Abstract 3113: Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in a genetically engineered mouse model of serous ovarian cancer

Abstract

Abstract Objectives: Anti-diabetic biguanide drugs have been shown to have anti-tumorigenic effects by behaving as AMPK activators and mTOR inhibitors. Phenformin is a biguanide that is associated with a higher risk of lactic acidosis than is seen in metformin. In vitro and in vivo studies suggest that phenformin may be more potent for inhibiting tumor growth than metformin. We sought to examine the efficacy of phenformin in primary cultures of human ovarian cancers (OCs) as well as in a genetically engineered mouse model of serous OC. Methods: Serous OC tumors were collected from consenting patients, at the time of their surgical debulking for primary culture. Cell proliferation was assessed by MTT assay. Apoptosis was evaluated by Annexin V-FITC assay using Cellometer. Effects of phenformin on phosphorylated-AMPK and -S6 expression was documented by Western immunoblotting. For the in vivo studies, we utilized the KpB++ serous OC mouse model. This is a more aggressive derivative of the K18-gT121+/-; p53fl/fl;Brca1fl/fl (KpB) genetically engineered OC mouse model. The KpB++ mice were treated with placebo or phenformin (2 mg/kg body weight in drinking water) following tumor onset for one month. Immunohistochemical analysis was performed on the ovarian tumors after treatment with placebo or phenformin, in regards to Ki-67 expression, cleaved caspase 3 (a marker of apoptosis), phosphorylated AMPK and phosphorylated S6. Individual slides were digitized using the Aperio ScanScope (Aperio Technologies, Vista, CA), and digital images were analyzed using Aperio ImageScope software. Results: Phenformin significantly inhibited cell proliferation in a dose-dependent manner in 100% (7/7) of the primary cultures of ovarian tumors, within 48 to 72 hours of exposure (p=0.00001-0.015, IC50 range of 0.1-5 mM). Treatment with phenformin resulted in the induction of apoptosis in 71.4% (5/7) of the ovarian tumors. Western immunoblot analysis demonstrated that phenformin increased phosphorylation of AMPK and decreased phosphorylation of S6. Phenformin significantly inhibited tumor growth in the KpB++ mice (n=8-13 animals per group) by 68% as compared to placebo treated controls, after one month of treatment (p=0.016). As compared to the placebo treated control animals, phenformin was found to statistically decrease Ki67 staining, inhibit phosphorylated S6 expression and increase both cleaved caspase 3 and phosphorylated AMPK expression (p= 0.01-0.04). Conclusions: In primary culture of human serous OC tumors, phenformin potently inhibited cell growth and increased apoptotic cell death through AMPK activation and mTOR pathway inhibition. In vivo studies using the KpB++ mouse model found that phenformin was highly efficacious in inhibiting tumor growth. Although the risk/benefit ratio clearly favors metformin over phenformin for the treatment of diabetes, this may not hold true for the treatment of cancer. Citation Format: Amanda L. Jackson, Xiaoyun Han, Joshua E. Kilgore, Chunxiao Zhou, Liza Makowski, Victoria Bae-Jump. Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in a genetically engineered mouse model of serous ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3113. doi:10.1158/1538-7445.AM2014-3113