molecular cancer therapeutics - ec-70124, a novel ......many cancer types (8–11). accordingly,...

Small Molecule Therapeutics

EC-70124, a Novel Glycosylated IndolocarbazoleMultikinase Inhibitor, Reverts Tumorigenic andStem Cell Properties in Prostate Cancer byInhibiting STAT3 and NF-kBGianluca Civenni1, Nicole Longoni1, Paula Costales2, Cecilia Dallavalle1,Cristina García Incl�an3, Domenico Albino1, Luz Elena Nu~nez2, Francisco Morís2,Giuseppina M. Carbone1,4, and Carlo V. Catapano1,4,5

Abstract

Cancer stem cells (CSC) contribute to disease progression andtreatment failure in prostate cancer because of their intrinsicresistance to current therapies. The transcription factors NF-kBand STAT3 are frequently activated in advanced prostate cancerand sustain expansion of prostate CSCs. EC-70124 is a novelchimeric indolocarbazole compound generated by metabolicengineering of the biosynthetic pathways of glycosylated indolo-carbazoles, such as staurosporine and rebeccamycin. In vitrokinome analyses revealed that EC-70124 acted as a multikinaseinhibitor with potent activity against IKKb and JAK2. In this study,we show that EC-70124blocked concomitantlyNF-kB and STAT3in prostate cancer cells and particularly prostate CSCs, whichexhibited overactivation of these transcription factors. Phosphor-ylation of IkB and STAT3 (Tyr705), the immediate targets of IKKb

and JAK2, respectively, was rapidly inhibited in vitro by EC-70124at concentrations that were well below plasma levels in mice.Furthermore, the drug blocked activation of NF-kB and STAT3reporters and suppressed transcription of their target genes. Treat-ment with EC-70124 impaired proliferation and colony forma-tion in vitro and delayed development of prostate tumor xeno-grafts. Notably, EC-70124 had profound effects on the prostateCSC subpopulation both in vitro and in vivo. Thus, EC-70124 is apotent inhibitor of the NF-kB and STAT3 signaling pathways andblocked tumor growth and maintenance of prostate CSCs. EC-70124 may provide the basis for developing new therapeuticstrategies that combine agents directed to the CSC componentand the bulk tumor cell population for treatment of advancedprostate cancer. Mol Cancer Ther; 15(5); 806–18. �2016 AACR.

IntroductionProstate cancer is the most common epithelial cancer and

the third leading cause of cancer-related death in men inwestern countries (1, 2). Substantial progress has been madein recent years to define the molecular and genetic mechanismsunderlying prostate carcinogenesis (3). Nevertheless, there hasbeen limited improvement in the management of patients withadvanced castration-resistant prostate cancer (CRPC) for whichthere are still few treatment options (4–6). There is increasing

evidence of intratumor heterogeneity in human cancers where-by distinct cell populations sharing common genetic makeupexhibit different tumor-initiating and metastasis-seeding capa-bility (7). A subpopulation of cancer cells, defined as cancerstem cells (CSC) or tumor-initiating cells, which display stemcell–like properties and high tumor-initiating potential, hasbeen implicated in progression and disease recurrence inmany cancer types (8–11). Accordingly, treatment failure inadvanced prostate cancer patients may be attributed to theintrinsic resistance of the subpopulation of prostate CSCs tocurrent therapies, such as androgen deprivation and cytotoxicdrugs (8, 10).

Development of new treatment strategies for prostate cancershould take in consideration the biologic complexity andcellular heterogeneity intrinsic to the disease. Drugs able toeliminate the prostate CSC subpopulation could have consid-erable impact on the management of prostate cancer (8). Inthis context, the signaling pathways controlling the expansionand maintenance of CSCs could provide the ideal targets fordevelopment of CSC-directed therapies (11, 12). The CSCsrelies both on cell-autonomous signals and the signals comingfrom the supportive niche formed by stromal cells within thetumor microenvironment (7, 11). Supportive cells includemacrophages, fibroblasts, and mesenchymal stem cells thatsecrete growth factors and cytokines, such as IL6, and create amilieu enabling the cancer cells to acquire and retain stem-like

1Tumor Biology and Experimental Therapeutics Program, Institute ofOncologyResearch (IOR), Bellinzona, Switzerland. 2Campus El Cristo,EntreChem, S.L., Edificio Científico Tecnol�ogico, Oviedo, Spain.3Department of Otolaryngology, IUOPA, Hospital Universitario Cen-tral deAsturias,Oviedo, Asturias, Spain. 4Oncology Institute of South-ern Switzerland (IOSI), Bellinzona, Switzerland. 5Department ofOncology, Faculty of Biology and Medicine, University of Lausanne,Lausanne, Switzerland.

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

Corresponding Author: Carlo V. Catapano, Institute of Oncology Research(IOR), Via Vela, 6, Bellinzona CH-6500, Switzerland. Phone: 4109-1820-0365;Fax: 4109-1820-0305; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0791

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(5) May 2016806

on March 20, 2021. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst January 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0791

http://mct.aacrjournals.org/

properties (13–15). Recent reports point to transcriptionalregulators, such NF-kB and STAT3, as key elements in the sig-naling pathways sustaining CSCs in human tumors (16–18).Constitutive activation of NF-kB is found in many tumors(19, 20). NF-kB and several of its target genes, including theproinflammatory cytokine IL6, promote cancer developmentand activation of prosurvival pathways (21). IL6 activatesthe JAK/STAT3 signaling, which promotes the production ofprosurvival, proangiogenic, and immunosuppressive factors(22–25). Activation of STAT3 is frequent in human cancersand is often concomitant with the activation of NF-kB (22).Notably, STAT3 and NF-kB induce highly overlapping setsof protumorigenic genes with important functions in tumor-igenesis and CSC biology (22,26–28). Furthermore, multiplepositive and negative feedback loops link the STAT3 andNF-kB pathways leading to reciprocal activation or inhibition,depending on the cell context and nature of the extracellularstimuli (29, 30).

