Molecular Mechanism Of Apoptosis Regulated By GMEB1 In Non-Small Cell Lung Cancer Cell Lines

Cancer has been a severe social problem and the number of people died of cancer is accelerating every year.It has been an urgent problem in science and medicine to overcome.In all kinds of cancer,lung cancer is receiving more and more attention because of its high pathogenicity and fatality rate.Lung cancer caused the most people death and it has been a very difficult problem in science.The death receptors,such as DR4 and DR5,are almost over expressed on the surface of tumor cells,which prompt scientists that treatment with ligands of these receptors to tumor could induce the apoptosis of tumor cells.But,the trait of TRAIL,ligand of DR5,got a failure as the weak ability of TRAIL to induce apoptosis.Otherwise,TRAIL facilitated the proliferation of tumor cells,which limits the use of TRAIL in cancer therapy.Many research shows that some proteins function as anti-apoptotic proteins in the apoptotic path way induced by TRAIL.These proteins concludes DcRl,DcR2,CFLARL,CFLARs,XIAP and so on.These proteins limit the pro-apoptotic function of TRAIL.Then,aimed to these proteins may sensitive the tumor cells to TRAIL treatment.GMEB1 is a protein that widely expressed in various organizations.It was identified as a transcription factor in the nucleus.GMEB1 can interact with GMEB2 and bind to the GME element in the promotor region of tyrosine aminotransferase(TAT)gene.The promotor region of TAT gene also possesses an element which called glucocorticoid response element(GRE).The GRE element is located downstream of GME.Glucocorticoid receptor(GR)can bind to the GRE and modulate the transcription of TAT gene.Otherwise,GMEB1 and GME element can inhibit the binding of GR to GRE and repress the transcription of TAT.So,GMEB1 can function as a repressor in the nucleus.Other study also showed that GMEB1 interacted with the transcription factor FOXL2 and regulated the apoptosis of cell.But the mechanism of this is still unclear.Beside the function as a transcription factor in the nucleus,GMEB1 also can localize in the cytoplasm.GMEB1 can interact with pro-caspases in the cytoplasm and inhibit the activation of pro-caspases.But the molecular mechanism of how to inhibit the activation of pro-caspases is still unclear now.The interaction of GMEB1 and pro-caspases is via the C terminal of GMEB1 and the DED domains of pro-caspases.Therefore,we guessed that CFLARL,which had a similar construction with pro-caspase 8,may be a protein that interacted with GMEB1.As CFLAR1 can inhibit the activation of pro-caspase 8 directly,GMEB1 might inhibit the activation of pro-caspase 8 via the function of CFLARL.As above,the purpose of this research are mainly on the next two aspects:first is to illustrate whether GMEB1 inhibit the activation of pro-caspase 8 via CFLAR1;and second is to understand the function of GMEB1 on the apoptosis induced by TRAIL.We want to find a target protein which can sense tumor cells to TRAIL treatment.And is GMEB1 the protein we found?We first analyzed the correlation between GMEB1 and CFLARL.Western blot results showed that GMEB1 protein levels positively corresponded to CFLARL protein levels in NSCLC cells.SAHA decreased both GMEB1 and CFLAR1 as a dose-dependent and time-dependent manners.GMEB1 didn't regulate CFLARL in the transcription levels but promoted the stability of CFLARL protein.Western blot analysis showed that overexpression of GMEB1 increased CFLARL and knockdown of GMEB1 decreased CFLARL.Decreasing of CFLARL could be rescued by the reverse overexpression of GMEB1.On the other hand,regulation of CFLARL by GMEB1 was mainly due to the impact on proteinase degradation pathway.GMEB1 overexpression inhibited poly-ubiquitination of CFLARL.All the results above indicated that GMEB1 regulated CFLARL by post-translational modification.To study the mechanism deeply,we conducted series co-IP,GST Pull-down and immunofluorescence experiments to study the interaction between GMEB1 and CFLARL in NSCLC cells.Results showed that GMEB1 interacted with CFLARL in the cytoplasm.And the interaction between GMEB1 and CFLARL was via the interaction of GMEB1 N terminal and CFLARL P20/P12 domains.As GMEB1 couldn't influence CFLAR1 protein levels directly,then we wanted to find a key