the stat3-mirna-92-wnt signaling pathway regulates ...regulatory pathways. targeting stat3 in...

Molecular and Cellular Pathobiology

The STAT3-miRNA-92-Wnt Signaling PathwayRegulates Spheroid Formation and MalignantProgression in Ovarian CancerMin-Wei Chen1, Shu-Ting Yang2, Ming-Hsien Chien3,4, Kuo-Tai Hua5, Chin-Jui Wu6,S.M. Hsiao7, Hao Lin8, Michael Hsiao9, Jen-Liang Su2,10,11,12, and Lin-Hung Wei1,6

Abstract

Ovarian cancer spheroids constitute a metastatic niche fortranscoelomic spread that also engenders drug resistance. Spher-oid-forming cells express active STAT3 signaling and display stemcell–like properties that may contribute to ovarian tumor pro-gression. In this study, we show that STAT3 is hyperactivated inovarian cancer spheroids and that STAT3 disruption in this settingis sufficient to relieve chemoresistance. In an NSG murine modelof human ovarian cancer, STAT3 signaling regulated spheroidformation and self-renewal properties, whereas STAT3 attenua-tion reduced tumorigenicity. Mechanistic investigations revealedthat Wnt signaling was required for STAT3-mediated spheroid

formation. Notably, the Wnt antagonist DKK1 was the moststrikingly upregulated gene in response to STAT3 attenuation inovarian cancer cells. STAT3 signaling maintained stemness andinterconnected Wnt/b-catenin signaling via the miR-92a/DKK1–regulatory pathways. Targeting STAT3 in combination with pac-litaxel synergistically reduced peritoneal seeding and prolongedsurvival in a murine model of intraperitoneal ovarian cancer.Overall, our findings define a STAT3–miR-92a–DKK1 pathway inthe generation of cancer stem–like cells in ovarian tumors, withpotential therapeutic applications in blocking their progression.Cancer Res; 77(8); 1955–67. �2017 AACR.

IntroductionEpithelial ovarian cancer (EOC) is the most lethal of all

gynecologic malignancies, and the majority of cases are discov-ered when the primary tumor has already metastasized. Despitethe high complete response rate after maximal debulking surgery

and platinum/taxane–combination chemotherapy, approximate-ly 75% of patients with advanced EOC develop recurrent diseasewithin 3 years of diagnosis (1). Recurrent disease is generally notcurable, and the relative survival rates at 10 years for stage III andIV disease are 23% and 8%, respectively (2).

The metastatic pattern of EOC differs from that of most otherepithelial malignant diseases. After direct extension, EOC mostfrequently disseminates via the transcoelomic route, with approx-imately 70% of patients having diffuse multifocal intraperitonealmetastasis and malignant ascites at staging laparotomy (3).Malignant cells are exfoliated as single cells and multicellularaggregates (spheroids) from the primary tumor to the peritonealcavity (4), where distribution is facilitated by the peritoneal fluid.The accumulation of carcinomatous ascites, comprised of cellularcomponents, membrane-bound vesicles, and soluble proteins,establishes a unique metastatic niche for the progression ofmetastatic disease (5). More importantly, the majority of currentchemotherapeutic agents are ineffective in inhibiting anchorage-independent growth associated with a three-dimensional struc-ture. It has become apparent that spheroids of malignant cellscontained within malignant ascites are a major source of diseaserecurrence and significantly impede efficacious treatment ofadvanced EOC (6).

The multicellular nature of the spheroids is thought to beattributable to adhesive molecules that mediate cell–cell andcell-secreted extracellular matrix (ECM) interactions (7, 8). It wasreported that the type I calcium-dependent cadherins, N- and E-cadherin, dominantly mediate spheroid compaction (9, 10).Indeed, an epithelial–mesenchymal transition (EMT) spectrumcandefine a spheroidogenic intermediatemesenchymal state, andsuch EMT gene expression signatures correlate with worse clinicaloutcomes (11–13). These EMT and cancer stem cell (CSC)-like

1Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan.2National Institute of Cancer Research, National Health Research Institute,Zhunan, Miaoli County, Taiwan. 3Graduate Institute of Clinical Medicine, Collegeof Medicine, Taipei Medical University, Taipei, Taiwan. 4Wan Fang Hospital,Taipei Medical University, Taipei, Taiwan. 5Graduate Institute of Toxicology,College of Medicine, National Taiwan University, Taipei, Taiwan. 6Department ofObstetrics & Gynecology, National Taiwan University Hospital, Taipei, Taiwan.7Department of Obstetrics andGynecology, Far EasternMemorial Hospital, NewTaipei, Taiwan. 8Department of Obstetrics and Gynecology, Kaohsiung ChangGung Memorial Hospital and Chang Gung University College of Medicine,Kaohsiung, Taiwan. 9Medical Biology, Genomics Research Center, AcademiaSinica, Taipei, Taiwan. 10Graduate Institute of Cancer Biology, China MedicalUniversity, Taichung, Taiwan. 11Department of Biotechnology, Asia University,Taichung, Taiwan. 12Center for Molecular Medicine, China Medical UniversityHospital, Taichung, Taiwan.

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

M.-W. Chen, S.-T. Yang, and M.-H. Chien contributed equally to this article.

Corresponding Authors: Lin-Hung Wei, Department of Oncology, NationalTaiwan University Hospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan.Phone: 8862-2312-3456, ext. 67140; Fax: 8862-2371-1174.E-mail: [email protected]; and Jen-Liang Su, National Institute of CancerResearch, National Health Research Institutes, No. 35, Keyan Road, Zhunan,Miaoli Country 35053, Taiwan. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1115

�2017 American Association for Cancer Research.

