Abstract POSTER-TECH-1123: Advancing metformin as therapeutic for ovarian cancer: metabolomic profiling of mouse ovarian tumors identifies metformin-induced global metabolic changes
Authors: Romero Iris L. , Liu Xiaojing , Mitra Anirban K , Lengyel Ernst , Locasale Jason W. 
Published: August 2015

Abstract
Background: Epidemiologic and preclinical studies indicate that use of metformin, a widely prescribed anti-diabetic drug, has protective effects in ovarian cancer (OvCa). One of the current challenges in the field is understanding which patients are most likely to benefit from metformin treatment. The aim of this study was to test the hypothesis that metformin would prevent OvCa progression in a mouse model and to identify metabolic changes induced in tumors by metformin. The long-term goal of these studies is to use the findings to inform the analysis of biospecimens in an ongoing prospective clinical trial of metformin in OvCa.
Methods: To test the effect of metformin on ovarian tumorigenesis, HeyA8 xenograft mice were treated with metformin (250 mg/kg/day) or control. At the time of sacrifice, mean tumor weight was determined and tumors were stained by immunohistochemistry for Cyclin D1. Metabolomic analysis was performed on serum and tumor samples from mice treated with metformin and compared to mice treated with control. Here, after precipitation in 80% methanol and reconstitution in LC/MS grade water/HPLC grade acetonitrile samples were analyzed using a Q-Exactive Mass Spectrometer coupled to HILIC chromatography that was able to quantify over 300 polar metabolites and over 5000 molecular features in each sample.
Results: The results indicate that in a mouse model metformin inhibits OvCa progression and induces global metabolic alterations. Metformin-treated mice had 68% lower mean tumor weight than controls (p=0.003). To understand how metformin inhibited tumor growth we stained tumors for Cyclin D1 to test whether metformin induced cell cycle arrest, a finding demonstrated in our prior studies (unpublished results). We found lower Cyclin D1 levels in tumors from metformin-treated mice than controls. Next, we measured the level of metformin in both the serum and tumors using mass-spectrometry. In serum, the results showed metformin levels in mice ranging from 1-5 nM; this was comparable to reports of serum levels of patients taking 1 gm of metformin daily. In tumors, there was trend toward smaller tumors having higher levels of metformin (Spearman, one-tailed R= -0.60, p=0.16). Finally, we used metabolomic profiling for a more global and unbiased assessment of metformin’s effect in OvCa. This analysis revealed two novel findings. First, an unbiased hierarchical clustering approach revealed metabolome profiles that could clearly predict the mice that had been treated with metformin. Second, global metabolic changes were noted in tumors from metformin treated mice. Metabolic pathways altered in metformin-treated mice included: alterations in glycolytic intermediates, induction of oxidative stress, decreased biosynthetic components of nucleotides, and changes in lipid metabolism.
Conclusion: These findings of decreased tumor burden in metformin-treated mice add to the growing body of evidence supporting repurposing metformin as a metabolic therapeutic for OvCa. In addition, the metabolic changes in tumors induced by metformin indicate that it might be possible to establish a metabolomic signature in ovarian tumors that is predictive of metformin response.
Citation Format: Iris L. Romero, Xiaojing Liu, Anirban K Mitra, Ernst Lengyel, Jason W. Locasale. Advancing metformin as therapeutic for ovarian cancer: metabolomic profiling of mouse ovarian tumors identifies metformin-induced global metabolic changes [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1123.