STAT1 Drives Tumor Progression in Serous Papillary ...and Vancouver General Hospital (Vancouver, Canada) includ-ed specimens from 460 endometrial cancers in ﬁve tissue microarrays

Molecular and Cellular Pathobiology

STAT1 Drives Tumor Progression in Serous PapillaryEndometrial Cancer

Budiman Kharma1, Tsukasa Baba1, Noriomi Matsumura1, Hyun Sook Kang1, Junzo Hamanishi1,Ryusuke Murakami1, Melissa M. McConechy2, Samuel Leung3, Ken Yamaguchi1, Yuko Hosoe1,Yumiko Yoshioka1, Susan K. Murphy4, Masaki Mandai5, David G. Hunstman2,3, and Ikuo Konishi1

AbstractRecent studies of the interferon-induced transcription factor STAT1 have associated its dysregulation

with poor prognosis in some cancers, but its mechanistic contributions are not well defined. In this study,we report that the STAT1 pathway is constitutively upregulated in type II endometrial cancers. STAT1pathway alteration was especially prominent in serous papillary endometrial cancers (SPEC) that arerefractive to therapy. Our results defined a "SPEC signature" as a molecular definition of its malignantfeatures and poor prognosis. Specifically, we found that STAT1 regulated MYC as well as ICAM1, PD-L1, andSMAD7, as well as the capacity for proliferation, adhesion, migration, invasion, and in vivo tumorigenecity incells with a high SPEC signature. Together, our results define STAT1 as a driver oncogene in SPEC thatmodulates disease progression. We propose that STAT1 functions as a prosurvival gene in SPEC, in amanner important to tumor progression, and that STAT1 may be a novel target for molecular therapy inthis disease. Cancer Res; 74(22); 6519–30. �2014 AACR.

IntroductionEndometrial cancer is one of the leading causes of gyne-

cologic malignancy. It is the fourth most common malig-nancy among women in the United States, with an estimated49,500 new cases and 8,200 deaths in 2013 (1), and in Japan,where the incidence has nearly doubled in the past decade(2). Most patients present with low-grade and early-stagedisease with favorable prognosis, but cases of high-gradetumors are aggressive and frequently diagnosed with tumorspread beyond the uterus (3).Endometrial cancers have generally been classified into

two groups, type I and type II (4). Type I endometrial cancersare low-grade endometrioid adenocarcinomas, which fea-ture high expression of estrogen receptor and a past historyof unopposed estrogen associated with anovulation or obe-

sity. Type I endometrial cancers usually have a favorableprognosis compared with type II endometrial cancers (5).Type I cancers often harbor alterations in oncogenes, suchas PTEN, PIK3CA, ARID1A, K-ras, b-catenin, and/or DNAmismatch repair genes (6). In contrast, type II endometrialcancers have worse outcomes and include histologic sub-types such as high-grade endometrioid adenocarcinoma,serous papillary and clear cell, which are estrogen-indepen-dent and more common in older, nonobese women. TP53,PPP2R1A, CHD4, FBXW7, SPOP mutations (3, 7), STK15 andHER2/neu amplification, p16 overexpression, downregula-tion or loss of E-cadherin, and also loss of heterozygosity(LOH) have all been reported in type II endometrial cancers(5). These hallmarks of type II cancers do not entirely explainthe aggressive nature of type II endometrial cancers, espe-cially for serous papillary endometrial cancers (SPEC). SPECis characterized by very aggressive progression with poorprognostic outcomes (5, 8). Recent genome-wide analyseshave revealed that SPEC exhibits gene expression profilesthat are distinct from the endometrioid histologic subtype(3). Thus, it is important to identify the SPEC driver genes orpathways responsible for the inherently aggressive pheno-types and to develop SPEC-specific therapies to target thesedriver genes or pathways.

Previous studies implicated the IFNg transcription factorsignal transducer and activator of transcription 1 (STAT1) as atumor suppressor, with transcription-dependent and tran-scription-independent mechanisms of regulation (9). STAT1has been classically defined as a Th1 proimmune and antitu-mor transcription factor, based on its canonical role in IFNgsignaling and on studies using STAT1�/� tumor cells andmouse models (10–12). STAT1 has also been described to

1Department of Gynecology and Obstetrics, Kyoto University GraduateSchool of Medicine, Kyoto, Japan. 2Department of Pathology and Labo-ratory Medicine, University of British Columbia, British Columbia CancerAgency, Vancouver, British Columbia, Canada. 3Genetic Pathology Eval-uation Centre, Vancouver General Hospital, Vancouver, British Columbia,Canada. 4Division ofGynecologic Oncology, Department ofObstetrics andGynecology, Duke University Medical Center, Durham, North Carolina.5Department of Obstetrics and Gynecology, Kinki University Faculty ofMedicine, Osaka, Japan.

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

Corresponding Author: Tsukasa Baba, Kyoto University, 54 ShogoinKawahara-cho, Sakyo-ku, Kyoto, Kyoto 606-8507, Japan. Phone: 81-75-751-3269; Fax: 81-75-761-3967; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-14-0847

�2014 American Association for Cancer Research.

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regulate DNA repair pathways and to be upregulated in seve-ral late-stage human cancers, including those of the breast,colon, glioblastoma, and soft tissue sarcoma (13–16). Over-expression of the IFN–STAT1 pathway is also associated withpoor prognosis in different types of cancer (17–20), especiallyin breast cancers in which increased IFN–STAT1 pathwayactivity is considered a marker predicting resistance tochemotherapy and radiotherapy (17, 21). The actual functionof STAT1 and associated regulatory mechanisms in cancerare not fully understood as well as the significance of theIFN–STAT1 pathway in SPEC.