One of the hallmark in prostate cancer is deregulated expres-sion of ETS transcription factors as consequence of genetic andepigenetic events (31–34). Recently, we reported the establish-ment of a feedback loop that links the ETS factor ESE1/ELF3with components of the NF-kB complex and leads to consti-tutive activation of NF-kB in a clinically aggressive subset ofprostate cancers (35). Notably, activation of the ESE1/ELF3/NF-kB axis promoted the production of inflammatory and protu-morigenic cytokines, including IL6 (35). The activation of NF-kB and cross-talks with the IL6/JAK/STAT3 signaling pathwaywere essential for the acquisition of the epithelial-to-mesen-chymal transition characteristics and the CSC phenotype inaggressive tumors (35). In such a context, combined targetingof NF-kB and STAT3 could provide a very effective strategy toeliminate prostate CSCs and prevent tumor progression.

Combinatorial biosynthesis by genetic rearrangements ofkey metabolic enzymes in microorganisms is a powerfulapproach to produce "chimeric" compounds that resemblenatural products but possess new and improved pharmaco-logic properties (36). Genetic manipulation of the biosyntheticpathways of the glycosylated indolocarbazoles, such as staur-osporine and rebeccamycin, was recently reported to give raiseto novel compounds retaining the indolocarbazole moiety ofrebeccamycin and glycosylation patterns similar to staurospor-ine (37). Many chimeric compounds acted as potent multi-kinase inhibitors with similar or higher activity compared withstaurosporine when assayed against a wide spectrum of humankinases (37) and unpublished data. In this series of novelglycosylated indolocarbazole compounds, EC-70124 (Fig. 1A)was of particular interest because it exhibited high activitytoward IKK and JAK kinases, such as IKKb and JAK2, with IC50

in the low nanomolar range (37, 38). IKKb phosphorylates IkB,the inhibitory subunit that maintains the NF-kB complex inthe inactive state in the cytoplasm (20). Inhibition of IKKbblocks degradation of IkB and nuclear translocation and acti-vation of NF-kB. JAK2 phosphorylates STAT3 at Tyr705 inresponse to IL6 and other cytokines and growth factors(39). Phosphorylation of Tyr705 is a critical step in theactivation of STAT3 enhancing the accumulation in the cellnucleus and transcriptional activity of STAT3 (24, 39). JAKinhibitors prevent Tyr705 phosphorylation and activation ofSTAT3 nuclear function. The possibility to target concurrentlyNF-kB and STAT3 with a single compound like EC-70124

could provide an innovative strategy to disrupt the protumori-genic cross-talks sustaining the prostate CSC phenotype andavoid the downsides associated with targeting individuallyeither NF-kB or STAT3 and activation of alternative survivalpathways. Therefore, in this study, we took advantage of theunique property of this novel compound to examine the effectsof dual inhibition of NF-kB and STAT3 on the CSC componentin human prostate cancer cell lines and tumor xenografts.

Materials and MethodsCell culture, cell transfection, luciferase reporter assays, andselection of stable cell clones

Prostate cancer cell lines were maintained as described pre-viously (34, 40). All cell lines were obtained from the ATCC,which performs cell line characterization based on DNA pro-filing (short tandem repeat analysis), and were used within 6months of culturing. ESE1/ELF3 expressing 22Rv1 cells weredescribed previously (35). Prostatosphere (PS) cultures wereestablished as described previously (40–42). Luciferase reporterassays from transient transfection were carried out using thepGL4.32(luc2P/NF-kB-RE/Hygro) and SIE pGL4[luc2P/SIE-RE/Hygro vectors along with pGL3-basic vector (Promega AG) ascontrol and pRL-TK vector to normalize for transfection effi-ciency as described previously (34). Stable DU145 cell lineswere generated with the NF-kB– and STAT3-responsive repor-ters using pGL4.32(luc2P/NF-kB-RE/Hygro) and precast lenti-viral particles with STAT3 reporter construct (Qiagen).

Cell proliferation and cell migrationCell growth was evaluated using the sulforhodamine B (SRB)

assay. Clonogenic, scratch/wound healing, and Boyden chamberassays were performed as described previously (35).

Cell cycle and apoptosis7-AAD (25 mg/mL, Sigma Aldrich) was used for cell-cycle

analysis by flow cytometry on a FACS Fortessa (BD Biosciences).Apoptosiswas evaluatedmeasuring the level of activatedCaspase-3 (BD Pharmingen Activated Caspase-3 MAb Apoptosis Kit).

Quantitative RT-PCRQuantitative real-time PCR (qRT-PCR) was performed on total

RNA using custom-made primers and analyzed as describedpreviously (35).

Immunoblotting and immunofluorescence microscopyCell lysates were prepared and analyzed as described previ-

ously (35). Lysates from tumor xenografts were prepared fromfreshly frozen tissue. Cytoplasmic and nuclear extract wereobtained using NE-PER Nuclear and Cytoplasmic ExtractionReagent (Thermo Scientific). For immunofluorescence micros-copy, cells grown on glass coverslips were incubated withprimary antibodies followed by incubation with anti-rabbitAlexa 488 or anti-mouse Alexa 594 (Invitrogen) secondaryantibodies (35). The following antibodies were used in thestudy: ESE1/ELF3 (ab1392, AbCam), p50, p65, b-tubulin (Cal-biochem), STAT3, pSTAT3 (Tyr705), pSTAT3 (Ser727), JAK2,and phosphorylated JAK2 (pJAK2; Tyr1007/1008; Cell Signal-ing Technology).

