STAT3/IRF1 pathway activation sensitizes cervical cancer cells to … · pre-treatment cervical...

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1 STAT3/IRF1 pathway activation sensitizes cervical cancer cells to chemotherapeutic drugs Barbara Walch-Rückheim 1+ , Jennifer Pahne-Zeppenfeld 2+ , Jil Fischbach 1 , Claudia Wickenhauser 3 , Lars Christian Horn 4 , Lars Tharun 5 , Reinhard Büttner 5 , Peter Mallmann 6 Peter Stern 7 , Yoo-Jin Kim 8 , Rainer Maria Bohle 8 , Christian Rübe 9 , Russalina Ströder 10 , Ingolf Juhasz-Böss 10 , Erich-Franz Solomayer 10 and Sigrun Smola 1 * 1 Institute of Virology, Saarland University, Homburg/Saar, Germany 2 Center for Molecular Medicine Cologne and Institute of Virology, University of Cologne, Germany 3 Institute of Pathology, University of Halle, Germany 4 Institute of Pathology, University of Leipzig, Germany 5 Institute of Pathology, University of Cologne, Germany 6 Department of Gynecology and Obstetrics, University of Cologne, Germany 7 Institute of Cancer Sciences, University of Manchester, UK 8 Institute of Pathology, Saarland University, Homburg/Saar, Germany 9 Department of Radiotherapy and Radiation Oncology, Saarland University, Homburg/Saar, Germany. 10 Department of Gynecology and Obstetrics, Saarland University, Homburg/Saar, Germany + shared first authorship Running title STAT3/IRF1 sensitizes cervical cancer for chemotherapy Research. on August 22, 2020. © 2016 American Association for Cancer cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 23, 2016; DOI: 10.1158/0008-5472.CAN-14-1306

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STAT3/IRF1 pathway activation sensitizes cervical cancer cells to

chemotherapeutic drugs

Barbara Walch-Rückheim1+, Jennifer Pahne-Zeppenfeld2+, Jil Fischbach1, Claudia

Wickenhauser3, Lars Christian Horn4, Lars Tharun5, Reinhard Büttner5, Peter Mallmann6

Peter Stern7, Yoo-Jin Kim8, Rainer Maria Bohle8, Christian Rübe9, Russalina Ströder10,

Ingolf Juhasz-Böss10, Erich-Franz Solomayer10 and Sigrun Smola1*

1Institute of Virology, Saarland University, Homburg/Saar, Germany

2Center for Molecular Medicine Cologne and Institute of Virology, University of Cologne,

Germany

3Institute of Pathology, University of Halle, Germany

4Institute of Pathology, University of Leipzig, Germany

5Institute of Pathology, University of Cologne, Germany

6Department of Gynecology and Obstetrics, University of Cologne, Germany

7Institute of Cancer Sciences, University of Manchester, UK

8Institute of Pathology, Saarland University, Homburg/Saar, Germany

9Department of Radiotherapy and Radiation Oncology, Saarland University,

Homburg/Saar, Germany.

10Department of Gynecology and Obstetrics, Saarland University, Homburg/Saar,

Germany

+shared first authorship

Running title

STAT3/IRF1 sensitizes cervical cancer for chemotherapy

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Key words

Cervical carcinogenesis, Human papillomavirus, STAT3, IRF1, radio/chemotherapy

Financial support

This work was supported by a grant of the Deutsche Krebshilfe (grant no. 109752) and

the Saarland Staatskanzlei to S. Smola.

*Corresponding author

Sigrun Smola

Institute of Virology, Saarland University

Kirrbergerstrasse, Building 47

D-66421 Homburg/Saar, Germany

Phone: ++49-6841-1623931

Fax: ++49-6841-1623980

E-mail: [email protected]

The authors have no conflict of interest.

Research article

Word count: 5000

Total number of figures and tables: 7

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Abstract

Neoadjuvant radio/chemotherapy regimens can markedly improve cervical cancer

outcome in a subset of patients, while other patients show poor responses but may

encounter severe adverse effects. Thus, there is a strong need for predictive biomarkers

to improve clinical management of cervical cancer patients. STAT3 is considered as a

critical anti-apoptotic factor in various malignancies. We therefore investigated STAT3

activation during cervical carcinogenesis and its impact on the response of cervical

cancer cells to chemotherapeutic drugs. Tyr705-phosphorylated STAT3 increased from

low-grade cervical intraepithelial neoplasia (CIN1) to pre-cancerous CIN3 lesions.

Notably, pTyr705-STAT3 activation significantly declined from CIN3 to invasive cancer,

also when compared in the same clinical biopsy. pTyr705-STAT3 was also low or absent

in cultured human cervical cancer cell lines consistent with the in vivo expression data.

