Drug-drug interaction between metformin and sorafenib alters antitumor effect in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is one of the leading causes of cancer-related deaths worldwide. The multi‐target inhibitor sorafenib is a first-line treatment for patients with advanced unresectable HCC. Recent clinical studies have evidenced that patients treated with sorafenib together with the anti-diabetic drug metformin have a survival disadvantage compared to patients receiving sorafenib only. Here, we examined whether a clinically relevant dose of metformin (50 mg/kg/d) could influence the antitumoral effects of sorafenib (15 mg/kg/d) in a subcutaneous xenograft model of human HCC growth using two different sequences of administration, i.e concomitant versus sequential dosing regimens. We observed that the administration of metformin six hours prior to sorafenib was significantly less effective in inhibiting tumor growth than concomitant administration of the two drugs. In vitro experiments confirmed that pretreatment of different human HCC cell lines with metformin reduced the effects of sorafenib on cell viability, proliferation and signaling. Transcriptomic analysis confirmed significant differences between xenografted tumors obtained under the concomitant and the sequential dosing regimens. Taken together, these observations call into question the benefit of parallel use of metformin and sorafenib in patients with advanced HCC and diabetes, as the interaction between the two drugs could ultimately compromise patient survival. To better characterize the molecular signatures driving the differential responses to concomitant and sequential biotherapies, we conducted a transcriptomic analysis on RNA extracted from tumor xenografts mice xenografed and treated with vehicle (control, n=3), metformin (50 mg/kg/day) combined to sorafenib (15 mg/kg/day) (concomitant schedule, n=3) or metformin (50 mg/kg/day) followed 6 hours later by sorafenib (15 mg/kg/day) (sequential schedule, n=3). Total RNA was extracted from tumors collected from mice xenografed subcutaneously in the right flank with PLC/PRF5 cells and treated with vehicle (control, n=3), metformin (50 mg/kg/day) combined to sorafenib (15 mg/kg/day) (concomitant schedule, n=3) or metformin (50 mg/kg/day) followed 6 hours later by sorafenib (15 mg/kg/day) (sequential schedule, n=3). Total RNA was amplified and labelled using the GeneChipTM WT PLUS Reagent Kit (ThermoFischer Scientific). Each RNA sample was hybridized to Human ClariomTM S GeneChip (ThermoFischer Scientific). Arrays were scanned, and images were analyzed and controlled for hybridization artefacts. The microarray data were normalized using Signal Space Transformation-RMA (SST-RMA) which is optimized for under-estimation of true fold changes. Following normalization, differential expression was carried out using eBayes function and One-Way Anova statistical analysis. The analysis was carried out using Transcriptome Analysis Console software (ThermoFischer Scientific, version 4.0.2) with p < 0.05 considered as statistically significant. The differentially expressed genes were then subjected to absolute GSEA searching through more than 10,000 different cellular pathways.