Mitochondrial oxidative metabolism contributes to a cancer stem cell phenotype in cholangiocarcinoma

BACKGROUND AND AIMS: Little is known about the metabolic regulation of cancer stem cells (CSC) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent metabolism and related signaling pathways contribute to stem state 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)-1alpha, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH by metformin, or PGC-1alpha 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 CCA patients, expression of PGC-1alpha was correlated to expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1alpha expression by immunostaining had lower overall survival (OS) and progression-free survival, higher angioinvasion and faster recurrence. In GSEA analysis, CCA patients with high levels of mitochondrial Complex II had shorter OS and time to recurrence.CONCLUSIONS: The CCA stem-subset has a more efficient respiratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1alpha to maintain CSC features.LAY SUMMARY: The growth of many cancers is sustained by a specific type of cells with more embryonic characteristics, termed as '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-1alpha, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treatments for cholangiocarcinoma.