Enzalutamide-Induced Feed-Forward Signaling Loop Promotes ... · receptorexpression...

MOLECULAR CANCER THERAPEUTICS | CANCER BIOLOGYAND TRANSLATIONAL STUDIES

Enzalutamide-Induced Feed-Forward Signaling LoopPromotes Therapy-Resistant Prostate Cancer GrowthProviding an Exploitable Molecular Target for Jak2InhibitorsVindhya Udhane1,2,3, Cristina Maranto1,2,3, David T. Hoang4, Lei Gu4, Andrew Erickson5,6, Savita Devi1,2,3,Pooja G. Talati4, Anjishnu Banerjee3,7, Kenneth A. Iczkowski1,3, Kenneth Jacobsohn3,8, William A. See3,8,Tuomas Mirtti5,6,9, Deepak Kilari3,10, and Marja T. Nevalainen1,2,3

ABSTRACT◥

The second-generation antiandrogen, enzalutamide, isapproved for castrate-resistant prostate cancer (CRPC) andtargets androgen receptor (AR) activity in CRPC. Despite initialclinical activity, acquired resistance to enzalutamide arises rap-idly and most patients develop terminal disease. Previous workhas established Stat5 as a potent inducer of prostate cancergrowth. Here, we investigated the significance of Jak2–Stat5signaling in resistance of prostate cancer to enzalutamide. Thelevels of Jak2 and Stat5 mRNA, proteins and activation wereevaluated in prostate cancer cells, xenograft tumors, and clinicalprostate cancers before and after enzalutamide therapy. Jak2 andStat5 were suppressed by genetic knockdown using lentiviralshRNA or pharmacologic inhibitors. Responsiveness of primaryand enzalutamide-resistant prostate cancer to pharmacologicinhibitors of Jak2–Stat5 signaling was assessed in vivo in micebearing prostate cancer xenograft tumors. Patient-derived pros-

tate cancers were tested for responsiveness to Stat5 blockade assecond-line treatment after enzalutamide ex vivo in tumorexplant cultures. Enzalutamide-liganded AR induces sustainedJak2–Stat5 phosphorylation in prostate cancer leading to theformation of a positive feed-forward loop, where activated Stat5,in turn, induces Jak2 mRNA and protein levels contributing tofurther Jak2 activation. Mechanistically, enzalutamide-ligandedAR induced Jak2 phosphorylation through a process involvingJak2-specific phosphatases. Stat5 promoted prostate cancergrowth during enzalutamide treatment. Jak2–Stat5 inhibitioninduced death of prostate cancer cells and patient-derived pros-tate cancers surviving enzalutamide treatment and blocked enza-lutamide-resistant tumor growth in mice. This work introduces anovel concept of a pivotal role of hyperactivated Jak2–Stat5signaling in enzalutamide-resistant prostate cancer, which isreadily targetable by Jak2 inhibitors in clinical development.

IntroductionThe standard treatment of locally advanced or metastatic prostate

cancer is androgen deprivation therapy (ADT; refs. 1, 2). Response toADT is limited, and prostate cancer becomes castrate-resistant (CR), astage for which there is no cure (1–4). ADT targets androgen receptor(AR), which translocates from cytoplasm to nucleus upon ligandbinding followed by chromatin engagement (5). ADT is carried outby luteinizing hormone–releasing hormone (LHRH) agonists/antago-nists or by antiandrogens, which block the AR activity at the tissuelevel (1–5). The first-generation antiandrogen, bicalutamide, compet-itively inhibits androgen binding to AR (1–6) and recruitment of ARcorepressors (6). The recently developed second-generation antian-drogen, enzalutamide (ENZ), has gained increasing dominance in theclinical space and is currently FDA approved as amonotherapy in bothpre- and postchemotherapy settings (7–10). Enzalutamide is a moreeffective competitive inhibitor of steroid binding to the androgen-binding pocket on the ligand-binding domain of theARand retainsARmore effective in the cytoplasmic compartment of prostate cancercells (11). However, despite its initial promise, enzalutamide providesan improvement in patient survival only by 4–6 months due to rapiddevelopment of resistance (8, 10, 12, 13).

Mechanisms underlying prostate cancer resistance to enzalutamideare incompletely understood. The current proposed mechanismsinclude emergence of AR splice variants (14, 15), glucocorticoid

1Department of Pathology, Medical College of Wisconsin Cancer Center,Medical College of Wisconsin, Milwaukee, Wisconsin. 2Department of Phar-macology and Toxicology, Medical College of Wisconsin Cancer Center,Medical College of Wisconsin, Milwaukee, Wisconsin. 3Prostate Cancer Cen-ter of Excellence at Medical College of Wisconsin Cancer Center, MedicalCollege of Wisconsin, Milwaukee, Wisconsin. 4Department of Cancer Biology,Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia,Pennsylvania. 5Department of Pathology, Medicum, University of Helsinki,Helsinki, Finland. 6Institute for Molecular Medicine Finland (FIMM), Helsinki,Finland. 7Institute for Health and Equity, Medical College of Wisconsin,Milwaukee, Wisconsin. 8Department of Urology and Medical College ofWisconsin Cancer Center, Medical College of Wisconsin, Milwaukee, Wis-consin. 9Department of Pathology, HUSLAB and Helsinki University Hospital,Helsinki, Finland. 10Department of Medicine, Medical College of Wisconsinand Milwaukee VA Medical Center, Milwaukee, Wisconsin.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

V. Udhane and C. Maranto contributed equally to this article.

Corresponding Author: Marja T. Nevalainen, Medical College of WisconsinCancer Center, Medical College of Wisconsin, 8701 Watertown Plank Rd.,Milwaukee, WI 53266. Phone: 414-955-2103; Fax: 414-955-6059; E-mail:[email protected]

Mol Cancer Ther 2020;19:231–46

doi: 10.1158/1535-7163.MCT-19-0508

�2019 American Association for Cancer Research.

AACRJournals.org | 231

on April 30, 2020. © 2020 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst September 23, 2019; DOI: 10.1158/1535-7163.MCT-19-0508

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-19-0508&domain=pdf&date_stamp=2019-12-14

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-19-0508&domain=pdf&date_stamp=2019-12-14

http://mct.aacrjournals.org/

receptor expression (16), a ligand-binding domain mutation F876L inthe AR that promotes an antagonist-to-agonist switch of enzaluta-mide (17, 18), and neuroendocrine differentiation (NE; ref. 19).Moreover, emergence of an AR-null, NE-null prostate cancer pheno-type driven by MAPK signaling pathway was recently reported (20).However, no single mechanism has been reliably shown to completelyaccount for progress to enzalutamide resistance in prostate cancer inexperimental models or in patients, and it is likely that additionalmechanisms are involved.

Stat5a/b (Stat5), which is both a signaling protein and a nucleartranscription factor (21, 22), sustains viability of prostatecancer (23–29). Blockade of Stat5 signaling induces apoptotic deathof hormone-na€�ve prostate cancer cells, suppresses growth of bothxenografted and autochthonous prostate cancer tumors as well asclinical patient-derived prostate cancers ex vivo in culture (23–28, 30).Conversely, overexpression of active Stat5 has been shown to induceproliferation of prostate cancer cells in vitro and growth of prostatecancer tumors in mice (31). Stat5 induces metastatic progression ofprostate cancer, as evidenced by Stat5 promotion of metastasis for-mation in vivo, and induction of hallmarks of EMT and stem-likecancer cell properties through induction of Twist1 and BMI1 expres-sion in prostate cancer (30, 32). In 30%–40% of advanced CRPCs, thechromosome 17 locus encompassing STAT5A and STAT5B genesundergoes amplification resulting in increased Stat5 protein levels (31).Notably, high nuclear Stat5 protein expression at the time of the initialprostate cancer treatment predicted recurrence of the disease in threeindependent cohorts totaling 1,035 patients (33, 34). The predictiverole of active Stat5 for clinical prostate cancer progression to lethal CRstate (33, 34) corroborates involvement of Stat5 in prostate cancerprogression in preclinical prostate cancer models.

Activation of Stat5 occurs in the cytoplasm through induciblephosphorylation of a conserved C-terminal tyrosine residue (21).Phosphorylated Stat5 (pY694/699) forms functional dimers thattranslocate to the nucleus and bind to specific DNA response elementsto regulate transcription (21). In prostate cancer, Stat5 is activatedpredominantly by the Jak2 tyrosine kinase (35), amember of Jak familyincluding Jak1, Jak3, and Tyk2 (36). The binding of cytokines,hormones, and growth factors to the specific receptor–Jak2 complexleads to a conformational change in Jak2, which results in autopho-sphorylation and kinase activation. Phosphorylation of amino acidresidues Tyr1007/1008 in the activation loop of the kinase domain isrequired for full catalytic activity of Jak2 (36). Besides canonicalcytokine-activated activation, Jak2 phosphorylation in nonhemato-poietic tissues is regulated by a set of Jak2-specific phosphatases, whichinclude SHP-2 (37), PTP1B (38), and PTBe (39, 40). In addition, Jak2phosphorylation state is affected by the levels of Jak2 protein in cellswhere overexpression of Jak2 leads to Jak2 autophosphorylation in theabsence of cytokine stimulation (41).

