Therapeutics, Targets, and Chemical Biology

STAT3/IRF1 Pathway Activation SensitizesCervical Cancer Cells to Chemotherapeutic DrugsBarbara Walch-R€uckheim1, Jennifer Pahne-Zeppenfeld2, Jil Fischbach1,Claudia Wickenhauser3, Lars Christian Horn4, Lars Tharun5, Reinhard B€uttner5,Peter Mallmann6, Peter Stern7, Yoo-Jin Kim8, Rainer Maria Bohle8, Christian R€ube9,Russalina Str€oder10, Ingolf Juhasz-B€oss10, Erich-Franz Solomayer10, and Sigrun Smola1

Abstract

Neoadjuvant radio/chemotherapy regimens can markedlyimprove cervical cancer outcome in a subset of patients, whileother patients show poor responses, but may encounter severeadverse effects. Thus, there is a strong need for predictivebiomarkers to improve clinical management of cervical cancerpatients. STAT3 is considered as a critical antiapoptotic factorin various malignancies. We therefore investigated STAT3activation during cervical carcinogenesis and its impact onthe response of cervical cancer cells to chemotherapeuticdrugs. Tyr705-phosphorylated STAT3 increased from low-grade cervical intraepithelial neoplasia (CIN1) to precancer-ous CIN3 lesions. Notably, pTyr705-STAT3 activation signif-icantly declined from CIN3 to invasive cancer, also whencompared in the same clinical biopsy. pTyr705-STAT3 wasalso low or absent in cultured human cervical cancer cell lines,consistent with the in vivo expression data. Unexpectedly,

IL6-type cytokine signaling inducing STAT3 activation ren-dered cervical cancer cells significantly more susceptible tochemotherapeutic drugs, that is, cisplatin or etoposide. Thischemosensitization was STAT3-dependent and we identifiedIFN regulatory factor-1 (IRF1) as the STAT3-inducible medi-ator required for cell death enhancement. In line with thesedata, pTyr705-STAT3 significantly correlated with nuclear IRF1expression in cervical cancer in vivo. Importantly, high IRF1expression in pretreatment cervical cancer biopsy cells wasassociated with a significantly better response to neoadjuvantradio/chemotherapy of the patients. In summary, our studyhas identified a key role of the STAT3/IRF1 pathway forchemosensitization in cervical cancer. Our results suggest thatpretherapeutic IRF1 expression should be evaluated as a novelpredictive biomarker for neoadjuvant radio/chemotherapyresponses. Cancer Res; 76(13); 3872–83. �2016 AACR.

IntroductionCervical cancer represents the third most common cause of

cancer-related death in women worldwide. Invasive cancerdevelops from persistent high-risk human papillomavirus(HPV) infection through well-defined stages of cervical intrae-

pithelial neoplasia (CIN1–3; ref. 1). This process takes years ordecades and it is assumed that further changes within the (pre)neoplastic cells and their microenvironment critically influencethe course of disease.

Cervical cancer therapy is still a major clinical challenge.Responses to neoadjuvant radio/chemotherapy vary greatly inpatients (2, 3). Intrinsic and acquired resistance of the neoplasticcells as well as substantial side effects from standard treatmentincluding platinum-based chemotherapy limit the options forescalation (4). Identification of patients that may best benefitfrom chemotherapy would be useful for improved clinical man-agement. This will require a better understanding of the mechan-isms influencing the balance between sensitivity and resistance tocervical cancer cell death.

The STAT3 transcription factor is commonly considered as asurvival or progression factor in different cancer types. Constitu-tive STAT3 activation is documented in various human malig-nancies including head and neck, brain, breast, lung, pancreas, aswell as prostate cancer and melanoma (summarized in ref. 5).STAT3 inhibition can affect tumor growth and enhance theresponse of certain tumors to chemotherapy directly or in animmune-dependent manner (6–8).

Notably, STAT3 activation also has profound direct effects onthe immune microenvironment (9). Our group has previouslydemonstrated pronounced tyrosine-phosphorylation of STAT3in cervical high-grade lesions (10). Strongly activated STAT3was detected within the inflammatory infiltrate of the lesions,

1Institute of Virology, Saarland University, Homburg/Saar, Germany.2Center for Molecular Medicine Cologne and Institute of Virology,University of Cologne, Cologne, Germany. 3Institute of Pathology,University of Halle, Halle, Germany. 4Institute of Pathology, Universityof Leipzig, Leipzig, Germany. 5Institute of Pathology, University ofCologne,Cologne,Germany. 6DepartmentofGynecologyandObstet-rics, University of Cologne, Cologne, Germany. 7Institute of CancerSciences, University of Manchester, Manchester, United Kingdom.8Institute of Pathology, Saarland University, Homburg/Saar, Germany.9Department of Radiotherapy and Radiation Oncology, Saarland Uni-versity, Homburg/Saar, Germany. 10Department of Gynecology andObstetrics, Saarland University, Homburg/Saar, Germany.

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

B. Walch-R€uckheim and J. Pahne-Zeppenfeld share first authorship of thisarticle.

Corresponding Author: Sigrun Smola, Institute of Virology, Saarland University,Kirrbergerstrasse, Building 47, Homburg/Saar D-66421, Germany. Phone: 49–6841–16–23931; Fax: 49–6841–16–23980; E-mail: sigrun.smola@uks.eu

doi: 10.1158/0008-5472.CAN-14-1306

�2016 American Association for Cancer Research.

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where it drives expression of the protumorigenic and vasculo-genic matrix metalloprotease MMP-9, as well as in the prema-lignant epithelial cells (10).

Major activators of the STAT3 pathway are members ofthe IL6-type cytokine family (11, 12). IL6 binds to the a-chainof the IL6R (IL6Ra, gp80), while the related cytokine oncos-tatin M (OSM), binds to the OSM receptor-b (OSMRb).Respective complexes then associate with the common recep-tor chain gp130, which recruits Janus kinases, leading tosubsequent STAT3-phosphorylation at tyrosine 705 (13).Whereas gp130 is ubiquitously expressed, IL6 signaling islimited by the availability of transmembrane gp80 or its naturallyoccurring soluble form sgp80,which induces "trans-signaling" viagp130 (14).

