Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs' ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.

Bioinformatics Unit Fondazione IRCCS Casa Sollievo della Sofferenza 71013 San Giovanni Rotondo Italy

Department of Biology Faculty of Medicine Masaryk University 62500 Brno Czech Republic

Department of Biomedical and Biotechnological Sciences University of Catania 95131 Catania Italy

Department of Medical and Surgical Sciences and Advanced Technologies GF Ingrassia University of Catania 95123 Catania Italy

Department of Medical Sciences and Chronobiology Laboratory Fondazione IRCCS Casa Sollievo della Sofferenza 71013 San Giovanni Rotondo Italy

ERA Chair in Translational Stem Cell Biology Medical University of Varna 9002 Varna Bulgaria

Institute for Liver and Digestive Health Division of Medicine University College London London NW32PF UK

International Clinical Research Center St Anne's University Hospital 65691 Brno Czech Republic

Research Area for Multifactorial Diseases Research Unit of Molecular Genetics of Complex Phenotypes Bambino Gesù Children's Hospital IRCCS 00165 Rome Italy

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