STAT3 and NF-kB Inhibition by EC-70124 Affects Prostate CSCs

www.aacrjournals.org Mol Cancer Ther; 15(5) May 2016 807




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Figure 1.EC-70124 blocks NF-kB and STAT3 activities in prostate cancer cells. A, structure of EC-70124. B, NF-kB and STAT3 transcriptional activities in 22Rv1 (black bars)and DU145 (white bars) determined using NF-kB– or STAT3-responsive luciferase reporters. C, evaluation of luciferase activity in DU145 stably expressing aNF-kB– and STAT3-responsive element luciferase reporters after treatment for 24 hours with EC-70124 (500 nmol/L). D, phosphorylation of JAK2 and STAT3evaluated by Western blot analysis after treatment of DU145 cells with EC-70124 (250 and 500 nmol/L). E, phosphorylation inhibition of STAT3 by EC-70124is reversible. DU145 cells were treated with EC-70124 (500 nmol/L) for 2 hours. Cells were lysed at different time points (0, 4, and 24 hours) and phosphorylationof STAT3 was evaluated by Western blot analysis. F, phosphorylation of NF-kB inhibitor IkBa evaluated by Western blot after treatment of DU145 cells withEC-70124 (250 nmol/L, 4 hours). G, level of p65 assessed by Western blot analysis in nuclear and citoplasmic fractions of DU145 cells after 4-hour treatmentwith EC-70124 (250 and 500 nmol/L). H, mRNA levels of ESE1/NF-kB target genes evaluated after 4 hours of incubation with EC-70124 (250 nmol/L). I,STAT3 and NF-kB transcriptional activities in 22Rv1 transiently cotransfected with a STAT3- and NF-kB–responsive element luciferase reporter and an ESE1expression vector (pESE1). J, expression of STAT3/NF-kB target genes (COX2, ST6GAL-5, and MMP10) in 22Rv1-pcDNA and 22RV1-pESE1 cells cultured inpresence of EC-70124 (250 nmol/L) was determined by qRT-PCR. P values were determined using t test. � , P < 0.01; �� , P < 0.05.

Civenni et al.

Mol Cancer Ther; 15(5) May 2016 Molecular Cancer Therapeutics808




Animal studiesStudy protocols involving animals were approved by the

Swiss Veterinary Authority. Experimental groups consisted of atleast 5 animals with equal age (8–10 weeks) and body weight(30–33 g). To monitor tumor growth athymic nude (Balb/cnu/nu) mice with subcutaneous tumor xenografts were treateddaily for 5 days for 2 consecutiveweekswith EC-70124 [40mg/kg,orally]. To assess the drug distribution, healthy CD-1 male micereceived a single dose of EC-70124 (40 mg/kg, orally) and druglevels in blood and tissues were measured by LC/MS. In vivoimaging was performed on an IVIS Spectrum Imaging System(Caliper LifeSciences; ref. 42). Details are provided in Supple-mentary Methods.

Statistical analysisDifferences between groups were assessed with an unpaired

two-tailed t test and were considered statistically significant forP < 0.01 (�) and < 0.05 (��). To calculate IC50 values, means andstandard deviations were plotted using GraphPad Prism Ver-sion 6. A sigmoidal 4PL model was used to interpolate the dataand calculate IC50. Each interpolation with R2 � 0.8 wasconsidered valid.

ResultsEC-70124 inhibits NF-kB and STAT3 in prostate cancer cells

Assays interrogating in vitro a large spectrum of kinases in thehuman kinome revealed that EC-70124 was highly activeagainst IKK and JAK kinases (37, 38). EC-70124 was alsoreported to inhibit NF-kB and JAK/STAT signaling, respectively,in glioblastoma cells (43) and in triple-negative breast cancercells (38). However, the effects of EC-70124 on STAT3 and NF-kB signaling and the consequences of concomitantly targetingthese key transcription factors in cancer cells with constitutiveactivation of both pathways have not been investigated. Toaddress this issue, we tested the effects of EC-70124 in DU145prostate cancer cells. DU145 cells are a model of androgenreceptor (AR) negative and androgen-independent prostatecancer. Furthermore, unlike the AR-dependent LNCaP cells,DU145 cells have constitutive activation of both STAT3 andNF-kB (30, 44, 45). These cells represent also a good model ofprostate tumors characterized by concomitant upregulation ofESE1/ELF3 and sustained activation of NF-kB (35).

Activity of transcription factors, such as NF-kB and STAT3,can be accurately assessed in cells using reporters with specificresponsive elements for the given factor. Using a reportersystem, we confirmed that both NF-kB and STAT3 were highlyactive in DU145 cells (Fig. 1B). For comparison, NF-kB– andSTAT3-responsive reporters had low activity in 22Rv1 prostatecancer cells in which these factors were not constitutivelyactive. To monitor the activity of EC-70124 on STAT3 andNF-kB signaling, we generated derivative DU145 cells withstable expression of the STAT3 (STAT3-RE DU145) and theNF-kB (NFKB-RE DU145) responsive reporters. The activity ofthe reporters reflected the level of the transcription factor andthe status of the signaling pathway responsible for its activa-tion. Treatment with EC-70124 inhibited both the STAT3- andNF-kB–responsive reporters (Fig. 1C).