The findings of Stat5 as a prostate cancer growth promoter and apredictor of prostate cancer recurrence implies involvement of Stat5 inthe development of CRPC, which led us to hypothesize that Jak2–Stat5signaling sustains viability of prostate cancer cells following disruptionof AR signaling by enzalutamide. We demonstrate, for the first time,that enzalutamide induces a robust increase in Stat5 activation inprostate cancer cells in vitro, in xenograft tumors in vivo, and inpatient-derived prostate cancers during enzalutamide treatment.Mechanistically, enzalutamide-liganded AR induces rapid and sus-tained Jak2 phosphorylation in prostate cancer cells via Jak2-specificphosphatases PTPe and SHP2. Enzalutamide-induced Jak2 activationleads to Stat5 phosphorylation, and the formation of a feed-forwardloop in prostate cancer cells where active Stat5 increases Jak2 mRNA

and protein levels.We further demonstrate that inhibition of Stat5 as asecond-line treatment induces extensive death of prostate cancer cellssurviving enzalutamide treatment. Stat5 blockade inhibited CR growthof prostate cancer xenograft tumors after enzalutamide resistancedeveloped and induced further death after enzalutamide treatmentin patient-derived prostate cancer s ex vivo in tumor explant cultures.In summary, this work supports the new concept of a critical role of ahyperactive Jak2–Stat5 signaling loop in promoting resistance ofprostate cancer to enzalutamide. Pharmacologic Jak2-Stat5 inhibitionmay provide an effective therapy for Stat5-positive advanced prostatecancer in combination with enzalutamide or after enzalutamide fails.

Materials and MethodsProstate cancers from enzalutamide-treated patients

Paraffin-embedded tissues sections of prostate cancers from enza-lutamide-treated patients were obtained from the pathology archivesat Helsinki University Hospital (Helsinki, Finland) and MedicalCollege of Wisconsin (MCW, Milwaukee, WI). In addition, tissuesections of hormone-na€�ve prostate cancers of corresponding histo-logic grades were from patients treated by radical prostatectomy (RP)without adjuvant hormone therapy (Helsinki University Hospital andMCW). Patient demographics and clinicopathologic data are pre-sented in Supplementary Table S1A. The study protocol for thesamples obtained from archives in Finland was approved by theEthical Committee of the University of Helsinki, and the NationalData Protection Ombudsman was notified about the collection of theinformation. Tissue sections of prostate cancers from patients beforeand after enzalutamide treatment were obtained from MCW and thepatient demographics and clinicopathologic data are presented inSupplementary Table S1B. These samples were deidentified archivaltissues and were granted an exemption from the MCW InstitutionalReview Board and is in compliance with federal regulations governingresearch [45 CFR 46102(f)]. Prostate cancer specimens for 3D explantcultures were obtained frompatients with localized or locally advancedprostate cancer undergoing RP at MCW (Supplementary Table S2)and were deidentified excess tissue available for research purposesgranted an exemption by theMCWInstitutional Review Board. All thestudies were conducted with the guidelines of Declaration of Helsinkiand U.S. Common Rule.

Ex vivo tumor explant cultures of clinical prostate cancersDetailed information is provided in Supplementary Materials and

Methods.

Cell lines and reagentsHuman prostate cancer cell lines LNCaP, PC-3, DU145 (from

ATCC), andCWR22Pc (42)were cultured inRPMI1640 growthmedia(Mediatech) containing 10% FBS (Quality Biological) and penicillin/streptomycin (50 IU/mL and 50 mg/mL, respectively; Mediatech).LAPC-4 cells (from Dr. Charles Sawyers, Memorial Sloan-KetteringCancer Center, New York, NY) were cultured under the sameconditions, with substitution of RPMI1640 for Iscove's modifiedDulbecco medium (Mediatech). LNCaP, LAPC-4, and CWR22Pccells were cultured in the presence of dihydrotestosterone (DHT)(Sigma-Aldrich; LNCap: 0.5 nmol/L, LAPC-4: 1 nmol/L, CWR22Pc:0.8 nmol/L). CWR22Pc subline expressing AR-F876L (from Dr.Charles Sawyers,Memorial Sloan-KetteringCancerCenter,NewYork,NY) was cultured in RPMI1640 supplemented with enzalutamide(10 mmol/L). All cell lines were regularly authenticated by observationof cell morphology, androgen responsiveness, and expression of cell

Udhane et al.

Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER THERAPEUTICS232




line–specific markers and tested forMycoplasma contamination (PCRMycoplasma Detection Set; Takara Bio Inc.) every 3 months. Enza-lutamide and AZD1480 were purchased from MedChem Express,MG132, and cyclohexamide from Calbiochem, LFA102 (43) fromNovartis, sodium orthovanadate from Sigma-Aldrich, and IST5-002was provided by Fox Chase Chemical Diversity Center (Doylestown,PA). Recombinant human prolactin (Prl) was obtained from NIDDKHormone and Peptide Program (Torrance, CA).

shRNA and cDNA constructs and lentiviral shRNA productionDetailed information is provided in Supplementary Materials and

Methods.

Adenovirus generationDetailed information is provided in Supplementary Materials and

Methods.

Protein solubilization, immunoprecipitation, andimmunoblotting

Detailed information is provided in Supplementary Materials andMethods and antibodies used are listed in Supplementary Table S3.

Quantitative real-time RT-PCRDetailed information is provided in Supplementary Materials and

Methods.

Cell viability analysisDetailed information is provided in Supplementary Materials and

Methods.

IHCDetailed information is provided in Supplementary Materials and

Methods.

Scoring of cell viability and nuclear Stat5a/bDetailed information is provided in Supplementary Materials and

Methods.

Immunofluorescence cytochemistryDetailed information is provided in Supplementary Materials and

Methods.

Human prostate cancer xenograft tumor studiesAll the animal studies have been conducted in accordance with and

approved Institutional Animal Care and Use Committee (IACUC).Castrated male athymic nudemice (Taconic) were cared for accordingto the institutional guidelines. Mice were implanted with sustainedrelease DHT pellets (60-day release, 1 pellet/mouse, InnovativeResearch of America) 7 days prior to prostate cancer cell inoculation.Briefly, 1.5� 107 CWR22Pc cells were mixed with 0.2 mL of Matrigel(BD Biosciences) and inoculated subcutaneously into flanks of nudemice (one tumor/mouse) as described previously (24, 29). Bicaluta-mide and enzalutamide were dissolved in 0.5% Tween 80 (Sigma-Aldrich)/PBS, AZD1480 in 0.1% Tween 80/0.5% hydroxypropylmethyl cellulose (HPMC, K4M prep, Dow Chemical)/H2O, andIST5-002 (IST5) in 0.3% hydroxypropyl cellulose (HPC, Sigma-Aldrich)/H2O.

For the monotherapy study (Fig. 6A), mice (5 mice/group) weretreated daily for 32 days by oral gavage with vehicle (0.5% Tween 80/PBS), bicalutamide (30 mg/kg) or enzalutamide (30 mg/kg), or byintraperitoneal injection with IST5-002 (50 mg/kg). DHT pellets were

removed from the mice in the castration group concurrently with thestart of the treatment period. Tumor dimensions were measured usingVernier calipers three times per week and tumor volumes calculatedusing the following formula: (3.14� length�width� depth)/6. Micewere sacrificed, and the tumor tissues were harvested at the end of the32-day treatment period, or prior to this endpoint if tumor sizesreached 15–20 mm in diameter, and tumor tissues were harvested.Tumor growth rates were calculated from the beginning of drugtreatment and are presented as fold changes in tumor volume of eachgroup.

For the sequential therapy study (Fig. 6B and C), mice (10 mice/treatment group) were treated daily for 13 days (phase I) by oral gavagewith vehicle (0.5% Tween 80/PBS) or enzalutamide (30 mg/kg) as thefirst-line therapy. On day 13, mice were randomly distributed into theindicated second-line therapy groups and treated daily for an addi-tional 18 days (phase II) by oral gavage with vehicle, enzalutamide(30 mg/kg), AZD1480 (30 mg/kg), or by intraperitoneal injection withIST5-002 (50 mg/kg), or by combination of enzalutamide (30 mg/kg)and IST5-002 (50mg/kg). Tumor dimensionsweremeasured twice perweek and tumor volumes calculated as described for the monotherapystudy. Mice were sacrificed when tumor sizes reached 15–20 mm indiameter in the vehicle-treated group (day 31), and the tumor tissueswere harvested.

Statistical analysesDetailed information is provided in Supplementary Materials and

Methods.

ResultsEnzalutamide-liganded AR induces phosphorylation andactivation of Stat5 in prostate cancer

IHC analyses of the activation status of Stat5 in prostate cancers ofpatients treated with enzalutamide show that the levels of nuclearactive Stat5 were significantly (P < 0.0001) elevated in biopsies ofenzalutamide-treated prostate cancers when comparedwith hormone-na€�ve prostate cancers of corresponding histologic grades (Fig. 1A;Supplementary Table S1A). In paired prostate cancer samples frompatients before and after enzalutamide treatment (SupplementaryTable S1B), active Stat5 levels were robustly elevated in the biopsiesafter enzalutamide treatment compared with the biopsies taken priorto enzalutamide treatment (Fig. 1B). In CWR22Pc and LAPC4 celllines, enzalutamide induced a rapid increase in Stat5 phosphorylationat 6 hours that was sustained and further increased at day 7 (Fig. 1C).At the same time, phosphorylation of Stat3 was not induced byenzalutamide in the same cell lines (Fig. 1C). In a CWR22Pc cellsubline expressing AR-F876L (CW22Pc-ENZ-R; ref. 17), whichemerges in CWR22Pc cells surviving extended treatment with enza-lutamide (�6months; ref. 18), active Stat5 levels weremarkedly highercompared with the parental CWR22Pc cells (Fig. 1C). Enzalutamideinduction of Stat5 phosphorylation resulted in nuclear translocation ofStat5 to an extent comparable with cytokine-induced Stat5 nucleartranslocation (Supplementary Fig. S1), indicating that enzalutamideinduces formation of Stat5 dimers capable of nuclear localization inprostate cancer cells.