In this study, we demonstrate constitutively active STAT1expression in SPEC as compared with other histologic sub-types of endometrial cancers. We also present novel findingsshowing that in SPECs, STAT1 functions as a driver thatmodulates expression of downstream genes such as MYC,which in turn promotes the cellular capacity for prolifera-tion, migration, invasion, and xenograft tumorigenecity.We therefore propose that STAT1 functions as a prosurvivalfactor in SPECs, in a manner important to tumor progres-sion, and may be a novel target for molecular therapy in thisdisease.

Materials and MethodsPatients and tissues

Clinicopathologic information (n ¼ 294) and specimens(n ¼ 91) from patients treated for endometrial cancerbetween 2004 and 2012 at Kyoto University Hospital wereobtained with written consent from each patient and usedunder protocols approved by the Kyoto University Insti-tutional Review Board.

Tissue microarrays obtained from the BC Cancer Agencyand Vancouver General Hospital (Vancouver, Canada) includ-ed specimens from 460 endometrial cancers in five tissuemicroarrays (355 endometrioid and 105 SPECs). These wereexamined independently and were used as an external valida-tion. All patients provided informed written consent and theresearch was approved under the University of British Colum-bia and BC Cancer Agency.

Cell lines and cultureHuman endometrial cancer cell lines, HEC1A (ATCC),

HEC50B (JCRB), Ishikawa (National Hospital Organization,Japan), SPAC-1L (The Cancer Institute of the Japanese Foun-dation for Cancer Research, Japan) were used for furtherfunctional assays as described precisely in the Online Repos-itory andwere regularly tested formycoplasma contamination,and were authenticated by STR analysis.

STAT1 knockdownSTAT1-specific short interfering RNAs (siRNA; FlexiTube

siRNA Qiagen; catalogue no. SI02662884), MYC-specific siRNA(FlexiTube siRNA Qiagen; catalogue no. GS4609), and negativecontrol siRNA (AllStars Negative Control siRNA; Qiagen) weretransfected into cell lines using HiPerFect TransfectionReagent (Qiagen) as previously described (22). For establishingSTAT1 stably suppressed cells, STAT1-shRNA (HuSH 29mer

shRNA pGFP-V-RS; Origene) and negative control shRNA(scrambled shRNA cassette; Origene) were transfected usingTurbofectin 8.0 Transfection Reagent, and stably transfectedcells were selected with puromycin treatment (0.5–1.0 mg/mL;Nacalai Tesque). A dominant-negative STAT1 DNA plasmid(pBOS-STAT1-DN; Osaka University Graduate School of Med-icine, Japan; ref. 23) was also transfected with Lipofectamine2000 for obtaining STAT1 dominant-negative cells, as describ-ed previously (24).

Microarray analysisGene expression microarray was generated from 63 endo-

metrial cancer samples (GSE56026) using Affymetrix U133 Plus2.0 gene chips (Affymetrix). The Significance Analysis ofMicro-arrays (SAM) software (http://statweb.stanford.edu/~tibs/SAM/) was used to detect genes distinguishing type II cancersfrom type I cancers as described previously (25, 26). Supervisedhierarchical clustering of these genes was performed andgraphically viewed as a dendogram and heatmap using Cluster3.0 and Java TreeView. The published microarray datasetTCGA UCEC_ 2013 (3) was also analyzed using this method,and the expression pattern for the group of genes commonlyup- or downregulated in type II cancers was designated as atype II signature. The cBioPortal for Cancer Genomics data-base (http://www.cbioportal.org/public-portal/) was used toanalyze genetic oncoprints of SPEC. Connectivity Map analysis(Cmap; http://www.broadinstitute.org/cmap/) was used tomine potential therapeutic agents for SPECs based on genesfor which expression was altered by STAT1 suppression. Usinga Bayesian binary regression model as previously reported (2),the MYC signature was generated and applied to our datasetsin vitro and in vivo for assessing MYC pathway activity inendometrial cancers.

In vivo experimentsSubcutaneous xenografts were established in the flanks of

female NOD-SCID mice (Nihon Clea) by inoculating 5 � 106

SPAC-1L cells with and without STAT1 alteration by domi-nant-negative and shRNA transduction methods. Tumorgrowth in inoculated mice was sequentially monitored twicea week for 8 weeks by measuring the volume of tumors.

Statistical analysisGroup comparisons were done using Mann–Whitney U

tests. Prognostic analysis was performed using the log-ranktest, Fisher exact test, and multivariate analysis. All statisticalanalyseswere done usingGraphPadPrism 5.5, SPSS ver. 22, andR software. Probability values below 0.05 were consideredsignificant.

A complete description of the materials and methods,and any associated references are available in the OnlineRepository.

ResultsPrognosis and gene profiling of endometrial cancers

A total of 294 patients were treated for endometrial cancersin Kyoto University from 2004 to 2012 (endometrioid grade 1,

Kharma et al.

