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

Background & Aims: Little is known about the metabolic regulation of cancer stem cells (CSCs) in cholangiocarcinoma (CCA). We analyzed whether mitochondrial-dependent meta- bolism 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)-1a, a master regulator of mitochondrial biogenesis. Targeting mitochondrial complex I in CCA-SPH using metformin, or PGC-1a 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 gener- ated 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-1acorrelated with expression of mitochondrial complex II and of stem-like genes. Patients with higher PGC-1a 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 respi- ratory phenotype and depends on mitochondrial oxidative metabolism and PGC-1a 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 fea- tures, and use mitochondria, an organelle located within the cells, as the major source of energy. We also identify PGC-1a, a molecule which regulates the biology of mitochondria, as a possible new target to be explored for developing new treat- ments for cholangiocarcinoma. (c) 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Automated summary

The study reveals that cancer stem cells in cholangiocarcinoma have a more efficient respiratory phenotype, relying on mitochondrial oxidative metabolism and PGC-1a to maintain CSC features. Targeting mitochondrial complex I or PGC-1a in CCA-SPH impairs spherogenicity and expression of markers related to CSC phenotype, pluripotency, and epithelial-mesenchymal transition. Administration of metformin or SR-18292 in mice with CCA-SPH xenografts significantly reduces tumor growth and reverts the phenotype to that of cells grown in monolayer.