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phenotypesmay facilitate chemoresistance in recurrent EOC(14).Genomic signature analysis identified several CSC markers thatwere upregulated in spheroids, including ALDH1A1, b-catenin,and c-KIT (15). These data strongly support the hypothesis thatEOC cells that form spheroids are enriched for CSCs, allowingtranscoelomic metastasis and persistence after chemotherapy.

Tumor cells isolated from the ascites of recurrent EOC patientsare enriched with tumor cells overexpressing EPCAM/STAT3compared with cells isolated from the ascites of chemotherapy-na€�ve patients (16). STAT3 activation has been implicated in theself-renewal and survival of embryonic, hematopoietic, and CSCs(17–20); however, its role in ovarian CSCs has not yet beendefined. We hypothesize that the inhibition of STAT3 disruptsspheroid formation, impairs ovarian CSCs, impacts drug sensi-tivity, and blocks cancer metastasis. In particular, we demonstratethat STAT3 directly activates the transcription ofmiRNAmiR-92a-1,which leads to the downregulation of theDICKKOPF-1 (DKK1)gene, a Wnt antagonist, in three-dimensional culture models.These STAT3-mediated regulatory circuits are required for spher-oid formation in diverse cell lines and intraperitoneal tumorgrowth in xenografts, supporting the idea that the inhibition ofthis pathway is vital to the discovery of a cure.

Materials and MethodsAntibodies and reagents

Antibodies against P-STAT3, STAT3, b-catenin, E-cadherin,OCT4, PARP, cleaved caspase-3, P-ERK, and ERK were purchasedfrom Cell Signaling Technology, and antibody against SOX2 wasobtained from GeneTex. All of the chemicals and McCoy 5Amedium were purchased from Sigma.

Specimens and IHCTissues were obtained from the Cancer Tissue Core of the

National Taiwan University Hospital (NTUH, Taipei, Taiwan)and Kaohsiung Chang Gung Memorial Hospital. Immunostain-ing was performed using the SuperPicture Kit (Life Technologies).Immunointensity was independently scored by two pathologistsbased on both nuclear immunoreactivity and extent. The per-centage of cells that stained positive within the tumor was scoredon a scale of 1–4where 1¼�10%, 2¼11%–50%, 3¼51%–75%,and 4¼ >75%.Written informed consent was obtained from eachpatient, and the use of the clinical samples was approved by theResearch Ethics Review Committee.

ImmunofluorescenceAdherent and nonadherent cells were plated on tissue culture–

treated chamber slides. Cells grown on chamber slides were fixedin 4% paraformaldehyde and permeabilized with ice-cold 100%methanol. Fixed cells were incubated in blocking buffer and thenwith anti-phospho-STAT3 (Alexa Fluor 488 conjugated). Finally,the slides were incubated with DAPI in PBS, and mounted withDako Fluorescent Mounting Medium.

Cell culturePrimary EOC cells were obtained from malignant ascites of

three consecutive relapsing patients. Written informed consentwas obtained from each patient, and the use of the clinicalsampleswas approved by the Institutional ReviewBoard atNTUH(Taipei, Taiwan). The SKOV3 and ES2 cells were obtained fromthe ATCC. The HeyA8 cells were obtained from Dr. Jean-Paul

Thiery (A�STAR, Singapore). All cell lines used in this study wereauthenticated by Promega using STR genotyping. The Tet-induc-ible STAT3 shRNA-expressing cells were established by infectionwith lentivirus. The luciferase-expressing cells were established byinfection with the pWPXL-Luc2-IRES-Puro–expressing lentivirus.

Vector constructionThe pLVTHM, pLV-tTRKRAB, and pWPXL constructs (Addgene

plasmids 12247, 12249, and 12257, respectively) were describedand provided by Dr. D. Trono (Global Health Institute, School ofLife Sciences, EPFL, Lausanne, Switzerland; ref. 21). To constructthe Tet-inducible shRNA lentiviral vector, the STAT3 shRNAsequences were synthesized, pair annealed, and subcloned intothe pLVTHM vector. The target sequences of STAT3 shRNA#1 andshRNA#2 are described in Supplementary Table S1. To constructpWPXL-Luc2-IRES-Puro, the sequence encoding Luc2 was excisedfrom pGL4 and inserted into pWPXL-IRES-Puro upstream of theIRES-Puro cassette.

Western blot analysisCells were lysed in NETN lysis buffer containing a protease

inhibitor cocktail (Sigma). Equal amounts of proteins wereseparated by SDS-PAGE and transferred to a polyvinylidenefluoride membrane. After blocking, the membrane-bound pro-teins were probed with the indicated primary antibodies. Afterwashing and incubating with secondary antibodies, antibody-bound proteins were detected using enhanced chemilumines-cence reagents (Millipore).

Anoikis assayCells were plated at 5� 105 cells/mL in growth medium in 3%

poly-HEMA–coated tissue culture plates. At the end of the indi-cated culture period, 150-mL aliquots were transferred to 96-wellplates. Cell survival was determined using the CellTiter AQueous

reagent (Promega).

Culture of ovarian cancer spheroidsCells (100 cells/mL) were seeded onto 96-well ultra-low plates

(Corning) in DMEM/F12 medium (Invitrogen) supplementedwith 20 ng/mL EGF, 20 ng/mLbFGF, and 5mg/mL insulin. Imagesof the spheroids were obtained, and the number of spheroids wascounted under a microscope 14 days after cell seeding.

