Chiara Raggi1, Maria Letizia Taddei2, Elena Sacco3, Nadia Navari2, Margherita Correnti4, Benedetta Piombanti2, Mirella Pastore2, Claudia Campani2, Erica Pranzini2, Jessica Iorio2, Giulia Lori2, Tiziano Lottini2, Clelia Peano5, Javier Cibella6, Monika Lewinska7, Jesper B Andersen7, Luca di Tommaso8, Luca Viganò9, Giovanni Di Maira2, Stefania Madiai2, Matteo Ramazzotti10, Ivan Orlandi3, Annarosa Arcangeli2, Paola Chiarugi11, Fabio Marra12. 1. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. Electronic address: chiara.raggi@unifi.it. 2. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy. 3. SYSBIO, Centre of Systems Biology, Milan, Italy; Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy. 4. Center for Autoimmune Liver Diseases, Humanitas Clinical and Research Center, Rozzano, Italy. 5. Genomic Unit, IRCCS, Humanitas Clinical and Research Center, Rozzano, Italy; Institute of Genetic and Biomedical Research, UoS Milan, National Research Council, Rozzano, Italy. 6. Genomic Unit, IRCCS, Humanitas Clinical and Research Center, Rozzano, Italy. 7. Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark. 8. Department of Pathology, Humanitas Clinical and Research Center, Rozzano, Italy; Department of Biomedical Sciences, Humanitas University, Rozzano, Italy. 9. Department of Biomedical Sciences, Humanitas University, Rozzano, Italy; Department of Hepatobiliary Surgery, Humanitas Clinical and Research Center, Rozzano, Italy. 10. Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy. 11. Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy; Excellence Center for Research, Transfer and High Education DenoTHE, Florence, Italy. 12. Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; Excellence Center for Research, Transfer and High Education DenoTHE, Florence, Italy. Electronic address: fabio.marra@unifi.it.
Abstract
BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.
BACKGROUND & AIMS: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stemness in CCA. METHODS: The stem-like subset was enriched by sphere culture (SPH) in human intrahepatic CCA cells (HUCCT1 and CCLP1) and compared to cells cultured in monolayer. Extracellular flux analysis was examined by Seahorse technology and high-resolution respirometry. In patients with CCA, expression of factors related to mitochondrial metabolism was analyzed for possible correlation with clinical parameters. RESULTS: Metabolic analyses revealed a more efficient respiratory phenotype in CCA-SPH than in monolayers, due to mitochondrial oxidative phosphorylation. CCA-SPH showed high mitochondrial membrane potential and elevated mitochondrial mass, and over-expressed peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1α silencing or pharmacologic inhibition (SR-18292), impaired spherogenicity and expression of markers related to the CSC phenotype, pluripotency, and epithelial-mesenchymal transition. In mice with tumor xenografts generated by injection of CCA-SPH, administration of metformin or SR-18292 significantly reduced tumor growth and determined a phenotype more similar to tumors originated from cells grown in monolayer. In patients with CCA, expression of PGC-1α correlated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1α expression by immunostaining had lower overall and progression-free survival, increased angioinvasion and faster recurrence. In GSEA analysis, patients with CCA and high levels of mitochondrial complex II had shorter overall survival and time to recurrence. CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1α to maintain CSC features. LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed 'cancer stem cells'. These cells have been described in cholangiocarcinoma, a type of liver cancer with poor prognosis and limited therapeutic approaches. We demonstrate that cancer stem cells in cholangiocarcinoma have different metabolic features, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1α, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.