Up-regulation Of Glycolysis Promotes The Stemness And EMT Phenotype In GEM-resistant PanCa Cells

Objective: Accumulating evidence suggests that cancer stem cells(CSCs)and epithelial–mesenchymal transition(EMT)-type cells are associated with chemoresistance,tumor recurrence,and metastasis in pancreatic cancer(Pan Ca).Previous findings show that metabolic reprogramming can determine cancer cell pluripotency.We aimed to investigate the mechanisms-particularly glycolysis-involved in the regulation of chemoresistance,CSC and EMT phenotypes in gemcitabine(GEM)resistant Pan Ca cells.Methods: A cell model of GEM resistance(GR)was established in Pan Ca.The glucose uptake of GR cells and their parental-GEM sensitive(GS)cells was examined using fluorescence microscopy and flow cytometry.Extracellular lactate was measured using lactic assay kit.In addition,the chemosensitivity toward 2-DG of Pan Ca cells was analyzed using MTT assay.Additionally,glycolysis-associated molecules such as GLUT1,HKII,PKM2,and LDHA were measured by Western blot and q RT-PCT analyses.Stemness-like characteristics were measured via examination of pluripotent markers(Nanog and Sox2),CSCs surface marker(CD24 and CD133)and sphere-formation assay.EMT phenotype was examined in terms of EMT markers such as E-cadherin,Vimentin and Snail1,cell morphology,migration and invasion abilities.Reactive oxygen species(ROS)levels in Pan Ca cells treated with or without 2-DG in the presence or absence of NAC were examined via flow cytometry;Meanwhile,Stemness-like and EMT phenotype in GR cellswere examined via inhibition of glycolysis,which was further validated by introduction of exogenous hydrogen peroxide(H2O2).We further evaluated the role of DCLK1 in the regulation of CSC and EMT phenotype.Moreover,we examined the relationship of DCLK1 with glycolysis and ROS.Finally,we evaluated the tumorigenesis and metastasis of GR cells treated with 2-DG or not in vivo.Results: GR cells were more resistant to GEM than their parental GS cells.GR cells were more glycolytic than GS cells.The levels of glucose uptake and extracellular lactate in GR cells were significantly higher than those in GS cells(both P < 0.05).GR cells showed higher protein and m RNA levels of GLUT1,HKII,PKM2 and LDHA than GS cells.GR cells were more dependent on glycolysis for survival.Cell viability of GR cells towards 2-DG were more significantly decreased compared with GS cells.In addition,the IC50 of GR cells towards GEM was more significantly decreased compared with GS cells when combined with 2-DG.GR cells showed CSC-like characteristics relative to GS cells.GR cells demonstrated higher expression of the stemness markers Nanog,Sox2 and acquired greater sphere-formation ability than GS cells.What's more,GR cells showed an obvious EMT phenotype with spindle-shaped morphology.The epithelial marker E-cadherin was decreased,while Vimential and Snail1 were up-regulated in GR cells compared with GS cells.Importantly,GR cells showed enhanced migratory and invasive ability relative to GS cells.Flow cytometry detected lower levels of ROS in GR than GS cells.However,inhibition of glycolysis using 2-DG increased ROS levels in GR cells,which was rescued by introduction of ROS scavenger NAC.Meanwhile,the stemness and EMT phenotype of GR cells was inhibited by 2-DG treatment with decreased expression of Nanog,Sox2,and weakened sphere-formation ability;and decreased mesenchymal markers Vimentin and Snail1,increased epithelial marker E-cadherin,decreased migratory and invasive ability,which was partially rescued by NAC.Interestingly,exogenous H2O2 induced similar biological effects like 2-DG.The stemness and EMT phenotype of GR cells was inhibitedby H2O2,which was also alleviated by NAC.GR cells had higher expression of DCLK1,a Pan Ca CSC marker.DCLK1 regulated the stemness and EMT phenotype of GR cells as knockdown of DCLK1 inhibited the stemness and EMT phenotype of GR cells.Importantly,DCLK1 was regulated by glycolysis linked with low ROS levels.GS and GR cells had similar tumorigenic ability in vivo,while DCLK1 was highly expressed in tumors formed by GR cells using immunohistochemistry staining.GR cells demonstrated enhanced metastatic ability compared with GS cells in vivo.However,inhibition of glycolysis weakened the tumorigenicity and metastasis of GR cells in vivo.Conclusions: GEM-resistant Pan Ca cells demonstrate enhanced glycolytic flux,stemness and EMT phenotypes compared with their parental cells.Glycolysis regulates the stemness and EMT phenotype via regulation of DCLK1,which was mediated by maintenance of lower ROS levels.The ROS-glycolysis-DCLK1 signaling axis might be promising targets for reversing chemoresistance,the stemness and EMT phenotype in PanCa.