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G1 n ¼ 148; endometrioid grade 2, G2 n ¼ 55; endometrioidgrade 3, G3 n ¼ 53; and SPEC n ¼ 38). The disease-specificsurvival rates of patients bearing G3 and SPEC cancers werelower than patients with G1–G2 cancers (P < 0.0001; Supple-mentary Fig. S1A) and extra-uterine spread was more fre-quently observed in G3 and SPEC than in G1–G2 (P < 0.0001and P < 0.0001, respectively; Supplementary Table S2). Thus,type II cancers, G3 and SPEC, exhibit unfavorable outcomewith highly progressive features.To investigate whether these distinct differences resulted

from specific gene profiles, gene expression microarrayanalysis was conducted for 63 endometrial cancers (G1n ¼ 22; G2 n ¼ 18; G3 n ¼ 11; and SPEC n ¼ 12). Un-supervised clustering analysis of gene expression revealedthat SPECs compose a distinct cluster apart from the G1–G2 enriched clusters (Supplementary Fig. S1B). Further-more, using supervised clustering with a total of 1,244genes, the expression of G3 and SPEC was statisticallydifferent from G1–G2 (t test, P < 0.005). The 63 endometrialcancer samples were divided into two clusters, but SPECswere enriched in only one of the subclusters (P <0.0001, Fig. 1A). The same result was found in the TCGAUCEC_ 2013 dataset. There were 416 overlapping genesbetween these datasets, whose gene expression profileswere subsequently designated as the "type II signature"(227 upregulated and 189 downregulated genes; Fig. 1B,Supplementary Fig. S1C, and Supplementary Table S3).Among these 227 upregulated genes, STAT1 and manySTAT1-associated genes such as MYC, ICAM1, and SMAD7were highly expressed in the SPEC-rich cluster (Fig. 1A and

B and Supplementary Table S3A). Conversely, ESR1 andPGR were included among the 189 downregulated genes(Fig. 1B and Supplementary Table S3B). SAM analysisconfirmed that STAT1 and its associated genes were highlyexpressed in the SPEC-rich cluster (Supplementary Fig. S1Dand Supplementary Table S4).

STAT1 expression and clinical significance inendometrial cancers

The expression of STAT1 in 91 patients with endometrialcancer (mean age, 59.1 years) was assessed by immuno-histochemistry (G1 n ¼ 35; G2 n ¼ 16; G3 n ¼ 18; and SPECn ¼ 22; Table 1). The expression of STAT1 was observedwithin the nucleus of cancer cells with weak staining inG1–G2, weak to intermediate staining in G3, and strongstaining in SPEC (Fig. 2A). The STAT1 expression score ofG3 tumors was higher than that of G1 tumors (P < 0.0001),but SPEC staining was much higher than that of G3 tumors(P < 0.01) and G1–G2 tumors (P < 0.0001, Fig. 2B). Thesefindings were also confirmed in the Vancouver tissuemicroarrays as an external validation, in which STAT1 washighly expressed in SPEC (P < 0.0001, Fig. 2C). The expres-sion of STAT1 mRNA was also higher in SPEC (P < 0.05),confirmed by similar results in our microarray data, theTCGA UCEC_2013 microarray dataset, and two additionaldatasets, GSE17025 and GSE24537 (Supplementary Fig.S2A–S2E). Intriguingly, around 77% (17 of 22) of SPECcases contain infiltrated CD8þ immune cells at the tumorfront with strongly positive staining of ICAM1 and PD-L1(Fig. 2A).

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Figure 1. Microarray gene expression across histologic subtypes of endometrial cancers. A, supervised analysis of 1,244 genes for which expressionin G3 and SPEC was significantly different from G1–G2 (t test, P < 0.005). Blue bar, G1 (n ¼ 22); green bar, G2 (n ¼ 18); yellow bar, G3 (n ¼ 11);and red bar, SPEC (n ¼ 12). SPECs are enriched in a subcluster within which STAT1 and associated genes ICAM1, PD-L1, MYC, IRF1, IFIT3, andSMAD7 are all highly expressed. B, a Venn diagram showing the overlap of 227 upregulated genes (red) and 189 downregulated genes (green) inthe SPEC-enriched clusters of both the Kyoto University human endometrial cancer samples (CC) and TCGA UCEC_2013 microarray datasets. STAT1and associated genes are included among the 227 upregulated genes.

STAT1 Is an Oncogene Driver in Serous Papillary Endometrial Cancer

www.aacrjournals.org Cancer Res; 74(22) November 15, 2014 6521




The disease-specific survival of patients bearing STAT1-high cancers was significantly shorter than those bearingSTAT1-low cancers (P < 0.0001, Fig. 2D), and this tendencywas also confirmed in the Vancouver setting (P < 0.05; Sup-plementary Fig. S2G). Furthermore, as shown in Table 1, highexpression of STAT1 was associated with known prognosticfactors of tumor progression, including deep myometrial inva-sion (P< 0.05), lymphovascular space invasion (P< 0.05), lymphnode metastasis (P < 0.0001), and consequently, high risk ofrecurrence (P < 0.0001). Multivariate analysis revealed thathigh STAT1 expression was a prognostic factor independentfrom histologic subtypes and FIGO stages (P < 0.05; Supple-mentary Table S5).

STAT1 pathway activity in endometrial cancer cellsTo investigate the STAT1 pathway in endometrial cancers,

mRNA levels of STAT1 and STAT1-associated genes inendometrial cancer cells with/without modulation of STAT1was assessed by quantitative real-time PCR (qRT-PCR).Ishikawa, HEC1A, HEC50B, and SPAC-1L cell lines wereused as representative of G1, G2, G3, and SPEC, respectively.STAT1 encodes two alternatively spliced isoforms, STAT1aand STAT1b, which is induced by homodimeric IFNg viaits receptors, IFNgR1 and IFNgR2 (18). In endometrialcancer cell lines, STAT1b and IFNgR2 mRNAs are generallyexpressed at higher levels than STAT1a and IFNgR1, andare highly expressed in SPAC-1L cells (Fig. 3A and E). STAT1mRNA expression was significantly upregulated by IFNgbut attenuated by siRNA-mediated suppression of STAT1.This modulation of STAT1 expression was most prominentin SPAC-1L cells at both mRNA and protein levels but not inother representative cell lines (Fig. 3A and D, SupplementaryFig. S5D and E). Furthermore, STAT1 expression was aug-mented by IFNg in a dose-dependent manner (Fig. 3D andSupplementary Fig. S3A).