Unexpectedly, IL-6-type cytokine signaling inducing STAT3 activation rendered cervical

cancer cells significantly more susceptible to chemotherapeutic drugs, i.e. cisplatin or

etoposide. This chemosensitization was STAT3-dependent and we identified interferon

regulatory factor-1 (IRF1) as the STAT3-inducible mediator required for cell death

enhancement. In line with these data, pTyr705-STAT3 significantly correlated with

nuclear IRF1 expression in cervical cancer in vivo. Importantly, high IRF1 expression in

pre-treatment cervical cancer biopsy cells was associated with a significantly better

response to neoadjuvant radio/chemotherapy of the patients. In summary, our study has

identified a key role of the STAT3/IRF1 pathway for chemosensitization in cervical

cancer. Our results suggest that pre-therapeutic IRF1 expression should be evaluated

as a novel predictive biomarker for neoadjuvant radio/chemotherapy responses.

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Introduction

Cervical cancer represents the third most common cause of cancer-related death in

women worldwide. Invasive cancer develops from persistent high-risk human

papillomavirus (HPV) infection through well-defined stages of cervical intraepithelial

neoplasia (CIN1-3) (1). This process takes years or decades and it is assumed that

further changes within the (pre)neoplastic cells and their microenvironment critically

influence the course of disease.

Cervical cancer therapy is still a major clinical challenge. Responses to neoadjuvant

radio/chemotherapy vary greatly in patients (2, 3). Intrinsic and acquired resistance of

the neoplastic cells as well as substantial side effects from standard treatment including

platinum-based chemotherapy limit the options for escalation (4). Identification of

patients that may best benefit from chemotherapy would be useful for improved clinical

management. This will require a better understanding of the mechanisms influencing the

balance between sensitivity and resistance to cervical cancer cell death.

The STAT3 transcription factor is commonly considered as a survival or progression

factor in different cancer types. Constitutive STAT3 activation is documented in various

human malignancies including head-and-neck, brain, breast, lung, pancreas as well as

prostate cancer and melanoma (summarized in (5)). STAT3 inhibition can affect tumor

growth and enhance the response of certain tumors to chemotherapy directly or in an

immune-dependent manner (6-8).

Notably, STAT3 activation has also profound direct effects on the immune

microenvironment (9). Our group has previously demonstrated pronounced tyrosine-

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phosphorylation of STAT3 in cervical high-grade lesions (10). Strongly activated STAT3

was detected within the inflammatory infiltrate of the lesions, where it drives expression

of the pro-tumorigenic and vasculogenic matrix-metalloprotease MMP-9, as well as in

the pre-malignant epithelial cells (10).

Major activators of the STAT3-pathway are members of the interleukin-6 (IL-6)-type

cytokine family (11, 12). IL-6 binds to the α-chain of the IL-6R (IL-6Rα, gp80), while the

related cytokine oncostatin M (OSM), binds to the OSM receptor-β (OSMRβ).

Respective complexes then associate with the common receptor chain gp130, which

recruits Janus kinases leading to subsequent STAT3-phosphorylation at tyrosine 705

(13). Whereas gp130 is ubiquitously expressed, IL-6 signaling is limited by the

availability of transmembrane gp80 or its naturally occurring soluble form sgp80, which

induces “trans-signaling” via gp130 (14).

In human squamous cell carcinoma (SCC) of the cervix uteri, IL-6 is strongly up-

regulated in-situ and in-vitro (15). IL-6 has a negative impact on the patient´s prognosis

(16). We have shown that it mainly acts in a paracrine manner and creates a pro-

tumorigenic and immunosuppressive microenvironment (10, 17, 18). Cervical cancer

cells display only low responses to autocrine IL-6 due to low gp80 expression levels (15,

19) but sgp80 can restore autocrine IL-6 signaling and induce pronounced STAT3

binding activity (15).

In this study, we investigated STAT3 activation during the different stages of cervical

carcinogenesis in more detail. In biopsies comprising pre-cancerous and cancerous

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lesions we demonstrate that STAT3 tyrosine-phosphorylation is highest in CIN3 and

significantly declines during progression to invasive cancer. Unexpectedly, when we

forced STAT3 activation with IL-6-type cytokines, cervical cancer cells were strongly

sensitized to the cytotoxic effects of chemotherapeutic drugs. We identified IRF1 as the

STAT3-inducible pro-apoptotic factor mediating chemosensitization. Notably, our data

show that pre-treatment IRF1 expression correlates with the response to

radio/chemotherapy in cervical cancer patients in-vivo.