To investigate whetherAR is required for enzalutamide induction ofStat5 signaling, we first analyzed enzalutamide activation of Stat5 inDU145 prostate cancer cell line which is AR-negative. In a parallel setof experiments, we inhibited AR expression in CWR22Pc and LAPC4cells by genetic knockdown using lentiviral expression of AR shRNA.In the absence of AR, enzalutamide failed to induce Stat5 activation

Jak2-Stat5 and Enzalutamide-Resistant Prostate Cancer

AACRJournals.org Mol Cancer Ther; 19(1) January 2020 233




Udhane et al.





(Fig. 1D) demonstrating that enzalutamide induction of Stat5 signal-ing is not an AR-independent off-target effect of the enzalutamidecompound. We next evaluated whether androgen deprivation, addi-tional androgen treatment, or genetic knockdown of AR would lead toan increase in Stat5 phosphorylation in prostate cancer cells.CWR22Pc and LAPC4 cell were androgen-deprived, cultured withadditional DHT supplementation (1.5 mmol/L), or AR was geneticallydepleted by lentiviral expression of AR shRNA (Fig. 1E). Androgendeprivation or AR knockdown did not stimulate Stat5 phosphoryla-tion levels in prostate cancer cells. In addition, when the AR wasliganded by DHT, Stat5 phosphorylation status remained unchanged.Taken together, these results indicate that enzalutamide-liganded ARinduces sustained Stat5 activation in human prostate cancer.

Enzalutamide-induced Stat5 activation in prostate cancer cellsoccurs via Jak2

To assess whether enzalutamide-induced Stat5 activation in pros-tate cancer cells is dependent on Jak2 signaling, Jak2 activity waspharmacologically inhibited by Jak2 inhibitor AZD1480 (44) duringenzalutamide treatment. AZD1480 blocked enzalutamide-inducedStat5 phosphorylation in CWR22Pc, LAPC4, and CWR22Pc-ENZ-R cells (Fig. 2A). To further assess the involvement of Jak2 inenzalutamide-induced Stat5 activation, Jak2 was genetically knockeddown by lentiviral expression of Jak2 shRNA inCWR22Pc and LAPC4cells for 3 days followed by enzalutamide treatment for 7 days(Fig. 2B). In the absence of Jak2, enzalutamide failed to induce Stat5phosphorylation in both cell lines. Unexpectedly, the protein levels ofJak2 on day 7 were robustly increased in enzalutamide-treatedCWR22Pc and LAPC4 cells compared with nontreated cells(Fig. 2B). Collectively, these results indicate that enzalutamide induc-tion of Stat5 activation in prostate cancer cells requires Jak2.

Wenext investigatedwhether enzalutamide-ligandedAR influencesthe phosphorylation status of Jak2 in prostate cancer cells. Enzaluta-mide induced a rapid phosphorylation of Jak2 already at 6 hours inboth CWR22Pc and LAPC4 cells to almost the same extent asstimulation of the cells with the cytokine prolactin (Prl; Fig. 2C).Enzalutamide-induced Jak2 phosphorylation was sustained at 12 daysand accompanied with a strong induction of Stat5 phosphorylation inboth CWR22Pc and LAPC4 cells (Figs. 2C). To further examinewhether AR is required for enzalutamide induction of Jak2 activation,AR was suppressed by lentiviral AR shRNA in CWR22Pc cells, whichblocked the enzalutamide-induced Jak2 activation (Fig. 2D). In addi-tion, AR agonists were not able to increase Jak2 phosphorylation inprostate cancer cells, because supplementation of culturemediumwithadditional DHT (1.5 nmol/L) for 6 hours and 7 days cell did not elevateJak2 phosphorylation in CWR22Pc prostate cancer cells (Fig. 2D,middle). To further understand whether enzalutamide-liganded AR

induction of Jak2–Stat5 activation requires the presence of a cytokinereceptor, we disrupted Prl receptor (PrlR) dimerization by a specific Prlantagonist LFA102 (43) for 6 hours and 7 days. As shown in Fig. 2D,enzalutamide failed to activate Jak2 and Stat5 in the presence ofLFA102 suggesting that docking of Stat5 and Jak2 to the cytokinereceptor complex is required for enzalutamide induction of Jak2–Stat5signaling. In summary, the data shown here demonstrate that AR-liganded enzalutamide induces rapid and sustained Jak2 phosphory-lation in prostate cancer cells that involves association of Jak2 with acytokine receptor.

Enzalutamide induction of Jak2 phosphorylation in prostatecancer cells involves Jak2 phosphatases

To investigate the mechanisms underlying enzalutamide activationof Jak2 in prostate cancer cells, CWR22Pc cells were treated withcycloheximide (35 mmol/L) for 24 hours followed by enzalutamide(40 mmol/L) for 6 hours, which blocked enzalutamide-induced Jak2phosphorylation (Fig. 2E). This suggests that enzalutamide inductionof Jak2–Stat5 signaling is mediated by a protein that is regulated byenzalutamide and, therefore, requires ongoing protein synthesis. Toevaluate whether enzalutamide induction of Jak2 phosphorylation isdependent on phosphatase activity in prostate cancer cells, we treatedprostate cancer cells with a broad-spectrum phosphatase inhibitorvanadate (VAN). Pretreatment of CWR22Pc with VAN led to ageneral increase in Jak2 phosphorylation, as expected, but also atten-uated the induction of Jak2 phosphorylation by enzalutamide (Fig. 2F)suggesting involvement of Jak2 phosphatases in enzalutamide induc-tion of Jak2 phosphorylation. The key known Jak2 phosphatasesexpressed in solid tumors are SHP-2 (37), PTPe (38), andPTP1B (39, 40), while SHP-1 is expressed in hematopoietic cells.Using lentiviral expression of shRNA, we depleted each of the threephosphatases for 3 days prior to enzalutamide treatment (6 hours) ofCWR22Pc and LAPC4 cells (Fig. 2G). In both prostate cancer celllines, enzalutamide-induced Jak2 phosphorylation was significantlyattenuated by depletion of either SHP-2 or PTPe. Genetic knockdownof PTPe resulted in generally elevated levels of Jak2 phosphorylation,which indicates that PTPe is a crucial negative regulator of Jak2phosphorylation in prostate cancer cells. Importantly, when PTPewas genetically depleted in prostate cancer cells, there was no differ-ence between enzalutamide treatment versus nontreated cells suggest-ing that the mechanism of action of enzalutamide on Jak2 phosphor-ylation involves suppression of PTPe activity (Fig. 2G, left). At thesame time, genetic depletion of SHP-2 decreased enzalutamide induc-tion of Jak2 phosphorylation while not increasing the general phos-phorylation levels of Jak2 (Fig. 2G, middle). This, in turn, suggests thatSHP-2 is a positive regulator of enzalutamide-induced Jak2–Stat5signaling and is required for enzalutamide induction of Jak2

Figure 1.Enzalutamide (ENZ) induces Jak2–Stat5a/b signaling in prostate cancer. A, Active Stat5 levels were robustly elevated in prostate cancers from patients treated byenzalutamide compared with hormone-na€�ve prostate cancers, demonstrated by immunostaining of paraffin-embedded tissue sections (image magnification 40�;scale bar, 50mm).B,Active Stat5 levelswere elevated in pairedbiopsies after enzalutamide treatment comparedwith biopsies taken prior to enzalutamide treatmentfrom the same patient (image magnification 40�; scale bar, 50 mm). C, Enzalutamide induces Stat5 phosphorylation in prostate cancer cells. Prostate cancer cellswere treatedwith enzalutamide or vehicle for the indicated periods of time. Expression levels of active Stat5 and Stat3were determined by immunoprecipitation (IP)of Stat5 and Stat3 followedby immunoblotting (WB) for pStat5a/b, pStat3, total Stat5, and total Stat3.Whole cell lysates (WCL)were immunoblotted for actin. Stat5phosphorylation levels are elevated in enzalutamide-resistant (R) (CWR22Pc AR-F876L) cells compared with parental CWR22Pc cells (right). D, AR is required forenzalutamide inductionof Stat5 phosphorylation. AR-negativeDU145 prostate cancer cellswere treatedwith enzalutamideor vehicle, andCWR22PcandLAPC4cellswere transduced with lentiviral AR shRNA (shAR) or lentiviral shCtrl for 3 days followed by treatment with enzalutamide or vehicle for 7 days at the indicatedconcentrations. E, DHT, androgen deprivation, or genetic AR suppression do not increase Stat5 phosphorylation levels in prostate cancer cells. Prostate cancer cellswere androgen deprived (charcoal-stripped FBS) or culturedwith additional DHT (1.5mmol/L) for 7 days. Alternatively, ARwas suppressedby lentiviral AR shRNA for7 days. Expression levels of active Stat5, Stat5, and Jak2 were determined by immunoprecipitation (IP) followed by immunoblotting (WB) with actin blotting asloading control.






Figure 2.

Enzalutamide (ENZ)-liganded androgen receptor induces Jak2 phosphorylation in prostate cancer cells. A, CWR22Pc, LAPC4, and ENZ-R cells werecultured with enzalutamide or Jak2 inhibitor AZD1480 alone or in combination for 12 days at indicated concentrations. Expression levels of active Stat5 weredetermined by immunoprecipitation (IP) of Stat5 followed by immunoblotting (WB) for pStat5a/b and total Stat5. Whole cell lysates (WCL) wereimmunoblotted for pStat3, Stat3 and actin. B, Genetic knockdown of Jak2 blocks enzalutamide-induced Stat5 phosphorylation in prostate cancercells. Jak2 was suppressed by lentiviral Jak2 shRNA versus shCtrl in prostate cancer cells for 3 days followed by enzalutamide or vehicle for 7 days. ActiveStat5, Stat5, and Jak2 levels were evaluated by IP and Western blot analysis, as depicted. C, CWR22Pc and LAPC-4 cells were treated with enzalutamide orvehicle for 6 hours at the indicated concentrations. Control cells were stimulated with prolactin (Prl; 10 nmol/L) for 20 minutes as control for cytokine-inducedJak2 phosphorylation. Alternatively, prostate cancer cells were treated with enzalutamide or vehicle for 12 days or stimulated with Prl for 20 minutes at theindicated concentrations. Stat5 and Jak2 were immunoprecipitated and immunoblotted (WB) for pStat5, pJak2, total Stat5, and total Jak2. WCLs wereimmunoblotted for actin. D, AR is required for enzalutamide induction of Jak2 activation. AR was suppressed in CWR22Pc cells by lentiviral AR shRNA (shAR)versus shCtrl for 3 days followed by enzalutamide for 6 hours. In parallel experiments, CWR22Pc cells were cultured with 1.5 mmol/L DHT for 6 hours or 7 days.Enzalutamide induction of Jak2–Stat5 activation requires a cytokine receptor shown by treatment of CWR22Pc cells with enzalutamide alone or incombination with a prolactin receptor antagonist LFA102 for 6 hours or 7 days at the indicated concentrations. (Continued on the following page.)