To determine whether these effects were due to interferencewith IKKb and JAK2 kinase, we assessed the phosphorylationstate of the immediate targets of these kinases by immuno-

blotting. EC-70124 reduced pJAK2 within 30 minutes of incu-bation (Fig. 1D). Concomitantly, phosphorylation of STAT3(pSTAT3) at Tyr705 was reduced with similar time and dosedependence. The rapid response of JAK2 and STAT3 (Tyr705)phosphorylation to EC-70124 suggested that the drug acted as adirect inhibitor of this kinase. JAK2 and STAT3 protein levelswere not affected. Furthermore, phosphorylation of STAT3at Ser727 was not reduced, consistent with selective inhibitionof the JAK2-mediated Tyr705 phosphorylation. Interestingly,the effects of EC-70124 on JAK2 and STAT3 phosphorylationwere rapidly reversed within 4 hours after drug washout(Fig. 1E), suggesting that continuous exposure would be likelyrequired to obtain more persistent inhibition of the target bythe drug. Treatment of DU145 cells with EC-70124 reducedalso phosphorylated IkBa (Fig. 1F) and the amount of theNF-kB subunit p65 translocated in the cell nuclei (Fig. 1G andSupplementary Fig. S1), a prerequisite for reduced NF-kB tran-scriptional activity. Notably, the effects on STAT3 and NF-kBsignaling were seen at nanomolar concentrations (250–500nmol/L) and relatively short incubation times, emphasizingthe potency of EC-70124 in these cellular assays. Moreover,inhibition of STAT3 and NF-kB activation led to significantdownregulation of various genes controlled by these transcrip-tion factors, such as ST6GAL-5, FN-1, ANGPTL-4, p50, ESE1/ELF3, and IL6 (Fig. 1H). These results support the efficacy ofEC-70124 as dual inhibitor of STAT3 and NF-kB signaling inthese cells.

We showed previously that overexpression of ESE1/ELF3 in22Rv1 cells resulted in activation of NF-kB and several of itstarget genes (35). We found that the activity of both NF-kB–and STAT3-responsive reporters was strongly induced byexpression of ESE1/ELF3 in 22Rv1 cells (Fig. 1I), indicatingconcomitant activation of these pathways in ESE1/ELF3 over-expressing 22Rv1 cells. Consistently, the STAT3 and NF-kBreporters were inhibited by EC-70124 in 22Rv1 cells expressingESE1/ELF3 (Fig. 1I). Furthermore, transcription of NF-kB andSTAT3 target genes, which were upregulated in ESE1/ELF3-22Rv1 cells, was inhibited by EC-70124 (Fig. 1J). Interestingly,EC-70124 had no effect on transcription in control 22Rv1 cellsindicating that the drug effect depended on the state of NF-kBand STAT3 in the cells.

EC-70124 affects proliferation of prostate cancer cellsInhibition of NF-kB and STAT3 individually is known to

affect proliferation of DU145 cells (30,44, 46, 47). Given theability of EC-70124 to block concomitantly both signalingpathways, we examined the compound's effect on the pheno-type of DU145 cells. Proliferation of DU145 cells grown asadherent monolayer was inhibited very effectively by treatmentwith EC-70124 for 72 hours (IC50 ¼ 127 � 18 nmol/L; Fig. 2A).Inhibition of cell proliferation increased drastically withincreasing treatment time from 24 to 72 hours (SupplementaryFig. S2). Consistently, monitoring the number of viable cellsover 7 days of continuous treatment showed that EC-70124inhibited cell proliferation in dose-dependent manner with>50% and >75% inhibition at 75 and 250 nmol/L, respectively(Fig. 2B), consistent with the need of prolonged and contin-uous drug exposure to affect cell proliferation. Other prostatecancer cell lines, like PC3 cells, without concomitant activ-ation of STAT3 and NF-kB, were slightly less sensitive (IC50 ¼394 � 36 nmol/L) to EC-70124 (Supplementary Fig. S3).






Treatment with EC-70124 suppressed also colony form-ation in adherence and in soft agar (Fig. 2C and D) and cellmigration in wound-healing assay (Fig. 2E). Interestingly, wedetected minimal or no induction of apoptotic cell death at thedose of 250 nmol/L that inhibited substantially cell prolifer-ation (Supplementary Fig. S4). At the dose of 250 nmol/L, therewas only a slight decrease of the fraction of cells in the Sphase of the cell cycle, consistent with partially reduced pro-

liferative capacity of DU145 cells treated with EC-70124 (Sup-plementary Fig. S5).

We also examined the effects of EC-70124 in ESE1/ELF3-22Rv1 cells. Overexpression of ESE1/ELF3 in 22Rv1 cells pro-moted cell proliferation, colony formation, and migration(35). EC-70124 was able to counteract the phenotypic effectsinduced by ESE1/ELF3 overexpression reducing cell migration(Fig. 2F and G) and colony formation in soft agar (Fig. 2H).

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Figure 2.EC-70124 decreases proliferation, migration, and colonies formation of prostate cancer cells. A, cell viability evaluated by SRB assay 72 hours after treatmentwith EC-70124 in DU145 cells. B, DU145 were treated with EC-70124 (75, 150, 250, and 500 nmol/L) and counted over a 7-day period. C, colonyformation in adhesive and in soft-agar conditions (D) after treatment EC-70124 (250 nmol/L) in DU145 cells. E, scratch-wound healing assay withDU145 cells following treatment with 250 nmol/L of NF-kB inhibitors for 4 hours. F, scratch-wound healing assay with control (pcDNA) and ESE1/ELF3-expressing (pESE1/ELF3) 22Rv1 cells after treatment EC-70124 (250 nmol/L) for 4 hours. G, modified Boyden chamber assay with pESE1/ELF3 andpcDNA 22Rv1 cell lines after treatment with EC-70124 (250 nmol/L). H, colony formation in soft-agar conditions after treatment EC-70124 (250 nmol/L)in control (pcDNA) and ESE1/ELF3-expressing (pESE1/ELF3) 22Rv1 cells. P values were determined using t test. � , P < 0.01.

Civenni et al.