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Materials and Methods

Immunohistochemical analysis

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 of Cologne,

Leipzig and Saarland University Hospitals, Germany. These included a subset of 24 pre-

treatment SCC biopsies from patients that had been subjected to neoadjuvant chemo- or

radio/chemotherapy. These tumors were pre- and post-therapeutically staged according

to the International Federation of Gynecology and Obstetrics (FIGO) or TNM categories

(Supplementary Table S1). Histological stainings, diagnosis and treatment responses

were assessed by expert pathologists (CW, LCH, LT, YJK, RMB). Staining of 5 µm

sections with antibodies (Abs) listed in Supplementary Table S2 was performed as

described (10, 20) and classified using the Immunoreactive Score (IRS) according to

Remmele & Stegner (21). Biopsies were evaluated with standardized settings with a

DMI 6000B microscope (Leica, Wetzlar, Germany) and Microsoft-Image-Composite-

Editor program. The retrospective study has been conducted according to Declaration of

Helsinki principles and was approved by the local Ethics Committees of the Cologne,

Leipzig and Saarland Universities (at the Saarland-Ärztekammer).

Cells and cell culture

Relatively 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 (ATCC

HTB-35) obtained from M. von Knebel Doeberitz before 2000 were last authenticated by

HPV16/18-E6/E7-qRT-PCR and multiplex human cell line authentication test

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(Multiplexion, DKFZ, Germany) in 04/2013 and cultured as previously described (17).

Normal human exocervical keratinocytes (NECK) isolated from hysterectomy specimens

according to (24) were cultured in supplemented KBM-Gold (Lonza, Basel, Switzerland)

(approved by the local Ethics Committee of the Saarland University at the Saarland-

Ärztekammer). Written informed consent was provided by the study participants.

Cell stimulation and Western blot analysis

Carcinoma cells or NECK were seeded at a density of 1.5 x 106 cells/6 cm culture dish.

24 h later they were incubated with medium, 10 ng/ml OSM, or 100 ng/ml IL-6

(PeproTech, Hamburg, Germany) in the presence of 500 ng/ml sgp80 (R&D Systems,

South Beloit, IL) (IL-6/sgp80) for the indicated time intervals. Whole cell or nuclear

extracts were prepared as in (25). Abs listed in Supplementary Table S2, secondary Abs

(Sigma-Aldrich) and ECL reagent (Roche, Mannheim, Germany) were used for detection

with ChemiDoc XRS+ Molecular Imager. All Western blots (WB) were performed under

standardized conditions. Expression was quantified with the Quantity One analysis

software (both BioRad, Philadelphia, PA).

Plasmids and transfections

IRF1 and IRF2 cDNAs (26) were amplified with primers listed in Supplementary

Table S3, cloned as NotI/SalI fragments in pCMV-Flag2 vector (Sigma-Aldrich) and

sequences were verified. 1.5 x 105 HeLa cells/6-well were transfected after 24 h with 0.0

(Mock control), 0.025 or 0.1 µg pCMV-Flag2-IRF1, or 0.6 or 0.8 µg pCMV-Flag2-IRF2

expression vector using Lipofectamine-2000 (Invitrogen). The total amount of DNA was

adjusted to 1 µg with empty pCMV-Flag2 vector. 6 or 10 pmol of indicated siRNAs (ON-

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TARGETplus Non-targeting siRNA #2, ON-TARGETplus siRNA #8 for human STAT3,

siRNA #6 for IRF1, all from Thermo Scientific, Bonn, Germany) were transfected with

Lipofectamine RNAiMax (Invitrogen) as described (20).

Cytotoxicity assays

In cytotoxicity assays with cisplatin (Hexal, Holzkirchen, Germany) or etoposide (Sigma-

Aldrich) cells were seeded in 96-well plates at a density of 1.0 x 104 cells/well,

stimulated with medium, OSM, or IL-6/sgp80 for 8 h and challenged for 48 h with serial

dilutions of chemotherapeutic drugs if not indicated otherwise. SiRNA-transfected cells

were pre-treated for 2 h and subsequently challenged with chemotherapeutic drugs for

20 h. Cell viability was assessed by the neutral-red-uptake-method as described

previously (27). For sequential staining with Annexin-V-APC (BD Bioscience,

Heidelberg, Germany) and propidium iodide (PI) (Sigma-Aldrich) assays were scaled up

to 24-well plates and analyzed by flow cytometry (FACSCalibur, BD Biosciences).

Quantitative real-time RT-PCR

HeLa and SW756 cells were stimulated for 2 h if not indicated otherwise. cDNA

synthesis, qRT-PCR and normalization to glyceraldehyde 3-phosphate dehydrogenase

(GAPDH) were performed as previously described (17, 25, 28). The 72-bp fragment of

IRF1 was detected with primers listed in Supplementary Table S3 and probe no. 36

(Roche Universal Probe Library; Roche).

