Inhibition Of DYRK1A-EGFR Axis By P53-MDM2 Cascade Mediates The Induction Of Cellular Senescence

Cellular senescence is characterized by irreversible cell-cycle arrest.Senescent cells manifest morphological changes(increase in cell size,flattened appearance),metabolic changes and a secretory phenotype(senescence-associated secretory phenotype).Telomere dysfunction,DNA damage,unscheduled oncogene activation or oxidative stress can each induce cellular senescence.Cellular senescence participates embryonic development and contributes to organ and tissue aging.Furthermore,cells with senescent cell properties can be found in the affected tissues of patients with age-related diseases such as osteoarthritis,pulmonary fibrosis,atherosclerosis,and Alzheimer's disease.Senescence also serves as a defense mechanism against malignant transformation.Thus,unraveling the mechanism of cellular senescence is critical for understanding oncotherapy and aging process.Cellular senescence is mainly established and maintained by p53 and pl6-pRb pathways.p53 induced senescence is mainly caused by activating its target genes.p16 inhibits cell cycle protein-dependent kinase.Upon encountering DNA damage,oncogene activation,oxidative stress or other stimuli,p53 is activated and transactivates a wide array of genes involved in cell cycle progression,redox homeostasis and metabolisms.p53 regulates its target genes such as p21,PML,CXCR2,PAI-1 and E2F7 to induce senescence.However,excessive activation of p53 can lead to cell death or cellular senescence.The activity of p53 is negatively regulated by MDM2,a p53 target itself.MDM2 ubiquitinates and destabilizes p53.p53 and MDM2 thus form a feedback loop.An antiterminating homozygous germline mutation in MDM2 was found to abrogate its activity to destabilize p53,thereby resulting in enhanced levels and stability of p53,causing segmental progeroid syndrome.Many literatures also reported that MDM2 catalyzes the ubiquitination and degradation of other proteins.The substrates ubiquitinated by MDM2 included FOXO3A,DHFR,SNAIL and H2A and were subjected to either polyubiquitination or monoubiquitination.Those substrates participated in diverse biological processes such as cell cycle progression,metabolism,redox homeostasis,epithelial-mesenchymal transition and epigenetic regulation.The known pathways that execute senescence program include p53-ROS,p53-PML,p53-CXCR2 and p53-p21.Activation of p53-ROS positive feedback loop induced senescence.p53 tumor suppressor promoted cell cycle arrest by promyelocytic leukemia gene(PML).CXCR2 was a critical mediator of cellular senescence activated by p53.Cells lacking p21,PML or CXCR2 showed a reduced propensity to undergo senescence in response to p53 activation,despite the induction of other p53 target genes.Although many pathways downstream of p53 activation have been identified to drive cellular senescence,it appears that each of those pathways may only operate in certain context or only account for the partial effect of p53 activation.Thus,additional senescence-driving pathways remain to be elucidated.Epidermal growth factor receptor(EGFR)is amplified or mutated in various human tumors,such as breast cancer,lung cancer,and prostate cancer.Upregulation of EGFR is associated with development and poor prognosis of many cancers.The dysfunction of EGFR affects downstream signaling,which eventually leads to proliferation,enhanced angiogenesis,necrosis and drug resistance.It was also reported that the transcription of EGFR was regulated by p53.Since p53 was reported to transactivate or repress EGFR expression,we investigated whether the positive or negative regulation of EGFR by p53 contributes to stress-induced senescence.PART ?Downregulation of EGFR mediates cellular senescence induced by p53 activationTranscriptional factor p53 can be activated by a variety of genotoxic stress such as DNA damage,hypoxia,and oxidative stress.p53 activation can inhibit the growth of a variety of cancer cells by inducing cell cycle arrest or cellular senescence.Nutlin-3a(Nut3a),an antagonist of MDM2,targets the interaction between p53 and murine double minute 2(MDM2),and inhibits ubiquitination and degradation of p53,which leads to p53 stabilization.In our study,we found that in U87,A172 cells and U20S,HT1080 cells,p53 stabilization and activation by Nut3a induced senescence.At the same time,p53 activation can inhibit the protein level of EGFR.To prove the downregulation of EGFR mediated the p53 induced senescence,we used EGFR pharmacological inhibitors or its small interference RNA to downregulate EGFR,and found that they all induced senescence.In addition,ectopic overexpression of EGFR rescued p53 induced cellular senescence.And berberine also induced senescence by downregulating EGFR-MEK-ERK signaling pathway.Next,we tested the transcription of EGFR when p53 was activated,and found that EGFR mRNA level was