Udhane et al.





phosphorylation in prostate cancer cells. A positive regulatory role ofSHP-2 for Jak2–Stat5 signaling has been previously reported forseveral different cell types (45, 46). Genetic knockdown of PTP1Bdid not affect Jak2 phosphorylation levels nor enzalutamide inductionof Jak2 phosphorylation (Fig. 2G, right). In conclusion, these dataindicate involvement of Jak2 phosphatases SHP-2 and PTPe inenzalutamide-induced Jak2 phosphorylation in prostate cancer cells.

Enzalutamide-induced Stat5 activation increases Jak2 mRNAand protein levels and a formation of a positive feed-forwardloop in prostate cancer cells

The finding demonstrating that enzalutamide may increase Jak2protein levels in CWR22Pc and LAPC4 cells (Fig. 2B), led us toevaluate the effects of enzalutamide on phosphorylation, proteinlevels, and mRNA levels of Jak2 and Stat5 during a time-courseranging from 2 hours to 6 days in CWR22Pc and LAPC4 cells(Fig. 3A and B). Enzalutamide increased phosphorylation of Jak2 inboth CWR22Pc and LAPC4 cells at 6 hours, which was accompa-nied by an increase in Stat5 phosphorylation. Activation of Stat5reached maximum at 12–16 hours and was sustained at 6 days ofexposure to enzalutamide (Fig. 3A). The levels of Jak2 protein, butnot Stat5, increased starting at approximately 16 hours (CWR22Pc)and 6 hours (LAPC4) after initiation of the enzalutamide treatment.Concomitant with enzalutamide-induced activation of Jak2 andStat5, Jak2 mRNA levels were increased by 70% at the 6-hour time-point of enzalutamide treatment, which was sustained at the day 6(Fig. 3B). In parallel experiments, prostate cancer cells resistant toenzalutamide (CWR22Pc-ENZ-R) displayed markedly higher levelsof Jak2 protein (Fig. 3A) and mRNA (Fig. 3B) in comparison withthe parental CWR22Pc cells. While inducing Jak2 levels, enzaluta-mide did not induce Jak1 levels in CWR22Pc or LAPC-4 cellsindicating specificity of enzalutamide regulation of the phosphor-ylation state of Jak2 (Fig. 3C).

To further understand the mechanisms underlying enzalutamideinduction of Jak2mRNA levels, we analyzed the significance of Stat5 inthis process by genetic knockdown of Stat5 by lentiviral shRNA inCWR22Pc and LAPC4 cells. In the absence of Stat5, enzalutamidefailed to induce Jak2 mRNA and protein expression and Jak2 mRNAlevels were generally decreased by more than 90% (Fig. 3D). Thisfinding suggests that Stat5 is critical for enzalutamide-induced upre-gulation of Jak2 mRNA expression in prostate cancer cells. BecauseStat5 has not been shown to upregulate Jak2 mRNA expression inother cell types, we investigated this in prostate cancer cells in moredetail. First, we suppressed Stat5 by lentiviral Stat5 shRNA inCWR22Pc and LAPC4 cells for 3 days and assessed Jak2 mRNA levelsby qRT-PCR. Depletion of Stat5 led to a 60%–80% decrease in Jak2mRNA levels in both CWR22PC and LAPC4 cells (Fig. 3E, left). Inparallel experiments, lentiviral expression of constitutively active (CA)Stat5 (32) in CWR22Pc and LAPC4 cells increased Jak2 mRNAexpression by 120%–160%, which was also reflected at Jak2 proteinlevels (Fig. 3E, middle). In addition, Prl-induced Stat5 activationincreased Jak2 mRNA levels by 80%–120% in prostate cancer cells,which was blocked by genetic knockdown of Stat5 by shRNA (Fig. 3E,

right). In summary, these results demonstrate that enzalutamideinduces Jak2 mRNA and protein levels by Stat5-driven mechanismsin prostate cancer. Collectively, these findings indicate that enzaluta-mide induces a positive Jak2–Stat5 feed-forward loop in prostatecancer, depicted in Fig. 3F. In this proposed scheme, enzalutamide-liganded AR induces Jak2 phosphorylation in prostate cancer cellsthrough regulation of the activity and function of Jak2 phosphatases.Activated Jak2 leads to phosphorylation of Stat5 which, in turn,increases Jak2 mRNA and protein levels. It is known that elevatedJak2 protein levels result in Jak2 autophosphorylation (41). In addi-tion, increased levels of Jak2 are likely to result in upregulation of Jak2–Stat5 activation in prostate cancer cells induced by enzalutamide.

Stat5 promotes viability of prostate cancer cells survivingenzalutamide treatment

Having established that enzalutamide induces Jak2–Stat5 signalingin prostate cancer, we investigated whether active Stat5 promotesCRPC growth during enzalutamide treatment. First, constitutivelyactive (CA) Stat5 (32) was expressed in CWR22Pc and LAPC4 cellsusing lentivirus during treatment of the cells with increasing doses ofenzalutamide ranging from 5 to 40 mmol/L for 6 days. Expression ofCAStat5 increased the fraction of viable CWR22Pc and LAPC4 cells atall concentrations of enzalutamide with themaximum increase of 60%(P < 0.0001) and 45% (P < 0.0001) in CWR22Pc and LAPC4 cells(Fig. 4A, B, D, and E). The efficacy of lentiviral CAStat5 expressionin prostate cancer cells was confirmed by immunoblotting (Fig. 4Cand F).

To investigate the efficacy of inhibition of Jak2–Stat5 signaling ineliminating prostate cancer cells that survive enzalutamide treatment,we used a specific Stat5 inhibitor IST5-002 (47) or Jak2 inhibitorAZD1480 (44). Prostate cancer cells were exposed to enzalutamide for5 days followed by treatment with IST5-002 or AZD1480 for 5 or10 days (Fig. 5A). Control cells were grown in culture medium(MOCK), in the presence of vehicle (VEH), enzalutamide, IST5-002(IST5), AZD148, or enzalutamide combined with IST5-002 (ENZ þIST5) or AZD1480 (ENZþAZD1480) for 5, 10, or 15 days, as depictedin Fig. 5A. In comparison with the control group (enzalutamidefollowed by vehicle: ENZ > vehicle), CWR22Pc cells treated at thestart of the experiment with enzalutamide and later switched to IST5-002 after 5 days (ENZ > IST5-002) incurred additional loss of viabilityat both time points with the maximal reduction of 50% at 10 days ofenzalutamide treatment (15-day timepoint; P < 0.001; Fig. 5B). Nota-bly, IST5-002 alone or in combination with enzalutamide was moreefficacious (P < 0.001) in reducing viability of CWR22Pc cells thanenzalutamide applied as a single treatment (Fig. 5B). This may be dueto enzalutamide induction of hyperactive Jak2–Stat5 signaling loop,which leads to Stat5 promotion of prostate cancer cell viability andgrowth. Moreover, when the cells were pretreated with enzalutamide,inhibition of Stat5 was less efficient in reducing prostate cancer cellviability compared with Stat5 inhibitionmonotherapy or combinationtreatment of the cells with both IST5-002 and enzalutamide (Fig. 5B).Similarly, this may be explained by enzalutamide activation of positiveJak2–Stat5 feed-forward loop, which sets a barrier for a second-line

(Continued.) E, Enzalutamide induction of Jak2–Stat5 activation requires ongoing protein synthesis in prostate cancer cells. CWR22Pc cells were treated withcycloheximide (CHX; 35 mmol/L) for 24 hours followed by enzalutamide treatment (40 mmol/L) for 6 hours. F, Phosphatase inhibitor vanadate (VAN)abolished enzalutamide induction of Jak2 phosphorylation. CWR22Pc cells were treated with enzalutamide (6 hours) or VAN (2 hours) or vehicle alone orpretreated with VAN (2 hours) followed by enzalutamide (6 hours). G, Enzalutamide-induced Jak2 phosphorylation is decreased by depletion of either PTPe orSHP-2 phosphatases. CWR22Pc and LAPC4 cells were infected with lentivirus expressing shRNA targeting PTPe, SHP-2, PTP1B, or shCtrl for 3 days followed byenzalutamide or vehicle treatment for 6 hours at the indicated concentrations. IPs and WBs in were conducted as described in A.






Udhane et al.





therapy to overcome. Viability of CWR22Pc in each group wasvisualized and quantified by cell-cycle analysis and crystal violetstaining (Fig. 5C and D).