Detection of ALDH1þ cellsALDH1enzyme activitywas detected using theALDEFLUORkit

(Aldagen). Briefly, cells were loaded with the enzyme substrateeither alone or in the presence of the specific enzyme inhibitordiethylamino-benzyaldehyde (DEAB). After 30 minutes at 37�C,the cells were analyzed using a BD FACSAria III flow cytometerwith a 488-nm blue laser and 530/30 bandpass filter. ALDH1þ

cells were identified as having greater fluorescence than cells inwhich enzyme activity was inhibited by DEAB.

Tumor formation assayVarying numbers of SKOV3/scramble and SKOV3/STAT3

shRNA#1 cells were prepared. The cells were serially diluted andresuspended in 100 mL of a 1:1 mixture of PBS and Matrigel andthen injected subcutaneously into the right and left flanks of 6-week-old female NOD/SCID/IL2rgnull (NSG)mice. Each uniquexenograft was treated as an individual experiment, and a totalof 20 mice (different number of cells were injected on each flank;

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Cancer Res; 77(8) April 15, 2017 Cancer Research1956




n ¼ 5 injections per group) were used to evaluate the clonogenicgrowth potential of each starting xenograft. Tumor formation wasconsidered positive if a mass greater than 1 cm in diameter wasdetected by palpation.

Quantitative RT-PCRReal-time PCR was performed using the Applied Biosystems

StepOne Real-Time PCR Systems (Applied Biosystems). All qPCRreactions were performed in duplicate and the amplificationsignal from the target gene was normalized to a GAPDH signal.For pri-miRNA and pre-miRNA detection, the sequences of theprimers are described in Supplementary Table S2. For the detec-tionofmaturemiR-92a, the TaqManMicroRNAassay kit (AppliedBiosystems) was used according to the manufacturer's instruc-tions. All qPCR reactions were performed in duplicate and theamplification signal from the target miRNA was normalized to aU6 signal. The average of three experiments each performed intriplicate with SEs is presented.

TaqMan qPCR array for human WNT pathwayTaqMan array plates of 92 genes to WNT signaling–associ-

ated genes and 4 assays to candidate endogenous control wereused to perform qPCR analysis (Applied Biosystems). Resultswere expressed as fold change, by comparing STAT3 knock-down SKOV3 cells with scramble control after correction forhousekeeping genes, using the threshold cycle (Ct) method andthe 2DDCt formula.

Luciferase assaysLuciferase reporter assays were carried out using the Luciferase

Assay System (Promega). The reporter gene construct and pTK-Renilla construct were cotransfected into cells, and luciferaseactivity was measured with the Dual-Luciferase Reporter AssaySystem (Promega). The results are expressed as luciferase/Renillaratios and represent the average� SDof at least three experiments,each performed in triplicate.

Animal studies and bioluminescenceAll of the procedures were carried out according to the animal

protocol approved by the National Taiwan University College ofMedicine Institutional Animal Care and Use Committee. Age-matched NOD/SCID female mice (6–8 weeks old) were used. Atotal of 3 � 106 luciferase-expressing SKOV3/tTR/STAT3 shRNAcells were intraperitoneally (i.p.) injected, and the mice wereseparated into 4 treatment groups. In the doxycycline treatmentgroup, a rodent diet with 625mg/kg doxycycline (Harlan-Teklad)was administered 14 days after the injection. In the paclitaxeltreatment group, beginning on day 14 after tumor inoculation,the mice were treated with paclitaxel (Sinphar) once a week for 6consecutive weeks. Tumor progression was monitored usingbioluminescence (IVIS Spectrum), and the survival was moni-tored daily. Tissue samples were collected on the indicated daysafter injection for pathologic analysis.

Statistical analysesData are expressed as the means � SD. Differences were

analyzed by one-way ANOVA. The median difference in paireddatawas tested by theWilcoxon signed rank test. Survival analyseswere conducted using the Kaplan–Meier method and log-ranktest. P < 0.05 was considered significant.

ResultsSTAT3 activation in chemoresistant ovarian carcinoma ascitesspheroids

To address whether STAT3 activation was associated with EOCprogression, we compared the expression levels of p-STAT3 in 31paired primary and recurrent tumor tissues. The clinicopathologiccharacteristics are presented in Supplementary Table S3. Figure 1Ashows representative examples of the differential nuclear p-STAT3staining between paired primary and recurrent tumors. Highlevels of STAT3 phosphorylation (p-STAT3 score � 3) wereobserved by IHC in 32.3% of primary (10/31) and 67.7% ofrecurrent (21/31) EOC tissues. Significantly higher levels of STAT3phosphorylation were found in recurrent tumors (P < 0.0001,Wilcoxon signed rank test; Fig. 1B). Indeed, STAT3 activation inovarian cancer was significantly associated with a reduced pro-gression-free interval (10.6 � 7.2 vs. 17.3 � 8.9 months, P <0.05; Fig. 1C).

Tumor cells in ascites, which survive as spheroids and exhibitchemoresistance (22, 23), are amajor source of disease recurrencein EOC patients. These nonadherent tumor cells that are isolatedfrom the ascites of recurrent EOC patients are enriched withphosphorylated STAT3 (Fig. 1D). Remarkably, we observed thattreatment with higher concentrations of paclitaxel resulted in amarginal increase in cell death of primary tumor spheroidsderived fromrecurrent EOCpatients,which couldbe substantiallyenhanced by concurrent STAT3 inhibition (Fig. 1E). Consistentwith these observations, phosphorylation of STAT3 increasedwhen the cells were grown in three-dimensional suspensioncultures compared with two-dimensional adherent cultures in apanel of EOC cell lines (SKOV3, HeyA8, and ES2; Fig. 1F). Wespeculated therefore that STAT3 activation is responsible foranchorage-independent EOC cell growth and chemoresistance.Accordingly, we found that knockdown of STAT3 (STAT3-KD)resulted in a significant reduction in the survival of EOC cellsgrown in three-dimensional suspension cultures (SupplementaryFig. S1). STAT3-KD significantly enhanced paclitaxel-inducedanoikis in EOC cells (Fig. 1G), which correlated with the down-regulation of various antiapoptotic gene products, including XIAPand Bcl-xL (Supplementary Fig. S2). In line with this, a nonpep-tide small-molecule inhibitor of STAT3 could quantitatively sen-sitize the response of EOC cells grown in three-dimensionalsuspension cultures to paclitaxel (Fig. 1H). Collectively, our datasuggest that the disruption of STAT3 could impact the chemore-sistance of these nonadherent EOC cells.