The mRNA expression levels of STAT1-associated genes,including CD274 (also known as PD-L1), ICAM1, IRF1, SMAD7,and CCL7 (also known as MCP3; refs. 27–29), were examinedby qRT-PCR following treatment with IFNg and/or STAT1-siRNA. Figure 3B and C show that IFNg treatment upregulatesPD-L1 and ICAM1mRNA expression in SPAC-1L cells (>60-foldand nearly 15-fold, respectively) and HEC50B cells (2.4-foldand 1.5-fold, respectively). Increased expression was observ-ed in a dose-dependent manner and was reversed by suppres-sion of STAT1 (Fig. 3D; Supplementary Fig. S3B and S3C).In SPAC-1L cells, similar IFNg augmentation of mRNA expres-sion was observed for IRF1, SMAD7, and MCP3 (�12-fold,2-fold, and 3-fold, respectively), and an observed attenuationof expression following treatment with STAT1-siRNA (Supple-mentary Fig. S3D–S3F). These results indicate that a SPECcell line, SPAC-1L, is highly responsive to IFNg inductionthrough IFNgR to activate STAT1-associated genes.

STAT1 functions as a tumor prosurvival andproprogression gene in SPEC cells

STAT1 has previously been considered to function as atumor suppressor to induce cell-cycle arrest and apoptosis invarious types of cancers (30, 31). To determine how STAT1contributes to cell growth and survival in SPEC, various in vitrofunctional assays were performed using SPAC-1L cells follow-ing manipulation of STAT1 activity. In addition to STAT1-siRNA transfected cells (STAT1-siRNA cells), SPAC-1L cellswith stable suppression of STAT1 were generated for furtherexamination by introducing STAT1-specific shRNA (STAT1-shRNA) or a dominant-negative plasmid (STAT1-DN3 andSTAT1-DN5 cells; Supplementary Fig. S4A–S4C).

To assess the potential role of STAT1 on prosurvival pro-perties, assays for proliferation and colony formation in softagar were performed. As a result, cellular proliferation wassuppressed in a time-dependent manner in both STAT1-siRNAcells (P < 0.0001, Fig. 4A) and STAT1-DN5 cells (P < 0.0001;Supplementary Fig. S4D). Similarly, anchorage-independent

Table 1. Clinicopathologic analysis of STAT1expression in 91 endometrial cancers alongwitheach known prognostic factor

Expressionof STAT1a

Characteristics Low High P

Age�60 30 20 0.1259>60 18 23

FIGO stageI 35 20II 7 1 0.0006III 4 14IV 2 8

HistologyG1 32 3G2 9 7 <0.0001G3 6 12Serous 1 21

Myometrial invasionb

No invasion 11 4 0.0229�1/2 (inner half) 19 9>1/2 (outer half) 16 24

Lymphovascular space invasionb

� 34 17 0.0193þ 12 18

Lymph node metastasisb

� 44 26 <0.0001þ 1 14

Ascitesb

� 38 25 0.1628þ 9 12

Recurrence riskLow 22 2 <0.0001Intermediate 17 13High 9 28

NOTE: Statistical significance was analyzed by the x2 test.aSTAT1 score: >1, high; �1, low.bSome data weremissing: 8 cases, 10 cases, 6 cases, and 7cases, respectively.

Kharma et al.





growth capacity was impaired in STAT1-shRNA cells (P <0.0005, Fig. 4B) and STAT1-DN5 cells (P < 0.0001; Supplemen-tary Fig. S4E).To assess the impact of STAT1 on progression, adhesion,

and migration, Boyden-chamber assays were performedwith IFNg induction. Adhesive capacity was augmented byIFNg (P < 0.0001), which was mitigated by STAT1 suppres-sion (P < 0.0005, Fig. 4C). Cellular adhesive capacity wasalso suppressed in STAT1-DN5 cells and STAT1-siRNA cells

(P < 0.0001), whereas STAT1-DN3 cells did not show thesame level of suppression (Supplementary Fig. S4F). The num-bers of SPAC-1L cells attached to the single-layered HUVECcells decreased with knockdown of STAT1, but attachmentwas recovered with concurrent IFNg treatment. Second,SPAC-1L cellular motility over a 24-hour tracking period wasaccelerated by IFNg treatment, although this accelerationwas not observed in STAT1-shRNA cells (Fig. 4D). Boyden-chamber assays demonstrated that cellular invasive activity

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Figure 2. Immunohistologic analysis of STAT1 expression in human endometrial cancers. A, representative micrographs of STAT1 expression in eachhistologic subtype (�400). Top, left, G1; middle top, G2; middle bottom, G3; right, SPEC. The expression of STAT1 was observed in the cancer cells;near negative-weak in G1–G2, weak–intermediate in G3, and positive in SPEC. Below dashed line, immune microenvironment of SPEC. Left, CD8þ

expression; mid, PD-L1 expression; right, ICAM1 expression. Scale bars, 20 mm. Arrow, abundant CD8þ cells surround the tumor invasive front withhigh expression of PD-L1 and ICAM1. B, the expression of STAT1 was scored on the basis of staining of cancer cells as follows: 0, negative; 1, weakpositive (weak intensity and�25% area stained); 2, intermediate (weak intensity and 25%–50% area stained); 3, positive (prominent intensity and 50%–