Given that enzalutamide induces a marked increase in Jak2 phos-phorylation in prostate cancer cells, we next assessed the efficacy of asequential application of enzalutamide followed by AZD1480 (44) as a

Figure 3.Enzalutamide (ENZ) induces a formation of a positive Jak2–Stat5 feed-forward loop in prostate cancer. A, Enzalutamide-induced Jak2 phosphorylation isaccompanied by an increase in Stat5 phosphorylation and Jak2 protein levels in prostate cancer cells. CWR22Pc, LAPC-4, and ENZ-R cells were treated withenzalutamide or vehicle during a time-course at the indicated concentrations. Prostate cancer cells were stimulatedwith Prl (10 nmol/L) as control for Jak2 and Stat5phosphorylation. Thephosphorylation status of Stat5 and Jak2was determinedby IPof Stat5 and Jak2 followedby immunoblotting for pStat5, total Stat5, pJak2, andtotal Jak2. WCLs were immunoblotted for actin. B, Enzalutamide induces Jak2 mRNA levels in prostate cancer cells. CWR22Pc and LAPC-4 cells were treated withenzalutamide or vehicle for indicated periods of time or infectedwith lentiviral Jak2 shRNA (shJak2) versus shCtrl (Jak2mRNA control; 3 days) followed byqRT-PCR.C, Jak2, but not Jak1, levels are induced by enzalutamide. Prostate cancer cells were treated with enzalutamide or vehicle for 12 days. Jak1 and Jak2 wereimmunoprecipitated and blotted for Jak1 and Jak2.WCLswere immunoblotted for actin.D, Enzalutamide increases Jak2 protein levels by Stat5-driven upregulationof Jak2mRNA expression in prostate cancer cells. When Stat5 was depleted in prostate cancer cells by lentiviral Stat5 shRNA (shStat5) or shCtrl for 3 days followedby enzalutamide or vehicle for 7 days, enzalutamide failed to increase Jak2 mRNA levels. For Jak2 mRNA control, Jak2 was suppressed by lentiviral Jak2 shRNA(shJak2) or shCtrl (left) for 3 days. Jak2mRNA levelsweredeterminedbyqPCRandprotein levels by IP andWesternblot analysis (WB) as described inA.E,CWR22PcandLAPC-4cellswere infectedwith lentiviral Stat5 shRNA (shStat5), shCtrl (top andbottompanels), constitutively active (CA) Stat5 orGFP for 3 days (middle). After3 days, prostate cancer cells were treated with Prl for 3 days (bottom). IP, immunoblotting (WB), and qPCR were conducted as described in A and B. F, Schematicrepresentation of the proposed enzalutamide induction of hyperactivated Jak2–Stat5 feed-forward loop in prostate cancer.

Figure 4.

Stat5 promotes viability of prostatecancer cells during enzalutamide (ENZ)treatment. Constitutively active (CA)Stat5 or GFP was lentivirally expressedinCWR22Pc and LAPC-4cells for 2 daysfollowed by enzalutamide treatment (5,10, 20, 30, and 40mmol/L) or vehicle for6 days (A andD). The fractions of viablecells were determined by countingthree separate fields from each of thethree parallel wells, and the averagesare indicated by a line graph with SDs.Crystal violet staining (B and E) andimmunoblotting of whole cell lysates(WCL) for Stat5 and actin on day 6 (Cand F).






Udhane et al.





representative of Jak2 inhibitors. Compared with control (ENZ >vehicle), CWR22Pc cells initially receiving enzalutamide at the startof the experiment for 5 days and switched to AZD1480 for 5 or 10 days(ENZ > AZD1480) exhibited additional loss of 15%–20% of cellviability at both time points (P < 0.05; Fig. 5E–G). Stat5 inhibitionwas verified by immunoblotting in cells receivingAZD1480, combinedenzalutamide and AZD1480, or enzalutamide followed by AZD1480(Fig. 5E). In summary, Jak2 inhibition by AZD1480 induced extensivedeath of CWR22Pc cells that survived enzalutamide treatment.

To investigate whether prostate cancer cells encompassing AR withthe mutation conferring enzalutamide resistance are sensitive toinhibition of Jak2–Stat5 signaling with cell viability as the endpoint,Jak2–Stat5a/b signaling was inhibited in both parental CWR22Pc andENZ-R (AR-F876L) cells by AZD1480 or IST5-002. While parentalCWR22Pc cells remained sensitive to enzalutamide, viability of enza-lutamide-resistant (AR-F876L) CWR22Pc cells was unaffected byenzalutamide, as expected (Fig. 5H). Viability of both parental andenzalutamide-resistant CWR22Pc cells was decreased by both IST5-002 (12.5 mmol/L; P < 0.001) or AZD1480 (P < 0.001; 0.8 mmol/L) at10 days, indicating that enzalutamide-resistant CWR22Pc cellsremained sensitive to Stat5a/b inhibition (Fig. 5H). Enzalutamide-resistant cells displayed uniformly high levels of active Stat5 (Fig. 5H).Collectively, these findings support the concept of an emergence ofsustained upregulation of Jak2–Stat5 activation during long-termenzalutamide treatment. To summarize, these data demonstrate thatactive Stat5 increases viability and growth of prostate cancer cellsduring enzalutamide treatment, and pharmacologic inhibition ofJak2–Stat5 signaling induces death of prostate cancer cells survivingenzalutamide treatment.

Stat5 inhibition decreases growth of enzalutamide-resistantprostate cancer xenograft tumors and patient-derived prostatecancers in tumor explant cultures ex vivo

Given that Stat5a inhibition potently induced apoptotic death ofprostate cancer cells depleted of AR signaling, we sought to extend ourin vitro findings to prostate cancer xenograft tumor growth in vivo.Castrated athymic nude mice, implanted with sustained-release DHTpellets to normalize circulating androgen levels, were inoculatedsubcutaneously with CWR22Pc cells. CWR22Pc tumors are knownto initially display androgen-sensitive growth and regress upon cas-tration, but later recur as castrate-resistant tumors (42). Themice weretreated with bicalutamide, enzalutamide, or IST5-002 daily for 32 dayswith vehicle or surgical castration as control groups (Fig. 6A). Tumorsin the castration group rapidly resumed the same growth rate astumors in the vehicle-treated mice, suggesting the presence of adrenal

and/or intracrine androgen synthesis. As expected, enzalutamide wasmore effective than bicalutamide in blocking androgen-sensitiveCWR22Pc tumor growth (P < 0.001; Fig. 6A). In line with the higherefficacy of IST5-002 than enzalutamide in inducing death of prostatecancer cells in vitro shown in Fig. 5, IST5-002 was superior toenzalutamide (P < 0.05) at suppressing in vivo androgen-sensitivegrowth of CWR22Pc xenograft tumors in the presence of circulatingandrogens (Fig. 6A). IHC analyses showed decreased nuclear Stat5content within tumor cells treated with IST5-002, while nuclear Stat5levels were elevated in tumors treated with enzalutamide (Supple-mentary Fig. S2A). Collectively, these results indicate that Stat5inhibition by IST5-002 blocked CWR22Pc xenograft tumor growthequally or more effectively than enzalutamide.

To assess therapeutic efficacy of sequential targeting ofAR and Stat5in prostate cancer tumors in vivo, we designed a two-phase in vivoexperiment, depicted in Fig. 6B. Mice received first-line therapy ofvehicle or enzalutamide daily until emergence of resistance to enza-lutamide occurred (day 13), followed by randomization and switch to asecond-line therapy (vehicle, enzalutamide, AZD1480, IST5-002, orENZ þ IST5-002) daily for additional 18 days. Tumors in micereceiving vehicle during the first phase represent androgen-sensitiveprostate cancer growth (Fig. 6C), and final fold changes in tumorvolume (volume at day 31/day 0� SEM) at the end of the second phase(day 31) were as follows: vehicle > vehicle, 41.6 � 1.9; vehicle > ENZ,29.8� 2.5; vehicle > AZD1480, 23.7� 0.9; vehicle > IST5-002, 18.9�2.0; vehicle > ENZ þ IST5-002, 14.6 � 2.0 (Fig. 6C). As expected,enzalutamide reduced androgen-sensitive CWR22Pc xenograft tumorgrowth significantly compared with vehicle-treated tumors (P¼ 0.05).At the same time, Stat5 inhibition by IST5-002 alone suppressedandrogen-sensitive prostate cancer xenograft tumor growth equallyeffectively as enzalutamide when compared with the control group(vehicle; P< 0.01). However, the growth of androgen-sensitive prostatecancer tumors was suppressed more effectively by combination ofIST5-002 with enzalutamide when compared with enzalutamide alone(P ¼ 0.02; Fig. 6C). In conclusion, while enzalutamide alone sup-pressed growth in the presence of androgens as expected, combinationof enzalutamide with IST5-002 was remarkably more effective thanenzalutamide alone.

Prostate cancer tumors in mice receiving enzalutamide during thefirst phase developed resistance as evidenced by regrowth of tumors inenzalutamide-treated mice starting on day 13. At the end of thesecond-line treatment on day 31, final fold changes in enzaluta-mide-resistant tumor volumes were as follows: ENZ > vehicle, 22.8� 2.3; ENZ > ENZ, 13.5 � 1.5; ENZ > AZD1480, 8.9 � 1.3; ENZ >IST5-002, 7.3 � 0.9; ENZ > ENZ þ IST5-002, 4.9 � 0.6 (Fig. 6D).