In human squamous cell carcinoma (SCC) of the cervix uteri,IL6 is strongly upregulated in situ and in vitro (15). IL6 has anegative impact on the prognosis of a patient (16). We haveshown that it mainly acts in a paracrine manner and creates aprotumorigenic and immunosuppressivemicroenvironment (10,17, 18). Cervical cancer cells display only low responses toautocrine IL6 due to low gp80 expression levels (15, 19) butsgp80 can restore autocrine IL6 signaling and induce pronouncedSTAT3-binding activity (15).

In this study, we investigated STAT3 activation during thedifferent stages of cervical carcinogenesis in more detail. Inbiopsies comprising precancerous and cancerous lesions, wedemonstrate that STAT3 tyrosine-phosphorylation is highestin CIN3 and significantly declines during progression toinvasive cancer. Unexpectedly, when we forced STAT3 activa-tion with IL6-type cytokines, cervical cancer cells were stronglysensitized to the cytotoxic effects of chemotherapeutic drugs.We identified IRF1 as the STAT3-inducible proapoptotic factormediating chemosensitization. Notably, our data show thatpretreatment IRF1 expression correlates with the response toradio/chemotherapy in cervical cancer patients in vivo.

Materials and MethodsImmunohistochemical analysis

Of note, 145 formalin-fixed paraffin-embedded (FFPE)anonymized lesions of the cervix uteri (22 CIN1/2, 29 CIN3,94 SCC) were retrieved from local pathology archives ofCologne, Leipzig, and Saarland University Hospitals, Germany.These included a subset of 24 pretreatment SCC biopsies frompatients that had been subjected to neoadjuvant chemo- orradio/chemotherapy. These tumors were pre- and post-thera-peutically staged according to the International Federation ofGynecology and Obstetrics (FIGO) or TNM categories (Sup-plementary Table S1). Histologic stainings, diagnosis, andtreatment responses were assessed by expert pathologists(C. Wickenhauser, L.C. Horn, L. Tharun, Y.-J. Kim, R.M. Bohle).Staining of 5-mm sections with Abs listed in SupplementaryTable S2 was performed as described previously (10, 20) andclassified using the Immunoreactive Score (IRS) according toRemmele and Stegner (21). Biopsies were evaluated with stan-dardized settings with a DMI 6000B microscope (Leica) andMicrosoft Image Composite Editor program. The retrospectivestudy has been conducted according to Declaration of Helsinkiprinciples and was approved by the local Ethics Committees ofthe Cologne, Leipzig, and Saarland Universities (at the Saar-land-€Arztekammer).

Cells and cell cultureRelatively low-passage cervical cancer cell lines 808 and 778

(22, 23) were last tested by short tandem repeat profiling in 2014.HPV18-positive cervical carcinoma cell lines SW756 (ATCC CRL-10302), HeLa (ATCC CCL-2), and HPV16-positive SiHa (ATCCHTB-35) obtained fromM. vonKnebelDoeberitz (Department ofApplied Tumor Biology, Institute of Pathology, University ofHeidelberg, Heidelberg, Germany) before 2000 were last authen-ticated by HPV16/18-E6/E7-qRT-PCR and multiplex human cellline authentication test (Multiplexion) in April 2013 and culturedas described previously (17). Normal human exocervical kerati-nocytes (NECK) isolated from hysterectomy specimens accordingto ref. 24 were cultured in supplemented KBM-Gold (Lonza;approved by the local Ethics Committee of the Saarland Univer-sity at the Saarland-€Arztekammer).Written informed consent wasprovided by the study participants.

Cell stimulation and Western blot analysisCarcinoma cells or NECK were seeded at a density of 1.5� 106

cells in a 6-cm culture dish. Twenty-four hours later, they wereincubated with medium, 10 ng/mL OSM, or 100 ng/mL IL6(PeproTech) in the presence of 500 ng/mL sgp80 (R&D Systems;IL6/sgp80) for the indicated time intervals. Whole cell or nuclearextracts were prepared as described in ref. 25. Abs listed inSupplementary Table S2, secondary Abs (Sigma-Aldrich), andECL reagent (Roche) were used for detection with ChemiDocXRSþMolecular Imager. All Western blots were performed understandardized conditions. Expression was quantified with theQuantity One analysis software (both Bio-Rad).

Plasmids and transfectionsIRF1 and IRF2 cDNAs (26) were amplified with primers listed

in Supplementary Table S3, cloned as NotI/SalI fragments inpCMV-Flag2 vector (Sigma-Aldrich) and sequences were verified.A total of 1.5 � 105 HeLa cells/6-well were transfected after 24hours with 0.0 (mock control), 0.025 or 0.1 mg pCMV-Flag2-IRF1,or 0.6 or 0.8 mg pCMV-Flag2-IRF2 expression vector using Lipo-fectamine 2000 (Invitrogen). The total amount of DNA wasadjusted to 1 mg with empty pCMV-Flag2 vector. Six or 10 pmolof indicated siRNAs (ON-TARGETplus Non-targeting siRNA #2,ON-TARGETplus siRNA #8 for human STAT3, siRNA #6 for IRF1,all from Thermo Scientific) were transfected with LipofectamineRNAiMax (Invitrogen) as described previously (20).

Cytotoxicity assaysIn cytotoxicity assays with cisplatin (Hexal) or etoposide (Sig-

ma-Aldrich) cells were seeded in 96-well plates at a density of1.0�104 cells/well, stimulated with medium, OSM, or IL6/sgp80for 8 hours and challenged for 48 hours with serial dilutions ofchemotherapeutic drugs if not indicated otherwise. siRNA-trans-fected cells were pretreated for 2 hours and subsequently chal-lenged with chemotherapeutic drugs for 20 hours. Cell viabilitywas assessed by the neutral red uptake method as describedpreviously (27). For sequential staining with Annexin-V–APC(BD Biosciences) and propidium iodide (PI; Sigma-Aldrich)assays were scaled up to 24-well plates and analyzed by flowcytometry (FACSCalibur, BD Biosciences).

Quantitative real-time RT-PCRHeLa and SW756 cells were stimulated for 2 hours if not

indicated otherwise. cDNA synthesis, qRT-PCR, and normalization

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to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) wereperformed as described previously (17, 25, 28). The 72-bpfragment of IRF1 was detected with primers listed in Supplemen-tary Table S3 and probe no. 36 (Roche Universal Probe Library;Roche).