STAT3 signaling regulates EOC stem cell–like propertiesTo determine whether STAT3 activation is indispensable in

EOC spheroid formation, we developed stable sublines of SKOV3cells that inducibly express shSTAT3 in the presence of tetracycline(Tet-on). Tight regulation of the gfpmarker was achieved throughthe incubation of each subline with 5 mg/mL of doxycycline,and the levels of STAT3 and p-STAT3 were readily suppressedafter 48 hours of repression (Fig. 2A and Fig. 5A). Culturesof both SKOV3/tTR/STAT3 shRNA#1 and SKOV3/tTR/STAT3shRNA#2 cells were dissociated, and single cells were then plated.Without doxycycline, the SKOV3/tTR/STAT3 shRNA#1 andSKOV3/tTR/STAT3 shRNA#2 cells grownonultra-lowattachmentculture plates formed compact spheroids after 12 days.In contrast, both cell lines hardly formed multicellular aggregatesor spheroids in the presence of doxycycline (Fig. 2B, top).

STAT3/miRNA-92/Wnt Signaling in Ovarian Cancer Spheroids

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Figure 1.

Activation of STAT3 in EOC. A, Representative expression of p-STAT3 in matched primary and recurrent ovarian cancers. p-STAT3 is overexpressed in therecurrent tumors with strong nuclear staining. B, Distribution of p-STAT3 immunohistochemical staining scores in 31 paired primary and recurrent ovarian cancers.C, Comparison of the disease-free intervals in ovarian cancer patients with or without STAT3 activation. D, Representative immunofluorescent images of tumorcells isolated from ascites associated with recurrent EOC in three-dimensional cultures. E, The selective STAT3 inhibitor Stattic (5 mmol/L) sensitized EOC spheroids topaclitaxel. Plot of viability, measured by the MTS assay (72 hours), of primary EOC cells derived from malignant ascites of recurrent EOC patients. All experimentswere performed in triplicate. F, Immunoblotting of various EOC cell lines showing the expression of p-STAT3 protein on monolayer or three-dimensionalcultures. a-Tubulin was used as a loading control. G, STAT3 silencing promotes anoikis in EOC cells exposed to paclitaxel. Top, expression of STAT3 and b-actin wasexamined by Western blot analysis. Bottom, SKOV3 and ES2 cells were cultured in poly-HEMA–coated plates and were treated with either paclitaxel alone ortogether with STAT3 knockdown in floating culture. After 72 h, MTS assay was performed. Error bars represent the SD from triplicate cultures. Each experiment wasrepeated at least three times with similar results. H, Combination of STAT3 inhibitor and paclitaxel effectively leads to anoikis in EOC cell lines. Cell lines grownin three-dimensional cultures were treated with paclitaxel, with or without 5 mmol/L Stattic for 96 hours, and cell viability was analyzed by an MTS assay. Results aremean � SD of three independent experiments. Across all panels, � , significant change t test P < 0.05; �� , P < 0.01; and ��, P < 0.001.

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Figure 2.

Activation of STAT3 in ovarian cancer spheroids regulates stem cell–like properties. A, Immunoblot analysis of STAT3/p-STAT3 levels in SKOV3 withdoxycycline (Dox)-inducible STAT3 knockdown expressing system at 3 days after doxycycline treatment. B, Top, representative spheroids formed by SKOV3transduced with the doxycycline-inducible vectors. Bottom, the doxycycline switches were performed at day 14. C, Plot of the number of spheroids formedper 1,000 cells in selected SKOV3. Error bars represent the SD from triplicate cultures. D, Plot of the number of spheroids formed by SKOV3 scramble/SKOV3STAT3-KD cells (per 1,000 cells) in the presence or absence of paclitaxel (100 nmol/L) or cisplatin (50 mmol/L). E,ALDEFLUOR staining of ALDHþ andALDH� SKOV3cells with DEAB controls. All data are representative of at least three independent experiments. F, Immunoblot analysis of SKOV3 scramble/SKOV3 STAT3-KDcells in spheroid culture for the expression of OCT4 and SOX2. a-Tubulin was used as a loading control. G, The incidence of mouse xenograft tumorsderived from SKOV3 scramble or SKOV3 STAT3-KD cells, following subcutaneous injection into NSG mice (n ¼ 5 injections per group) at different cellnumbers (1 � 105, 1 � 104, 1 � 103, and 1 � 102 cells). Across all panels, �� , significant change t test P < 0.01.






