High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

RESEARCH ARTICLE High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer John J. Wallbillich¤, Srirama Josyula, Uksha Saini, Roman A. Zingarelli, Kalpana Deepa Priya Dorayappan, Maria K. Riley, Ross A. Wanner, David E. Cohn, Karuppaiyah Selvendiran* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Wallbillich JJ, Josyula S, Saini U, Zingarelli RA, Dorayappan KDP, Riley MK, et al. (2017) High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer. PLoS ONE 12(1): e0170318. doi:10.1371/journal. pone.0170318 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: October 18, 2016 Accepted: January 3, 2017 Published: January 23, 2017 Copyright: © 2017 Wallbillich et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by the Division of Gynecologic Oncology Startup Fund and The Ohio State University Comprehensive Cancer Center Startup Fund, an internal grant from the Ohio State University Comprehensive Cancer Center (OSU CCC) to DEC, and a startup fund from the Division of Gynecologic Oncology to KS. The funders had Division of Gynecologic Oncology, Comprehensive Cancer Center, The Ohio State University Wexner Medical Center, Columbus, Ohio, United States of America ¤ Current address: Section of Gynecologic Oncology, Department of Obstetrics and Gynecology, Augusta University, Augusta, Georgia, United States of America * Abstract Objectives STAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin. Methods In Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the in vivo efficacy of metformin. Results Expression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. In vitro, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. In vivo, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins. PLOS ONE | DOI:10.1371/journal.pone.0170318 January 23, 2017 1 / 16 Metformin Targets STAT3 no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Conclusions These results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin. Introduction Endometrial cancer is the most common gynecologic cancer and the fourth-most common cancer among women in the United States. 60,050 new cases of endometrial cancer and 10,470 deaths due to the disease are estimated for 2016. The incidence of endometrial cancer has been rising, with a 2.3% annual increase reported from 2006–2012 [1]. This increase has been attributed to the most common subtype of endometrial cancer, type 1 [2]. Type 1 endometrial cancer is comprised of grades 1 and 2 endometrioid adenocarcinoma [3]. Risk factors for type 1 endometrial cancer include obesity and diabetes, which have been increasing in prevalence in the United States[4, 5]. Further, in women with endometrial cancer, obesity leads to a 6.25 fold increased risk of dying due to the disease [6]. Regarding diabetes, while the disease is frequently co-morbid with obesity, it has been shown to independently increase the risk of endometrial cancer [7, 8]. Insulin resistance, insulin-like growth factors, hyperinsulinemia, and hyperglycemia have been implicated as potential etiologic agents in diabetes-associated endometrial cancer. However, existing data is conflicted as to the exact role, if any, of such diabetes-associated factors in the pathogenesis of endometrial cancer [9–14]. Given that STAT3 is over-expressed in endometrial cancer [15] and hyperglycemia has been shown to activate the STAT3 pathway in another disease site [16], we investigated whether high glucose concentrations might have an impact on STAT3 in endometrial cancer. Based on our pilot data that showed a high glucose-mediated increase in STAT3 in grade 1 endometrial cancer cells, we aimed to evaluate a potential inhibitor of such STAT3 activation. Metformin, a medication commonly used in the treatment of type II diabetes, has gained recent traction as potential therapeutic agent for endometrial cancer [17, 18]. Epidemiologic studies have shown that metformin may lower risk of cancer and cancer-related death among patients with diabetes [19–21]. In vitro data has shown that metformin can inhibit cell proliferation in endometrial cancer cell lines [22, 23]. Based on these data, a phase II/III study is currently underway to evaluate metformin vs. placebo in patients receiving paclitaxel and carboplatin advanced or recurrent endometrial cancer (NCT02065687). Although metformin has been found to inhibit PI3K/AKT/mTOR and K-Ras signaling in endometrial cancer, the precise mechanism of the drug’s action against the disease has not been fully delineated [22, 24]. While the influence of metformin on STAT3 in endometrial cancer is unknown, metformin has been shown to inhibit STAT3-mediated cell proliferation in breast, esophageal, and bladder cancers [25–27]. Based on these reports as well as our preliminary results, we investigated whether metformin impacts high glucose-mediated STAT3 expression in type 1 endometrial cancer. Materials and Methods Materials Metformin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sterile water (...truncated)