ESPE Abstracts (2018) 89 P-P2-170

Development of Resistance to Sorafenib, a Multikinase Inhibitor, in Hepatocellular Carcinoma is Mediated by SIRT

Antje Gartena,b, Theresa Grohmanna, Anja Barnikol-Oettlera, Katharina Kluckovab, Gareth Laveryb, Wieland Kiessa,c,d & Melanie Penkea

aCenter for Pediatric Research Leipzig, Leipzig, Germany; bInstitute of Metabolism and Systems Research, Birmingham, UK; cLIFE-Child- Leipzig Research Center for Civilization Diseases, Leipzig, Germany; dUniversity Hospital for Children and Adolescents, Leipzig, Germany

Background/aim: Sorafenib® is a multi-kinase inhibitor and one of the few systemic treatment options for patients with advanced hepatocellular carcinomas (HCCs). Resistance to Sorafenib® develops frequently and could be mediated by the NAD dependent deacetylase sirtuin (SIRT) 1, a master regulator of cellular energy metabolism and stress responses. We aimed to find out if Sorafenib® treatment depends on changes in cellular NAD levels, SIRT1 activity and the cellular energy sensor adenosine monophosphate kinase (AMPK).

Methods: Hepatocarcinoma cells (HCC) (HepG2, Hep3B und Huh7) were stimulated with different concentrations of Sorafenib® (0 to 5 μM) and/or NMN (0.2 mM). Apoptosis was measured by AnnexinV-FITC/PI staining. NAD levels were determined by reversed phase HPLC. ATP levels were measured by CellTiter-Glo® Luminescent Cell Viability Assay. Protein and mRNA levels were analysed by Western blotting and qPCR, respectively. Mitochondrial activity was measured by high resolution respirometry.

Results: We could show that Sorafenib® treatment of HCC cell lines induced apoptosis after stimulation with 5 μM Sorafenib® (HUH7: 6.0±1.5-fold; Hep3B: 2.4±0.4 fold; HepG2 1.9±0.3 fold). Sirt1 protein was downregulated to 60.7±5.4%. Cellular NAD concentrations were significantly decreased from 6.8±0.7 to 2.3±0.5 nmol/mg protein in Huh7 cells after exposure to Sorafenib® for 24h. ATP levels were decreased to 32.4±12.6%. Concomitant to increasing phosphorylation of AMP kinase (3.4±0.5 fold), activity of its downstream target mammalian target of rapamycin (mTOR) was decreased to 83.4±7.2% after Sorafenib® treatment, which could indicate energy deprivation. Oxygen flow in permeabilised cells was lower and citrate synthase activity was inhibited by 28.4±7.8% after Sorafenib® treatment (1 μM, 24 h). While pharmacologically inhibiting NAMPT by FK866 or knockdown of SIRT1 by siRNA did not sensitise HCC to Sorafenib® treatment, transient overexpression of SIRT1 decreased Sorafenib®-induced apoptosis via normalisation of p53 acetylation. Both number of apoptotic cells and effects on AMPK/mTOR phosphorylation were reversed by supplementation of nicotinamide mononucleotide (NMN), the enzyme product of NAMPT. However, mitochondrial activity could not be rescued by NMN supplementation.

Conclusion: We can therefore conclude that Sorafenib® influences SIRT1 and that overexpression of SIRT1 could be an underlying mechanism of resistance to Sorafenib® treatment in HCC.

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