P < 0.001).

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association of the induction of LIF and ZBTB46 expressionwith aggressive prostate cancer.

LIF-induced neuroendocrine differentiation is ZBTB46-dependent

We hypothesized that LIF stimulates malignant progressionthrough the upregulation of ZBTB46 after ADT. To address thishypothesis, we measured ZBTB46 and LIF expression in avariety of nonmetastatic and metastatic prostate cancer celllines. We found that AR-negative PC3 and RasB1 cells, whichreadily metastasize to bone (28, 33), had higher ZBTB46 andLIF expression levels than cell lines that do not metastasize,such as AR-positive LNCaP, C4-2, and 22Rv1 cells (Fig. 5A).

Regarding our recent study demonstrating the induction ofZBTB46 following ADT (24), we next examined whetherADT-induced ZBTB46 was associated with LIF abundance andneuroendocrine differentiation following ADT. We found thatlong-term treatment with CSS-containing medium induced LIFand was associated with ZBTB46, p-STAT3, and neuroendocrinemarker stimulation in C4-2 cells (Fig. 5B). We further con-firmed that such signaling activation was mediated by ZBTB46,as we found that ZBTB46 knockdown was able to inhibit theexpression of LIF and neuroendocrine markers, even in cellstreated with LIF protein (Supplementary Fig. S6A). We thenanalyzed the expression of LIF and neuroendocrine markersafter stably introducing a ZBTB46 cDNA vector into C4-2 cells.

Figure 3.

LIF promotes tumor growth and enzalutamide resistance, and LIF inhibitor reduces neuroendocrine marker expression. A and B, Proliferation assay in C4-2 cellstreated with androgen withdrawal [A, charcoal-stripped serum (CSS)] or the combination of androgen withdrawal and enzalutamide (10 mmol/L; B,CSSþMDV3100; n¼ 8). C,Quantification and images of the colony formation of C4-2 cells with androgen withdrawal (CSS) or combined androgen withdrawalwith MDV3100 (10 mmol/L) treatment (CSSþMDV) for 6 days following empty vector (EV) or LIF-expressing vector overexpression. D, Proliferation assays inLNCaP, C4-2, PC3, and NCI-H660 cells treated with an LIF inhibitor (EC330) at the indicated concentrations for 24 hours (n¼ 8). E, Colony formation of PC3 andNCI-H660 cells with EC330 (35 nmol/L) treatment for 6 days. F–H, Tumor growth analysis of NCI-H660 cells subcutaneously inoculated into male nude micefollowed by treatment with EC330 (2.5 mg/kg). Tumor sizes were monitored twice a week (F), and images (G) and tumor weights (H) were also measured at theend of the experiment. n¼ 6mice per group. I, IHC staining of subcutaneous tumors with antibodies specific for p-STAT3, CgA, NSE, Ki67, and cleaved caspase-3in tumor-bearing mice from F. Scale bars represent 100 mm. Data from the proliferation and colony formation assays are presented as the mean� SEM from threeindependent experiments (� , P < 0.05; �� , P < 0.01; ��� , P < 0.001).

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Increased ZBTB46 expression significantly induced the expres-sion of LIF and neuroendocrine marker mRNAs, and betterinduction was observed in cells treated with LIF protein (Sup-plementary Fig. S6B). Immunoblots of extracts from ZBTB46-knockdown C4-2 cells confirmed the reduction of LIF, p-STAT3,and neuroendocrine markers, regardless of whether the cellswere treated with LIF protein (Fig. 5C). Interestingly, we alsoobserved a positive feedback of the LIF–STAT3 pathway ininducing ZBTB46 abundance because increased LIF was foundto induce ZBTB46 expression in control cells (Fig. 5C; Supple-

mentary Fig. S6A and S6B). These results indicate that stimu-lating the LIF–STAT3 pathway promotes neuroendocrine dif-ferentiation, which is associated with ZBTB46 expression inprostate cancer cells.