75% area stained); and 4, strong positive (prominent intensity and �75% area stained). STAT1 expression was significantly higher in high-gradeendometrial cancers (G3 and SPEC; �, P < 0.05; ��, P < 0.0001); k-agreement between observers are 0.823, 0.848, and 0.822, respectively. C, theexpression of STAT1 from the Vancouver endometrial cancer tissue microarrays was scored on the basis of staining of cancer cells. STAT1 expressionwas higher in SPECs (P < 0.0001); k-agreement between observers are 0.892, 0.982, and 0.910, respectively. D, Kaplan–Meier analysis ofdisease-specific survival for 91 patients with endometrial cancers in the Kyoto cohort with respect to intensity of STAT1 expression. STAT1-L,expression score �1; STAT1-H >1. The prognostic outcome is worst in STAT1-H (P < 0.0001).






was augmented by IFNg (P < 0.0005), but this augmentationwas remarkably suppressed by STAT1 knockdown (P < 0.0001).Indeed, cellular invasion was suppressed in all cells withSTAT1 knockdown (Fig. 4E and Supplementary Fig. S4G).These in vitro results suggest that high expression of STAT1in SPEC might contribute to aggressive cell behavior viaenhanced proliferation and capacity for disease progression.

STAT1 pathway significance in tumorigenesisTo further determine the roles of the STAT1 pathway

in vivo, NOD-SCID mice were used to compare the tumor-

igenic capacity of STAT1-shRNA cells with that of theparental SPAC-1L cells. Among 15 mice inoculated withSTAT1-shRNA cells, xenograft tumors were observed in onlyfour mice, all less than 50 mm3 in size, whereas all 10 miceinoculated with SPAC-1L cells formed large tumors (P <0.0001, Fig. 5A). This tumorigenic inhibition was alsoobserved for STAT1-DN5 cells (P < 0.0001; SupplementaryFig. S5A). Thus, the suppression of STAT1 expression inhib-ited tumor growth in NOD-SCID mice, likely via attenuationof oncogene function. The Connectivity Map (Cmap) ana-lysis showed gene expression changes resulting from STAT1

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Figure 3. STAT1 pathway activity in endometrial cancer cells. A–C, expression of STAT1, ICAM1, and PD-L1mRNAs in endometrial cancer cell lines wasassessed by qRT-PCR. Blue bar, mock-treated; red bar, STAT1-suppressed with siRNA; green bar, IFNg treated; and purple bar, treated withboth STAT1-siRNA and IFNg . SPAC-1L, a cell line derived from SPEC, showed high responsiveness to IFNg treatment with induction of STAT1(�16), ICAM1 (�15), and PD-L1 (�60) mRNAs. STAT1-siRNA treatment suppressed STAT1, ICAM1, and PD-L1 mRNA expression in HEC50B(G3 cell line) and SPAC-1L. D, expression of STAT1 and proteins from STAT1-associated genes, ICAM1 and PD-L1, in SPAC1-L cells by Westernblotting with/without STAT1-siRNA and/or IFNg treatment. E, IFNg receptor status within endometrial cancer cell lines. The expression of IFNgreceptor1 (IFNGR1) and IFNg receptor2 (IFNGR2) was examined in four endometrial cancer cell lines by qRT-PCR.

Kharma et al.





suppression were not associated with changes induced bydoxorubicin or paclitaxel but were associated with thoseinduced by some bioactive molecules including sirolimus asthe top-ranked candidate compound (Supplementary TableS6 and Supplementary Fig. S5B).The MYC promoter region has STAT1 binding sites, and

STAT1 has been reported to maintain basal expression ofMYC during tumorigenesis (32). Both MYC and STAT1 werehighly expressed in SPECs (Fig. 1B), although the functionalimplications for these findings have not yet been clarified.We therefore investigated whether STAT1 regulates theoncogenic function of MYC. Similar to other genes, MYCmRNA expression was induced 12-fold following treatmentwith IFNg (P < 0.0001; Fig. 5B and Supplementary Fig. S5G),but this induction was remarkably attenuated in STAT1-shRNA cells not only in vitro (P < 0.0001), but also in

xenograft tumors (23-fold, P < 0.0001, Fig. 5C). Westernblotting showed that induction of STAT1 was accompaniedby upregulation of MYC, whereas suppression of STAT1resulted in decreased MYC expression in vitro and in vivo(Fig. 5D). This was also true for the STAT1-DN5 cells, as MYCexpression was not induced by IFNg treatment (Supplemen-tary Fig. S5C). In contrast, MYC expression was not remark-ably changed in non-SPEC cell lines by STAT1 manipulation(Supplementary Fig. S5D and S5F–S5G). A predictive MYCsignature score can be used to represent MYC pathwayactivity for a given specimen based on the expression levelsof known MYC target genes (33). The MYC signature scoresof STAT1-siRNA cells were significantly lower than that ofSPAC-1L cells (P < 0.05), whereas that in SPECs was higherthan that of endometrioid adenocarcinomas (P < 0.05 Sup-plementary Fig. S5D and S5E). To clarify the functional role