Statistical analysisTo evaluate the statistical differences between analyzed groups,

two-sided t test was applied. Significances are indicated by aster-isks (�, P < 0.05; ��, P < 0.01; ���, P < 0.001). Correlations ofpTyr705-STAT3 IRS with IRF1 IRS or with FIGO stages of SCCswere investigated using Spearman rank correlation.

ResultsDecline of STAT3 tyrosine-phosphorylation during malignantprogression in human cervical carcinogenesis in situ

In tissue sections from the different stages of human cervicalcarcinogenesis, we found activated STAT3 throughout all pre-malignant stages, however with different intensities. In low-grade CIN, staining was mostly confined to the cells of thesuprabasal layers (Fig. 1A). In contrast, in CIN3 pTyr705-STAT3staining was detected in all epithelial layers and was strongestwhen a stromal inflammatory STAT3-positive infiltrate waspresent (Fig. 1A; ref. 10). The pTyr705-STAT3 staining scorewas significantly higher in CIN3 than in CIN1 or CIN2 (Fig. 1Aand C; P < 0.0001), also in biopsies where both, low- and high-grade CIN, were present (Fig. 1D).

Unexpectedly, 49 of 94 (52.1%) SCCs displayed only weakpTyr705-STAT3 staining, 39 of 94 (41.5%) were rated negative,

and 6 of 94 (6.4%) showed a moderate IRS (Fig. 1C). PositivepTyr705-STAT3 staining in SCCs was preferentially detected atthe tumor borders adjacent to stroma. The pTyr705-STAT3 IRSdid not correlate with FIGO stage (Supplementary Fig. S1). Innegative SCCs, proper staining was ascertained by positivelystained stromal infiltrating or endothelial cells. Notably, epi-thelial pTyr705-STAT3 staining was significantly weaker inSCCs than CIN3 (Fig. 1B and C; P < 0.0001) and this was alsoobserved, when both SCC and CIN3 were present in the samebiopsy (Fig. 1D).

These results demonstrated that STAT3 is activated in humancervical epithelium during carcinogenesis peaking in CIN3. How-ever, during progression from CIN3 to invasive cancer, STAT3activation significantly declines or is lost.

OSM and IL6/sgp80 signaling activate STAT3 in cervicalcarcinoma cells

The same pTyr705-STAT3–specific Ab was used to analyzeSTAT3 phosphorylation in the established cervical SCC cell linesSW756 and SiHa, the adenocarcinoma cell lineHeLa, as well as inthe more recently generated cervical cancer cell lines 808 and 778(22, 23). In medium controls, only faint constitutive STAT3activation was observed. Stimulation with OSM or IL6/sgp80,led to a pronounced STAT3 phosphorylation in cervical cancercells (Fig. 2), whichwas generally stronger than inNECK as shownin prolonged exposures of the Western blots (Supplementary Fig.S2). Thus, constitutive cell-autonomous STAT3 phosphorylationin cervical cancer cells is low or absent but strongly inducible byIL6-type cytokine signaling.

Figure 1.

pTyr705-STAT3 expression in CIN1-3 and cervical SCCs. Human FFPE-sections were stained with anti-pTyr705-STAT3 Ab (brown). A, biopsy containing CIN1, CIN2,and CIN3. B, biopsy containing CIN3 and SCC (all �200). Bars, 100 mm. Asterisks in B indicate SCC. C, IRS of pTyr705-STAT3 staining in CIN1/2 (n ¼ 22),CIN3 (n ¼ 29), and SCC (n ¼ 94). Mean, blue line. D, IRS of pTyr705-STAT3 staining in a subgroup of biopsies containing two stages of cervical carcinogenesis(linked by lines), CIN1/2 and CIN3 (n ¼ 8), or CIN3 and SCC (n ¼ 19); statistical analysis: two-sided t test. �� , P < 0.01; ��� , P < 0.001.

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OSM and IL6/sgp80 signaling sensitize cervical cancer cells tochemotherapeutic drug–induced cell death

As STAT3 protects various cell types from cell death (6, 29), wewere interested whether OSM or IL6 trans-signaling influencedcell death induction by chemotherapeutic drugs in cervical cancercells. Initially, HeLa cells were prestimulated with OSM fordifferent time intervals, whichdidnot significantly change cellularviability, and subsequently treated with cisplatin, the most com-mon drug used in cervical cancer chemotherapy. Unexpectedly,OSM preactivation did not prevent but enhanced cisplatin-mediated cell death in HeLa cells as judged by combinedAnnexin-V/PI flow cytometry (Fig. 3A). Two hours of OSM pre-treatment were sufficient for significant sensitization to cisplatin-induced cell death (P < 0.0001), which was further increased by15% (P ¼ 0.001) after 8 hours of pretreatment (Fig. 3B).

We therefore used an 8-hour cytokine pretreatment schedulefor our series of cervical cancer cell lines. StimulationwithOSMorIL6/sgp80 alone neither altered cellular viability (Fig. 3 and 4) norproliferation (Supplementary Fig. S3) under any condition tested.Cisplatin killed the individual cell lines at different concentra-tions. Notably, as a uniform response in all tested cancer cell lines,we observed a significant increase in cisplatin-induced cell deathafter pretreatment with IL6/sgp80 (26%–54% increase for thehighest cisplatin dose, P values from 0.0002 to 0.0079) or OSM(19%–52% increase, P values 0.0003–0.0038; Fig. 4A). Similarobservationsweremade for etoposide, another chemotherapeuticdrug in clinical use for cervical cancer treatment (30). Etoposide-induced cell death increased by 64% or 55% in HeLa (P values0.0001 or 0.003) and 72% or 69% (P values 0.0001 or 0.0004) inSW756 after pretreatment with OSM or IL6/sgp80, respectively(Fig. 4B). In contrast, NECK were not significantly sensitized(P > 0.3917) to either of the chemotherapeutic drugs by IL6/sgp80 or OSM (Fig. 4A and B).

Cervical cancer cells produce IL6 but their autocrine IL6response is limited due to low expression of the receptor gp80(15, 19). To restore autocrine signaling, HeLa and SW756 cellswere pretreated with sgp80 alone without exogenous IL6. Nota-

bly, sgp80 was sufficient to strongly sensitize both cell lines tocisplatin- (Fig. 4C; P¼ 0.021 or 0.0015) or etoposide-induced celldeath (Fig. 4D; P < 0.0001).

These data demonstrated that reconstitution of autocrine IL6signaling, IL6 trans-signaling, orOSM can sensitize cervical cancercells but not normal human exocervical keratinocytes for chemo-therapeutic drugs.