Figure 5.Pharmacologic inhibition of Jak2–Stat5 signaling increases death of prostate cancer cells when used in combination with enzalutamide (ENZ) or alone afterenzalutamide.A, The experimental design for a sequential treatment of prostate cancer cells with AR inhibition followed by genetic or pharmacologic knockdown ofStat5. B, Stat5 inhibitors alone or in combination with enzalutamide are more effective than enzalutamide alone in suppressing prostate cancer cell viability.Sequential therapy with enzalutamide followed by IST5-002 decreases viability of prostate cancer cells surviving enzalutamide treatment. CWR22Pc cells weretreated at the start of the experiment (day 0) with vehicle, enzalutamide (40 mmol/L) for 5 days, IST5-002 (12.5 mmol/L) alone or in combination, as indicated or leftuntreated (MOCK). For sequential treatment, cells were treated at the start of the experiment (day 0) with enzalutamide (40 mmol/L) followed by treatment withvehicle (ENZ > vehicle) or IST5-002 (12.5 mmol/L; ENZ > IST5-002) for 5 or 10 days. The fractions of viable cells are indicated by columnswith SDs. Activation of Stat5was determined by IP of followed by immunoblotting (WB) for pStat5 and total Stat5.WCLswere immunoblotted for actin.C, FACS analysis of cell-cycle distributionat the indicated timepoints.D,Pictures of prostate cancer cells stainedwith crystal violet after the treatments, as described inB.E,Combination of enzalutamidewithAZD1480 is more effective than enzalutamide alone in decreasing viability of prostate cancer cells surviving enzalutamide treatment. Sequential treatment ofCWR22Pc cells with enzalutamide (40 mmol/L) for 5 days followed by AZD1480 (0.8 mmol/L) for 5 or 10 days suppresses viability of prostate cancer cells survivingenzalutamide treatment. CWR22Pc cells were treated and analyzed for viable cells as described in A. F, FACS analysis of cell-cycle distribution at the indicatedtimepoints.G, Crystal violet staining of the parallel wells.H, IST5-002 and AZD1480 decreased viability of both parental CWR22Pc cells and ENZ-R cells (AR-F876L)after 5 and 10 days of treatment. Enzalutamide decreased viability of parental cells but did not affect viability of ENZ-R cells after 5 or 10 days of treatment. Thefractions of viable cells are indicated by columns with SDs.






Udhane et al.





Importantly, IST5-002 alone suppressed enzalutamide-resistant pros-tate cancer tumor growth. At the same time, combined enzalutamidewith IST5-002 was most effective in suppressing enzalutamide-resistant CRPC tumor growth when compared with continued enza-lutamide treatment (P< 0.01). IHC analysis of Stat5 demonstrated thatenzalutamide increased nuclear Stat5 in both androgen-induced andcastrate-resistant tumors (Supplementary Fig. S2). In conclusion,growth of enzalutamide-resistant CRPC xenograft tumors was mosteffectively suppressed by a second-line therapy of combined enzalu-tamide and IST5-002.

To evaluate the efficacy of combined enzalutamide and IST5-002as a second-line therapy in clinical patient-derived prostate cancers,we utilized an ex vivo 3D tumor explant culture system of patient-derived prostate cancers, which we have established and charac-terized previously (25, 32, 47–49). Crucial interactions betweenprostate epithelium and stroma are retained in tumor explantcultures and, therefore, it is considered a more physiologic modelof prostate cancer growth than cell lines or primary cell cultures.Prostate cancers from seven individual patients (SupplementaryTable S2) were cultured ex vivo in tumor explant cultures withenzalutamide alone for 4 days followed by combined enzalutamideand IST5-002 for 4 days with explants cultured with enzalutamideor vehicle alone for 8 days as control groups. Prostate cancersreceiving combined enzalutamide and IST5-002 as the second-linetherapy demonstrated extensive loss of viable epithelium at the endof the culture period, with only 15% viable cells at the end of thetreatment period compared with enzalutamide monotherapy (30%;P < 0.05) or vehicle (80%; P < 0.0001; Fig. 6E). Nuclear levels ofactive Stat5 evaluated by IHC, were increased in the explants treatedwith enzalutamide compared with vehicle-treated explants (P ¼0.05). At the same time, treatment of the explants with the com-bination of enzalutamide and IST5-002 as a second-line therapyreduced the nuclear active Stat5 levels compared with enzalutamide(P ¼ 0.05; Fig. 6F). Overall, these data suggest that second-linetherapy with combined enzalutamide and IST5-002 was moreeffective than enzalutamide alone in inducing epithelial cell deathin patient-derived clinical prostate cancers ex vivo.

DiscussionProstate cancer cells require androgen receptor (AR) signaling for

growth and survival, a dependency exploited by androgen deprivationtherapy (ADT) for locally advanced, recurrent, or metastatic prostatecancer. A more potent second-generation AR antagonist, enzaluta-mide, was developed to retarget the persistent AR activity in CRPCtumors and has become standard-of-care in this setting (7–9). Enza-lutamide is approved as first-line therapy for metastatic and nonmeta-static CRPC (8, 10). However, despite these advances, CRPC remains auniformly lethal disease due to rapid development of resistance.Identification of molecular mechanisms and pathways that drivegrowth of enzalutamide-resistant prostate cancer cells holds potentialfor development of effective first-line combination therapies thatsuppress development of enzalutamide resistance or second-linetherapies once enzalutamide fails. In this study, we show, for the firsttime, that enzalutamide induces a hyperactive Jak2–Stat5 signalingloop in prostate cancer through mechanisms that involve Jak2 phos-phatases PTPe and SHP-2. Active Stat5 increased prostate cancer cellsurvival during enzalutamide treatment and, conversely, Stat5 inhi-bition blocked growth of prostate cancer cells surviving enzalutamidetreatment. Pharmacologic inhibition of Stat5 suppressed enzaluta-mide-resistant growth of xenograft tumors that emerged duringenzalutamide treatment and reduced cell viability in patient-derivedclinical prostate cancers cultured ex vivo in tumor explant cultures.Pharmacologic inhibitors of Jak2–Stat5 signaling may be effectiveagents to overcome enzalutamide-resistant CRPCs driven by Jak2–Stat5.

One of the key results of the work presented here is thatenzalutamide induced a hyperactive Jak2–Stat5 feed-forward sig-naling loop in prostate cancer, as demonstrated in prostate cancercell lines, prostate cancer xenograft tumors, and clinical prostatecancers from patients treated with enzalutamide. Eight of 8 enza-lutamide-treated clinical prostate cancer specimens examinedshowed high Stat5 signaling. This finding has high translationalsignificance because Stat5 is a potent inducer of prostate cancergrowth and a therapeutically targetable survival factor in prostate

Figure 6.Second-line treatment by pharmacologic inhibitors of Jak2–Stat5 signaling suppresses growth of both androgen-dependent and enzalutamide (ENZ)-resistant xenograft tumors in nude mice and in patient0-derived prostate cancers ex vivo in tumor explant cultures. A, Inhibition of Stat5 by IST5-002suppressed androgen-sensitive CWR22Pc xenograft tumor growth more effectively than bicalutamide (BIC) or enzalutamide, as shown by tumor growthcurves (top) and representative tumor images (bottom). CWR22Pc cells were inoculated subcutaneously (s.c.) into flanks of castrated athymic nude micesupplied with sustained release DHT pellets. Mice were surgically castrated or treated daily with vehicle, bicalutamide (30 mg/kg), enzalutamide (30 mg/kg),or IST5-002 (IST5; 50 mg/kg), and tumor growth rates were calculated for each treatment group and are presented as fold changes in tumor volume (volumeat timepoint/volume at treatment start). B, Experimental design for the sequential therapy of the xenograft tumors in vivo. A two-phase in vivo experimentusing vehicle or enzalutamide as first-line therapy (phase I, 13 days) and vehicle, enzalutamide, AZD1480, IST5-002, or ENZþ IST5-002 as second-line therapy(phase II, 18 days). On day 31, mice were sacrificed and CWR22Pc xenograft tumors collected for analyses. Sequential, second-line therapy with IST5-002(IST5) or ENZ þ IST5-002 (IST5) effectively suppressed the growth of androgen-sensitive (left; C) and enzalutamide-resistant (right; D). CWR22Pc xenografttumors, as shown by tumor growth curves (top) and representative tumor images (bottom). CWR22Pc cells were inoculated subcutaneously into flanks ofcastrated athymic nude mice supplied with sustained release DHT pellets. Mice were treated with vehicle or enzalutamide (30 mg/kg) for 13 days. On day 13,mice were randomly distributed into subgroups and treated daily for an additional 18 days with vehicle, enzalutamide (30 mg/kg), AZD1480 (30 mg/kg), IST5-002 (IST5; 50 mg/kg), or enzalutamide (30 mg/kg) þ IST5-002 (IST5; 50 mg/kg). Tumor growth rates are calculated and presented as described in A. E, Totest the efficacy of a second-line therapy with combined enzalutamide and IST5-002 (IST5) versus enzalutamide monotherapy in clinical prostate cancers,seven localized prostate cancers obtained from radical prostatectomies were cultured ex vivo in tumor explant cultures in the presence of vehicle (8 days),enzalutamide (40 mmol/L) alone (8 days), or enzalutamide (40 mmol/L) as first-line therapy (4 days) followed by enzalutamide (40 mmol/L) and IST5-002(IST5; 25 mmol/L) as a second-line therapy (4 days). Second-line therapy with combined enzalutamide and IST5-002 (IST5) induced cell death in clinicalprostate cancers more effectively than enzalutamide alone, as demonstrated by extensive loss of viable acinar epithelium. The number of viable epithelial cellsin the explants at the end of the cultures was counted and presented as percentage per epithelial cells prior to culture. Representative histologies are shown(image magnification 40�; scale bar, 50 mm). F, Levels of nuclear Stat5 in the prostate cancer explants were determined by immunostaining using biotin–streptavidin–amplified peroxidase-antiperoxidase immunodetection at the end of the cultures and expressed as percentages of Stat5-positive cells per 100epithelial cells in explants for each treatment group (12–20 explants; image magnification 40�; scale bar, 50 mm).






cancer (23–28, 32). We show here that the initial step in thehyperactive Jak2–Stat5 signaling in enzalutamide-treated prostatecancer cells was induction of Jak2 phosphorylation by enzaluta-mide-liganded AR. Enzalutamide-induced Jak2 activation wasaccompanied by simultaneous and robust upregulation of Stat5phosphorylation in prostate cancer. In addition, the levels of bothJak2 mRNA and protein were increased by exposure of prostatecancer cells to enzalutamide, as demonstrated by parallel evaluationof Stat5 and Jak2 phosphorylation, protein levels, and Jak2 mRNAexpression in two prostate cancer cell lines during a time-courseranging from 2 hours to 6 days. Enzalutamide induction of Jak2mRNA levels preceded enzalutamide induction of Jak2 proteinexpression, which suggests that Jak2 mRNA levels are predomi-nantly induced by enzalutamide and lead to increased Jak2 proteinlevels in prostate cancer. When Stat5 was genetically suppressed,enzalutamide failed to induce Jak2 mRNA and protein levels. Thisdemonstrates that enzalutamide-induced Stat5 activation is crucialfor enzalutamide-induced upregulation of Jak2 in prostate cancer.Separate evaluation of Stat5 regulation of Jak2 mRNA and proteinexpression by genetic knockdown or overexpression approachesrevealed that Jak2 mRNA levels are tightly regulated by Stat5 inprostate cancer. Future work will need to determine whether Stat5induces Jak2 mRNA stability and/or the transcription of the Jak2gene in prostate cancer and the associated enhancer elements andtheir genomic locations.