ZBTB46 binds directly to the LIF regulatory sequence andenhances its transcription

Wenext examinedwhether the LIF abundance is upregulated byZBTB46. Using ZBTB46-specific siRNA in RasB1 and PC3 cells, weobserved a reduction in LIF mRNA (Supplementary Fig. S6C),

Figure 4.

LIF is positively associated with the induction of ZBTB46 and neuroendocrine markers in clinical samples.A, IHC staining of ZBTB46 and LIF of a prostate cancerTMA from Duke University School of Medicine. Scale bars, 100 mm. B, Relative intensities of the ZBTB46- and LIF-positive staining statuses in prostate cancerTMA sections containing normal tissues (n¼ 16), adenocarcinomas with a Gleason score� 7 (n¼ 81), adenocarcinomas with a Gleason score� 8 (n¼ 19), andSCNCs (n¼ 8) from Duke University School of Medicine by H-score analysis. C, IF staining of a CRPC TMA from Duke University School of Medicine showingcoexpression of LIF and CgA in the same tumor cells. Scale bars, 50 mm. Z-score analyses (D) and GSEA (E) of the Taylor prostate dataset showing that higher LIFexpression in prostate tissues was positively associated with a CRPC-NE response signaling gene set (D) and neuronal development signature (E). F and G,Pearson correlation analysis of LIFwith ZBTB46, NE markers, and androgen-response gene mRNA levels in clinical tissue samples from the Taylor and TCGAprostate cancer datasets. Significance was determined using a two-tailed test. (� versus LIF; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001; ���� , P < 0.0001).H, Survivalanalysis of patients in the Taylor dataset (n¼ 111) based on LIF expression. A log-rank test was used for the survival curve analysis.

ZBTB46-LIF Drives CRPC-NE

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whereas overexpressionof ZBTB46 cDNA in LNCaP andC4-2 cellsresulted in an abundance of LIFmRNA (Supplementary Fig. S6D).We hypothesized that ZBTB46 could transcriptionally activateLIF in prostate cancer cells by directly binding to the ZBTB46response element (TGACGTC; ref. 34) on the LIFpromoter.Whenanalyzing the putative ZBTB46 response element in the LIFregulatory sequence, we found a candidate response element for

ZBTB46 at nucleotide -6543 relative to the LIF transcriptional startsite (Fig. 5D). To test whether ZBTB46 directly bound to LIF, weperformed ChIP assays in PC3 cells and found increased ZBTB46-binding activity at the putative ZBTB46-binding site (ZBE) on theLIF regulatory sequence and at a positive ZBE compared with anon-ZBTB46-binding site (non-ZBE; Fig. 5E). Importantly,decreased ZBTB46-binding activity was found at the putative ZBE

Figure 5.

The induction of LIF is regulated by ZBTB46 in prostate cancer cells following ADT. A,Western blotting of ZBTB46 and LIF from a panel of prostate cancer cells.B,Western blotting of ZBTB46, LIF, p-STAT3, STAT3, CgA, and NSE in C4-2 cells after treatment with androgen withdrawal (CSS) at each time point. C,Abundances of ZBTB46, LIF, p-STAT3, STAT3, CgA, and NSE protein levels in control (Luc) and ZBTB46-knockdown C4-2 cells and those treated with LIF protein(200 ng/mL) or vehicle (PBS) for 24 hours. D, Schematic of the predicted ZBTB46 response element (ZBE) or a non-ZBTB46 response element (non-ZBE) andan introduced binding site mutant in regulatory sequence reporter constructs of human LIF. E, ChIP assays in PC3 cells. Antibodies against H3K4me3 and GAPDHserved as the positive and negative controls, respectively. Precipitated DNA was quantified by qRT-PCR of ZBE, non-ZBE, and positive ZBE sites. Enrichment ispresented as a percentage of the total input followed by normalization to immunoglobulin G (IgG). � versus non-ZBE. F, ChIP assays in PC3 cells following control(Luc) or ZBTB46 knockdown. � versus shLuc.G and H, ChIP assays in C4-2 cells following MDV3100 (10 mmol/L, left) or DHT (10 nmol/L, right) treatment for 10hours. I and J, Relative MFIs of the wild-type (WT) andmutant (M) LIF reporter in LNCaP and C4-2 cells after treatment with MDV3100 (I) and DHT (J). RelativeWT and M LIF reporter activities in response to ZBTB46 overexpression in LNCaP and C4-2 cells (K) or ZBTB46 knockdown in RasB1 and PC3 cells (L).Quantification of the mRNA and ChIP data and MFIs are presented as the mean� SEM from three independent experiments (� , P < 0.05; �� , P < 0.01;��� , P < 0.001).