The activity of EC-70124 in parental 22Rv1 cells was minimalin these assays, indicating that ESE1/ELF3 overexpression andconsequent activation of STAT3 and NF-kB determined highersensitivity to the drug.

EC-70124 inhibits STAT3 and NF-kB in prostate CSC cellsCulturing prostate cancer cells, like DU145 cells, at low cell

density in nonadherent conditions and serum-free media allowsthe selection of single-cell–derived floating colonies, calledprostate spheres (PS), that are enriched of cancer cells withstem-like and tumor-initiating properties (40–42). PS-formingDU145 cells have high expression of CSC-specific markers andproduce tumors in immunodeficient mice with much higherefficiency compared with the bulk cancer cell population grownas adherent monolayer (40–42). Using the stable reporter celllines, we examined the activity of STAT3 and NF-kB in the CSC-enriched PS and bulk DU145 cells. The activity of both STAT3and NF-kB reporters was strikingly higher in PS-forming cellscompared with adherent DU145 cells (Fig. 3A). The level oftarget genes, like COX2, IL6, and ESE1, was also increased in thePS-forming cell subpopulation compared with bulk adherentDU145 cells (Fig. 3B). These data suggested that hyperactivity ofSTAT3 and NF-kB could drive the expansion of CSCs in prostatecancers. Consistently, treatment with EC-70124 drasticallyreduced STAT3 and NF-kB reporter activity in CSC-enrichedDU145 PS cells (Fig. 3C). Furthermore, EC-70124 led to asignificant decrease of COX2, IL6, and ESE1 expression in PS-derived cells (Fig. 3D). These results indicated that EC-70124effectively blocked STAT3 and NF-kB signaling in the CSCsubpopulation in this prostate cancer cell line.

Next, we examined whether treatment with EC-70124 affect-ed the number of PS-forming cells in prostate cancer cellcultures. STAT3 and NF-kB are hyperactive in the prostate CSCsubpopulation and likely provide survival and growth signalsfor maintaining the CSC compartment. DU145 cells culturedas adherent monolayer were dissociated, plated in PS-formingconditions and then incubated in the presence or absence ofEC-70124 (Fig. 3E). We observed dose-dependent reduction inthe number of PS in the presence of EC-70124 compared withcontrol cells (IC50 ¼ 139 nmol/L). Thus, treatment with EC-70124 impaired the ability of the CSC subpopulation toexpand and generate single-cell–derived PS. To determinewhether EC-70124 affected the CSC fraction also within theheterogeneous bulk cancer cell population, we treated DU145cells in adherent monolayer cultures with the drug for 24hours. Then, cells were harvested, washed, and plated in PS-forming conditions. Strikingly, the number of PS-forming cellswas strongly reduced by the pretreatment with EC-70124compared with control cells (>90% inhibition; Fig. 3F), sug-gesting that treatment with EC-70124 and consequent inhibi-tion of NF-kB and STAT3 had a prominent effect on theprostate CSC subpopulation.

Oral EC-70124 has favorable pharmacokinetic andpharmacodynamic profile in mice

To assess the activity of EC-70124 in mouse xenograft mod-els, we examined the compound pharmacokinetic and phar-macodynamic properties to define optimal delivery and sched-ule of administration. Prior studies indicated that the drugcould be given safely by intravenous injection (38). Toxicity

studies indicated that EC-70124 was could also be well toler-ated when given orally. Therefore, we explored the option ofgiving EC-70124 orally, which could be particularly attractivebecause of the likely need of continued treatment to maintaindurable target inhibition. We assessed the pharmacokineticprofile of EC-70124 given orally at a single dose of 40 mg/kg.Measurement of the drug level in the plasma showed an initialpeak after 2 hours and a slow decline of the level of EC-70124in the following 24 hours (Fig. 4A). Importantly, the concen-tration of EC-70124 in plasma was 1,107 ng/g (2.4 mmol/L)and 425 ng/g (0.9 mmol/L) at 4 and 8 hours, respectively, wellabove the drug concentration (250–500 nmol/L) required fortarget and cell growth inhibition in vitro. Furthermore, the levelof EC-70124 in mouse tissues (e.g., prostate) was comparablewith the level in the plasma (Fig. 4A), indicating that the drugwas rapidly distributed from the bloodstream and efficientlytaken up in organs reaching levels compatible with targetinhibition.

To verify whether EC-70124 was able to inhibit the ex-pected targets in vivo, we used the reporter cell line andperformed in vivo luciferase reporter assays. The NFKB-REDU145 reporter cells were implanted subcutaneously to gen-erate tumors in mice. Constitutive activity of NF-kB in DU145cells ensured elevated activity of the luciferase reporter inthe subcutaneous tumor xenografts detectable by in vivo bio-luminescence imaging. Mice bearing tumors were treated withEC-70124 (40 mg/kg, orally) at day 1. Reporter activity wasevaluated in control and drug-treated mice at various timesafter treatment (2–48 hours; Fig. 4B). Remarkably, a singledose of EC-70124 on day 1 profoundly reduced the activityof the NF-kB reporter within 2 hours (85 % of reduction;Fig. 4C). The inhibition of the NF-kB reporter was notsustained and recovered completely at 24 hours. After asecond dose of EC-70124 on day 3, the reporter activity wasmaximally reduced at 6 hours (96% of reduction) andreturned slowly to the control level within 24 to 48 hours(Fig. 4B). Thus, the pharmacodynamic behavior was consis-tent with the pharmacokinetic profile of EC-70124 showingthat levels of the drug sufficient to engage the target wereachieved within few hours and were maintained for severalhours after repeated oral administration.