Statistical analysis

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To evaluate the statistical differences between analyzed groups, two-sided t-test was

applied. Significances are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001).

Correlations of pTyr705-STAT3 IRS with IRF1 IRS or with FIGO stages of SCCs were

investigated using Spearman’s rank correlation.

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Results

Decline of STAT3 tyrosine-phosphorylation during malignant progression in

human cervical carcinogenesis in-situ

In tissue sections from the different stages of human cervical carcinogenesis 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 the suprabasal layers

(Figure 1A). In contrast, in CIN3 pTyr705-STAT3 staining was detected in all epithelial

layers and was strongest when a stromal inflammatory STAT3-positive infiltrate was

present (Figure 1A) (10). The pTyr705-STAT3 staining score was significantly higher in

CIN3 than in CIN1 or CIN2 (Figure 1AC, p<0.0001), also in biopsies where both, low-

and high-grade CIN, were present (Figure 1D).

Unexpectedly, 49/94 (52.1%) SCCs displayed only weak pTyr705-STAT3 staining, 39/94

(41.5%) were rated negative and 6/94 (6.4%) showed a moderate IRS (Figure 1C).

Positive pTyr705-STAT3 staining in SCCs was preferentially detected at the tumor

borders adjacent to stroma. The pTyr705-STAT3 IRS did not correlate with FIGO stage

(Supplementary Figure S1). In negative SCCs, proper staining was ascertained by

positively stained stromal infiltrating or endothelial cells. Notably, epithelial pTyr705-

STAT3 staining was significantly weaker in SCCs than CIN3 (Figure 1BC, p<0.0001)

and this was also observed, when both SCC and CIN3 were present in the same biopsy

(Figure 1D).

These results demonstrated that STAT3 is activated in human cervical epithelium during

carcinogenesis peaking in CIN3. However, during progression from CIN3 to invasive

cancer STAT3 activation significantly declines or is lost.

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OSM and IL-6/sgp80 signaling activate STAT3 in cervical carcinoma cells

The same pTyr705-STAT3-specific Ab was used to analyze STAT3-phosphorylation in

the established cervical SCC cell lines SW756 and SiHa, the adenocarcinoma cell line

HeLa, as well as in the more recently generated cervical cancer cell lines 808 and 778

(22, 23). In medium controls only faint constitutive STAT3 activation was observed.

Stimulation with OSM or IL-6/sgp80, led to a pronounced STAT3-phosphorylation in

cervical cancer cells (Figure 2), which was generally stronger than in normal human

exocervical keratinocytes NECK as shown in prolonged exposures of the WBs

(Supplementary Figure S2). Thus, constitutive cell-autonomous STAT3-phosphorylation

in cervical cancer cells is low or absent but strongly inducible by IL-6-type cytokine

signaling.

OSM and IL-6/sgp80 signaling sensitize cervical cancer cells to chemotherapeutic

drug-induced cell death

Since STAT3 protects various cell types from cell death (6, 29), we were interested

whether OSM or IL-6 trans-signaling influenced cell death induction by

chemotherapeutic drugs in cervical cancer cells. Initially, HeLa cells were pre-stimulated

with OSM for different time intervals, which did not significantly change cellular viability,

and subsequently treated with cisplatin, the most common drug used in cervical cancer

chemotherapy. Unexpectedly, OSM pre-activation did not prevent but enhanced

cisplatin-mediated cell death in HeLa cells as judged by combined Annexin-V/PI flow

cytometry (Figure 3A). 2 h of OSM pre-treatment were sufficient for significant

sensitization to cisplatin-induced cell death (p<0.0001), which was further increased by

15% (p=0.001) after 8 h of pre-treatment (Figure 3B).

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We therefore used an 8 h cytokine pre-treatment schedule for our series of cervical

cancer cell lines. Stimulation with OSM or IL-6/sgp80 alone neither altered cellular

viability (Figure 3 and 4) nor proliferation (Supplementary Figure S3) under any condition

tested. Cisplatin killed the individual cell lines at different concentrations. Notably, as a

uniform response in all tested cancer cell lines, we observed a significant increase in

cisplatin-induced cell death after pre-treatment with IL-6/sgp80 (26-54% increase for the

highest cisplatin dose, p-values from 0.0002 to 0.0079) or OSM (19-52% increase, p-

values 0.0003 to 0.0038) (Figure 4A). Similar observations were made for etoposide,

another chemotherapeutic drug in clinical use for cervical cancer treatment (30).