increased in U20S cells,but decreased in U87,HT1080 and A172 cells.These results suggested that although repression of EGFR transcription may contribute to the downregulation of EGFR when p53 was activated,reduction in EGFR can also be accompanied by an increased EGFR transcription.These results suggested that post-transcriptional regulation likely plays an important role in determining the eventual amount of EGFR,and the regulation of EGFR by p53 maybe cell type specific.PART ?EGFR-stabilizing DYRK1A is negatively regulated by p53 Activation of p53 downregulated EGFR protein level,but increased the transcription level,it suggested that the p53 may reduce the stability of EGFR.It was reported that DYRK1A was critical in maintaining the stability of EGFR,and inhibition of DYRK1A reduced the stability of EGFR and inhibited the growth of glioblastoma.Another article reported that p53 decreased DYRK1A by miR-1246.Therefore,we decided to test whether DYRK1A was involved in the negative regulation of EGFR by p53.Indeed,depletion of DYRK1A by RNAi as well as its inhibition by harmine resulted in a significant reduction in EGFR.Consequently,the percentage of senescent cells was significantly increased by either treatment.Importantly,DYRK1A was negatively regulated by p53,activation of p53 by Nut3a leaded to a significant reduction in DYRX1A.Ectopic expression of p53 similarly decreased the level of DYRK1A.As expected,the reduction in DYRK1A caused by Nut3a treatment was greatly attenuated in p53 knockdown cells.Furthermore,while DYRK1A and EGFR were both reduced by Nut3a,they occurred sequentially,with the reduction of DYRK1A preceding that of EGFR,in agreement with the interpretation that EGFR change is consequential to the downregulation of DYRK1A.As expected,ectopic expression of DYRK1A blocked the reduction in EGFR caused by p53 activation.Furthermore,DYRK1A greatly rescued p53 activation-induced cellular senescence,further supporting the notion that inhibition of DYRK1A-EGFR axis mediated the induction of senescence caused by p53 activation.It was previously reported that p53 can downregulate DYRK1A by transactivating miR-1246.But in our system,p53-induced downregulation of DYRK1A was not mediated by miR-1246.PART IIINegative regulation of DYRK1A by p53 is mediated by MDM2 E3 ubiquitin ligaseThe downregulation of DYRK1A by p53 activation was not due to decreased transcription.But MG132 could indeed attenuate the downregulation of DYRK1A caused by p53 activation;therefore the reduction of DYRK1A may be due to increased proteosomal degradation.Because MDM2,a p53 transcriptional target,is an ubiquitin ligase,promotes p53 ubiquitin and degradation.We here demonstrated DYRK1A was a novel substrate of MDM2.Strikingly,ectopic expression of MDM2 nearly abolished DYRK1A.Depletion of MDM2 by RNAi,on the other hand,blocked the effect of Nut3a.These results suggested that the negative regulation of DYRK1A by p53 was mediated by MDM2.We next determined whether MDM2 physically interacts with DYRK1A by transfecting cells with His-tagged DYRK1A and Myc-tagged MDM2 expression vectors,and we found that the two proteins could indeed be reciprocally co-immunoprecipitated.We next determined whether MDM2 could polyubiquitinate DYRK1A.We found that the ectopic expression of wild-type MDM2 significantly increased the amount of polyubiquitinated DYRK1A,but not that of mutant MDM2(C464A).Moreover,the ubiquitination of DYRK1A was greatly increased by Nut3a.However,when MDM2 was depleted,the DYRK1A ubiquitination was also reduced,further supporting that the increased ubiquitination of DYRK1A caused by p53 activation was dependent on MDM2.These results indicated that the downregulation of DYRK1A by p53 was mediated by MDM2.PART IVNut3a downregulates DYRK1A-EGFR axis and induces senescence of glioblastoma in vivo Glioblastoma multiforme(GBM)is the most aggressive form of gliomas.DYRK1A was highly expressed in GBM and regulated EGFR.Therefore,inhibition of DYRK1A could represent a promising therapeutic intervention for EGFR-dependent GBM.We tested whether p53 activation would reduce tumor growth in vivo and whether it would also lead to a downregulation of DYRK1A-EGFR signaling cascade.Significant increase in senescence was detected in tumors of the Nut3a treatment group.Importantly,EGFR was nearly absent in the tumors in treatment group.Correspondingly,DYRK1A was drastically downregulated.But ectopic expression of EGFR significantly attenuated tumor shrinkage and senescence induced by Nut3a.Taken together,these results indicate that the negative regulation of DYRK1A-EGFR axis by p53 also operates in vivo and may suppress tumor growth by promoting cellular senescence.Because EGFR is commonly upregulated in glioblastoma,the demonstration of effective inhibition of EGFR by p53 activation points to the possibility of employing Nut3a in the treatment of patients with glioblastoma.