functional molecular by analyze different de-ubiquitinases protein levels in NSCLC cells.Our results indicated that USP40 might be the molecular we found.In NSCLC cells,USP40 protein levels positively corresponded to GMEB1 and CFLAR1.USP40 promoted the stability of CFLAR1.USP40 also regulated CFLAR1 protein levels and USP40 overexpression could increase CFLAR1 protein levels while USP40 knockdown could decrease CFLAR1 protein levels.USP40 interacted with CFLAR1 in the cytoplasm via P20 and P12 domains of CFLAR1.Additionally,USP40 overexpression decreased the ubiquitination of CFLAR1 while USP40 knockdown increased the ubiquitination of CFLAR1.Specially,USP40 targeted CFLAR1 for K48-linked poly de-ubiquitination.Enzyme dead mutation USP40 could interact with CFLAR1 but couldn't increase CFLARL and target CFLARL for de-ubiquitination.Results above indicated that USP40 was one of the de-ubiquitinases targeted for CFLARL and USP40 could regulate CFLAR1,directly.Next,we evaluated the correlations among GMEB1/USP40/CFLARL.Co-IP results showed that GMEB1 could interact with both USP40 and CFLAR1.GMEB1 acted as an adaptor protein for USP40 and CFLAR1,GMEB1 promoted the interaction between USP40 and CFLARL.Additionally,USP40 overexpression could slightly increase CFLARL while GMEB1 was knocked down in NSCLC cells.Otherwise,GMEB1 overexpression could hardly increase CFLARL,while USP40 was knocked down.All the results above indicated that GMEB1 regulated CFLAR1 via the function of USP40.We had elucidated the molecular mechanism that GMEB1 regulated CFLAR1,then we began to study the effect of GMEB1 on apoptosis.Firstly,co-IP assays showed that GMEB1 knockdown decreased the CFLARL composition in DISC.And GMEB1 knockdown increased caspase-8 activity as well as protein levels of cleaved CASP8,CASP3 and PARP induced by TRAIL or SAHA.Also,GMEB1 knockdown promoted cell apoptosis induced by TRAIL or SAHA.Our results also showed that CFLARL could inhibit the function of GMEB1 knockdown in NSCLC cells,which indicated that GMEB1 inhibited apoptosis via the function of CFLARL.We still understood little about the mechanism how GMEB1 inhibited CASP8 activity,though we had been clear that GMEB1 regulated CFLAR1 via USP40 and GMEB1 inhibited apoptosis via CFLAR1.Then we evaluated the interactions among GMEB1,USP40 and CFLAR1.Results showed that CFLAR1 overexpression promoted the interaction between GMEB1 and CASP8,but GMEB1 overexpression didn't influence the interaction between CFLAR1 and CASP8.These results indicated that CFLAR1 was important for the interaction between GMEB1 and CASP8.GMEB1 might interacted with CASP8 via the adaptor function of CFLAR1,which might be an explanation for how GMEB1 inhibited the activity of CASP8.At last,we constructed A549 cell lines that GMEB1 was stably knocked down.Then we conducted nude mouse xenograft tumor experiments and found GMEB1 knockdown inhibited tumor growth.GMEB1 might influenced tumor growth via its function on apoptosis.Statistic obtained from TCGA indicated that GMEB1 and USP40 overexpression both related to bad prognosis of lung cancer patients.In general,our experiments indicated that GMEB1 and USP40 both could interacted with CFLAR1 in the cytoplasm in NSCLC cells.USP40 was one of the de-ubiquitinases of CFLARL and it could targeted CFLAR1 for K48-linked poly-ubiquitination.USP40 promoted CFLARL stability and increased CFLAR1.GMEB1 regulated CFLARL by promoting the interaction of USP40 and CFLAR1.GMEB1 also inhibited apoptosis induced by TRAIL or SAHA via regulation of CFLARL.CFLAR1 promoted the interaction of GMEB1 and CASP8 while GMEB1 had no effect on the interaction of CFLAR1 and CASP8,which indicated that GMEB1 interacted CASP8 via CFLAR1 and GMEB1 inhibited CASP8 activity also via regulation of CFLAR1.In nude mouse xenograft tumor model,GMEB1 knockdown inhibited tumor growth.Statistic obtained from TCGA indicated that GMEB1 and USP40 overexpression both related to bad prognosis of lung cancer patients.Our study indicated that GMEB1 inhibited the pro-caspase 8 via the direct function of CFLARL.We firstly identified CFLARL as a target protein of the de-ubiquitinase USP40.Our study also indicated that knocked down of GMEB1 sensed tumor cells to the apoptosis induced by TRAIL,which may be a new target for cancer therapy.