EC-70124 inhibits tumor growth and depletes CSC in prostatetumor xenografts in mice

Orally administered EC-70124 reached the tumor andinhibited the target as assessed by the in vivo reporter cellassays. The effect, although profound, did not persist suggest-ing that repeated dosing were required to obtain prolongedinhibition of the target and induce an antitumor response. Toevaluate the in vivo antitumor effect of EC-70124, the com-pound (40 mg/kg) was administered daily for 5 days in 2consecutive weeks to mice carrying subcutaneous DU145tumor xenografts. The treatment led to a significant delay oftumor growth (Fig. 5A). EC-70124 was well tolerated and noweight loss or other adverse events were observed. At the lastday of treatment, pIkBa and pSTAT3 (Tyr705) were signifi-cantly reduced in tumors from EC-70124–treated mice com-pared with control mice (Fig. 5B and C). In line with the dualinhibition of NF-kB and STAT3 signaling, expression of NF-kBand STAT3 target genes was significantly decreased in EC-70124–treated tumors (Fig. 5D).






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We further tested the activity of EC-70124 in tumor xenograftsformed by ESE1/ELF3 overexpressing and control 22Rv1 cells. Wereported previously that ESE1/ELF3-22Rv1 cells formed largertumors than control 22Rv1 cells (35). ESE1/ELF3 overexpressing22Rv1 cells exhibited constitutive activation of NF-kB and upre-gulation of NF-kB and STAT3 target genes (35). Furthermore, invitro the effects of EC-70124 depended on the state of activation oftheNF-kB and STAT3pathways, beingmore pronounced in ESE1/ELF3-22Rv1 than control 22Rv1 cells. Notably, treatment withEC-70124 delayed substantially the growth of 22RV1-pESE1xenografts, while 22Rv1-pcDNA xenografts minimally affected(Fig. 6A). Similar to the DU145 tumor xenografts, the level ofpIkBa, pSTAT3 (Tyr705; Fig. 6B and C), and expression of ESE1,COX2, MMP10, and IL6 (Fig. 6D) were reduced in 22Rv1-pESE1tumors frommice treated with EC-70124 compared with tumorsfrom control mice.

In line with the in vitro data, the pattern of inhibition oftumor growth induced by EC-70124 in vivo suggested that thedrug had a cytostatic rather than cytotoxic effect, with the druginducing perhaps a more differentiated phenotype and pro-gressively reducing the fraction of proliferating tumor cellsrather than inducing rapid growth arrest or cell death. In linewith this hypothesis, the number of tumor cells positive for theproliferation marker Ki67 in both tumor xenograft models wasnot significantly different between EC-70124–treated and con-trol mice at the end of the treatment (Supplementary Fig. S6).Thus, there was no massive reduction of proliferating cells inthe bulk tumors based on Ki67 staining. This pattern could becompatible with the hypothesis that the drug was hitting moreeffectively the CSC subpopulation than the bulk proliferatingtumor cells and progressively depleting the CSC component.This was consistent with the in vitro data showing the highefficacy of EC-70124 on the CSC-enriched PS-forming cellswithin the bulk tumor cell population (Fig. 3F). This hypoth-esis was further supported by the significant reduction of Mycand Nanog, two genes that we found consistently associatedwith the CSC phenotype, in tumor xenografts of DU145 and22Rv1-ESE1 treated with EC-70124 (Fig. 5E and 6E). To furtherinvestigate this aspect, we performed ex vivo PS-forming assayswith cells isolated from tumor xenografts at the end of the 2-week treatment with EC-70124. Tumors were excised anddissociated to isolate single cells that were then plated in PS-forming conditions to determine the fraction of cancer cellsthat retained CSC-like properties in control and drug-treatedtumor xenografts. Consistent with our hypothesis, the PS-forming ability of DU145 tumor xenografts from EC-70124-treated mice were significantly reduced compared with controlxenografts (Fig. 5F). We observed a similar reduction of ex vivoPS-forming ability in 22Rv1-ESE1 tumor xenografts treatedwith EC-70124 compared with control xenografts (Fig. 6F).Collectively, these results indicated that treatment with EC-

70124 led to inhibition of STAT3 and NF-kB signaling path-ways, delayed tumor growth and induced a concomitant deple-tion of the CSC subpopulation in the tumor xenografts byimpairing their self-renewal ability.

DiscussionThe transcription factors NF-kB and STAT3 are frequently and

concomitantly activated in many human cancers (19, 39). Inprostate cancer, activation of these transcription factors has beenassociated with disease progression and poor clinical outcome(28, 46, 48). Both STAT3 and NF-kB have been linked todevelopment of castration resistance and poor responses totargeted therapeutics and cytotoxic drugs. NF-kB and STAT3contribute to tumor progression also by influencing the inter-cellular communications between cancer cells and cells in thesurrounding stroma and establishing an inflammatory andprotumorigenic tumor microenvironment (21, 22, 39). More-over, both pathways are found to be hyperactive in the CSCsubpopulation and have been implicated in the acquisition andmaintenance of the CSC phenotype in human tumors (11).Thus, targeting these transcription factors might provide aneffective way to contrast the expansion of the highly tumorigenicstem-like tumor cells in advanced prostate cancer. CSCs areintrinsically resistant to androgen deprivation therapy and manycytotoxic drugs causing treatment failures and disease recurrence(8–10, 49). CSC-targeted agents could be most effective incombination with current therapies to increase response ratesand prolong survival in advanced prostate cancer patients (11,12). However, drugs that could selectively and effectively affectprostate CSC behavior are not currently available. CSCs upre-gulated multiple mechanisms that ensure survival and resistanceto treatment (7, 11). Therefore, combined targeting of multiplekey oncogenic pathways might be beneficial to effectively blockthe expansion and survival of CSCs.