Etoposide-induced cell death increased by 64 or 55% in HeLa (p-values 0.0001 or

0.003) and 72 or 69% (p-values 0.0001 or 0.0004) in SW756 after pre-treatment with

OSM or IL-6/sgp80, respectively (Figure 4B). In contrast, NECK were not significantly

sensitized (p>0.3917) to either of the chemotherapeutic drugs by IL-6/sgp80 or OSM

(Figure 4AB).

Cervical cancer cells produce IL-6 but their autocrine IL-6 response is limited due to low

expression of the receptor gp80 (15, 19). To restore autocrine signaling, HeLa and

SW756 cells were pre-treated with sgp80 alone without exogenous IL-6. Notably, sgp80

was sufficient to strongly sensitize both cell lines to cisplatin- (Figure 4C, p-values 0.021

or 0.0015) or etoposide-induced cell death (Figure 4D, p<0.0001).

These data demonstrated that reconstitution of autocrine IL-6 signaling, IL-6 trans-

signaling or OSM can sensitize cervical cancer cells but not normal human exocervical

keratinocytes for chemotherapeutic drugs.

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STAT3 mediates sensitization for cell death by OSM and IL-6/sgp80 signaling in

cervical cancer cells

To investigate the involvement of STAT3 in chemosensitization in cervical cancer cells,

STAT3-specific siRNA was used to knock-down STAT3 expression in HeLa

(Figure 5ACD) and SW756 cells (Figure 5BEF). In both cell lines, STAT3 knock-down

significantly reverted OSM- or IL-6/sgp80-mediated sensitization to cell death induced by

cisplatin (reversion up to 97%, Figure 5CE, p>0.0021) or etoposide (reversion up to

96%, Figure 5DF, p>0.0012), while control siRNA did not. Similar results were obtained

with an independent STAT3 siRNA (Supplementary Figure S4).

To further substantiate this finding, HeLa cells were transiently transfected with a

dominant-negative version of STAT3 interfering with phosphorylation at Tyr705

(dnSTAT3-Y705F, STAT3F), treated with OSM and subsequently challenged with

etoposide. Transfected cells were visualized by EGFP co-expression. STAT3F

overexpression completely restored cellular viability further underlining that STAT3

activation is required for cell death sensitization (Supplementary Figure S5).

These data provided evidence that chemosensitization by IL-6-type cytokines depends

on the STAT3-pathway in cervical cancer cells.

OSM and IL-6/sgp80 signaling in cervical cancer cells strongly induce IRF1 in a

STAT3-dependent manner

To further elucidate the molecular mechanism underlying cell death sensitization, we

performed mRNA gene expression analysis in SW756 cells after OSM or IL-6/sgp80

stimulation. Strong up-regulation of CCL2 confirmed our previously published results

(15) and validated the assay (data not shown). Besides CCL2, the transcription factor

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IRF1, which has pro-apoptotic activities (31) was significantly up-regulated after IL-

6/sgp80 or OSM stimulation. qRT-PCR revealed a rapid (within 2 h) and strong (10 to

12-fold) induction of IRF1 mRNA after OSM or IL-6/sgp80 stimulation in HeLa and

SW756 cells (Figure 6A, p-values <0.0001). IRF1 protein induction after 2 h of IL-

6/sgp80 or OSM stimulation was demonstrated in nuclear extracts of both cervical

cancer cell lines (Figure 6B) but not in NECK (Supplementary Figure S6).

To verify nuclear IRF1 expression in-vivo, IHC-staining was performed in a subset

(n=79) of cervical cancers (Figure 6C) and compared with pTyr705-STAT3 staining

results. Notably, the IRS of both factors, pTyr705-STAT3 and nuclear IRF1, significantly

correlated with each other (Figure 6D, r=0.3984, p=0.0003). These results indicated that

both factors might be functionally linked to each other and prompted us to analyze a

causal role of STAT3 for IRF1 induction. Indeed, as shown with both, HeLa and SW756

cells, STAT3 knock-down significantly suppressed OSM-induced IRF1 induction

(Figure 6E).

These data provided evidence for a STAT3-dependent mechanism of IRF1 induction in

cervical cancer cells.

IRF1 mediates sensitization for cell death by OSM and IL-6/sgp80 signaling in

cervical cancer cells

To investigate a direct impact on cell death sensitization of cervical cancer cells, IRF1 or

its functional antagonist IRF2 were transiently overexpressed as confirmed by WB

(Figure 7AB). Neither IRF1, IRF2 (in the absence or presence of OSM) nor empty

expression vector alone significantly altered cellular viability (Supplementary Figure S7).