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Figure 4. STAT1 functions to promote tumor cell survival and progression in SPEC cells. WT, nontreated cells; mock, negative control siRNA/shRNA–treated cells; STAT1-siRNA, STAT1-siRNA–treated cells; IFNg , IFNg-treated cells. A, STAT1 regulates cell proliferation. Proliferation in SPAC-1Lcells was assessed by WST-1 assays in quintuplicate with or without STAT1-siRNA. Cell proliferation decreased significantly in STAT1-siRNA cells(P < 0.0001). B, STAT1 inhibition decreases anchorage-independent growth. SPAC-1L cell colony formation in soft agar was assessed with orwithout STAT1 knockdown. Left, colony numbers are significantly decreased by downregulating STAT1 gene expression with STAT1-shRNA (n ¼ 10,P ¼ 0.0003). Right, representative pictures of cells cultured in soft agar. C, STAT1 modulates cell adhesion. After coculturing with HUVEC cells for4 hours, cell adhesion was measured by spectrophotometer as relative fluorescence units (RFU). Left, downregulating STAT1 expression resultsin reduced SPAC-1L adhesion (n ¼ 10; ��, P < 0.0001) and mitigates the inducing effect of IFNg (��, P ¼ 0.0005). Right, representative pictures ofattached cells in each condition. D, STAT1 promotes cell migration. The effect of STAT1 on migration was assessed using wound-healingassays. The migration rate was evaluated in quadruplicate by measuring the gap between the cells most closely spaced on each leading edge at 0,6, 12, and 24 hours postwounding. STAT1 knockdown impairs migration of SPAC-1L cells and also lessens the inducing effect of IFNg . E,STAT1 modulates invasion. Invasive potential of SPAC-1L cells was assessed using Boyden-chamber assays. Left, by downregulating STAT1using STAT1-shRNA, the number of invading cells was decreased (n ¼ 10; ��, P < 0.0005) and reduced the promoting effect of IFNg (��, P < 0.0001).Right, representative micrographs of hematoxylin-stained cells that have invaded through the membrane.






of the IFNg–STAT1–MYC axis, cell proliferation assays wereperformed under conditions of cosuppression of STAT1 andMYC in vitro. As shown in Fig. 5E, MYC suppression led toa significant reduction of SPAC-1L cellular proliferation (P <0.0001). This inhibitory effect was more prominent inSTAT1-shRNA cells (P < 0.0001). Cosuppression of STAT1and MYC showed no significant difference in inhibitoryeffect as compared with STAT1 suppression alone. Theseresults suggest that STAT1 contributes to regulation of

oncogenic MYC expression to promote cancer cell prolifer-ation and tumor growth.

DiscussionSPECs account for only 4% to 10% of endometrial cancer,

are highly aggressive, and are difficult to treat effectively,whereas low-grade endometrioid adenocarcinoma com-prises 80% of endometrial cancer and is frequently diagnosed

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Figure 5. Significant role of the STAT1 pathway in tumorigenesis. A, STAT1 downregulation inhibits tumorigenesis in vivo. NOD-SCID mice weresubcutaneously inoculated with 5 � 106 SPAC-1L cells with and without STAT1 knockdown. Tumor growth was inhibited in mice inoculated withSTAT1-shRNA cells (n ¼ 25, P < 0.0001). Right, arrows indicate growing tumors of SPAC-1L cells. B, STAT1 regulates the MYC oncogene. STAT1knockdown suppressed MYC mRNA expression (n ¼ 5; P < 0.0001) and diminished the inducing effect of IFNg (P < 0.0001). C, MYC mRNAexpression in xenograft tumors. MYC expression was repressed in STAT1-suppressed xenograft tumors (P < 0.0001). D, STAT1 regulates MYCin vitro and in vivo. MYC expression was assessed by Western blotting. MYC expression was suppressed by prior STAT1 knockdown, in vitro andin vivo. E, proliferative capacity is regulated by STAT1 via MYC. Proliferation is inhibited by MYC-siRNA–mediated knockdown (n ¼ 10; P < 0.0001),but double knockdown of STAT1 and MYC showed no additive effect as compared with STAT1 knockdown alone in suppression of cellularproliferation (n ¼ 10, ns).

Kharma et al.





at an early stage, at which point it is surgically curable (3, 34).As SPEC is chemorefractory and patients bearing SPECusually exhibit unfavorable outcomes with multiple metas-tases and/or recurrence, SPEC has attracted a great deal ofattention to determine characteristic malignant features thatare distinct from endometrioid adenocarcinoma. We havepreviously described fludarabine as a potent therapeuticcandidate for chemorefractory endometrial cancer (2). Itspredicted efficacy was higher for G3 and SPEC than for G1–G2, but the precise mechanism explaining fludarabine effi-cacy or its therapeutic target(s) in this setting is unknown.Recent integrated genomic analysis demonstrated that SPECexhibited specific genomic features that were quite differentfrom the endometrioid subtype, but rather were shared withovarian serous and basal-like breast carcinomas (3). How-ever, the representative genes or pathways responsible forSPEC's aggressive malignant phenotype were not well clar-ified from these studies. Thus, further study of SPEC withregard to these findings is needed to determine the principalunderlying genetic signature to not only improve under-standing of SPEC-specific tumor biology but, more impor-tantly, for developing novel targeted therapies based on thisbiology.In our cohort setting, patients with SPEC exhibited poor

outcome (Supplementary Fig. S1A) and a distinct geneexpression profile (Fig. 1A) as compared with other subtypesof endometrial cancer. These results were compatible withthose of the TCGA UCEC_2013 dataset (3); therefore, wesought to identify SPEC-specific signature genes that werecommonly activated in the SPECs in our dataset and in theTCGA dataset. As the Venn diagram demonstrates in Fig. 1B,227 genes were highly expressed in SPECs, including STAT1and STAT1-associated genes such as MYC, SMAD7, andIFIT3, which have been reported to be involved in carcino-genesis or cancer progression (32, 35, 36), and these geneswere also detected as SPEC-specific upregulated genes inanother SAM analysis (Supplementary Fig. S1D and Supple-mentary Table S4). In contrast, ESR1 and PGR were amongthe downregulated genes, which is compatible with thehormone-independent feature of SPEC. Several genome-wide analyses have described distinctive gene expressionpatterns in SPEC from analysis of a single dataset withoutany external validation (37, 38); nevertheless, the SPEC drivergenes or pathways were not conclusively identified. At thesame time, interobserver reproducibility in subtype classi-fication of type II endometrial cancers was poor (3, 39) andtraditional subtype classification itself sometimes failed todistinguish SPEC from high-grade endometrioid endometri-al carcinoma, as several G3 cases and two SPEC cases werenot classified with their counterparts by clustering analysisin this study (Fig. 1A and Supplementary Fig. S1B and S1C).To address this issue, we identified STAT1 as a key moleculeof the SPEC-signature that was commonly upregulated inboth the datasets studied, and this was accompanied by theupregulation of putative downstream STAT1 gene targets.We confirmed high STAT1 expression in SPEC by externalvalidation using GSE17025 and GSE24537 (SupplementaryFig. S2D and S2E; refs. 37, 38).