Jak2 phosphorylation triggered by enzalutamide in prostatecancer cells was rapid occurring already at 6 hours and was furtherincreased over the 12-day period tested here. The fact that ongoingprotein synthesis was required for enzalutamide induction of Jak2phosphorylation and that a broad-spectrum phosphatase inhibitorblocked enzalutamide-induced Jak2 phosphorylation both suggestmechanistic involvement of Jak2 phosphatases in this process.Genetic AR knockdown or androgen depletion failed to increaseJak2–Stat5 signaling, which indicates that phosphorylation of Jak2is not triggered by cellular stress of androgen pathway suppressionin general. We further showed that genetic depletion of PTPe, butnot SHP-2 or PTP1B, resulted in elevated levels of Jak2 phosphor-ylation, suggesting that PTPe is a key phosphatase and a negativeregulator of Jak2 phosphorylation in prostate cancer. PTPe knock-down abolished the difference in Jak2 phosphorylation levels inenzalutamide -treated versus nontreated cells, which implies thatenzalutamide-liganded AR directly or indirectly suppresses theactivity of PTPe in prostate cancer cells. At the same time, depletionof SHP-2 levels decreased enzalutamide induction of Jak2 phos-phorylation. This is consistent with a previously reported role ofSHP-2 as a positive regulator of Jak2 activation (45, 46) and isrequired for enzalutamide induction of Jak2 phosphorylation inprostate cancer cells. The finding that AR liganded by DHT did notincrease phosphorylation of either Stat5 or Jak2 implies thatenzalutamide induction of Jak2 phosphorylation may be causedby cytoplasmic actions of the AR. Future studies will need toestablish the mechanisms underlying enzalutamide regulation ofenzymatic activity of PTPe as well as the mechanistic role of SHP-2in enzalutamide-enhanced coupling of Jak2–Stat5 signaling inprostate cancer.

Active Stat5 increased the viability of prostate cancer cells duringenzalutamide treatment and, conversely, Stat5 inhibition induceddeath of the residual prostate cancer cells surviving enzalutamidetherapy. In addition, pharmacologic inhibition of Stat5 in combi-nation with enzalutamide displayed greater efficacy in suppressing

growth of prostate cancer in vitro and in vivo than enzalutamidealone in both androgen-sensitive and castrate-resistant settings.Combined enzalutamide and IST5-002 as second-line therapy wasmore effective than enzalutamide monotherapy in suppressingin vivo growth of CRPC xenograft tumors and ex vivo growth ofclinical patient-derived prostate cancers in tumor explant cultures.Importantly, Stat3 has been implicated in enzalutamide-resistantCRPC growth in different preclinical models than those used in thisstudy (50). Evaluation of the efficacy of Jak2 inhibitors that arecurrently in the clinical development for myeloproliferative dis-orders for blocking enzalutamide-resistant prostate cancer tumorgrowth will be essential for transition to phase I/II studies inprostate cancer and may provide efficacious second-line treatmentfor enzalutamide-resistant prostate cancer. Clinical evaluation ofthe efficacy of Jak2–Stat5 inhibitors in enzalutamide-resistant pros-tate cancers should utilize positive Stat5 activation status as abiomarker for patient selection for accrual.

In summary, this work introduces a concept of a hyperactiveJak2–Stat5 signaling loop as a mechanism mediating resistance ofprostate cancer to enzalutamide. The Jak2–Stat5 pathway providesan attractive therapeutic target for enzalutamide-resistant prostatecancer as a second-line treatment or for first-line therapy incombination with enzalutamide. Future work will need to evaluatethe efficacy of Jak2 inhibitors in the treatment of enzalutamide-resistant prostate cancer with positive status for active Stat5 as oneof the inclusion criteria.

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

Authors’ ContributionsConception and design: D.T. Hoang, M.T. NevalainenDevelopment of methodology: V. Udhane, C. Maranto, D.T. Hoang, L. Gu,P.G. Talati, M.T. NevalainenAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): V. Udhane, C. Maranto, D.T. Hoang, L. Gu, A. Erickson, S. Devi,P.G. Talati, K.A. Iczkowski, K. Jacobsohn, W.A. See, T. Mirtti, D. Kilari,M.T. NevalainenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V. Udhane, C. Maranto, S. Devi, A. Banerjee,K.A. Iczkowski, T. Mirtti, D. Kilari, M.T. NevalainenWriting, review, and/or revision of the manuscript: V. Udhane, C. Maranto,A. Banerjee, K.A. Iczkowski, K. Jacobsohn, W.A. See, T. Mirtti, M.T. NevalainenAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): V. Udhane, C. Maranto, A. Erickson, K.A. Iczkowski,T. Mirtti, D. Kilari, M.T. NevalainenStudy supervision: M.T. Nevalainen

AcknowledgmentsThis work was financially supported by an NCI/NIH Research Project Grant

(2RO1CA11358-06), an NCI/NIH Exploratory/Developmental Research Grant(1R21CA178755-01), Advancing a Healthier Wisconsin (#5520368), and WisconsinCancer Showhouse Grant (#15437; to M.T. Nevalainen). D.T. Hoang was supportedby an NCI/NIH Predoctoral Individual National Research Service Award (NRSA)Fellowship (1F31CA180626-01), and A. Banerjee was supported by an NCI/NIHResearch Project Grant (R21CA231892).

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 May 14, 2019; revised August 16, 2019; accepted September 17, 2019;published first September 23, 2019.

Udhane et al.





References1. Attard G, Parker C, Eeles RA, Schroder F, Tomlins SA, Tannock I, et al. Prostate

cancer. Lancet 2016;387:70–82.2. Litwin MS, Tan HJ. The diagnosis and treatment of prostate cancer: a review.

JAMA 2017;317:2532–42.3. Yap TA, Zivi A, Omlin A, de Bono JS. The changing therapeutic landscape of

castration-resistant prostate cancer. Nat Rev Clin Oncol 2011;8:597–610.4. Wong YN, Ferraldeschi R, Attard G, de Bono J. Evolution of androgen receptor

targeted therapy for advanced prostate cancer. Nat Rev Clin Oncol 2014;11:365–76.

5. Labrie F, Belanger A, Simard J, Labrie C, Dupont A. Combination therapy forprostate cancer. Endocrine and biologic basis of its choice as new standard first-line therapy. Cancer 1993;71:1059–67.

6. Masiello D, Cheng S, Bubley GJ, Lu ML, Balk SP. Bicalutamide functions as anandrogen receptor antagonist by assembly of a transcriptionally inactive recep-tor. J Biol Chem 2002;277:26321–6.

7. Beer TM, Armstrong AJ, Rathkopf DE, Loriot Y, Sternberg CN, Higano CS, et al.Enzalutamide in metastatic prostate cancer before chemotherapy. N Engl J Med2014;371:424–33.

8. Ning YM, PierceW,Maher VE, Karuri S, Tang SH, Chiu HJ, et al. Enzalutamidefor treatment of patientswithmetastatic castration-resistant prostate cancer whohave previously received docetaxel: U.S. Food and Drug Administration drugapproval summary. Clin Cancer Res 2013;19:6067–73.

9. Beer TM, Armstrong AJ, Rathkopf D, Loriot Y, Sternberg CN, Higano CS, et al.Enzalutamide in men with chemotherapy-naive metastatic castration-resistantprostate cancer: extended analysis of the phase 3 PREVAIL study. EurUrol 2017;71:151–4.

10. Ning YM, BraveM,Maher VE, Zhang L, Tang S, Sridhara R, et al. U.S. Food andDrug Administration approval summary: enzalutamide for the treatment ofpatients with chemotherapy-naive metastatic castration-resistant prostate can-cer. Oncologist 2015;20:960–6.

11. Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Arora V, et al. Development of asecond-generation antiandrogen for treatment of advanced prostate cancer.Science 2009;324:787–90.

12. Claessens F, Helsen C, Prekovic S, Van den Broeck T, Spans L, Van Poppel H,et al. Emerging mechanisms of enzalutamide resistance in prostate cancer.Nat Rev Urol 2014;11:712–6.

13. Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance toandrogen receptor inhibitors in prostate cancer.Nat RevCancer 2015;15:701–11.

14. Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR-V7and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med2014;371:1028–38.

15. Dehm SM, Schmidt LJ, Heemers HV, Vessella RL, Tindall DJ. Splicing of anovel androgen receptor exon generates a constitutively active androgenreceptor that mediates prostate cancer therapy resistance. Cancer Res 2008;68:5469–77.