However, IRF1 overexpression was sufficient to sensitize the cancer cells to etoposide-

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mediated cell death in a dose-dependent manner (Figure 7A). Correspondingly, IRF2

significantly interfered with OSM-mediated sensitization for etoposide-mediated cell

death (Figure 7B). Knock-down of endogenous IRF1 with IRF1-specific siRNA in HeLa

(Figure 7CD) and SW756 cells (Figure 7EF) strongly suppressed OSM-mediated IRF1

protein induction (92% or 78.5%) (Figure 7CE) and significantly reverted sensitization to

etoposide- or cisplatin-induced cell death in both cell lines (Figure 7DF). Similar results

were obtained with a second independent IRF1-specific siRNA (Supplementary Figure

S8).

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

cancer patients

We then evaluated IRF1 expression in human cervical cancer biopsies prior to

neoadjuvant radio/chemotherapy in relation to the patient´s individual response to

therapy (Supplementary Table S1). Patients with complete response to

radio/chemotherapy displayed significantly higher scores of pre-therapeutic nuclear IRF1

expression in neoplastic cells than patients with only partial response to therapy

(Figure 7G, left panel, p=0.0378). This was also observed when total IRF1 expression

(nuclear and cytoplasmic) was evaluated (Figure 7G, right panel, p=0.0033).

These data identified IRF1 as a key regulator of chemosensitivity in cervical cancer cells

and demonstrate a clear association between pre-treatment IRF1 expression and

response to radio/chemotherapy in-vivo.

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Discussion

Cervical cancer therapy is still a major clinical challenge, since patients substantially

differ in their response to standard treatments including platinum-based

radio/chemotherapy. Clinically, it is highly desirable to identify those patients that may

benefit from neoadjuvant radio/chemotherapy and to distinguish them from putative

partial- or non-responders.

Our study has unraveled a key role of the STAT3-dependent IRF1-pathway for

chemosensitization. This was unexpected, since STAT3 is considered as a survival

factor in various other cancer types. IRF1 was inducible by IL-6-type cytokines via

STAT3 activation in cervical cancer cells and in-vivo, nuclear IRF1 and pSTAT3

expression scores significantly correlated with each other. Importantly, our data show

that cervical cancer patients with higher IRF1 expression scores responded significantly

better to radio/chemotherapy. This may have major implications for personalization of

cervical cancer chemotherapy.

Our study has unraveled novel findings on the activation and function of the transcription

factor STAT3 in cervical carcinogenesis. Comparing different human CIN stages, we

found that STAT3-tyrosine-phosphorylation dramatically increased from low-grade CIN1

to pre-invasive CIN3 lesions. This was in line with observations in various murine tumor

models (32, 33) and supports the notion that STAT3 activation has an important role at

pre-cancerous stages of cervical carcinogenesis. Unexpectedly, in biopsies comprising

both CIN3 and SCC, we observed a strong decline of STAT3 activation during

progression to invasive cancer. The pTyr705-STAT3 staining score was weak in more

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than half or negative in more than 40% of invasive cervical cancers. Positive staining

was mostly confined to the tumor borders adjacent to the stroma suggesting a paracrine

mode of STAT3 activation. In line with our results, another study found pTyr705-STAT3

in less than 10% of cells in most cervical cancer cases (34) and two previous studies

detected pTyr705-STAT3 (35) or stronger nuclear STAT3 staining (36, 37) in 22-24% of

cervical cancers. Only one group detected higher percentages of STAT3-

phosphorylation in cervical cancers in an Indian population (38). Whether the results of

the latter study are due to ethnical, environmental or technical issues remains to be

determined. Higher FIGO stages in Indian patients can probably be excluded as the

bases for these differences, since we did not observe a correlation between pTyr705-

STAT3 IRS and FIGO stage. To our knowledge, our study is the first report on the direct

comparison of pre-cancerous and invasive cervical cancer lesions within the same

clinical specimen and the first to demonstrate a strong decline of STAT3 activation

during progression to malignancy. Whether this particular reaction pattern is a

consequence of the viral etiology of cervical carcinogenesis will be of high interest for

future research. From our data we conclude that in most cervical cancers STAT3

activation is balanced at a low level. A high level of STAT3 activation is obviously not

required for the malignant cells and may be counter selected during progression to SCC.

In fact, our attempts to overexpress a constitutively active form of STAT3 in cervical

cancer cells failed and did not give rise to cell clones (unpublished observation).