Immunohistochemical (IHC) staining confirmed highSTAT1 expression in SPEC and demonstrated the associa-tion of STAT1 expression with worse clinical outcomeaccompanied by prognosis indicators, including deep myo-metrial invasion, lymphovascular space invasion, and lymphnode metastasis (Fig. 2, Table 1). The most robust findings,that STAT1 was expressed significantly higher in SPECs thanin the endometrioid subtype and that STAT1-high endome-trial cancers had worse prognostic outcome (Fig. 2B and D),were externally validated by IHC finding in a large Vancou-ver cohort (Fig. 2C and Supplementary Fig. S2G). Thus, highexpression of STAT1 is an indicator of recurrence, and isindependently related to shorter disease-free survival, whichis consistent with previous reports showing STAT1 over-expression or genes induced by IFNg , is associated with poorclinical outcomes of breast cancers (19, 40). STAT1 expres-sion in a SPEC cell line, SPAC-1L, was constitutively high,and augmented by IFNg in a dose-dependent manneraccompanied with similar augmentation of expression ofseveral STAT1-associated genes (Fig. 3). STAT1-associatedgenes, such as ICAM1,MYC, PD-L1, are known to play roles inthe progression and metastasis of other types of cancerthrough mechanisms specific to the type of malignancy(14, 29, 36, 41, 42). This augmentation by IFNg was specif-ically prominent in SPAC-1L, and attenuated by suppressingSTAT1 expression. These results imply that overexpressionof STAT1 might be associated with the clinical aggres-siveness of SPECs and that the IFNg signal was transducedthrough STAT1, although the source of IFNg in vivo isunknown.

Among the STAT1-associated genes, ICAM-1, PD-L1, andSMAD7 are involved in cancer immunity (43), and proteomicanalysis revealed multiple proteins associated with inflam-mation are overexpressed in the uterus with endometrialcancer (44). Immunohistochemical staining demonstratedthat SPEC has a characteristic tumor microenvironment atits invasive front that is fully infiltrated by CD8þ immunecells (Fig. 2A), which are a known source of IFNg (41). AsSPAC-1L cells have relatively high transcription of IFNgreceptors 1 and 2, the tumor microenvironment could bea potential source of persistent IFNg in vivo that results inthe constitutive activation of the STAT1 pathway for atten-uating cancer immunity to promote progression in SPEC.Further experiments are required to determine the reasonwhy this scenario is not the case for the endometrioidsubtypes.

Previous studies showed that STAT1 activates antipro-liferative and proapoptotic genes as a tumor suppressor (45);in contrast, STAT1 in SPEC appears to function as a tumorprosurvival gene. Alteration of STAT1 function using adominant-negative plasmid and/or siRNA decreased themalignant characteristics of SPAC-1L cells, while restorationof these features occurred with IFNg treatment to activatethe STAT1 pathway (Fig. 4 and Supplementary Fig. S4D–S4G). With regard to IFNg–STAT1 pathway genes, ICAM1is involved in sphere formation and metastatic potential(46), and increased expression at invasive fronts is positive-ly correlated with invasion and metastasis (47). SMAD7 is






involved in tumorigenesis of mesenchymal stem cells con-comitant with upregulation of MYC under prolonged inflam-matory IFNg exposure (48). Cellular capacity for migration,anchorage-independent growth, and attachment are impor-tant prerequisites for tumor invasion and metastasis (49).The highly progressive features of SPEC might be partiallydue to constitutively high STAT1 expression and consequentupregulation of downstream STAT1 target genes under ahighly orchestrated series of tumor microenvironment com-ponents, and we propose that constitutively high STAT1expression in SPEC has a tumor-promoting role rather thana tumor-suppressing role. This is further supported by ourobservation that xenograft tumor growth was remarkablyinhibited by repression of STAT1 expression. Althoughmetastasis was not observed in the NOD-SCID mouse model,this might be because progression outside of the primarytumor requires exogenous IFNg . From these experiments,however, we can conclude that STAT1 pathway activation inSPEC cells is essential for tumor growth.

We then investigated the mechanism by which STAT1works as a tumor-promoting gene as well as potential targetmolecules. MYC is one of the Yamanaka genes essential forcellular proliferation, and both STAT1 and STAT3 compet-itively bind to the MYC promoter to regulate expression

(9, 32). Because the gene expression microarray datarevealed that STAT3 is not highly expressed in SPEC, it wasreasonable to consider that MYC might be regulated bySTAT1 in SPEC as a growth-promoting driver gene. Tosupport this notion, 108 of the 227 upregulated genes inthe SPEC signature harbor the MYC binding site motif byGATHER analysis (V$MYCMAX_B; http://gather.genome.duke.edu/), and the predictive MYC activity signature scorewas statistically higher in SPEC than in other subtypes ofendometrial cancers, whereas that in SPAC-1L cells wasdiminished with STAT1 knockdown (Fig. 5 and Supplemen-tary Fig. S5). As the suppressive effect on proliferation bydouble knockdown of STAT1 and MYC was not superior toknockdown of STAT1 alone (Fig. 5E), STAT1 may function asthe master modulator of MYC activity. The cBioPortal forCancer Genomics database indicated that aberration of theSTAT1–MYC axis was more frequently found in SPECS (51%)than in endometrioid subtypes (17.6%; data not shown),whereas there were no SPEC samples with downregulationof STAT1 or MYC. As MYC upregulation was not observed inendometrioid cell lines (Supplementary Fig. S5D, S5F, andS5G), these results imply that more than half of SPECs haveaberrant upregulation of the STAT1–MYC axis as a partic-ular driver oncoprint of their aggressiveness. This STAT1-

