GZN1201804). Authors contributions X.X., Q.L., and C.S. when ectopically expressed, can attenuate p53 activation-induced EGFR reduction and cellular senescence. We further showed that the increased degradation of DYRK1A caused by p53 activation was mediated by MDM2. MDM2 was found to physically interact with and ubiquitinate DYRK1A, ultimately leading to its proteosomal degradation. Importantly, administration of Nutlin-3a, which disrupts the binding of MDM2 to p53, but not that of MDM2 to DYRK1A, reduced the levels of DYRK1A and EGFR, induced senescence, and inhibited growth of tumor xenografts formed by U87 glioblastoma cells. Ectopic expression of EGFR in tumor xenografts attenuated senescence and tumor reduction caused by Nultin-3a. Our findings thus established a novel link between p53 and EGFR and may have implications in p53 activation-based therapies. Introduction Upregulation of epidermal growth factor receptor (EGFR), in the forms of amplification and activating point mutation, was commonly detected in lung cancer1C3, gliblastomas4, esophageal squamous cell cancers5, and many other types of cancer6. The gain of function in EGFR plays a critical role in driving the Meclofenoxate HCl proliferation and survival of many types of cancer cells, via upregulating the AKT and MAPK pathways. Correspondingly, treatment of lung cancers bearing EGFR mutations with EGFR tyrosine kinase inhibitors Gefitinib and Erlotinib has been shown to be much more effective than chemotherapy7C9. In addition, upregulation of EGFR in tumor stroma also mediates angiogenesis and resistance to vascular endothelial growth factor (VEGF) inhibitor10. Cancer cells can even transfer activated EGFR to macrophages and thereby suppress innate immunity11. Therefore, inhibition of EGFR signaling by RTK inhibitor or Rabbit polyclonal to ABHD12B antibodies has far-reaching clinical implications. is the most commonly mutated tumor suppressor gene in human cancer12. p53, the protein encoded by has been shown to be either up- or downregulated by p53 at the transcription level, depending on cell lines or cell types under study22C25. Many factors were also identified to regulate EGFR turnover at protein level26C28. Dual-specificity tyrosine-phosphorylated and tyrosine-regulated kinase 1A, or DYRK1A, was shown to promote the stabilization of EGFR by phosphorylating SPRY2, which interferes with the Cbl-mediated ubiquitination of Meclofenoxate HCl EGFR29. Interestingly, DYRK1A can be negatively regulated by p53 via miR-124630. Therefore, diverse mechanisms may govern the regulation of EGFR by p53. Downregulation of EGFR-MEK-ERK signaling pathway is sufficient to induce cellular senescence in glioblastoma cells31. In an effort to elucidate the mechanisms underlying the cellular senescence induced by p53 activation, we found that downregulation of EGFR can also mediate p53-induced senescence in a subset of cancer cell lines. The downregulation of EGFR by p53 is achieved at both the transcriptional level and protein level. Even in cells in which transcription is enhanced by p53 activation, EGFR protein level can still be reduced. DYRK1A, which is required for the maintenance of EGFR stability, is downregulated by p53. We further showed that the downregulation of DYRK1A is mediated by p53 target gene was increased. A luciferase reporter containing EGFR promoter showed a reduction in luciferase activity when treated by Nutlin-3a (Fig.?S3A), indicating that p53 could negatively regulate transcription. However, in contrast to the reduction of EGFR at the protein level, transcription showed a positive response to p53 activation in U2OS and A2780 cells (Fig.?S3B and S3C). mRNA levels were reduced by Nutlin-3a in A172 and HT1080 cells (Fig.?S3D and S3E). These results suggest that while repression of transcription may contribute to the downregulation of EGFR when p53 is activated, reduction in EGFR can occur in the presence of increased transcription. On the other Meclofenoxate HCl hand, while the protein amount of EGFR was elevated in A549 cells in response to Nutlin-3a treatment, mRNA level was reduced (Fig.?S4). These results suggest that post-transcriptional regulation likely plays an important role in determining the eventual amount of EGFR. Downregulation of EGFR mediates cellular senescence induced by p53 activation The activation of p53 can either lead to apoptosis or cellular senescence depending on cell types. We next examined the fates of the cells in which EGFR was downregulated by p53 activation. Nutlin-3a treatment strikingly induced cellular senescence in U87 and U2OS cells, as shown by positive senescence-associated -galactosidase (SA–gal) staining, reduction of lamin B1, and reduced 5-ethynyl-2-deoxyuridine (EdU) incorporation, p16 (Fig.?2aCc, Figs.?S5AC5D). Consistently, depletion of p53 by RNA interference (RNAi) greatly attenuated the Nutlin-3a-induced senescence (Fig.?2d, e). No increase in the level of apoptosis was detected (data not shown). The senescent cells appeared to be arrested at the G0 or G2 phase (data not shown). We previously showed that berberine-induced senescence of U87 glioblastoma cells is mediated by the downregulation of EGFR and RNAi of EGFR alone can induce.