Furthermore, the treatment of intact SKOV3/tTR/STAT3shRNA#1 and SKOV3/tTR/STAT3 shRNA#2 spheroids withdoxycycline led to the dissociation of the established spheroids(Fig. 2B, bottom). We observed that Tet-on STAT3-KD in theSKOV3 cells resulted in a significant decrease in the formationof spheroids (Fig. 2C). Similar results were observed in otherEOC cell lines (Supplementary Fig. S3). Moreover, treatmentwith paclitaxel or cisplatin resulted in a 25%–50% decrease inspheroid formation, which was significantly enhanced by con-current STAT3 inhibition (Fig. 2D). These observations suggestthat the activation of STAT3 is involved in EOC spheroidformation and that STAT3-KD could overcome spheroid-relat-ed chemoresistance. One of the striking features of EOC spher-oids is the enrichment of cells with a CSC-like phenotype. Wemeasured ALDH1 activity to assess whether STAT3 activation inEOC cells could affect characteristic traits of tumor-initiatingcells. Flow cytometry analysis showed a reduced number ofAldefluorþ cells in STAT3-KD SKOV3 spheroids compared withparental cell spheroids (0.03% and 0.2% vs. 4.5%; Fig. 2E).Additional immunoblotting analyses validated the decreasedexpression of OCT4 and SOX2 in STAT3-KD EOC spheroids(Fig. 2F). As the number of tumor-initiating cells correlates withtumorigenic capacity in animals, we next measured the effect ofSTAT3-KD on EOC tumorigenicity in NSG mice. Notably,STAT3-KD either led to a reduced number of tumor-bearingmice or a reduction in the average size of the primary tumors,indicating reduced tumorigenicity (Fig. 2G and data not shown).

Regulation of Wnt/b-catenin signaling by STAT3 in EOCspheroids

It is known that the Wnt/Wingless pathway contributes to theregulation of CSCs in different organs and has a crucial role in celltransformation and tumor progression of EOC (24). Activation ofb-catenin regulates the tumor-initiating capacity of EOC and isinstrumental in EOC spheroid formation and tumor metastases(15). The Wnt antagonist compound C59, which interferes withzygotic Wnt ligand secretion, was used to determine whether theWnt/b-catenin pathway mediates STAT3-induced spheroid for-mation in EOC. Overexpression of STAT3 in SKOV3 cells remark-ably induced spheroid formation, whereas C59 treatment signif-icantly diminished STAT3-induced spheroid formation capacity(Fig. 3A). To explore the potential regulation of the Wnt pathwayby STAT3 in EOC spheroid formation, qPCR analysis using aTaqMan low-density array was used. STAT3-KD mediated thedifferential expression of genes associated with Wnt signaling,which are listed in Fig. 3B.Among them, theWnt antagonistDKK1was the most strikingly upregulated gene in response to STAT3-KD in SKOV3 cells (Fig. 3C). In addition, we analyzed DKK1protein levels in cell lysates and conditioned media from three-dimensional cancer spheroid cultures and found that the DKK1protein level was significantly increased in STAT3-KD SKOV3 cellscompared with parental cells (Fig. 3D). Consistent with thesuppression of b-catenin in SKOV3/STAT3-KD spheroids (Fig.3D), functional canonical Wnt signaling was significantly inhib-ited in response to STAT3-KD compared with control shRNA cellsbased on a TCF/LEF reporter assay (Fig. 3E). To further define therole of DKK1 in decreasing spheroid formation in this context, weinfected SKOV3/STAT3-KD cells with lentiviral shRNAs targetingDKK1 or a scramble control. Silencing of DKK1 remarkablyreversed the sphere-forming ability of SKOV3/STAT3-KD cellscompared with cells infected with a scramble control (Fig. 3F).

miR-92a-1 is regulated by STAT3 and targets DKK1Next, we used computational target prediction to identify the

DKK1 gene as a potential target ofmiR-92a. It is known that STAT3transcriptionally upregulates miR-92a in cancer cells (25). There-fore, we examined whether STAT3 suppresses DKK1 expressionthrough the upregulation ofmiR-92a. The significant reduction ofmaturemiR-92a, pre-miR-92a-1, andpri-miR-92a-1was observedin STAT3-KD SKOV3 and HeyA8 cells (Fig. 4A and B). The STAT3inhibitor S3I-201 suppressed miR-92a expression in a dose-dependent manner in SKOV3 cells (Fig. 4C). To confirm thetranscriptional control of miR-92a by STAT3, the promoterregion, which contains numerous putative STAT3-binding sites,was investigated by the luciferase reporter assay. The resultsshowed that knockdown or overexpression of STAT3 markedlyinhibited or enhanced miR-92a promoter activity, respectively(Fig. 4D). These findings demonstrated that STAT3 transcription-ally controls miR-92a expression. Because DKK1 was predicted asa target ofmiR-92a and is suppressed by STAT3, we found that the30UTR of DKK1 encompasses a complementary binding regionagainst miR-92a. To verify the role of miR-92a in DKK1 expres-sion, the DKK1 30UTR fragment harboring the putative miRNA-binding sites (wild-type and mutant) was cloned into the lucif-erase reporter gene. Luciferase activity of WT-DKK1 30UTR wassignificantly reduced by miR-92a in a dose-dependent manner,whereas miR-92a abrogated the reduction of luciferase activity oftheMT-DKK130UTR (Fig. 4E).We thendetermined the expressionofDKK1 in shSTAT3- ormiR-92a–expressing SKOV3 cells. Expres-sion of DKK1 was increased by STAT3-KD, while overexpressionof miR-92a markedly inhibited shSTAT3-induced DKK1 expres-sion (Fig. 4F), suggesting that the suppression of DKK1 by STAT3is through the upregulation of miR-92a. To further validate thefunctional role of miR-92a in ovarian cancer spheroids, wedetermined the spheroid formation in STAT3-KD cells and foundthat the decreased spheroid formation by STAT3 inhibition wasrecovered by overexpression of miR-92a (Fig. 4G). Consistentwith the in vitro results, enhanced tumorigenicity was observed inmice bearing miR-92a–overexpressing SKOV3/STAT3-KD xeno-grafts (Fig. 4H). Taken together, these observations showed thatSTAT3-induced miR-92a expression is required for spheroid for-mation and tumor growth.