STAT3 is generally considered as an anti-apoptotic factor and the IL-6 signaling

pathway can promote resistance to chemotherapeutic drugs in different cancer types (6,

7). In contrast, we found that cervical cancer cells were not protected but sensitized for

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chemotherapeutic drugs by IL-6-type cytokine-induced STAT3 signaling as shown via

STAT3 knock-down or dominant-negative STAT3F (13). In our study we used the

alkylating agent cisplatin, the chemotherapeutic drug most commonly used against

cervical cancer. Moreover, a higher sensitivity was also observed for the topoisomerase

II inhibitor etoposide used in recurrent cervical cancer (30) indicating that the

sensitization was not restricted to a single class of chemotherapeutic drugs. Although

the IL-6-/STAT3-pathways appear to be attractive pharmacological targets to improve

the immune microenvironment (9, 10, 17, 18), our data suggest that one should be

cautious to combine STAT3-inhibitors with cisplatin or etoposide in cervical cancer

patients.

In search of the mechanism underlying IL-6-type cytokine-induced STAT3-dependent

chemosensitization, we identified IRF1, a pro-apoptotic factor, anti-oncogene and

regulator of autophagy (39-42). Notably, there are complex interactions between HPV

and IRFs. IRF1 and 2 can activate the HPV16 oncogene promoter (43, 44) and the E5

oncoprotein induces IRF1 (45). In contrast, the E7 oncoprotein can suppress IFN-γ-

induced IRF1 expression and interferes with the effectiveness of the immune response

(46, 47). Our data clearly show that IL-6/sgp80- or OSM-induced STAT3 signaling

sensitizes HPV-positive cervical cancer cells for chemotherapeutic drugs via IRF1 up-

regulation. This suggests that STAT3 activation may override IRF1 inhibition by HPV.

HPV-transformation may be necessary in keratinocytes for the unusual STAT3 effect

mediating IRF1 induction but it is apparently not essential in other cell types. It was

observed before in murine myeloid leukemic M1 cells, where STAT3/IRF1-signaling is

associated with growth arrest and differentiation (13, 48, 49). The consequences of

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STAT3/IRF1 pathway activation in cervical cancer cells in the absence of cancer therapy

are under current investigation in our laboratory.

Since IRF1 was sufficient to render cervical cancer cell lines more susceptible to

chemotherapeutic drugs, we were interested in the in-vivo expression of IRF1 in human

cervical cancer. In patient samples nuclear IRF1 correlated significantly with pTyr705-

STAT3 activation. This was interesting, since IRF1 can also be induced by pathways

other than STAT3 (39). Clinically most important, we detected the highest IRF1 pre-

treatment expression scores in cancers of those patients who responded completely to

neoadjuvant or radio/chemotherapy. In contrast, partial- or non-responders displayed

significantly lower IRF1 expression scores. The results were similar, irrespective of

whether nuclear or total IRF1 expression in the neoplastic cells were taken into account.

This is plausible, since higher basal IRF1 expression levels, which may be determined at

the genetic level (50), can also contribute to a stronger IRF1 activation during the course

of chemotherapy.

In summary, our data provide novel mechanistic insight on the contributory role of

STAT3 in inducing IRF1 and thereby chemosensitization of cervical cancer cells. Based

on these results, pre-treatment IRF1 expression should be evaluated as a predictive

biomarker for the individual response of cervical cancer patients to neoadjuvant

radio/chemotherapy in prospective clinical studies.

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Acknowledgments

We thank U. Sandaradura de Silva, T. Tänzer 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 and C. Herr for help with slide scanning.

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Figure Legends

Figure 1

pTyr705-STAT3 expression in CIN1-3 and cervical SCCs. Human FFPE-sections

were stained with anti-pTyr705-STAT3 Ab (brown color). (A) Biopsy containing CIN1,

CIN2 and CIN3. (B) Biopsy containing CIN3 and SCC (all 200x). SCC is marked by

asterisk (*). Bars: 100 µm. Asterisks in (B) indicate SCC. (C) IRS of pTyr705-STAT3

staining in CIN1/2 (n=22), CIN3 (n=29) and SCC (n=94). Mean is indicated by 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=8), or CIN3 and SCC

(n=19); statistical analysis: two-sided t-test.

Figure 2

OSM or IL-6/sgp80 strongly activate STAT3 in cervical cancer cells. HeLa, SW756,

SiHa, 808, 788 cells and NECK were stimulated with medium, OSM or IL-6/sgp80 for 15

min. Whole cell extracts were analyzed by WB using anti-pTyr705-STAT3-, STAT3- or

β-actin-specific Ab.

Figure 3

OSM sensitizes HeLa cells for cisplatin-induced cell death in a time-dependent

manner. (A) HeLa cells were pre-stimulated with medium or OSM for 8 h and treated

with 25 µg/ml cisplatin for further 12 h. Cells were stained with PI and Annexin-V and

analyzed by flow cytometry. Left panel shows one experiment out of n=3, right panel

summarizes 3 experiments. (B) HeLa cells were pre-stimulated with medium (squares,

black line) or OSM (circles, grey line) for 2, 4 or 8 h and then treated with medium (open

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symbols) or 1.56 µg/ml cisplatin (filled symbols) for 48 h. Cell viability was assessed by

the neutral-red-uptake-method, n=2 performed in triplicates; statistical analysis: two-

sided t-test.