STAT3 mediates sensitization for cell death by OSM and IL6/sgp80 signaling in cervical cancer cells

To investigate the involvement of STAT3 in chemosensitiza-tion in cervical cancer cells, STAT3-specific siRNA was used toknockdown STAT3 expression in HeLa (Fig. 5A, C, and D) andSW756 cells (Fig. 5B, E, and F). In both cell lines, STAT3knockdown significantly reverted OSM- or IL6/sgp80-mediatedsensitization to cell death induced by cisplatin (reversion up to97%; P > 0.0021; Fig. 5C and E) or etoposide (reversion up to96%, P > 0.0012; Fig. 5D and F), while control siRNA did not.Similar results were obtained with an independent STAT3siRNA (Supplementary Fig. S4).

To further substantiate this finding, HeLa cells were transientlytransfectedwith a dominant-negative version of STAT3 interferingwith phosphorylation at Tyr705 (dnSTAT3-Y705F, STAT3F), trea-ted with OSM, and subsequently challenged with etoposide.Transfected cells were visualized by EGFP coexpression. STAT3Foverexpression completely restored cellular viability furtherunderlining that STAT3 activation is required for cell deathsensitization (Supplementary Fig. S5).

These data provided evidence that chemosensitization byIL6-type cytokines depends on the STAT3 pathway in cervicalcancer cells.

OSM and IL6/sgp80 signaling in cervical cancer cells stronglyinduce IRF1 in a STAT3-dependent manner

To further elucidate the molecular mechanism underlying celldeath sensitization, we performed mRNA gene expression anal-ysis in SW756 cells after OSM or IL6/sgp80 stimulation. Strong

Figure 2.

OSM or IL6/sgp80 strongly activate STAT3 incervical cancer cells. HeLa, SW756, SiHa, 808,788 cells, and NECK were stimulated with medium,OSM, or IL6/sgp80 for 15 minutes. Whole cellextracts were analyzed by Western blot analysisusing anti-pTyr705-STAT3-, STAT3-, orb-actin–specific Ab.

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upregulation of CCL2 confirmed our previously published results(15) and validated the assay (data not shown). Besides CCL2, thetranscription factor IRF1, which has proapoptotic activities (31)was significantly upregulated after IL6/sgp80orOSMstimulation.qRT-PCR revealed a rapid (within 2 hours) and strong (10- to 12-fold) induction of IRF1 mRNA after OSM or IL6/sgp80 stimula-tion in HeLa and SW756 cells (Fig. 6A; P <0.0001). IRF1 proteininduction after 2 hours of IL6/sgp80 or OSM stimulation wasdemonstrated in nuclear extracts of both cervical cancer cell lines(Fig. 6B) but not in NECK (Supplementary Fig. S6).

To verify nuclear IRF1 expression in vivo, IHC staining wasperformed in a subset (n ¼ 79) of cervical cancers (Fig. 6C) andcompared with pTyr705-STAT3 staining results. Notably, the IRSof both factors, pTyr705-STAT3 and nuclear IRF1, significantlycorrelated with each other (Fig. 6D; r¼0.3984, P¼ 0.0003). Theseresults indicated that both factors might be functionally linked toeach other and prompted us to analyze a causal role of STAT3 forIRF1 induction. Indeed, as shown with both, HeLa and SW756cells, STAT3 knockdown significantly suppressed OSM-inducedIRF1 induction (Fig. 6E).

These data provided evidence for a STAT3-dependent mecha-nism of IRF1 induction in cervical cancer cells.

IRF1 mediates sensitization for cell death by OSM and IL6/sgp80 signaling in cervical cancer cells

To investigate a direct impact on cell death sensitization ofcervical cancer cells, IRF1 or its functional antagonist IRF2 were

transiently overexpressed as confirmed by Western blot analysis(Fig. 7A and B). Neither IRF1, IRF2 (in the absence or presence ofOSM), nor empty expression vector alone significantly alteredcellular viability (Supplementary Fig. S7). However, IRF1 over-expression was sufficient to sensitize the cancer cells to etoposide-mediated cell death in a dose-dependent manner (Fig. 7A).Correspondingly, IRF2 significantly interfered with OSM-medi-ated sensitization for etoposide-mediated cell death (Fig. 7B).Knockdown of endogenous IRF1 with IRF1-specific siRNA inHeLa (Fig. 7C and D) and SW756 cells (Fig. 7E and F) stronglysuppressed OSM-mediated IRF1 protein induction (92% or78.5%; Fig. 7C and E) and significantly reverted sensitization toetoposide- or cisplatin-induced cell death in both cell lines (Fig.7D and F). Similar results were obtained with a second indepen-dent IRF1-specific siRNA (Supplementary Fig. S8).

IRF1 expression is associated with response to radio/chemotherapy in cervical cancer patients

We then evaluated IRF1 expression in human cervical cancerbiopsies prior to neoadjuvant radio/chemotherapy in relation tothe patient's individual response to therapy (SupplementaryTable S1). Patients with complete response to radio/chemother-apy displayed significantly higher scores of pretherapeutic nuclearIRF1 expression in neoplastic cells than patients with only partialresponse to therapy (Fig. 7G, left; P ¼ 0.0378). This was alsoobserved when total IRF1 expression (nuclear and cytoplasmic)was evaluated (Fig. 7G, right; P ¼ 0.0033).

Figure 3.

OSM sensitizes HeLa cells for cisplatin-induced cell death in a time-dependent manner. A, HeLa cells were prestimulated with medium or OSM for 8 hoursand treated with 25 mg/mL cisplatin for further 12 hours. Cells were stained with PI and Annexin-V and analyzed by flow cytometry. Left, one experiment out of n¼ 3;right, three experiments. B, HeLa cells were prestimulated with medium (squares, black line) or OSM (circles, gray line) for 2, 4, or 8 hours and then treatedwith medium (open symbols) or 1.56 mg/mL cisplatin (filled symbols) for 48 hours. Cell viability was assessed by the neutral red uptake method, n ¼ 2, performedin triplicates; statistical analysis: two-sided t test. � , P < 0.05; ��� , P < 0.001. n.s., nonsignificant.

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Figure 4.