Interrupting the EOC stem cell pathway by targeting STAT3suppresses peritoneal seeding and overcomes chemoresistancein vivo

Our cell-based studies demonstrated that STAT3-KD preventedspheroid formation and enhanced cytotoxicity of chemotherapyagents in vitro (Figs. 1 and 2). To determine the relevance of thesefindings in vivo, we examined Tet-inducible STAT3 inhibition in amurine model of intraperitoneal SKOV3 tumors. We previouslyshowed that SKOV3 tumors exhibit active STAT3 signaling anddisplay extensive intraperitoneal tumor seeding and signs ofascites formation following inoculation (26). In mice receivingSKOV3/tTR/STAT3 shRNA#2 xenografts, adding doxycycline tothe food resulted in robust GFP expression in the tumors com-pared with the xenografts in the absence of doxycycline, reflectingthe tight control of a gfpmarker in vivo (Fig. 5A). Paclitaxel alonesubstantially inhibited ascites formation compared with the con-trol group, and treatment with the STAT3-KD/paclitaxel combi-nation completely inhibited the formation of measurable ascites(data not shown). During postmortem examination, a widespread of seededmetastases was observed throughout the control

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Figure 3.

Wnt signaling is critical for the STAT3-mediated stem cell-like phenotype. A, Spheroid-forming ability of the STAT3-overexpressing SKOV3 cells treated withthe Wnt inhibitor C59 by quantifying the number of tumor spheroids. B, The Wnt pathway gene expression levels are represented as a heatmap. C, qPCRanalysis of genes involved in theWnt signaling pathway in SKOV3 spheroids. D, Top, Western blot analysis of STAT3, b-catenin, DKK1, and a-tubulin in SKOV3 cellsinfected with scramble or STAT3 shRNAs. Bottom, ELISA analysis of DKK1 levels in conditioned media of SKOV3 cells infected with scramble or STAT3 shRNAs.E, Either TOP or FOP TCF luciferase along with Renilla (RL) luciferase were transfected into stable SKOV3 cells expressing shDKK1, shSTAT3, or both.Luciferase reporter activity was calculated by dividing the ratio TOP/RL by the FOP/RL ratio. � , P < 0.05 compared with the control group; #, P < 0.05 comparedwith the STAT3-KD group. F, Spheroid-forming ability of STAT3-KD SKOV3 cells expressing control or DKK1 shRNA by quantifying the number of tumorspheroids. Across all panels, � , significant change t test P < 0.05; �� , P < 0.01.






Figure 4.

Epigenetic silencing of DKK1 by mir-92a. A and B, The expression of mature miR-92a, pre-miR-92a-1, and pri-miR-92a-1 was detected in STAT3-KD SKOV3and HeyA8 cells by qRT-PCR analysis. C, Assessment of the expression of mi-92a and subsequent qRT-PCR analysis were performed after SKOV3 cells weretreated with various doses of the STAT3 inhibitor S3I-201. D, Luciferase reporter assays were carried out using SKOV3 cells overexpressing STAT3-C or the vectorcontrols or cells expressing either a control shRNA or a shRNA targeting STAT3. The results showed that miR-92a promoter activity was regulated by STAT3expression. E, DKK1 30-UTR is the direct target of miR-92a. The diagram shows miR-92a and its putative binding site at the 30-UTR of DKK1 and the sequencesof WT and MT DKK1 30-UTR. A luciferase reporter assay was used to assess whether miR-92a can directly bind to the 30-UTR of DKK1. F, The expressionof DKK1 is suppressed by STAT3 through the upregulation of miR-92a. Western blot analysis was used to detect the protein expression of DKK1 in SKOV3 cells inwhich STAT3 was knocked down using shRNA or miR-92a was overexpressed. Tubulin was used as an internal control. G, Spheroid formation was determinedin SKOV3 and HeyA8 cells in which STAT3 was knocked down with shRNA and/or miR-92a was overexpressed. H, The incidence of xenograft tumor wasincreased by STAT3 through miR-92a. NSG mice were separated into groups and injected subcutaneously with shSTAT3-, shSTAT3/miR-92a-, or vectorcontrol–expressing SKOV3 cells to observe tumorigenicity. Across all panels, � , significant change t test P < 0.05; �� , P < 0.01; and �� , P < 0.001.

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mesentery compared with the STAT3-KD- and/or paclitaxel-trea-ted mesentery, where the spread of tumor seeding decreased (Fig.5B). The number of small (�1mm), large (>1mm,�3mm), andbulk (>3 mm) peritoneal tumor seedings was quantified. In thepaclitaxel-treated group, the number of bulk tumor seedings wassignificantly lower than that in the untreated group (P < 0.05);however, the number of small tumor seedings was not affected. In

contrast, STAT3-KD substantially reduced the number of smalltumor seedings but was not able to suppress the tumor growth ofestablished peritoneal implants. Furthermore, the combinationof STAT3-KD and paclitaxel significantly reduced the intra-abdominal tumor burden such that bulk tumor seedings wereabolished, and the number of small tumor seedings was signi-ficantly lower than that in the paclitaxel-treated mice (P < 0.05;

Figure 5.

Targeting STAT3 suppresses peritoneal seeding and overcomes chemoresistance in vivo. A, Conditional knockdown of STAT3 concomitant with GFP expressionin vivo. A doxycycline (Dox)-containing diet was administered to mice 2 weeks after the intraperitoneal injection, and GFP fluorescence was analyzed 1 weekafter the administration of the doxycycline-containing diet. B, The visible peritoneal seeding of the mesentery was substantially reduced in the STAT3knockdown (Doxþ) and/or Taxol treatment groups. C, Quantification of peritoneal metastases. Peritoneal tumor seedings: small (�1 mm), large (>1 mm, �3 mm),and bulk (>3 mm). D, Hematoxylin and eosin (H&E) staining and IHC with antibodies against p-STAT3 and cleaved caspase-3.