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in the presence of ZBTB46-knockdown (Fig. 5F). Moreover, theZBTB46-binding signal was enriched in C4-2 cells afterMDV3100treatment (Fig. 5G) but was reduced after DHT treatment ofthe cells (Fig. 5H), which is consistent with our hypothesisthat ADT-regulated ZBTB46 mediates LIF transcription. We nextperformed reporter assays with an LIF regulatory sequence con-struct containing the ZBE (Fig. 5D). We generated a mutation atthe putative ZBE and performed reporter assays in RasB1 and PC3cells and found that compared with the ZBE wild-type, the ZBEmutant reduced the reporter activity (Supplementary Fig. S6E).We also found significantly increased reporter activity inMDV3100-treated LNCaP and C4-2 cells (Fig. 5I), and decreasedreporter activity was observed upon DHT treatment (Fig. 5J).Moreover, the ZBE mutant disrupted the ability of MDV3100and DHT, respectively, to induce and reduce reporter activity inthe promoter assays (Fig. 5I and J). Furthermore, the ZBE mutantabolished the ability of ectopic ZBTB46 to induce reporter activityin LNCaP and C4-2 cells (Fig. 5K) and disrupted the ability ofZBTB46 knockdown to reduce reporter activity in RasB1 and PC3cells (Fig. 5L). These data are consistent with a mechanismwhereby ZBTB46-enhanced LIF transcription is site-specific andAR signaling-dependent.

LIF titers in serum as a diagnostic and prognostic biomarker forprostate cancer

To definitively assess whether LIF is a diagnostic and prognosticbiomarker for prostate cancer, we validated LIF titers in thesera from a cohort including healthy donors (n ¼ 20), patientswith BPH (n ¼ 20), patients with prostate tumors (n ¼ 20),and patients with prostate cancer withmetastatic tumors (n¼ 20)from the Chinese PLA General Hospital (Beijing, China).

We found that healthy donors and patients with BPH displayeda low level of LIF in serum (<200 pg/mL), whereas thepatients with tumors displayed a higher level of LIF in serum(>400 pg/mL; Fig. 6A). Interestingly, patients with bone metas-tasis showed robust LIFquantities in serum(>700pg/mL; Fig. 6A).These data revealed that LIF titration of serum can serve asa biomarker to predict patients with high-grade or metastaticprostate cancer. To better understand the connection between NEmarkers in prostate tumors and LIF levels in serum, we collectedprostate tissue samples for LIF and neuroendocrine marker exam-ination from the same patients who had been tested for LIF serumlevels. The results demonstrated that comparedwithBPHpatients,prostate cancer patients with higher LIF serum levels were posi-tively associated with induced LIF and neuroendocrine markerexpression in their prostate tissues (Fig. 6B and C). In summary,LIF titers in serum were associated with prostate cancer progres-sion and could help in classifying patients with prostate cancerwith metastasis or CRPC-NE development. Our data support theconnection between ADT-induced ZBTB46 and STAT3 pathwaystimulation in therapeutic resistance and neuroendocrinedifferentiation of prostate cancer through activated LIF. Mecha-nistically, the abundance of LIF was upregulated by ADT resis-tance-induced ZBTB46 by direct and physical interaction withLIF (Fig. 6D).

DiscussionConsistent with our earlier studies of patients with ADT,

prostate adenocarcinoma cells are likely to change into a neuro-endocrine-like phenotype, which is AR signaling suppressed andterminally resistant to AR-targeted therapies (2, 35, 36). Herein,

Figure 6.