OSM or IL6/sgp80 sensitize cervical cancer cells to chemotherapeutic drugs. A, HeLa, SW756, SiHa, 808, 778 cells, and NECK were prestimulated withmedium (black squares or bars), OSM (dark gray circles or bars), or IL6/sgp80 (light gray triangles or bars) and then treated with cisplatin at the indicatedconcentrations. B, HeLa, SW756 cells, and NECK were stimulated as above and treated with etoposide. C and D, HeLa and SW756 cells were prestimulated withmedium (black squares or bars) or sgp80 (gray triangles or bars) and treated with cisplatin (C) or etoposide (D). Cell viability was assessed by the neutral reduptake method. Shown is one experiment out of n ¼ 3 performed in triplicates. Histograms summarize n ¼ 3 for cells treated with medium or the highestchemotherapeutic drug concentration; statistical analysis: two-sided t test. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

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Figure 5.

STAT3 mediates OSM- or IL6/sgp80-induced cell death sensitization. HeLa (A) or SW756 (B) cells were transfected with 10 pmol/1.5 � 105 cells of humanSTAT3-specific siRNA or mock siRNA. Cells were stimulated with OSM for 15 minutes. Whole-cell extracts were analyzed by Western blot analysis usinganti-pTyr705-STAT3-, STAT3-, or b-actin–specific Ab. Shown is one experiment out of n¼ 3. Diagram summarizes three experiments. Expression of the respectivecontrols was set at 100%. HeLa (C and D) or SW756 (E and F) cells were transfected as in A, prestimulated with OSM or IL6/sgp80, and then treated withtwo concentrations of cisplatin (C and E) or etoposide (D and F), respectively. Cell viability was assessed by the neutral red uptake method. Shown is n ¼ 3performed in triplicates; statistical analysis: two-sided t test. � , P < 0.05; �� , P < 0.01; ���, P < 0.001.

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These data identified IRF1as a key regulator of chemosensitivityin cervical cancer cells and demonstrate a clear associationbetween pretreatment IRF1 expression and response to radio/chemotherapy in vivo.

DiscussionCervical cancer therapy is still a major clinical challenge, as

patients substantially differ in their response to standard treat-ments including platinum-based radio/chemotherapy. Clinically,

Figure 6.

IRF1 is induced by OSM or IL6/sgp80 in cervical cancer cells in a STAT3-dependent manner. A, HeLa and SW756 cells were stimulated with medium, OSM,or IL6/sgp80. After 2, 4, and 8 hours, RNA was isolated and IRF1-specific mRNA was quantified by qRT-PCR in relation to GAPDH; n ¼ 2 performed in triplicates.B, HeLa and SW756 cells were stimulated as in A for 2 hours. Nuclear IRF1 expression was analyzed by Western blot analysis. C, 79 cervical SCCs werestained with anti-pTyr705-STAT3 or anti-IRF1 Ab. Shown is a representative pSTAT3- and IRF1-positive SCC in the left panel (brown; all�200; bars, 50 mm). IRS ofnuclear staining (right); means, blue lines. D, correlation of both IRS with each other. E, HeLa or SW756 cells were transfected with 10 pmol/1.5 � 105 cells ofhuman STAT3-specific siRNA or mock siRNA and stimulated with medium or OSM for 2 hours. Nuclear extracts were analyzed by Western blot for IRF1expression. Shown is one experiment out of n ¼ 3. Diagram summarizes three experiments; statistical analysis: two-sided t test. ��� , P < 0.001. n.s., nonsignificant.

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Figure 7.

Association between IRF1 expression and chemotherapy response. HeLa cells were transfected with the indicated amounts of pCMV-Flag2-IRF1 (A) orpCMV-Flag2-IRF2 (B). In B, cells were prestimulated after 16 hours with OSM for 2 hours and in A and B treated with serial dilutions of etoposide for 24 hours. HeLa(C and D) and SW756 (E and F) cells were transfected with 6 pmol of human IRF1-specific siRNA or mock siRNA/1.5 � 105 cells and stimulated with OSM.C and E, nuclear extractswere analyzed byWestern blot for IRF1 expression. D and F, transfected cellswere incubatedwith serial dilutions of etoposide or cisplatin. A,B, D, and F, cell viability was assessed by the neutral red uptake method; n ¼ 3 performed in triplicates. G, IRS of nuclear (left) or total (right) IRF1 expressionwas determined in pretherapeutic biopsies of n ¼ 24 cervical SCCs and compared with the individual patient's response (non, partial, or complete response)to neoadjuvant chemotherapy (triangles) or radio/chemotherapy (circles); statistical analysis: two-sided t test. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

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it is highly desirable to identify those patients that may benefitfrom neoadjuvant radio/chemotherapy and to distinguish themfrom putative partial- or nonresponders.

Our study has unraveled a key role of the STAT3-dependentIRF1 pathway for chemosensitization. This was unexpected, asSTAT3 is considered as a survival factor in various other cancertypes. IRF1 was inducible by IL6-type cytokines via STAT3 acti-vation in cervical cancer cells and in vivo, nuclear IRF1 and pSTAT3expression scores significantly correlated with each other. Impor-tantly, our data show that cervical cancer patientswith higher IRF1expression scores responded significantly better to radio/chemo-therapy. This may have major implications for personalization ofcervical cancer chemotherapy.

Our study has unraveled novel findings on the activation andfunction of the transcription factor STAT3 in cervical carcinogen-esis. Comparing different human CIN stages, we found thatSTAT3-tyrosine-phosphorylation dramatically increased fromlow-grade CIN1 to preinvasive CIN3 lesions. This was in linewith observations in various murine tumor models (32, 33) andsupports the notion that STAT3 activationhas an important role atprecancerous stages of cervical carcinogenesis. Unexpectedly, inbiopsies comprising both CIN3 and SCC, we observed a strongdecline of STAT3 activation during progression to invasive cancer.The pTyr705-STAT3 staining score was weak in more than half ornegative in more than 40% of invasive cervical cancers. Positivestaining wasmostly confined to the tumor borders adjacent to thestroma, suggesting a paracrine mode of STAT3 activation. In linewith our results, another study found pTyr705-STAT3 in less than10% of cells in most cervical cancer cases (34) and two previousstudies detected pTyr705-STAT3 (35) or stronger nuclear STAT3staining (36, 37) in 22%–24%of cervical cancers. Only one groupdetected higher percentages of STAT3-phosphorylation in cervicalcancers in an Indian population (38). Whether the results of thelatter study are due to ethnical, environmental, or technical issuesremains to be determined. Higher FIGO stages in Indian patientscan probably be excluded as the basis for these differences, as wedid not observe a correlation between pTyr705-STAT3 IRS andFIGO stage. To our knowledge, our study is the first report on thedirect comparison of precancerous and invasive cervical cancerlesions within the same clinical specimen and the first to dem-onstrate a strong decline of STAT3 activation during progressionto malignancy. Whether this particular reaction pattern is aconsequence of the viral etiology of cervical carcinogenesis willbe of high interest for future research. Fromour data, we concludethat inmost cervical cancers STAT3 activation is balanced at a lowlevel. A high level of STAT3 activation is obviously not required forthe malignant cells and may be counter selected during progres-sion to SCC. In fact, our attempts to overexpress a constitutivelyactive formof STAT3 in cervical cancer cells failed and did not giverise to cell clones (unpublished observation).