Figure 6. Schematic showing STAT1 roles as a driver gene of SPEC in modulating "transcriptional prosurvival pathways" to enhance malignant capacity.In response to IFNg stimulation, dimerization of the IFN receptor allows phosphorylation and activation of JAKs (JAK 1 and JAK 2), and this isfollowed by STAT1 activation through phosphorylation, leading to the formation of a STAT1 homodimer. The STAT1 homodimer activates geneexpression by binding to the IFNg responsive element, GAS, as previously reported (9). This binding leads to activation of transcriptional activityin promoting some STAT1-associated genes, including MYC, IRF1, PD-L1, SMAD7, and ICAM1. MYC activation positively affects cell-cycleregulation; ICAM-1, PD-L1, and IRF1 are involved in cancer immunity (41) in the tumor microenvironment; SMAD7 functions in TGFb signaling, andICAM1 and SMAD7 affect metastatic capability. These effects suggest that STAT1 functions as a driver gene in a tumor prosurvival pathway. Incontrast, reciprocal regulation between STAT1 and STAT3 has also been described as a "Death Signaling Pathway" (9); TP53 mutation is commonin SPEC and is expected to inhibit this death signaling pathway.

Kharma et al.





MYC signature may be viewed as a SPEC-specific target.Cmap analysis based on SPEC's specific gene signaturepredicted doxorubicin and paclitaxel as not effective forSTAT1-high tumors, and many SPECs are indeed clinicallyresistant to these drugs. In contrast, sirolimus was identifiedas the top-most candidate drug for STAT1-high tumors outof the 72 candidates identified (Supplementary Table S6 andSupplementary Fig. S5B). As temsirolimus, a derivative ofsirolimus, was identified as a potential drug for chemore-fractory SPECs in our prior study (2), sirolimus might be anovel candidate for SPEC although further studies arewarranted.In summary, a genome-wide analysis together with func-

tional assays revealed that STAT1 is constitutively activated inSPEC. This activation may be the result of the tumor micro-environment and results in promotion of tumor growth andextra-uterine spread via sequential activation of STAT1 down-stream genes. Generally, STAT1 has been considered as atumor suppressor involved in the "death signaling pathway,"but in this study of SPEC, STAT1 was identified as a mastergene modulating "transcriptional prosurvival pathways" toenhance multiple malignant characteristics (Fig. 6). SPEC ishighly aggressive and chemorefractory such that patients withSPECoften have poor outcomes, thus the development of noveltreatment strategies based on the biology of this disease is anurgent need. Our findings support that targeting STAT1, theSPEC driver gene, may provide the means to improve pooroutcomes for patients with SPEC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.Novelty and Impact: We declare that all data are novel, developed by our

own experiments, and have not been published or submitted for publication.We

have revealed new insights regarding STAT1 as a driver of tumor progression inrefractory serous papillary endometrial cancers (SPEC). Our study, based onbioinformatic analysis and in vitro/in vivo experiments, also suggests STAT1functions as a tumor prosurvival gene rather than a tumor suppressor gene.These findings will inform much needed therapeutic strategies for SPEC bytargeting the SPEC driver gene, STAT1, to improve the poor outcome of thisdisease.

Authors' ContributionsConception and design: B. Kharma, T. Baba, N. Matsumura, M. Mandai,I KonishiDevelopment of methodology: B. Kharma, T. Baba, D.G. HunstmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Kharma, T. Baba, R. Murakami, M.M. McConechyAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Kharma, T. Baba, R. Murakami, S. Leung,K. Yamaguchi, M. Mandai, I. KonishiWriting, review, and/or revision of the manuscript: B. Kharma, T. Baba,M.M. McConechy, S.K. MurphyAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B. Kharma, T. Baba, J. Hamanishi,S. Leung, K. Yamaguchi, Y. Hosoe, I. KonishiStudy supervision: B. Kharma, T. Baba, N. Matsumura, H.S. Kang, K. Yama-guchi, Y. Yoshioka, M. Mandai, I. Konishi

AcknowledgmentsThe authors thank all patients and families who contributed to this study;

Prof. Remi Fagard, French Institute of Health and Medical Research, Paris,France, for suggestions regarding the study; Prof. Toshio Hirano, Osaka Univer-sity, for kindly providing pBOS-STAT1-DN; Prof. Tokuichi Kawaguchi, the CancerInstitute of the Japanese Foundation for Cancer Research, for kindly providingthe SPAC-1L cell line. The authors also thankChristineChowandShermanLau atthe Vancouver Genetic Pathology Education Centre for their assistance in thisstudy.

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 indicate thisfact.

Received April 2, 2014; revised August 7, 2014; accepted August 31, 2014;published OnlineFirst September 29, 2014.

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2014;74:6519-6530. Published OnlineFirst September 29, 2014.Cancer Res Budiman Kharma, Tsukasa Baba, Noriomi Matsumura, et al. CancerSTAT1 Drives Tumor Progression in Serous Papillary Endometrial

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STAT1 Drives Tumor Progression in Serous Papillary ...and Vancouver General Hospital (Vancouver, Canada) includ-ed specimens from 460 endometrial cancers in ﬁve tissue microarrays