Figure 4

OSM or IL-6/sgp80 sensitize cervical cancer cells for chemotherapeutic drugs. (A)

HeLa, SW756, SiHa, 808, 778 cells and NECK were pre-stimulated with medium (black

squares or bars), OSM (dark grey circles or bars) or IL-6/sgp80 (light grey triangles or

bars) and then treated with cisplatin at the indicated concentrations. (B) HeLa, SW756

cells and NECK were stimulated as above and treated with etoposide. (C, D) HeLa and

SW756 cells were pre-stimulated with medium (black squares or bars) or sgp80 (grey

triangles or bars) and treated with cisplatin (C) or etoposide (D). Cell viability was

assessed by the neutral-red-uptake-method. Shown is one experiment out of n=3

performed in triplicates. Histograms summarize n=3 for cells treated with medium or the

highest chemotherapeutic drug concentration; statistical analysis: two-sided t-test.

Figure 5

STAT3 mediates OSM- or IL-6/sgp80-induced cell death sensitization. HeLa (A) or

SW756 (B) cells were transfected with 10 pmol/1.5 x 105 cells of human STAT3-specific

siRNA or mock siRNA. Cells were stimulated with OSM for 15 min. Whole cell extracts

were analyzed by WB using anti-pTyr705-STAT3-, STAT3- or β-actin-specific Ab.

Shown is one experiment out of n=3. Diagram summarizes 3 experiments. Expression of

the respective controls was set at 100%. HeLa (C, D) or SW756 (E, F) cells were

transfected as in (A), pre-stimulated with OSM or IL-6/sgp80 and then treated with two

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concentrations of cisplatin (C, E) or etoposide (D, F), respectively. Cell viability was

assessed by the neutral-red-uptake-method. Shown is n=3 performed in triplicates;

statistical analysis: two-sided t-test.

Figure 6

IRF1 is induced by OSM or IL-6/sgp80 in cervical cancer cells in a STAT3-

dependent manner. (A) HeLa and SW756 cells were stimulated with medium, OSM or

IL-6/sgp80. After 2, 4 and 8 h RNA was isolated and IRF1-specific mRNA was quantified

by qRT-PCR in relation to GAPDH, n=2 performed in triplicates. (B) HeLa and SW756

cells were stimulated as in (A) for 2 h. Nuclear IRF1 expression was analyzed by WB.

(C) 79 cervical SCCs were stained with anti-pTyr705-STAT3 or anti-IRF1 Ab. Shown is a

representative pSTAT3- and IRF1-positive SCC in the left panel (brown color; all 200x,

bars: 50 µm). IRS of nuclear staining (right panel); means are indicated by blue lines.

(D) Correlation of both IRS with each other. (E) HeLa or SW756 cells were transfected

with 10 pmol/1.5 x 105 cells of human STAT3-specific siRNA or mock siRNA and

stimulated with medium or OSM for 2 h. Nuclear extracts were analyzed by WB for IRF1

expression. Shown is one experiment out of n=3. Diagram summarizes 3 experiments;

statistical analysis: two-sided t-test.

Figure 7

Association between IRF1 expression and chemotherapy response. (A) HeLa cells

were transfected with the indicated amounts of pCMV-Flag2-IRF1 or (B) pCMV-Flag2-

IRF2. In (B) cells were pre-stimulated after 16 h with OSM for 2 h and in (A, B) treated

with serial dilutions of etoposide for 24 h. HeLa (C, D) and SW756 (E, F) cells were

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32

transfected with 6 pmol of human IRF1-specific siRNA or mock siRNA/1.5 x 105 cells

and stimulated with OSM. (C, E) Nuclear extracts were analyzed by WB for IRF1

expression. (D, F) Transfected cells were incubated with serial dilutions of etoposide or

cisplatin. In (A, B, D and F) cell viability was assessed by the neutral-red-uptake-

method, n=3 performed in triplicates. (G) IRS of nuclear (left panel) or total (right panel)

IRF1 expression was determined in pre-therapeutic biopsies of n=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.

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Published OnlineFirst May 23, 2016.Cancer Res   Barbara Walch-Rückheim, Jennifer Pahne-Zeppenfeld, Jil Fischbach, et al.   to chemotherapeutic drugsSTAT3/IRF1 pathway activation sensitizes cervical cancer cells

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