STAT3 is generally considered as an antiapoptotic factor and theIL6 signaling pathway can promote resistance to chemotherapeu-tic drugs in different cancer types (6, 7). In contrast, we found thatcervical cancer cells were not protected but sensitized for chemo-therapeutic drugs by IL6-type cytokine-induced STAT3 signalingas shown via STAT3 knockdown or dominant-negative STAT3F(13). In our study we used the alkylating agent cisplatin, thechemotherapeutic drug most commonly used against cervicalcancer. Moreover, a higher sensitivity was also observed for thetopoisomerase II inhibitor etoposide used in recurrent cervicalcancer (30), indicating that the sensitizationwasnot restricted to a

single class of chemotherapeutic drugs. Although the IL6-/STAT3-pathways appear to be attractive pharmacologic targets toimprove the immunemicroenvironment (9, 10, 17, 18), our datasuggest that one should be cautious to combine STAT3 inhibitorswith cisplatin or etoposide in cervical cancer patients.

In search of the mechanism underlying IL6-type cytokine-induced STAT3-dependent chemosensitization, we identifiedIRF1, a proapoptotic factor, antioncogene, and regulator of autop-hagy (39–42). Notably, there are complex interactions betweenHPV and IRFs. IRF1 and 2 can activate the HPV16 oncogenepromoter (43, 44) and the E5 oncoprotein induces IRF1 (45). Incontrast, the E7 oncoprotein can suppress IFNg-induced IRF1expression and interferes with the effectiveness of the immuneresponse (46, 47). Our data clearly show that IL6/sgp80- or OSM-induced STAT3 signaling sensitizes HPV-positive cervical cancercells for chemotherapeutic drugs via IRF1 upregulation. Thissuggests that STAT3 activation may override IRF1 inhibition byHPV. HPV transformation may be necessary in keratinocytes forthe unusual STAT3 effect mediating IRF1 induction but it isapparently not essential in other cell types. It was observed beforein murine myeloid leukemic M1 cells, where STAT3/IRF1 signal-ing is associated with growth arrest and differentiation (13, 48,49). The consequences of STAT3/IRF1 pathway activation incervical cancer cells in the absence of cancer therapy are undercurrent investigation in our laboratory.

As IRF1 was sufficient to render cervical cancer cell lines moresusceptible to chemotherapeutic drugs, we were interested in thein vivo expression of IRF1 in human cervical cancer. In patientsamples, nuclear IRF1 correlated significantly with pTyr705-STAT3 activation. Thiswas interesting, as IRF1 can also be inducedby pathways other than STAT3 (39). Clinically most important,we detected the highest IRF1 pretreatment expression scores incancers of those patients who responded completely to neoadju-vant or radio/chemotherapy. In contrast, partial- or nonrespon-ders displayed significantly lower IRF1 expression scores. Theresults were similar, irrespective of whether nuclear or total IRF1expression in the neoplastic cells were taken into account. This isplausible, as higher basal IRF1 expression levels, which may bedetermined at the genetic level (50), can also contribute to astronger IRF1 activation during the course of chemotherapy.

In summary, our data provide novel mechanistic insight on thecontributory role of STAT3 in inducing IRF1 and thereby chemo-sensitization of cervical cancer cells. On the basis of these results,pretreatment IRF1 expression should be evaluated as a predictivebiomarker for the individual response of cervical cancer patients toneoadjuvant radio/chemotherapy in prospective clinical studies.

Disclosure of Potential Conflicts of InterestP. Stern has received speakers bureau honoraria from GlaxoSmithKline. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: B. Walch-R€uckheim, S. SmolaDevelopment of methodology: B. Walch-R€uckheim, J. Fischbach, C. Wicken-hauser, R. B€uttner, S. SmolaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Walch-R€uckheim, J. Pahne-Zeppenfeld, C. Wick-enhauser, L.C.Horn, L. Tharun, R. B€uttner, P.Mallmann, P. Stern, Y.-J. Kim, R.M.Bohle, C. R€ube, R. Str€oder, I. Juhasz-B€oss, E.-F. Solomayer, S. SmolaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Walch-R€uckheim, J. Pahne-Zeppenfeld, C. Wick-enhauser, L.C. Horn, L. Tharun, Y.-J. Kim, S. Smola

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STAT3/IRF1 Sensitizes Cervical Cancer to Chemotherapy

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Writing, review, and/or revision of the manuscript: B. Walch-R€uckheim,J. Pahne-Zeppenfeld, L.C. Horn, R. B€uttner, P. Stern, Y.-J. Kim, I. Juhasz-B€oss,E.-F. Solomayer, S. SmolaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Walch-R€uckheim, P. Mallmann, R.M. Bohle,C. R€ube, R. Str€oder, E.-F. Solomayer, S. SmolaStudy supervision: B. Walch-R€uckheim, E.-F. Solomayer, S. Smola

AcknowledgmentsThe authors thank U. Sandaradura de Silva, T. T€anzer, and B. Glombitza

for excellent technical assistance, Drs. G.S. Stein, T. Hirano, M. Hibi, and

K. Nakajima for generously providing cDNA constructs, and Drs. R. Bals andC. Herr for help with slide scanning.

Grant SupportThis work was supported by a grant from the Deutsche Krebshilfe (grant no.

109752) and the Saarland Staatskanzlei to S. Smola.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, 2014; revised March 18, 2016; accepted March 21, 2016;published OnlineFirst May 23, 2016.

