The Function And Mechanism Of Carnitine Palmitoyltransferase 1A On Maintaining The Stemness Of Glioma Stem Cells

Glioblastoma?GBM?,one of the most common primary malignant tumors of the central nervous system,exhibits a high incidence of recurrence and poor prognosis.Glioma stem cells?GSCs?are a subpopulation of glioma cells,characterized by the abilities of self-renewal,multi-differentiation and initiation of tumorigenesis.They sustain stemness features and could differentiate into glioma cells upon tumor microenvironmental stimulation,thus contribute to tumor heterogenicity.Meanwhile,GSCs are highly resistant to radiotherapy and chemotherapy due to their high invasive nature,quiescent phenotype and preferential activation of anti-apoptotic/pro-survival pathways.Therefore,GSCs are regarded as the"seeds"of gliomagenesis,recurrence and treatment resistance.Investigations of the molecular mechanisms underlying GSC phenotypes and specific therapeutic targets against GSCs are crucial for GBM therapy.Metabolic dysfunction is one of the hallmarks of malignant tumors.Recent studies have shown that tumor cells may hijack lipid metabolism for tumor progression.Whereas lipid molecules could hardly be observed in normal brain cells,aberrant lipid accumulation are frequently found in glioma cells.In silico analyses of TCGA database also identify the distinct expressions of lipid metabolization-related genes in different grades of gliomas.However,whether lipid metabolism is pivotal for maintaining GSC features is still unknown.CPT1A is a member of CPT1,which located at the outer membrane of mitochondria and majorly expressed in the liver and brain.Aberrant expression of CPT1A in GBMs has been reported,but its role in regulating GSC stemness and the associated clinical significance are still unknown.Herein,we investigated the effect of CPT1A on regulating the lipid metabolism in GSCs.We also investigated the expression and clinicopathological significance of CPT1A in human GBMs.1.We establish in vitro GSC differentiation model,and identify fatty acid accumulation during GSC differentiation.We established an in vitro GSC differentiation model as previously described.The cells were harvested on day 0,1,3,5 and 7 upon serum stimulation,and the expressions of astrocyte marker GFAP,and GSC markers SOX2 and OLIG2 were determined by Western blot.The results showed that GFAP expression increased,whereas the expressions of SOX2 and OLIG2 were gradually decreased during GSC differentiation.These data indicated that the in vitro GSC differentiation model was successfully established.Using the established in vitro GSC differentiation model,we observed the accumulation of lipid droplets and fatty acid in cells at different time of GSC differentiation by BODIPY^TM fluorescence staining.Lipid droplets could hardly be detected in GSCs,and the accumulation of lipid droplets increased over time during GSC differentiation.To note,on day 7 following GSC differentiation,the content of fatty acids in differentiated GSCs increased about twice than that on the starting day.Collectively,these results indicate that lipid droplets and fatty acid accumulation increased continuously during GSC differentiation.2.Long-chain fatty acids are the major accumulated fatty acid during GSC differentiation.We further identified the diversity of the accumulated fatty acids in GSCs and differentiated non-GSCs on days 0,1,3,5 and 7 post-differentiation by mass spectrometry.The majority of accumulated fatty acids in GSCs were long-chain fatty acids,including pentadecanoic acid,heptadecenoic acid,linoleic acid,arachidonic acid,and hexadecenoic acid.3.Exogenous fatty acids promote GSC differentiation.To investigate the function of fatty acid in GSC differentiation,we added extra fatty acids to GSC medium.The addition of exogenous fatty acids resulted in increased accumulation of lipid droplets in GSC.Western blot assay was carried out to detect the expression of GSC stemness markers and differentiation markers.Data shows that in comparison with control group,the expression of GFAP increased,while SOX2 and OLIG2 were decreased during GSC differentiation.These data suggested that fatty acids could promote GSC differentiation.4.Knockdown CPT1A inhibits GSC differentiation.Lentivirus was applied to knockdown CPT1A.Western blot analysis was carried out to detect the expression of GSC stemness markers and differentiation markers.The expression of SOX2 and OLIG2 were significantly increased after CPT1A knockdown,whereas the expression of GFAP was decreased.Limiting dilution experiments further confirmed that the self-renewal ability of GSCs was significantly increased in CPT1A-knockdown cells.These results indicated that CPT1A knockdown inhibited GSC differentiation.We labeled mitochondria with TOM20,a mitochondrial outer membrane protein,in CPT1A-knockdown cells or CPT1A inhibitor Etomoxir?ETO?-treated cells,followed by evaluating area,and perimeter of mitochondria.As compared with the control group,the CPT1A knockdown groups displayed the mitochondrial area by 34%and 27%,and the mitochondrial perimeter by 34%and 25%,respectively.The same tendency was observed in ETO-treated cells.Collectively,the data demonstrate that the area and perimeter of mitochondria are all reduced by inhibiting the expression or activity of CPT1A,suggesting that CPT1A impairment promotes mitochondrial fission.5.CPT1A expression is increased in CD133-negative non-GSCs,and negatively correlates with glioma grades.Western blot analyses showed that CPT1A expression was decreased in GBM cells relative to control astrocytes.Immunofluorescent staining demonstrated a reduced level of CPT1A in CD133⁺GSCs in comparison with CD133^-non-GSCs.Bioinformatic analyses of the TCGA database demonstrated that CPT1A expression was negatively correlated with glioma grades.In summary,our data reveals the accumulation of fatty acid,especially the long-chain fatty acids during GSC differentiation.Disruption of CPT1A promotes mitochondrial fission,maintains GSC stemness.Our results indicate that CPT1A is a potential therapeutic target for GBMs.