Overcoming sorafenib resistance in hepatocellular carcinoma by fasting


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Jelena Krstic1,Isabel Reinisch1,Maria Depaoli1,Natascha Berger2,Christoph Noessing3,Markus Galhuber1,Ines Anders4,Martina Auer1,Elisabeth Moyschewitz1,Beate Rinner5,Martin Pichler6,Roland Malli1,Andreas Prokesch1
1Medical University of Graz, Graz, Austria, Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Graz, Austria,2Department of Obstretics and Gynaecology, Medical University of Graz, Graz, Austria,3Cancer Research UK Beatson Institute, London, UK,4Medical University of Graz, Graz, Austria, Core Facility Alternative Biomodels and Preclinical Imaging, Graz, Austria,5Division of Biomedical Research, Medical University of Graz, Graz, Austria,6Division of Oncology, Medical University of Graz, Graz, Austria

Abstract

Background

Fasting is suggested as adjuvant for cancer treatment, since it increases cancer cell sensitivity to chemo- and targeted therapy in various cancer models. The mechanisms of this pleiotropic effect of fasting (i.e. starvation) are not fully elucidated, especially in therapy-resistant cancers. We investigated whether starvation can sensitize therapy-resistant hepatocellular carcinoma (HCC) cells to sorafenib, the only first-line treatment currently in use.

Method

HepG2 cells were grown in growth or starvation medium and treated with sorafenib for 24h. Cell viability was analyzed by colorimetric assay and by flow cytometry. Energy metabolism was assayed using XF96 Seahorse analyzer. Components of growth pathways (mTOR and Ras/Raf/MEK/ERK) were analyzed by western blot. p53 knock-out cells were generated using Crispr/Cas9 system. For xenograft assays, cells were injected subcutaneously into the flanks of NMRI-Foxn1nu mice, which were then treated with vehicle or sorafenib and fed ad libitum or intermittently fasted during four weeks. 

Results

Sorafenib-resistant HepG2 cells were sensitized by starvation, with viability decreasing below 15% after 24h of combined treatment. Similar effect was observed in xenografts: tumors grew at a slower rate only when sorafenib-treated mice were exposed to an intermittent fasting regimen. Mechanistically, bioenergetic profiling suggested abrogated oxidative phosphorylation in HepG2 cells, as an early event in sensitization. Starvation-supported sorafenib also blocked the two major growth pathways, mTOR and Ras/Raf/MEK/ERK. Furthermore, the sensitizing effect of starvation was blunted in p53-deficient cells indicating dependency on p53-mediated cell death.

Conclusion

We tested whether starvation can augment the effects of sorafenib in therapy-resistant HCC. Combined treatment could sensitize resistant cancer cells in vitro as well as in xenografts and resulted in complete inhibition of mitochondrial respiration and ATP production, multiple growth pathway blockage, and subsequent p53-mediated cell death. Thus, our study suggests fasting and sorafenib treatment as potential polytherapy for HCC.