In this study, we show that the multikinase inhibitor EC-70124 is able to block concurrently NF-kB and STAT3 signalingin human prostate cancer cells. This approach was particularlyeffective in the subpopulation of highly tumorigenic and stemcell–like prostate cancer cells that exhibited hyperactivity of theSTAT3/NF-kB axis. Drugs targeting prostate CSCs and inducingtheir elimination or differentiation may represent a majoradvance for management of CRPC. The availability of suchdrugs should permit the implementation of combined treat-ment regimens with agents directed to the CSC componentsand bulk proliferating cancer cells. We showed recently thatblocking c-Myc in prostate CSCs shut down a key oncogenicpathway and led to the progressive loss of self-renewal capacityand induction of cell senescence resulting in effective CSCelimination and inhibition of tumor development in mousexenografts (42).

Figure 3.EC-70124 decreases the prostate cancer stem cell compartment. A, STAT3 and NF-kB transcriptional activities in DU145 cells cultured in adherent (Adh,white bars) sphere-forming (PS, gray bars) conditions. B, expression of STAT3/NF-kB target genes (COX2, ESE1, and IL6) in DU145 cells cultured inadherent (white bars) or sphere-forming (gray bars) conditions determined by qRT-PCR. C, NF-kB transcriptional activities after treatment withEC-70124 (500 nmol/L, 24 hours). D, expression of COX2, ESE1, and IL6 mRNA in DU145 cells cultured in sphere-forming conditions after treatmentwith EC-70124 (250 nmol/L) for 24 hours determined by qRT-PCR. E, sphere-forming capacity was evaluated after 10 days of treatment with EC-70124at different concentrations in DU145 cells. F, sphere-forming capacity of DU145 cells treated for 24 hours in adherence conditions. P values weredetermined using t test. �, P < 0.01.






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Figure 4.Pharmacokinetics andpharmacodynamics of EC-70124 inmice. A, after administrating EC-70124orally, plasma and prostate tissueswere collected at different time pointsand the levels of the drug wereevaluated. B, xenografts generated byDU145 cells constitutively engineeredwith a NF-kB–responsive elementluciferase reporter were treated withEC-70124 [40 mg/kg, orally (PO), redline] and compared with mock-treatedmice (black line). Using an IVISSpectrum Imaging System (CaliperLifeSciences), NF-kB transcriptionalactivitywas evaluated after 2, 6, 24, and48 hours. At 48 hours, a second dose ofEC-70124 (40 mg/kg, orally) was givenand, similarly, luciferase activity in vivowas detected after 2, 6, 24, and 48hours. C, representative pictures of amouse treated with EC-70124 at time 0and after 2, 24, and 54 hours oftreatment. P values were determinedusing t test. � , P < 0.01.

Civenni et al.





Using a similar approach, we examined the effects of EC-70124on the NF-kB/STAT3 axis in the bulk and CSC-enriched cellsubpopulation derived from human prostate cancer cell lines.PS formed in vitro by single cells grown in nonadherent andserum-free culture conditions are enriched of prostate CSCs andrepresent an elective model to study the biology of CSCsand their response to drugs (42, 50). Notably, PS derived fromhuman prostate cancer cell lines, like DU145 cells, exhibitedhyperactivity of NF-kB and STAT3 and overexpression of numer-ous target genes compared with the bulk cancer cells, despite theenhanced activity of these pathways in the bulk tumor cellpopulation. Indeed, hyperactivity of these signaling pathways isa common trait in CSC subpopulations derived from differentprostate cancer cell models, although the underlyingmechanismsof activation may vary (35, 40). Treatment with EC-70124 inhib-ited STAT3 and NF-kB transcriptional activity and target geneexpression. These effects were linked to reduced IkB, JAK2, and

STAT3 (Tyr705) phosphorylation, sustaining the hypothesis ofdual targeting of IKKb and JAK2 kinases in these cells. Moreover,EC-70124 effectively blocked STAT3 and NF-kB signaling inthe CSC-enriched subpopulation and reversed the CSC pheno-type reducing in vitro PS formation. EC-70124 exerted similareffects in vivo in tumor xenograft models of prostate cancer whereit reduced IkB and STAT3 (Tyr705) phosphorylation and inhib-ited NF-kB and STAT3 transcriptional activity. Inhibition ofthe NF-kB/STAT3 axis was accompanied by significantly impair-ed development of subcutaneous tumors. Moreover, treatmentwith EC-70124 reduced the CSC component in the tumor xeno-grafts as demonstrated by ex vivo PS-forming assays.

Enhanced activity of NF-kB and STAT3 is common and can bedriven by multiple mechanisms in prostate tumors. We recentlyidentified a subgroup of prostate tumors representing 20% to25% of primary tumors in which upregulation of the ETS factorESE1/ELF3 was associated with prominent activation of NF-kB

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Figure 5.EC-70124 blocks growth of xenografts generated by ESE1/ELF3 high prostate cancer cells. A, DU145 cells were injected subcutaneously into nude mice (n¼ 5)in the right flank (5 � 106 cells/injection). Treatment with EC-70124 started after 27 days from the subcutaneous injections. EC-70124 (40 mg/kg) wasgiven orally for 5 consecutive days for 2 weeks. At the end of the treatment, mice were sacrificed and tumor tissues were collected. Statisticalsignificance of the differences was evaluated at the end of experiment. B, pIKB and IKB levels were assessed by immunofluorescence in tumor tissuesfrom mice treated as indicated in A and collected at the end of the experiment. pSTAT3 and STAT3 levels were assessed by IHC. C, percentage of cellspositive for pIKB and pSTAT3 in control and EC-70124–treated mice are shown in box plots. D, mRNA levels of STAT3/NF-kB targets were evaluatedby qRT-PCR in DU145 xenografts treated with EC-70124 (white bars) and control (black bars). E, c-Myc and Nanog expression determined by qRT-PCRin control (black bars) and drug (white bars) treated DU145 tumor xenografts. F, sphere forming capacity was estimated ex vivo in cells derived fromDU145 tumor xenografts generated in control and EC-70124 (40 mg/kg) treated mice. P values were determined using t test. � , P < 0.01.






