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 antiapoptotic 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 lowgrade cervical intraepithelial neoplasia (CIN1) to precancerous 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, IL6-type cytokine signaling inducing STAT3 activation rendered cervical cancer cells significantly more susceptible to chemotherapeutic drugs, that is, cisplatin or etoposide. This chemosensitization was STAT3-dependent and we identified IFN 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 pretreatment 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 pretherapeutic IRF1 expression should be evaluated as a novel predictive biomarker for neoadjuvant radio/chemotherapy responses. Cancer Res; 76(13); 3872-83. Ó2016 AACR.

Abstract. Cervical cancer stage-dependent therapies include surgery, chemotherapy, radiotherapy and chemoradiotherapy. Concurrent cisplatin-based chemoradiotherapy (CCRT) is the standard therapy for locally advanced cervical carcinoma (FIGO>IIB), however therapy resistance in a subset of patients is still a major clinical challenge. The present study aimed to analyze the impact of Oncostatin M (OSM) stimulation on CCRT-induced cell death. The present study used cells derived from cervical squamous cell carcinomas (SW756, 808, CaSki and 879) and adenocarcinoma (HeLa). The cervical carcinoma cells were HPV18-positive (HeLa, SW756, 808) or HPV16-positive (CaSki, 879). In addition to the established cell lines HeLa, SW756 and CaSki, the more recently generated cervical cancer cells 808 and 879 were also used. To analyze their radiosensitivity, cells were treated with increasing doses of irradiation (0-8 Gy). To mimic chemotherapy, radiotherapy or CCRT in vitro, the cells were challenged with 0.975 µg/ml cisplatin, irradiated with 6 Gy or a combination. A total of 10 ng/ml OSM was applied for 2 h prior to the respective therapy. The responsiveness toward radiation alone varied among the cervical carcinoma cells. CaSki, 808 and 879 cells were resistant to irradiation up to 8 Gy. OSM pre-treatment sensitized two out of five cell lines (HeLa and 879) to irradiation. Notably, all tested cells were sensitized by OSM for CCRT-treatment, particularly in the less radiosensitive cells. Cell death enhancement was dependent on phosphorylated signal transducer and activator of transcription 3 (STAT3; Tyr705) signaling activation as demonstrated with a dominant-negative version of STAT3 interfering with phosphorylation at Tyr705 (dnSTAT3-Y705F). In conclusion, OSM pre-treatment was able to override resistance to CCRT via the STAT3 signaling pathway.

<p>1.5 x 105 HeLa cells/6-well were either left untransfected or were transfected with (A) 0 µg, 0.025 µg or 0.1 µg of pCMV-Flag2-IRF1 or (B) 0 µg, 0.6 µg or 0.8 µg of pCMV-Flag2-IRF2.</p>

<p>Legends to Supplementary Figures S1-S8.</p>

<p>Pre- and Post-therapeutically staged tumors according to the International Federation of Gynecology and Obstetrics or TNM categories (S1); List of used antibodies (S2); Primers for amplification of cDNAs of IRF1 and IRF2 (S3).</p>