References1. zurHausenH. Papillomaviruses in the causation of human cancers - a brief

historical account. Virology 2009;384:260–5.2. Benedetti Panici P, Palaia I, Marchetti C, Ruscito I, Fischetti M, Musella

A, et al. Dose-dense neoadjuvant chemotherapy plus radical surgery inlocally advanced cervical cancer: a phase II study. Oncology 2015;89:103–10.

3. Schreuder SM, Lensing R, Stoker J, Bipat S. Monitoring treatment responsein patients undergoing chemoradiotherapy for locally advanced uterinecervical cancer by additional diffusion-weighted imaging: a systematicreview. J Magn Reson Imaging 2015;42:572–94.

4. Maduro JH, Pras E, Willemse PH, de Vries EG. Acute and long-term toxicityfollowing radiotherapy alone or in combination with chemotherapy forlocally advanced cervical cancer. Cancer Treat Rev 2003;29:471–88.

5. Tan FH, Putoczki TL, Stylli SS, Luwor RB. The role of STAT3 signaling inmediating tumor resistance to cancer therapy. Curr Drug Targets 2014;15:1341–53.

6. Sansone P, Bromberg J. Targeting the interleukin-6/Jak/stat pathway inhuman malignancies. J Clin Oncol 2012;30:1005–14.

7. Ara T, Nakata R, Sheard MA, Shimada H, Buettner R, Groshen SG, et al.Critical role of STAT3 in IL-6-mediated drug resistance in human neuro-blastoma. Cancer Res 2013;73:3852–64.

8. Yang H, Yamazaki T, Pietrocola F, Zhou H, Zitvogel L, Ma Y, et al. STAT3Inhibition enhances the therapeutic efficacy of immunogenic chemother-apy by stimulating type 1 interferon production by cancer cells. Cancer Res2015;75:3812–22.

9. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immunecells: role of STAT3 in the tumour microenvironment. Nat Rev Immunol2007;7:41–51.

10. Schroer N, Pahne J, Walch B, Wickenhauser C, Smola S. Molecular patho-biology of human cervical high-grade lesions: paracrine STAT3 activationin tumor-instructed myeloid cells drives local MMP-9 expression. CancerRes 2011;71:87–97.

11. Kishimoto T. INTERLEUKIN-6: from basic science to medicine-40 years inimmunology. Annu Rev Immunol 2005;23:1–21.

12. Taniguchi K, Karin M. IL-6 and related cytokines as the critical lynchpinsbetween inflammation and cancer. Semin Immunol 2014;26:54–74.

13. Nakajima K, Yamanaka Y, Nakae K, Kojima H, Ichiba M, Kiuchi N, et al. Acentral role for Stat3 in IL-6-induced regulation of growth and differen-tiation in M1 leukemia cells. EMBO J 1996;15:3651–8.

14. Peters M, Muller AM, Rose-John S. Interleukin-6 and soluble interleukin-6receptor: direct stimulation of gp130 and hematopoiesis. Blood 1998;92:3495–504.

15. Hess S, SmolaH, SandaraduraDe SilvaU,HadaschikD, KubeD, Baldus SE,et al. Loss of IL-6 receptor expression in cervical carcinoma cells inhibitsautocrine IL-6 stimulation: abrogation of constitutive monocyte chemoat-tractant protein-1 production. J Immunol 2000;165:1939–48.

16. Srivani R, Nagarajan B. A prognostic insight on in vivo expression ofinterleukin-6 in uterine cervical cancer. Int J Gynecol Cancer 2003;13:331–9.

17. Pahne-Zeppenfeld J, Schroer N, Walch-Ruckheim B, Oldak M, Gorter A,Hegde S, et al. Cervical cancer cell-derived interleukin-6 impairs CCR7-dependent migration of MMP-9-expressing dendritic cells. Int J Cancer2014;134:2061–73.

18. Walch-Ruckheim B, Mavrova R, Henning M, Vicinus B, Kim YJ, Bohle RM,et al. Stromal fibroblasts induce CCL20 through IL6/C/EBPbeta to support

the recruitment of Th17 Cells during cervical cancer progression. CancerRes 2015;75:5248–59.

19. Bauknecht T, Randelzhofer B, Schmitt B, Ban Z, Hernando JJ, Bauknecht T.Response to IL-6 of HPV-18 cervical carcinoma cell lines. Virology1999;258:344–54.

20. Schmidt SV, Seibert S, Walch-Ruckheim B, Vicinus B, Kamionka EM,Pahne-Zeppenfeld J, et al. RIPK3 expression in cervical cancer cells isrequired for PolyIC-induced necroptosis, IL-1alpha release, and efficientparacrine dendritic cell activation. Oncotarget 2015;6:8635–47.

21. Remmele W, Stegner HE. [Recommendation for uniform definition of animmunoreactive score (IRS) for immunohistochemical estrogen receptordetection (ER-ICA) in breast cancer tissue]. Pathologe 1987;8:138–40.

22. Brady CS, Bartholomew JS, Burt DJ, Duggan-Keen MF, Glenville S, TelfordN, et al. Multiple mechanisms underlie HLA dysregulation in cervicalcancer. Tissue Antigens 2000;55:401–11.

23. SteenbergenRD, KramerD, Braakhuis BJ, Stern PL, Verheijen RH,Meijer CJ,et al. TSLC1 gene silencing in cervical cancer cell lines and cervicalneoplasia. J Natl Cancer Inst 2004;96:294–305.

24. Smola H, Thiekotter G, Fusenig NE. Mutual induction of growth factor geneexpression by epidermal-dermal cell interaction. J Cell Biol 1993;122:417–29.

25. Sperling T, Oldak M, Walch-Ruckheim B, Wickenhauser C, Doorbar J,Pfister H, et al. Human papillomavirus type 8 interferes with a novel C/EBPbeta-mediated mechanism of keratinocyte CCL20 chemokine expres-sion and Langerhans cell migration. PLoS Pathog 2012;8:e1002833.

26. Vaughan PS, Aziz F, van Wijnen AJ, Wu S, Harada H, Taniguchi T, et al.Activation of a cell-cycle-regulated histone gene by the oncogenic tran-scription factor IRF-2. Nature 1995;377:362–5.

27. Hess S, EngelmannH. A novel function of CD40: induction of cell death intransformed cells. J Exp Med 1996;183:159–67.

28. Oldak M, Tolzmann L, Wnorowski A, Podgorska MJ, Silling S, Lin R, et al.Differential regulation of human papillomavirus type 8 by interferonregulatory factors 3 and 7. J Virol 2011;85:178–88.

