Autophagy Suppresses Self-renewal Ability And Tumorigenicity Of Glioma-initiating Cells And Promotes Notch1 Degradation

Glioblastomas(GBMs)are the most common and primary malignant central nervous system tumors and have a poor prognosis.The current standard-of-care treatment consists of maximal surgical safe resection followed by concurrent chemo-radiotherapy and subsequent sequential temozolomide administration.Even with advances in targeted therapies and immunotherapies,the median survival of GBM patients is only 14.6 months.Glioma stem cells,i.e.,glioma initiating cells(GICs),which are capable of self-renewal,infinite proliferation,multiple potential differentiation,and vigorous tumorigenicity,are closely associated with GBM resistance to chemotherapy and radiotherapy.Notch pathway plays a important role in maintaining GIC self-renewal and tumorigenicity.The GIC stemness phenotype maintains as a result of Notch pathway activation.Although targeting the Notch pathway in the early stage of therapy can suppress the formation of a hypoxic tumor microenvironment and promote cell apoptosis,it has no significant benefit for GBM patients undergoing long term treatment.Targeting Notch pathway alone may be not adequate,GIC may survive supported by other potential activated pathway.The novel mechanism underlying Notch-pathway-dependent therapy will be discussed in this study.Autophagy is an evolutionarily conserved lysosome-dependent process that involves degradation of long-lived proteins and dysfunctional organelles and contributes to cell metabolism.In cancers,autophagy has pivotal functions since it prevents tumor progression.Recently,autophagy has been shown to promote differentiation and attenuate self-renewal of GICs.However,how autophagy regulates self-renewal and tumorigenicity of GICs is not well understood.In the present study,we evaluated the association between autophagy and the Notch1 pathway in the context of GIC self-renewal.Our findings for the first time have shown that autophagy suppressed GIC self-renewal and tumorigenicity.Also,our data reveal that autophagy inhibit Notch1 pathway activation by up-regulating Notch1 degradation.Therefore,autophagy-induced Notch1 degradation could be a promising therapeutic strategy for preventing GBM progression.This study mainly investigated the role and underlying mechanism of autophagy on maintaining GICs self-renewal and tumorigenicity,including three parts:1.Screening and identification of CD133 + glioma cells.To investigate the mechanism underlying maintenance of stemness in GICs,we established a CD133+ glioma neurosphere model in vitro: Magnetic-activated cell sorting(MACS)was used to collect CD133+ cells from U87 and U251 glioma cells;flow cytometry was then performed to quantify the CD133+ cells in the MACS+ population to confirm the effectiveness of the sorting.CD133 and Nestin were selected as markers to assess GIC stemness.Notch1 pathway activation was evaluated based on expression of NICD and target gene Hes1.Cell differentiation was assessed by expression of the astrocyte marker,glial fibrillary acidic protein(GFAP).The results showed that after MACS sorting,the percentage of CD133+ cells was significantly higher;the expressions of Nestin,CD133,Notch1 and NICD in CD133 + cells was positive;the CD133+ cells were then cultured in stem cell medium and formed neurospheres,while cells not sorted on the basis of CD133 positivity failed to develop spheroids under the same culture conditions.2.Inhibition of mTOR pathway induced autophagy to suppress self-renewal ability of GICs.Two classical mTOR pathway inhibitors,AZD8055 and Rapamycin,were selected to detect their effects on GIC survival.Western Bloting and immunofluorescence staining were used to detect the expressions of mTOR pathway-related proteins mTOR,p-mTOR,Akt,p-Akt and autophagy-related proteins p62 and LC3 B in GIC,which treated with AZD8055 and Rapamycin.After AZD8055 and rapamycin treatment,limiting dilution,neurosphere formation,and cell viability assays were performed to detect GIC self-renewal and proliferation.AZD8055+3-MA combination was used to confirm the direct autophagy-dependent GIC self-renewal inhibition.The expressions of Nestin,CD133 and GFAP were also detected by Western Blot and immunofluorescence staining.The results showed that treatment with different concentrations of AZD8055 and rapamycin for different durations decreased GIC viability;both AZD8055 and rapamycin suppressed the mTOR pathway and induced autophagy,Nestin and CD133 expression decreased,GFAP expression increased,and The difference in protein expression between each concentration group has a dose-dependent manner(P <0.05).With the increase of drug concentration,the self-renewal ability of GIC decreased;the number and capacity of neurospheres decreased;the proliferation of GIC decreased;the above differences were statistically significant(P <0.05).3.Autophagy inhibited Notch1 pathway activation and GIC tumorigenicity.First of this part,the expressions of Notch1 pathway related proteins Notch1,NICD and Hes1 were detected after AZD8055 and rapamycin treatment.The results suggested that expressions of Notch1 pathway-associated protein decreased in a dose-dependent manner(P<0.05).Further study showed that autophagy regulated Notch1 Degradation.After AZD8055 and rapamycin treatment,Notch1 expression on the membrane of GIC was detected by Western Blot.Co-localization of intracellular Notch1 and LC3 B was detected by immunofluorescence staining.The results showed that Notch1 expression decreased and Dll1 expression did not change on the plasma membrane(P<0.05).Co-localization of Notch1 with LC3 B were showed in the cytoplasm.Further more,to investigate the potential effects of AZD8055 and rapamycin on GIC tumorigenicity,an intracranial orthotopic xenograft model was generated by implanting U87 GICs into the brain.Seven days after implantation,DMSO,AZD8055 or rapamycin was injected intraperitoneally five days a week for 3 weeks.Bioluminescence imaging and hematoxylin-eosin staining revealed that GIC tumorigenicity was lower in the AZD8055 and rapamycin groups than that in the DMSO group;in particular,AZD8055 treatment resulted in smaller tumor volumes than rapamycin treatment.AZD8055-and rapamycin-treated mice survived markedly longer than DMSO-treated mice,where AZD8055-treated mice harbored the longest survival among the cohorts.These results show that AZD8055-and rapamycin-induced autophagy suppressed tumorigenicity of U87 GICs in vivo.in conclusion: 1.Inhibition of mTOR pathway could induce autophagy,and reduce the self-renewal ability of GICs;2.Autophagy inhibits the activation of Notch1 pathway;3.Autophagy suppresses Notch1 pathway activity via the autophagic vesicle engulfing and degrading Notch1;4.In vivo experiments shows that induced autophagy can inhibit the tumorigenicity of GICs.