Glutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells

BACKGROUND Cellular metabolism regulates stemness in health and disease.A reduced redox state is essential for self-renewal of normal and cancer stem cells(CSCs).However,while stem cells rely on glycolysis,different CSCs,including pancreatic CSCs,favor mitochondrial metabolism as their dominant energy-producing pathway.This suggests that powerful antioxidant networks must be in place to detoxify mitochondrial reactive oxygen species(ROS)and maintain stemness in oxidative CSCs.Since glutathione metabolism is critical for normal stem cell function and CSCs from breast,liver and gastric cancer show increased glutathione content,we hypothesized that pancreatic CSCs also rely on this pathway for ROS detoxification.AIM To investigate the role of glutathione metabolism in pancreatic CSCs.METHODS Primary pancreatic cancer cells of patient-derived xenografts(PDXs)were cultured in adherent or CSC-enriching sphere conditions to determine the role of glutathione metabolism in stemness.Real-time polymerase chain reaction(PCR)was used to validate RNAseq results involving glutathione metabolism genes in adherent vs spheres,as well as the expression of pluripotency-related genes following treatment.Public TCGA and GTEx RNAseq data from pancreatic cancer vs normal tissue samples were analyzed using the webserver GEPIA2.The glutathione-sensitive fluorescent probe monochlorobimane was used to determine glutathione content by fluorimetry or flow cytometry.Pharmacological inhibitors of glutathione synthesis and recycling[buthionine-sulfoximine(BSO)and 6-Aminonicotinamide(6-AN),respectively]were used to investigate the impact of glutathione depletion on CSC-enriched cultures.Staining with propidium iodide(cell cycle),Annexin-V(apoptosis)and CD133(CSC content)were determined by flow cytometry.Self-renewal was assessed by sphere formation assay and response to gemcitabine treatment was used as a readout for chemoresistance.RESULTS Analysis of our previously published RNAseq dataset E-MTAB-3808 revealed upregulation of genes involved in the KEGG(Kyoto Encyclopedia of Genes and Genomes)Pathway Glutathione Metabolism in CSC-enriched cultures compared to their differentiated counterparts.Consistently,in pancreatic cancer patient samples the expression of most of these up-regulated genes positively correlated with a stemness signature defined by NANOG,KLF4,SOX2 and OCT4 expression(P<10-5).Moreover,3 of the upregulated genes(MGST1,GPX8,GCCT)were associated with reduced disease-free survival in patients[Hazard ratio(HR)2.2-2.5;P=0.03-0.0054],suggesting a critical role for this pathway in pancreatic cancer progression.CSC-enriched sphere cultures also showed increased expression of different glutathione metabolism-related genes,as well as enhanced glutathione content in its reduced form(GSH).Glutathione depletion with BSO induced cell cycle arrest and apoptosis in spheres,and diminished the expression of stemness genes.Moreover,treatment with either BSO or the glutathione recycling inhibitor 6-AN inhibited self-renewal and the expression of the CSC marker CD133.GSH content in spheres positively correlated with intrinsic resistance to gemcitabine treatment in different PDXs r=0.96,P=5.8×1011).Additionally,CD133+cells accumulated GSH in response to gemcitabine,which was abrogated by BSO treatment(P<0.05).Combined treatment with BSO and gemcitabine-induced apoptosis in CD133+cells to levels comparable to CD133-cells and significantly diminished self-renewal(P<0.05),suggesting that chemoresistance of CSCs is partially dependent on GSH metabolism.CONCLUSION Our data suggest that pancreatic CSCs depend on glutathione metabolism.Pharmacological targeting of this pathway showed that high GSH content is essential to maintain CSC functionality in terms of self-renewal and chemoresistance.

Lipid droplets as metabolic determinants for stemness and chemoresistance in cancer

Previously regarded as simple fat storage particles,new evidence suggests thatlipid droplets(LDs)are dynamic and functional organelles involved in keycellular processes such as membrane biosynthesis,lipid metabolism,cellsignalling and inflammation.Indeed,an increased LD content is one of the mostapparent features resulting from lipid metabolism reprogramming necessary tosupport the basic functions of cancer cells.LDs have been associated to differentcellular processes involved in cancer progression and aggressiveness,such astumorigenicity,invasion and metastasis,as well as chemoresistance.Interestingly,all of these processes are controlled by a subpopulation of highly aggressivetumoral cells named cancer stem cells(CSCs),suggesting that LDs may befundamental elements for stemness in cancer.Considering the key role of CSCs onchemoresistance and disease relapse,main factors of therapy failure,the design ofnovel therapeutic approaches targeting these cells may be the only chance forlong-term survival in cancer patients.In this sense,their biology and functionalproperties render LDs excellent candidates for target discovery and design ofcombined therapeutic strategies.In this review,we summarise the currentknowledge identifying LDs and CSCs as main contributors to cancer aggressiveness,metastasis and chemoresistance.

Metabolic determinants of stemness in medulloblastoma

Medulloblastomas(MBs)are the most prevalent brain tumours in children.They are classified as grade IV,the highest in malignancy,with about 30%metastatic tumours at the time of diagnosis.Cancer stem cells(CSCs)are a small subset of tumour cells that can initiate and support tumour growth.In MB,CSCs contribute to tumour initiation,metastasis,and therapy resistance.Metabolic differences among the different MB groups have started to emerge.Sonic hedgehog tumours show enriched lipid and nucleic acid metabolism pathways,whereas Group 3 MBs upregulate glycolysis,gluconeogenesis,glutamine anabolism,and glutathione-mediated anti-oxidant pathways.Such differences impact the clinical behaviour of MB tumours and can be exploited therapeutically.In this review,we summarise the existing knowledge about metabolic rewiring in MB,with a particular focus on MB-CSCs.Finally,we highlight some of the emerging metabolism-based therapeutic strategies for MB.

Mitochondrial determinants of chemoresistance

Chemoresistance constitute nowadays the major contributor to therapy failure in most cancers.There are main factors that mitigate cell response to therapy,such as target organ,inherent sensitivity to the administered compound,its metabolism,drug efflux and influx or alterations on specific cellular targets,among others.We now know that intrinsic properties of cancer cells,including metabolic features,substantially contribute to chemoresistance.In fact,during the last years,numerous reports indicate that cancer cells resistant to chemotherapy demonstrate significant alterations in mitochondrial metabolism,membrane polarization and mass.Metabolic activity and expression of several mitochondrial proteins are modulated under treatment to cope with stress,making these organelles central players in the development of resistance to therapies.Here,we review the role of mitochondria in chemoresistant cells in terms of metabolic rewiring and function of key mitochondria-related proteins.