LIF is a potent diagnostic andpredictive biomarker for prostatecancer. A,Measurement of LIFlevels in human serum from healthydonors (n¼ 20), patients with BPH(n¼ 20), prostate cancer (n¼ 20),and patients with bone metastatictumors (n¼ 20) collected from theChinese PLA General Hospital.� versus Normal. IHC staining (B)and relative intensities (C) ofsamples from 8 patients with BPHand 18 patients with prostate cancercollected from (A) for LIF, CgA,and NSE examination. Scale bars,100 mm. �� , P < 0.01; ��� , P < 0.001.D, Proposed model of LIF-mediatedtherapeutic resistance andneuroendocrine differentiation ofprostate cancer. An anti-androgenor AR antagonist inactivates ARsignaling and induces ZBTB46expression. Induced ZBTB46enhances malignant progressionand is involved in thedevelopment of CRPC-NE by anactivated LIF–STAT3 pathway.

ZBTB46-LIF Drives CRPC-NE

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we identified that the ADT resistance–induced ZBTB46–LIF path-way is involved in therapeutic resistance and neuroendocrinedifferentiation of prostate cancer. We provide evidence showingthat ZBTB46 is involved through the inactivation of AR signalingand activation of the LIF–STAT3 pathway in prostate cancer cells.This finding, together with the observation that induction ofLIF is related to increased malignancy in vitro and in vivo andneuroendocrine marker expression, suggests that LIF might act asan important regulator in CRPC-NE development. These resultsare consistent with previous findings in which LIF was originallydemonstrated to play an important role in the differentiation andproliferation of murine macrophage cells and to belong toa neuropoietic cytokine family that contributes to neuraldevelopment (37).

We have previously shown that ZBTB46 overexpression canpromote AR-independent proliferation under conditions ofandrogen withdrawal or in the presence of enzalutamide (24),which provides insights into the oncogenic role of ZBTB46 inprostate cancer after ADT. We investigated the mechanismsunderlying how hormonal therapy enhances the overexpres-sion of ZBTB46 in malignant progression. Our study confirmeda novel positive association between ZBTB46 activity and LIFlevels in prostate cancer tissues and cells. Under androgenregulation, low levels of ZBTB46 are an essential transcriptionalfactor for maintaining LIF–STAT3 signaling, while the lossof androgen signaling or inhibition of AR signaling causesLIF-enhanced therapeutic resistance and CRPC characteristicsthrough the upregulation of ZBTB46. We also found that LIFactivation drives malignant progression and neuroendocrine-like reprogramming in prostate cancer by activating STAT3signaling. Consistently, our results support that LIF functionsas an oncogene and participates in the progression of manymalignancies (38, 39).

Prostate cancer frequently becomes refractory to hormonetherapy because the AR is often mutated or absent fromrecurrent tumors, thus rendering androgen-independent pros-tate cancer (1, 40). STAT signaling normally activates anandrogen response in prostate cancer epithelial cells, andandrogen-independent prostate cancer cells exhibit aberrationsin STAT3 signaling that promote cell proliferation (41, 42). Ourstudy elucidated a link between neuroendocrine differentiationand ADT-induced ZBTB46 that is associated with activation ofthe LIF–STAT3 pathway. Although we demonstrated that LIF isa direct target of ZBTB46, our results show that an increase inLIF protein can also induce ZBTB46, suggesting a positivefeedback loop between ZBTB46 and LIF–STAT3 signaling. Inaddition to the involvement of the IL6 cytokine axis in prostatecancer resistance to AR antagonists (43, 44), we showed thattargeting AR activity induces both ZBTB46 and LIF; it is possiblethat factors downstream of STAT3 signaling are involved inthe androgen resistance phenotype, partly through induction ofZBTB46. Because of the oncogenic function of ZBTB46observed in prostate cancer cells (24), the induction of LIFexpression by suppression of AR signaling would provide aselective advantage to cells that stimulate STAT3 signalingduring CRPC-NE progression.