(35). The establishment of this positive feedback loop led toconstitutive activation of NF-kB and increased production of theproinflammatory and protumorigenic cytokine IL6. Interestingly,human prostate cancer cell lines that model this tumor subgrouphad concomitant activation of STAT3 and were highly responsiveto EC-70124, suggesting that this compound could be veryeffective in this subgroup of aggressive prostate tumors. Consis-tently, EC-70124was remarkably active in vivo in tumor xenograftsderived from these cell line models.

EC-70124 is a product of metabolic engineering of the staur-osporine and rebeccanycin biosynthetic pathways. In biochem-ical kinase assays, EC-70124 acted as a highly effective multi-kinase inhibitor (37). We show here that both in cells and tumorxenografts, the efficacy of EC-70124 depended on the cellcontext and activation state of the STAT3 and NF-kB pathways.The treatment with EC-70124 was more effective in DU145 and22Rv1-ESE1 cells with hyperactive NF-kB and STAT3 signaling

and high dependency on these pathways. However, because ofthe inherent promiscuity of a multikinase inhibitor and thepossibility of undesired off-target and toxic effects, appropriatetreatment regimens will need to be accurately defined to favoreffective and selective inhibition of the proper intracellulartargets. In our preclinical trial, we approached this issue bydefining the dose and schedule of administration required toobtain a specific biologic response (i.e., inhibition of the NF-kBreporter) in mice with subcutaneous prostate tumor xenografts.Pharmacokinetic and pharmacodynamic studies showed thatsufficient drug levels and effective target inhibition wereobtained by single oral administration of EC-70124 at a rela-tively low dose (40 mg/kg), below the MTD of >100 mg/kg.Repeated daily treatment at this dose was well tolerated with nosigns of systemic toxicity and resulted in significant targetinhibition, antitumor activity, and depletion of the CSC com-ponent in tumor xenograft tissues in mice.

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Figure 6.EC-70124 highly reduces tumor growth of xenografts generated by ESE1 overexpressing 22RV1. A, 22Rv1-pcDNA and 22RV1-pESE1 cells were injectedsubcutaneously into nude mice (n ¼ 5) in the right flanks (5.0 � 106 cells/injection). Treatment with EC-70124 (red lines, 40 mg/kg) or vehicle (black lines)started after 12 days from the subcutaneous injections. EC-70124 (40 mg/kg) was given orally for 5 consecutive days for 2 weeks. At the end of thetreatment, mice were sacrificed and tumor tissues were collected. Statistical significance of the differences was evaluated at the end of experiment. B, pIKBand IKB levels were assessed by immunofluorescence in tumor tissues from mice treated as indicated in A and collected at the end of the experiment.pSTAT3 and STAT3 levels were assessed by IHC. C, percentage of pIKB and pSTAT3 positive cells is shown in box plots. D, mRNA levels of ESE1targets were evaluated by qRT-PCR in 22RV1-pESE1 xenografts treated with EC-70124 and control. E, c-Myc and Nanog expression determined by qRT-PCRin control (black bars) and drug (white bars) treated DU145 tumor xenografts. F, sphere-forming capacity was estimated ex vivo in cells derived from22RV1-pESE1 tumor xenografts generated in control and EC-70124 (40 mg/kg) treated mice. P values were determined using t test. � , P < 0.01.

Civenni et al.





Collectively, these findings underscore the ability of EC-70124to act as a dual inhibitor of STAT3 andNF-kB signaling in vitro andin vivo. This novel kinase inhibitor with its action on two keysignaling pathways may represent the prototype of a new class ofanticancer drugs with ability to interfere with CSCs in prostatetumors providing tools for innovative approaches for treatment ofadvanced prostate cancer. In many other tumor types, CSCsexhibit activation and dependency on NF-kB and STAT3 signal-ing. Therefore, EC-70124 might be similarly effective in othermalignancies.

Disclosure of Potential Conflicts of InterestF. Moris has ownership interest (including patents) in EntreChem SL. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: G. Civenni, P. Costales, F. Morís, G.M. Carbone,C.V. CatapanoDevelopment of methodology: G. Civenni, P. Costales, C. Dallavalle,C.V. CatapanoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Civenni, N. Longoni, L. Elena Nu~nez,C.V. Catapano

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Civenni, N. Longoni, P. Costales, C.V. CatapanoWriting, review, and/or revision of the manuscript: G. Civenni, P. Costales,F. Morís, G.M. Carbone, C.V. CatapanoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Civenni, F. Morís, C.V. CatapanoStudy supervision: G. Civenni, G.M. Carbone, C.V. Catapano

AcknowledgmentsThe authors thank Enrica Mira Cato and Sandra Pinton for the support with

in vivo experiments and immunohistochemistry.

Grant SupportThis work was supported by the Ticino Foundation for Cancer Research, the

Virginia Boeger Foundation, and EntreChem SL (to C.V. Catapano); SwissNational Science Foundation (toG.M.Carbone); and FICYTMOBILITYGRANTIT13-017 (to P. Costales).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 30, 2015; revised January 8, 2016; accepted January 8,2016; published OnlineFirst January 29, 2016.

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Civenni et al.




2016;15:806-818. Published OnlineFirst January 29, 2016.Mol Cancer Ther Gianluca Civenni, Nicole Longoni, Paula Costales, et al.

BκCancer by Inhibiting STAT3 and NF-ProstateInhibitor, Reverts Tumorigenic and Stem Cell Properties in

EC-70124, a Novel Glycosylated Indolocarbazole Multikinase

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