Fig. 5C).Notably, p-STAT3 expressionwas persistently increased aweek after paclitaxel treatment in the SKOV3 tumors. In responseto the activation of STAT3 shRNA expression with doxycycline invivo, we observed an increase in the number of apoptotic cells inresponse to the combination therapy compared with paclitaxeltreatment or STAT3-KD alone (Fig. 5D). These data reinforce theimportance of the STAT3 pathway in the peritoneal metastasis ofovarian cancer and, in particular, the role of STAT3-KD in con-ferring sensitivity to paclitaxel therapy.

Blocking STAT3 and paclitaxel in combination suppresses EOCtumor growth and improves survival in mice

To evaluate the combinatorial effect of STAT3 inhibition andchemotherapy on EOC growth and progression, we treatedmice bearing intraperitoneal EOC xenografts with STAT3-KDand paclitaxel, according to the depicted schema (Fig. 6A).Intraperitoneal implantation of SKOV3/tTR/STAT3 shRNA#2cells resulted in the development of tumor foci throughout theperitoneal cavity as monitored with in vivo bioluminescenceimaging (Fig. 6B). Paclitaxel substantially suppressed tumorgrowth as a single agent, whereas conditional STAT3-KD begin-ning on day 14 after tumor inoculation had no significant effecton tumor reduction as measured by bioluminescent imaging(Fig. 6C). Notably, the combination of the two treatmentssuppressed tumor growth even further (Fig. 6C). Importantly,combined STAT3-KD and paclitaxel treatment prolonged sur-vival of mice with implanted EOC tumors from a mediansurvival of 58 days for control animals to 80 days for thecombination of paclitaxel and STAT3-KD (P < 0.0001, log-ranktest). In addition, in this model, the STAT3-KD/paclitaxelregimen significantly increased survival compared with pacli-taxel alone (log-rank test P < 0.01; HR, 4.101; Fig. 6D). Theseresults suggested that STAT3 inhibition might potentially com-bine with current standard-of-care chemotherapy.

DiscussionExtrinsic tumor microenvironmental factors in EOC, such as

inflammatory cytokines, growth factors, and oxidative stress, canactivate STAT3 through different mechanisms (27–29). The datapresented herein demonstrate the enhancement of STAT3 phos-phorylation in tumors frompatientswith recurrent ovarian cancerand in residual xenografts harvested after paclitaxel treatment,suggesting that STAT3 activation may essentially contribute toovarian cancer progression. In this article, we demonstrate that theactivation of STAT3 has a central role in the generation of EOCspheroids. Moreover, we found that STAT3 signaling intercon-nects Wnt/b-catenin signaling, a known pathway regulating ovar-ian CSCs, in EOC spheroids. Inhibition of STAT3 signalingeffectively eliminates the formation of the metastatic niche andsuppresses its persistence after chemotherapy. These featurespoint to new directions for targeting STAT3 for EOC therapy.

EOC spheroids serve as the vehicle for ovarian cancer celldissemination in the peritoneal cavity and represent a significantimpediment in the efficacy of chemotherapy agents (6). Thedisruption of STAT3 signaling alters homotypic cell–cell adhesioncomplexes by increasing the tyrosine phosphorylation of b-cate-nin, leading to the loss of b-catenin–cadherin associations (30)and highlighting STAT3 signaling in the self-assembly of EOCcells into spheroids. Notably, the current data reveal that cellsexhibiting endogenous STAT3 phosphorylation readily aggregate

from single cells into spheroids. It is noteworthy that STAT3signaling may be involved in the promotion of the CSC pheno-type of EOC. Studies have indicated that ovarian CSCs areinvolved in chemoresistance, metastasis, and tumor recurrence(31).We and others have suggested that targeting STAT3 signalingor its crucial upstream activators results in the disruption of CSCmaintenance and dramatically reduces the chemoresistance andtumor metastases of EOC (32, 33). These findings are in accor-dance with previous studies, which revealed that STAT3 signalingis necessary for the growth of several types of CSCs, includingcolon, breast, and prostate CSCs (19, 34, 35).

The precisemechanismbywhich STAT3 signaling promotes theself-renewal of CSC in human cancers is not completely under-stood. In malignant gliomas, STAT3 signaling in neural stem cellsprevents neural differentiation and triggers reprogrammingtoward an aberrant mesenchymal lineage (36). More recentstudies suggested that STAT3 can recruit G9a histone methyl-transferase to coordinately mediate epigenetic silencing of aspecific cohort of gene targets involved in stemness differentiationplasticity (37). In accordance with the findings of these studies,our study provides the first evidence showing that STAT3 canactivate Wnt signaling through epigenetic inactivation of the Wntantagonist DKK1. DKK1 was discovered due to its ability tospecifically bind and modulate the Wnt coreceptors Lrp5 and6, which are indispensable for routing theWnt signal to b-catenin(38). This canonical Wnt pathway results in b-catenin nuclearaccumulation and transcriptional activation of target genes,which contribute to the maintenance of EOC spheroids (15).Studies have shown that several Wnt antagonist genes were low inEOC and epigenetic silencing by DNA methylation (16, 39).Deregulation of canonicalWnt/b-catenin signaling by these genescontributes to CSC populations, chemoresistance, and the aggres-siveness of EOC (39, 40). Here, we found that DKK1 exhibited themost decreased expression as a result of STAT3 signaling. STAT3silencing negatively regulates the canonicalWnt pathway, leadingto increased phosphorylated b-catenin, decreased nuclear b-cate-nin levels, and inhibition of b-catenin–dependent TCF/LEF tran-scription activity. In particular, our data indicate that in vitrospheroidogenesis and in vivo tumorigenicity mediated by STAT3signaling in EOC were substantially reduced in DKK1 transfec-tants. These results are in line with earlier studies in breast andcolon cancer (41, 42), indicating a tumor suppressor function ofDKK1 and its potential role in STAT3-regulated CSC character-istics in EOC.