16. Arora VK, Schenkein E, Murali R, Subudhi SK, Wongvipat J, Balbas MD, et al.Glucocorticoid receptor confers resistance to antiandrogens by bypassingandrogen receptor blockade. Cell 2013;155:1309–22.

17. Joseph JD, Lu N, Qian J, Sensintaffar J, Shao G, Brigham D, et al. A clinicallyrelevant androgen receptor mutation confers resistance to second-generationantiandrogens enzalutamide and ARN-509. Cancer Discov 2013;3:1020–9.

18. Korpal M, Korn JM, Gao X, Rakiec DP, Ruddy DA, Doshi S, et al. An F876Lmutation in androgen receptor confers genetic and phenotypic resistance toMDV3100 (enzalutamide). Cancer Discov 2013;3:1030–43.

19. Beltran H, Prandi D, Mosquera JM, Benelli M, Puca L, Cyrta J, et al. Divergentclonal evolution of castration-resistant neuroendocrine prostate cancer.Nat Med 2016;22:298–305.

20. Bluemn EG, Coleman IM, Lucas JM, Coleman RT, Hernandez-Lopez S, Thara-kan R, et al. Androgen receptor pathway-independent prostate cancer issustained through FGF signaling. Cancer Cell 2017;32:474–89.

21. Levy DE, Darnell JE, Jr. Stats: transcriptional control and biological impact.Nat Rev Mol Cell Biol 2002;3:651–62.

22. Liu X, Robinson GW, Gouilleux F, Groner B, Hennighausen L. Cloning andexpression of Stat5 and an additional homologue (Stat5b) involved in prolactinsignal transduction inmousemammary tissue. ProcNatl Acad SciU SA1995;92:8831–5.

23. Ahonen TJ, Xie J, LeBaron MJ, Zhu J, Nurmi M, Alanen K, et al. Inhibition oftranscription factor Stat5 induces cell death of human prostate cancer cells. J BiolChem 2003;278:27287–92.

24. Dagvadorj A, Kirken RA, Leiby B, Karras J, NevalainenMT. Transcription factorsignal transducer and activator of transcription 5 promotes growth of humanprostate cancer cells in vivo. Clin Cancer Res 2008;14:1317–24.

25. Maranto C, Udhane V, Hoang DT, Gu L, Alexeev V, Malas K, et al. STAT5A/Bblockade sensitizes prostate cancer to radiation through inhibition of RAD51and DNA Repair. Clin Cancer Res 2018;24:1917–31.

26. Kazansky AV, Spencer DM, Greenberg NM. Activation of signal transducer andactivator of transcription 5 is required for progression of autochthonous prostatecancer: evidence from the transgenic adenocarcinoma of the mouse prostatesystem. Cancer Res 2003;63:8757–62.

27. Thomas C, Zoubeidi A, Kuruma H, Fazli L, Lamoureux F, Beraldi E, et al.Transcription factor Stat5 knockdown enhances androgen receptor degradationand delays castration-resistant prostate cancer progression in vivo. Mol CancerTher 2010;10:347–59.

28. Tan SH, Dagvadorj A, Shen F, Gu L, Liao Z, Abdulghani J, et al. Transcriptionfactor Stat5 synergizes with androgen receptor in prostate cancer cells.Cancer Res 2008;68:236–48.

29. Gu L, Dagvadorj A, Lutz J, Leiby B, Bonuccelli G, LisantiMP, et al. Transcriptionfactor Stat3 stimulatesmetastatic behavior of humanprostate cancer cells in vivo,whereas Stat5b has a preferential role in the promotion of prostate cancer cellviability and tumor growth. Am J Pathol 2010;176:1959–72.

30. Gu L, Vogiatzi P, Puhr M, Dagvadorj A, Lutz J, Ryder A, et al. Stat5 promotesmetastatic behavior of human prostate cancer cells in vitro and in vivo.Endocr Relat Cancer 2010;17:481–93.

31. Haddad BR, Gu L,Mirtti T, Dagvadorj A, Vogiatzi P, HoangDT, et al. STAT5A/B gene locus undergoes amplification duringhumanprostate cancer progression.Am J Pathol 2013;182:2264–75.

32. Talati PG, Gu L, Ellsworth EM, Girondo MA, Trerotola M, Hoang DT, et al.Jak2-Stat5a/b signaling induces epithelial-to-mesenchymal transition and stem-like cell properties in prostate cancer. Am J Pathol 2015;185:2505–22.

33. Li H, Zhang Y, Glass A, Zellweger T, Gehan E, Bubendorf L, et al. Activation ofsignal transducer and activator of transcription-5 in prostate cancer predictsearly recurrence. Clin Cancer Res 2005;11:5863–8.

34. Mirtti T, Leiby BE, Abdulghani J, Aaltonen E, Pavela M, Mamtani A, et al.Nuclear Stat5a/b predicts early recurrence and prostate cancer-specific death inpatients treated by radical prostatectomy. Hum Pathol 2013;44:310–9.

35. Dagvadorj A, Collins S, Jomain JB, Abdulghani J, Karras J, Zellweger T, et al.Autocrine prolactin promotes prostate cancer cell growth via Janus kinase-2-signal transducer and activator of transcription-5a/b signaling pathway. Endo-crinology 2007;148:3089–101.

36. Ghoreschi K, Laurence A, O'Shea JJ. Janus kinases in immune cell signaling.Immunol Rev 2009;228:273–87.

37. Yin T, Shen R, Feng GS, Yang YC. Molecular characterization of specificinteractions between SHP-2 phosphatase and JAK tyrosine kinases. J Biol Chem1997;272:1032–7.

38. MyersMP,Andersen JN, ChengA, TremblayML,HorvathCM, Parisien JP, et al.TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B. J Biol Chem2001;276:47771–4.

39. Tanuma N, Nakamura K, Shima H, Kikuchi K. Protein-tyrosine phosphatasePTPepsilon C inhibits Jak-STAT signaling and differentiation induced byinterleukin-6 and leukemia inhibitory factor in M1 leukemia cells. J Biol Chem2000;275:28216–21.

40. Lee JH, Kim C, Baek SH, Ko JH, Lee SG, Yang WM, et al. Capsazepine inhibitsJAK/STAT3 signaling, tumor growth, and cell survival in prostate cancer.Oncotarget 2017;8:17700–11.

41. Duhe RJ, Farrar WL. Characterization of active and inactive forms of the JAK2protein-tyrosine kinase produced via the baculovirus expression vector system.J Biol Chem 1995;270:23084–9.

42. Dagvadorj A, Tan SH, Liao Z, Cavalli LR, Haddad BR, Nevalainen MT.Androgen-regulated and highly tumorigenic human prostate cancer cell lineestablished from a transplantable primary CWR22 tumor. Clin Cancer Res 2008;14:6062–72.

43. Damiano JS, Rendahl KG, Karim C, Embry MG, Ghoddusi M, Holash J, et al.Neutralization of prolactin receptor function bymonoclonal antibody LFA102, anovel potential therapeutic for the treatment of breast cancer. Mol Cancer Ther2013;12:295–305.

44. HedvatM,Huszar D, HerrmannA, Gozgit JM, Schroeder A, Sheehy A, et al. TheJAK2 inhibitor AZD1480 potently blocks Stat3 signaling and oncogenesis insolid tumors. Cancer Cell 2009;16:487–97.






45. Futami M, Zhu QS, Whichard ZL, Xia L, Ke Y, Neel BG, et al. G-CSF receptoractivation of the Src kinase Lyn is mediated by Gab2 recruitment of the Shp2phosphatase. Blood 2011;118:1077–86.

46. Pazdrak K, Adachi T, Alam R. Src homology 2 protein tyrosine phos-phatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosinephosphatase is a positive regulator of the interleukin 5 receptor signaltransduction pathways leading to the prolongation of eosinophil sur-vival. J Exp Med 1997;186:561–8.

47. Liao Z, Gu L, Vergalli J, Mariani SA, De Dominici M, LokareddyRK, et al. Structure-based screen identifies a potent smallmolecule inhibitor of Stat5a/b with therapeutic potential for pros-

tate cancer and chronic myeloid leukemia. Mol Cancer Ther 2015;14:1777–93.

48. Nevalainen MT, Harkonen PL, Valve EM, Ping W, Nurmi M, Martikainen PM.Hormone regulation of human prostate in organ culture. Cancer Res 1993;53:5199–207.

49. NevalainenMT, Valve EM, Ingleton PM, NurmiM,Martikainen PM, HarkonenPL. Prolactin and prolactin receptors are expressed and functioning in humanprostate. J Clin Invest 1997;99:618–27.

50. Liu C, Zhu Y, Lou W, Cui Y, Evans CP, Gao AC. Inhibition of constitutivelyactive Stat3 reverses enzalutamide resistance in LNCaP derivative prostatecancer cells. Prostate 2014;74:201–9.


Udhane et al.




2020;19:231-246. Published OnlineFirst September 23, 2019.Mol Cancer Ther Vindhya Udhane, Cristina Maranto, David T. Hoang, et al. Exploitable Molecular Target for Jak2 InhibitorsTherapy-Resistant Prostate Cancer Growth Providing an Enzalutamide-Induced Feed-Forward Signaling Loop Promotes

Updated version

10.1158/1535-7163.MCT-19-0508doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2019/09/21/1535-7163.MCT-19-0508.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/19/1/231.full#ref-list-1

This article cites 50 articles, 25 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/19/1/231To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-19-0508

http://mct.aacrjournals.org/content/suppl/2019/09/21/1535-7163.MCT-19-0508.DC1

http://mct.aacrjournals.org/content/19/1/231.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/19/1/231


Enzalutamide-Induced Feed-Forward Signaling Loop Promotes ... · receptorexpression...

Documents

Transcript of Enzalutamide-Induced Feed-Forward Signaling Loop Promotes ... · receptorexpression...