EGFR-ERK/CSN6 Regulates PD-L1 Expression In Glioblastoma

Objective:Glioblastoma?GBM?is the most aggressive and most common primary brain tumor in adults,with a very poor prognosis,due to limited therapeutic efficacy of available treatments.Recently,it has been found that PD-L1/PD-1 pathway,the important immune block checkpoints,could also exist in central neuron system,which may contribute to tumor immune escape.PD-L1 protein on immune cells engaged with PD-1 on the surface of T cells causing apoptosis of cytotoxic CD8+T cells,thereby generating immune escape and promoting tumor progression.Thus,PD-1/PD-L1 immune checkpoint inhibitors may improve cancer therapy in GBM.However,the mechanism of PD-L1 upregulation on glioblastoma is still unclear.Recently,COP9 signalosome family?CSN?was shown to regulate PD-L1expression since COP9 signalosome 5?CSN5?,induced by NF-kB p65,is required for TNF-?mediated PD-L1 stabilization in cancer cells.In colorectal cancer,EGFR-ERK-related pathways in colorectal cancer can maintain PD-L1protein stability by regulating CSN6.Since it has been shown that overexpression of CSN6 in glioblastoma might be associated with tumor progression.Thus it is necessary to explore whether EGFR-CSN6 regulates PD-L1 expression through affecting PD-L1 stabilization in GBM.Therefore,this study aims to provide new ideas for glioblastoma-related immunotargeting therapy.Therefore,human glioma specimens and in vitro glioblastoma tumor cells lines were applied in this study to investigate whether the EGFR-ERK pathway contributes to PD-L1 upregulation in glioblastoma by regulating CSN6 expression.Methods:1.Human paraffin-embedded cancer samples were obtained from the Department of Pathology,second hospital of Hebei Medical University.The expression of EGFR,CSN6 and PD-L1 in human glioma tissues samples was measured by immunohistochemical staining and immunofluorescence analysis.The correlation among the EGFR,CSN6 and PD-L1 expression and their relationship with clinical prognosis were measured.2.Human glioblastoma cells U87 and U251 were cultured,and treated with EGF.The activation of EGFR-ERK pathway as well as CSN6 and PD-L1expression was detected by Western Blot.After ERK pathway blocking or CSN6 siRNA treatment,the expression of PD-L1 or CSN6 upon EGFR activation were measured.Results:1.EGFR,CSN6 and PD-L1 expression as well as their relationship with clinical prognosis in different grades of human glioma tissue samplesThe data from the TCGA dataset showed that the expression level of EGFR in glioblastoma at the mRNA level in human glioma tissue samples was the highest[Normal brain:0.0055?-0.0004,0.0117?vs astrocytoma:0.0763?0.0222,0.4098?vs glioblastoma:0.6787?0.3756?,3.1276)]?P<0.0001?,EGFR high expression was associated with poor prognosis?P<0.05?.CSN6has the highest expression level in glioblastoma[Normal brain:0.0051?0.0013,0.0103?vs astrocytoma:0.0837?0.0209,0.3874?vs glioblastoma:0.4406?0.2292,0.5442?]?P<0.0001?,CSN6 high expression was associated with poor prognosis?P<0.05?.The expression level of PD-L1 in glioblastoma was higher than that of astrocytoma[Astrocytoma:-0.1306?-0.4890,0.0171?vs glioblastoma:0.1608?-0.1411,0.5999?]?P<0.05?,PD-L1 high expression was associated with poor prognosis?P<0.05?.Our IHC data showed that that the expression of EGFR protein in human glioblastoma tissue specimens is higher than that of astrocytoma,IHC scores[Astrocytoma:1?0,4?vs glioblastoma:4?2,9?]?P<0.0001?,the expression of EGFR protein in astrocytoma was higher than that in normal brain tissue,IHC scores[Normal brain:0?0,0?vs astrocytoma:1?0,4?]?P=0.0002?;There was no statistical difference in EGFR protein expression between GBM and anaplastic astrocytoma IHC scores[Anaplastic astrocytoma:6?3,12?vs glioblastoma:4?2,9?]?P=0.1344?.High expression of EGFR protein was positively correlated with poor prognosis?P<0.0001?.The expression of CSN6 protein in human glioblastoma tissue samples was higher than that in astrocytoma,IHC scores[Astrocytoma:0?0,2?vs glioblastoma:4?1,6?]?P<0.0001?,There was no statistical difference between the expression of CSN6 in astrocytoma and normal brain tissue in patient specimens,IHC scores[Normal brain:0?0,1?vs astrocytoma:0?0,2?]?P=0.0969?;The expression of CSN6 protein in glioblastoma was higher than that in anaplastic astrocytoma,IHC scores[Anaplastic astrocytoma:4?2,8?vs glioblastoma:4?1,6?]?P=0.0388?.High expression of CSN6 protein was positively correlated with poor prognosis?P<0.0001?.The expression of PD-L1 protein in human glioblastoma tissue specimens was higher than that in astrocytoma,IHC scores[Astrocytoma:1?0,2?vs glioblastoma:4?1,9?]?P=0.0002?,the expression of PD-L1 protein in astrocytoma patients was higher than that in normal brain tissues,IHC scores[Normal brain:0?0,0.25?vs astrocytoma:1?0,2?]?P=0.0036?;there was no statistical difference between the expression of PD-L1 protein in anaplastic astrocytoma and glioblastoma,IHC scores[Anaplastic astrocytoma:4?2,9?vs glioblastoma:4?1,9?]?P=0.2226?.High expression of PD-L1 protein was significantly correlated with poor prognosis?P=0.0019?.2.Correlation between EGFR,CSN6 and PD-L1 expression in human GBMImmunofluorescence staining indicated that EGFR and CSN6 or CSN6and PD-L1 proteins could co-express in GBM.Immunohistochemistry results showed that there was a positive correlation between EGFR,CSN6 and PD-L1expression in GBM.3.The correlation between PD-L1 expression and CD8⁺T cell infiltration number in tumor microenvironment.PD-L1 protein expression was significantly negatively correlated with CD8⁺T cell infiltration in tumor microenvironment.PD-L1^LessCD8⁺T^Highigh patients showed the best prognosis.Thus,the combination of PD-L1 protein expression and CD8⁺T infiltration could be a good marker for the prognosis of patients.4.CSN6,activated by EGFR-ERK pathway,is involved in the regulation of PD-L1 protein stability in GBM in vitroEGF stimulation activated the EGFR-ERK pathway and up-regulated CSN6 and PD-L1 expression in vitro;Blocking ERK pathway inhibited EGF-induced CSN6 and PD-L1 upregulation.Knockdown of CSN6 protein by siRNA inhibited PD-L1 upregulation upon the EGFR-ERK pathway.After inhibiting the synthesis of new protein by the half-life experiment,EGF still maintained the existence of PD-L1 protein,which suggests that CSN6 by EGFR-ERK pathway increase the stability of PD-L1 protein in GBM.Conclusions:1.High expression of EGFR,CSN6 and PD-L1 protein in human GBM was positively related to poor prognosis.EGFR and CSN6 could be positive related with PD-L1 expression in human GBM.Higher expression of PD-L1protein was significantly negatively correlated with CD8⁺T cell infiltration in glioblastoma tumor microenvironment.PD-L1^LowCD8⁺T^More patients had the best prognosis.2.In vitro,activation of EGFR-ERK signaling pathway can up-regulate the expression of CSN6 in glioblastoma,and maintain the stability of PD-L1protein.