000099089 001__ 99089
000099089 005__ 20210902121942.0
000099089 0247_ $$2doi$$a10.4252/wjsc.v12.i11.1410
000099089 0248_ $$2sideral$$a122331
000099089 037__ $$aART-2020-122331
000099089 041__ $$aeng
000099089 100__ $$aJagust, P.
000099089 245__ $$aGlutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells
000099089 260__ $$c2020
000099089 5060_ $$aAccess copy available to the general public$$fUnrestricted
000099089 5203_ $$aBACKGROUND
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 up-regulation 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.
000099089 536__ $$9info:eu-repo/grantAgreement/ES/MS-ISCIII-FSE/CP16-00121$$9info:eu-repo/grantAgreement/ES/ISCIII-FSE/El FSE invierte en tu futuro$$9info:eu-repo/grantAgreement/ES/ISCIII/FIS/PI17-00082$$9info:eu-repo/grantAgreement/EC/FP7/602783/EU/Novel therapy for pancreatic cancer/CAM-PAC$$9info:eu-repo/grantAgreement/ES/FEDER/Una manera de hacer Europa$$9info:eu-repo/grantAgreement/EC/ERC/Pa-CSC-233460
000099089 540__ $$9info:eu-repo/semantics/openAccess$$aby-nc$$uhttp://creativecommons.org/licenses/by-nc/3.0/es/
000099089 590__ $$a5.326$$b2020
000099089 591__ $$aCELL & TISSUE ENGINEERING$$b14 / 29 = 0.483$$c2020$$dQ2$$eT2
000099089 591__ $$aCELL BIOLOGY$$b72 / 195 = 0.369$$c2020$$dQ2$$eT2
000099089 592__ $$a0.907$$b2020
000099089 593__ $$aCell Biology$$c2020$$dQ2
000099089 593__ $$aGenetics$$c2020$$dQ2
000099089 593__ $$aMolecular Biology$$c2020$$dQ2
000099089 593__ $$aHistology$$c2020$$dQ2
000099089 593__ $$aGenetics (clinical)$$c2020$$dQ2
000099089 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000099089 700__ $$aAlcalá, S.
000099089 700__ $$aJr, B.S.
000099089 700__ $$aHeeschen, C.
000099089 700__ $$0(orcid)0000-0002-8624-8757$$aSancho, P.
000099089 773__ $$g12, 11 (2020), 1410-1428$$pWorld j. stem cells$$tWorld Journal of Stem Cells$$x1948-0210
000099089 8564_ $$s833739$$uhttps://zaguan.unizar.es/record/99089/files/texto_completo.pdf$$yVersión publicada
000099089 8564_ $$s2420002$$uhttps://zaguan.unizar.es/record/99089/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000099089 909CO $$ooai:zaguan.unizar.es:99089$$particulos$$pdriver
000099089 951__ $$a2021-09-02-11:01:38
000099089 980__ $$aARTICLE