Our studies identify a previously unrecognized mechanism bywhich STAT3 signaling downregulates DKK1 expression. Theepigenetic silencing ofDKK-1 expression is linked to DNA hyper-methylation, histone deacetylation, and histone methylation(43–45). Interestingly, we found that STAT3-mediated DKK1silencing was regulated via the DKK1 30 UTR by miRNA in EOC.Combining miRNA microarray analysis, computational targetprediction, and functional analysis, we observed that miR-92anegatively regulates protein levels of DKK1 by targeting a specificbinding site in the DKK1 30 UTR sequence. Through inactivationof the canonical Wnt antagonist DKK1, miR-92a may activateWnt/b-catenin signaling and regulate CSC characteristics in EOC.Wu and colleagues reported that themiR-17-92 clustermay targetthe E2F1 and HIPK1 proteins, which suppress Wnt/b-cateninsignaling (46). It has also been shown that the miR-17-92 clusterwas highly induced during early reprogramming stages in inducedpluripotent stem cells (47). Consistent with these observations,

Chen et al.





our study demonstrates the ability of miR-92a to mediate STAT3-regulated self-renewal of ovarian CSCs based on in vitro and in vivoassays.While STAT3andmiR-92a are associatedwithhallmarks ofstemness in ovarian cancer cells, regulatory pathways directlyconnecting them are largely unknown. It has been suggested thatSTAT3 is an upstream activator of miR-92a (25). Here, we dem-

onstrate that STAT3 directly binds specific sites in the miR-92apromoter region and that the promoter activity was decreasedafter themutation of putative STAT3-binding sites, indicating thatSTAT3 is required for the transcriptional induction of miR-92a.Moreover, miR-92a is critically involved in the downregulation ofDKK1 by STAT3 and is required for the maintenance of ovarian

Figure 6.

STAT3 silencing combines with paclitaxel to inhibit tumor growth of EOC cell xenografts and promote the survival of mice. SKOV3/tTR/STAT3 shRNA2 EOCcells were intraperitoneally implanted into NOD/SCID mice. A, Mice were separated into four treatment groups and treated according to the schema depicted.B, Weekly representative bioluminescence images of animals in each group are shown depicting tumor burden. C, Bioluminescence-based tumor growth(measured by the photon counts of the xenografts on the 7th week divided by the corresponding photon counts on day 0) was evaluated by changes in relativebioluminescence. Significance testing was performed by one-way ANOVA with Tukey post hoc multiple pairwise testing. D, Effects of STAT3 knockdown(n¼ 10; median survival¼ 58 days), paclitaxel (PTX; n¼ 10; median survival¼ 70 days), and combination paclitaxelþ STAT3 knockdown (n¼ 10; median survival¼80 days) on survival of implanted EOC tumors compared with control-treated animals (n ¼ 12; median survival ¼ 58 days). Significant differences ofsurvival between groups were determined by a log-rank test (�� , P < 0.01; �� , P < 0.0001). Dox, doxycycline.






cancer stemness in vitro as well as tumorigenicity in vivo. Notably,DKK1 is a target gene for activation by b-catenin due to thepresence of TCF-binding elements (TBE) in the DKK1 promoter(48). Accordingly, additional mechanistic insight shows that thenegative feedback loop in Wnt signaling may be lost in thepresence of STAT3 activation.

In summary, given the efficient tumor-initiating capacity, met-astatic niches, and chemoresistance of ovarian CSCs, therapeuticstrategies that can eliminate these cells are urgently needed. Ourresults provide evidence that STAT3 signaling regulates ovarianCSCs by targeting miR-92a/DKK1 and subsequently activatingWnt/b-catenin signaling. We propose that inhibition of STAT3maybe a rational option for EOC-targeted therapy against ovarianCSCs in combination with the current standard chemotherapy toblock EOC progression.

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

Authors' ContributionsConception and design: M.-W. Chen, M.-H. Chien, J.-L. Su, L.-H. WeiDevelopment of methodology: M.-W. Chen, M.-H. Chien

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.-W. Chen, C.-J. Wu, H. Lin, L.-H. WeiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.-W. Chen, S.-T. Yang, K.-T. Hua, S.-M. Hsiao,M. Hsiao, J.-L. SuWriting, review, and/or revision of the manuscript: M.-W. Chen, S.-T. Yang,M.-H. Chien, J.-L. Su, L.-H. WeiStudy supervision: L.-H. Wei

AcknowledgmentsWe would like to thank Dr. Yung-Ming Cheng and Kuan-Ting Kuo for their

help with the histopathological staining and interpretation.We also thankMin-Liang Kuo for critical feedback and technical assistance.

Grant SupportThis work was funded by NSC102-2628-B-002-050 and NHRI-EX-10133BI

(L.-H. Wei).The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 29, 2016; revised December 22, 2016; accepted January 9,2017; published OnlineFirst February 16, 2017.

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2017;77:1955-1967. Published OnlineFirst February 16, 2017.Cancer Res Min-Wei Chen, Shu-Ting Yang, Ming-Hsien Chien, et al. Formation and Malignant Progression in Ovarian CancerThe STAT3-miRNA-92-Wnt Signaling Pathway Regulates Spheroid

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