29. Borsellino N, Belldegrun A, Bonavida B. Endogenous interleukin 6 is aresistance factor for cis-diamminedichloroplatinum and etoposide-medi-ated cytotoxicity of human prostate carcinoma cell lines. Cancer Res1995;55:4633–9.

30. WatanabeY,HoshiaiH,Nakanishi T, KawamuraN, TanakaN, Isaka K, et al.Evaluation of oral etoposide in combination with cisplatin for patientswith recurrent cervical cancer: long-term follow-up results of a Japanesemulticenter study. Anticancer Res 2011;31:3063–7.

31. Kim PK, Armstrong M, Liu Y, Yan P, Bucher B, Zuckerbraun BS, et al. IRF-1expression induces apoptosis and inhibits tumor growth in mouse mam-mary cancer cells invitro and invivo. Oncogene 2004;23:1125–35.

32. Chan KS, Sano S, Kiguchi K, Anders J, Komazawa N, Takeda J, et al.Disruption of Stat3 reveals a critical role in both the initiation and thepromotion stages of epithelial carcinogenesis. J Clin Invest 2004;114:720–8.

33. De Andrea M, Ritta M, Landini MM, Borgogna C, Mondini M, Kern F, et al.Keratinocyte-specific stat3 heterozygosity impairs development of skintumors in human papillomavirus 8 transgenic mice. Cancer Res 2010;70:7938–48.

34. Takemoto S, Ushijima K, Kawano K, Yamaguchi T, Terada A, Fujiyoshi N,et al. Expression of activated signal transducer and activator of transcrip-tion-3 predicts poor prognosis in cervical squamous-cell carcinoma. Br JCancer 2009;101:967–72.

Walch-R€uckheim et al.

Cancer Res; 76(13) July 1, 2016 Cancer Research3882

on September 29, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 23, 2016; DOI: 10.1158/0008-5472.CAN-14-1306

35. Chen CL, Hsieh FC, Lieblein JC, Brown J, Chan C, Wallace JA, et al. Stat3activation in human endometrial and cervical cancers. Br J Cancer 2007;96:591–9.

36. Chen HH, Chou CY, Wu YH, Hsueh WT, Hsu CH, Guo HR, et al.Constitutive STAT5 activation correlates with better survival in cervicalcancer patients treated with radiation therapy. Int J Radiat Oncol Biol Phys2012;82:658–66.

37. Yang SF, Yuan SS, Yeh YT, Hung SC, Wu MT, Su JH, et al. Positiveassociation between STAT3 and Ki-67 in cervical intraepithelial neoplasia.Kaohsiung J Med Sci 2006;22:539–46.

38. Shukla S, Shishodia G, Mahata S, Hedau S, Pandey A, Bhambhani S, et al.Aberrant expression and constitutive activation of STAT3 in cervical car-cinogenesis: implications in high-risk human papillomavirus infection.Mol Cancer 2010;9:282.

39. Tamura T, Yanai H, Savitsky D, Taniguchi T. The IRF family transcriptionfactors in immunity and oncogenesis. Annu Rev Immunol 2008;26:535–84.

40. Bouker KB, Skaar TC, Riggins RB, Harburger DS, Fernandez DR, Zwart A,et al. Interferon regulatory factor-1 (IRF-1) exhibits tumor suppressoractivities in breast cancer associated with caspase activation and inductionof apoptosis. Carcinogenesis 2005;26:1527–35.

41. Rettino A, Clarke NM. Genome-wide identification of IRF1 binding sitesreveals extensive occupancy at cell death associated genes. J CarcinogMutagen 2013;S6–009. doi: 10.4172/2157-2518.S6-009.

42. Schwartz-Roberts JL, Cook KL, Chen C, Shajahan-Haq AN, Axelrod M,Warri A, et al. Interferon regulatory factor-1 signaling regulates the switchbetween autophagy and apoptosis to determine breast cancer cell fate.Cancer Res 2015;75:1046–55.

43. LaceMJ, Anson JR, Klingelhutz AJ, HaradaH, Taniguchi T, Bossler AD, et al.Interferon-beta treatment increases human papillomavirus early genetranscription and viral plasmid genome replication by activating interferonregulatory factor (IRF)-1. Carcinogenesis 2009;30:1336–44.

44. LaceMJ, Anson JR,Haugen TH, Turek LP. Interferon regulatory factor (IRF)-2 activates the HPV-16 E6-E7 promoter in keratinocytes. Virology2010;399:270–9.

45. Muto V, Stellacci E, Lamberti AG, Perrotti E, Carrabba A, Matera G, et al.Human papillomavirus type 16 E5 protein induces expression of betainterferon through interferon regulatory factor 1 in human keratinocytes.J Virol 2011;85:5070–80.

46. Zhou F, Chen J, Zhao KN. Human papillomavirus 16-encoded E7 proteininhibits IFN-gamma-mediated MHC class I antigen presentation and CTL-induced lysis by blocking IRF-1 expression in mouse keratinocytes. J GenVirol 2013;94:2504–14.

47. Um SJ, Rhyu JW, Kim EJ, Jeon KC, Hwang ES, Park JS. Abrogation of IRF-1response byhigh-riskHPVE7protein invivo. Cancer Lett 2002;179:205–12.

48. Minami M, Inoue M, Wei S, Takeda K, Matsumoto M, Kishimoto T, et al.STAT3 activation is a critical step in gp130-mediated terminal differenti-ation and growth arrest of a myeloid cell line. Proc Natl Acad Sci U S A1996;93:3963–6.

49. Yoshida T, Iwamoto T, Adachi K, Yokota T, Miyake Y, Hamaguchi M.Functional analysis of the effect of forced activation of STAT3 onM1mouseleukemia cells. Int J Mol Med 2005;15:269–75.

50. Cavalli LR, Riggins RB, Wang A, Clarke R, Haddad BR. Frequent loss ofheterozygosity at the interferon regulatory factor-1 gene locus in breastcancer. Breast Cancer Res Treat 2010;121:227–31.

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2016;76:3872-3883. Published OnlineFirst May 23, 2016.Cancer Res   Barbara Walch-Rückheim, Jennifer Pahne-Zeppenfeld, Jil Fischbach, et al.   Chemotherapeutic DrugsSTAT3/IRF1 Pathway Activation Sensitizes Cervical Cancer Cells to

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