Our results not only have potential therapeutic implications bytargeting intracellular ZBTB46-LIF signaling in prostate cancer,but also clinical implications by suggesting that LIF titrationcan serve as a diagnostic and predictive marker in serum.We demonstrated that the increased LIF titers in serum were

associated with prostate cancer progression and could aid inclassifying patients with prostate cancer in terms of metastasisor NE differentiation. Although most CRPC-NE patients do notrespond to androgen-targeted therapies and chemotherapeuticagents (5, 45), our study provides the potential to develop newdiagnostic and therapeutic options for patients CRPC by provid-ing a rationale for theuseof an LIF inhibitor to combat therapeuticresistance and NE differentiation of prostate cancer. AlthoughLIF is broadly expressed in the gall bladder, appendix, urinarybladder, and some other organs (46–48), we showed that LIF isvariably expressed in high-grade and metastatic prostate cancercompared with the expression in the normal prostate or BPH.Because abnormal LIF was also shown to be expressed duringthe progression of other tumors or under inflammatory condi-tions (49, 50), we should pay attention to potentially falsepositive results when patients present prostate cancer and otherdiseases such as appendicitis or cholecystitis at the same time. Insummary, our study supports a molecular and therapeutic basisfor the ability of LIF to regulate drug resistance and neuroendo-crine-like differentiation in prostate cancer. We determined amolecular mechanism of prostate cancer lineage plasticity thatelucidates an abnormal LIF–STAT3 pathway to facilitate NEdifferentiation through the activation of ADT-induced ZBTB46signaling.

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

Authors' ContributionsConception and design: Y.-N. Liu, J. HuangDevelopment of methodology: Y.-N. Liu, S. Niu, W.-Y. Chen, W.-H. Chen,K.-C. Jiang, J. HuangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.-N. Liu, S. Niu, W.-Y. Chen, Y. Tao, X. Chen,A. Liu, Y. Li, J. HuangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.-N. Liu, S. Niu, W.-Y. Chen, Q. Zhang, Q. Zhang,W.-H. Chen, K.-C. Jiang, X. Chen, J. HuangWriting, review, and/or revision of the manuscript: Y.-N. Liu, J. HuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y.-N. Liu, S. Niu, X. Chen, Y. Li, Y.-C. LeeStudy supervision: Y.-N. Liu, X. ZhangOther (pathology support): H. Shi, J. Li

AcknowledgmentsThis work was jointly supported by grants from the NIH (1R01 CA172603-

01A, 1R01 CA205001-01, 1R01 CA181242-01A1, 1U54 CA217297-01, 1R01CA212403-01A1, 1R01 CA200853-01A1, and 1R01 CA195505), Departmentof Defense Prostate Cancer Research Program (W81XWH-11-1-0227,W81XWH-12-1-0206, PC150382, and PC150382), Prostate Cancer Founda-tion, UCLA SPORE in Prostate Cancer (2P50CA092131-11A1), and the Stand-up-to-Cancer Dream Team award (to J. Huang); the Ministry of Science andTechnology of Taiwan (MOST105-2628-B-038-006-MY3, MOST106-2918-I-038-001, and MOST107-2628-B-038-001), the National Health Research Insti-tute of Taiwan (NHRI-EX108-10702BI), and the "TMU Research Center ofCancer Translational Medicine" from The Featured Areas Research CenterProgram within the framework of the Higher Education Sprout Project by theMinistry of Education (MOE) in Taiwan (to Y.N. Liu).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 2, 2018; revised February 22, 2019; accepted March 29,2019; published first April 8, 2019.

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2019;25:4128-4140. Published OnlineFirst April 8, 2019.Clin Cancer Res   Yen-Nien Liu, Shaoxi Niu, Wei-Yu Chen, et al.   Cancer and Neuroendocrine Differentiation by Activated ZBTB46Leukemia Inhibitory Factor Promotes Castration-resistant Prostate

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