Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma

// Milica Stefanovic 1 , Anna Tutusaus 1 , Guillermo A. Martinez-Nieto 1 , Cristina Barcena 1 , Estefania de Gregorio 1 , Catia Moutinho 2 , Elisabet Barbero-Camps 1 , Alberto Villanueva 3 , Anna Colell 1 , Montserrat Mari 1 , Carmen Garcia-Ruiz 1, 4 , Jose C. Fernandez-Checa 1, 4, 5 , Albert Morales 1 1 Department of Cell Death and Proliferation, IIBB-CSIC, IDIBAPS, Barcelona, Catalonia, Spain 2 Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute, Barcelona, Catalonia, Spain 3 Translational Research Laboratory, Catalan Institute of Oncology - Bellvitge Biomedical Research Institute, Barcelona, Catalonia, Spain 4 Liver Unit, Hospital Clinic, CIBEREHD, Barcelona, Catalonia, Spain 5 Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA Correspondence to: Albert Morales, e-mail: amorales@clinic.ub.es Jose C. Fernandez-Checa, e-mail: checa229@yahoo.com Keywords: liver cancer, chemotherapy, mitochondria, ceramide, mouse model Received: July 27, 2015      Accepted: January 13, 2016      Published: January 22, 2016 ABSTRACT Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated.