| Melanoma is a melanocyte derived cancer which leads to the highest lethality in multiple skin cancers.In the past decades,the morbidity of melanoma in China and other countries is continuously increasing.Although immunotherapy,especially the widely use of checkpoint blockades such as anti-PD-1/PD-L1 antibody and anti-CTLA-4 antibody,have been demonstrated as the first-line drugs in treating melanoma,data analysis shows low clinical response rate of anti-PD-1/PD-L1 therapy in melanoma patients.In fact,around 60-70%of melanoma patients have complained with poor clinical response or acquired drug resistance to anti-PD-1/PD-L1immunotherapy after a period of treatment.In many cases,the blocking antibodies cannot achieve optimized effect,which prompts us to explore the mechanism underlying the immunotherapy resistance,to discover the new drug target and update the present therapeutic strategy.By now,it has become the crucial problem in melanoma treatment.Checkpoint molecules on the membrane of tumor cells or immune cells such as CTLA-4 and PD-1/PD-L1 are key regulators of tumor immune escape.The checkpoint molecule PD-L1 on tumor cells can bind with PD-1 on CD8~+T cells to activate them and lead the exhaustion.Therefore,tumor cells exhaust immune cells through over-expressing checkpoint molecules,which is also the critical reason for the resistance to immunotherapy.To be specific,the abnormal expression of PD-L1has been documented to prominently intervene the efficacy of anti-PD-1/PD-L1antibodies.Tumor-infiltrating CD8~+T cells can secrete IFN-γto induce the up-regulation of PD-L1 on tumor cells to render the therapeutic resistance.However,the underlying mechanism remains far from understood.Ubiquitin-editing enzyme A20(Tumor necrosis factor alpha-induced protein 3,TNFAIP3)is firstly identified in vascular endothelial cells,of which the expression can be robustly induced by tumor necrosis factor.A20 has an OTU domain in N terminal and a Zn figure structure in C terminal,providing this protein with both ubiquitin ligase function and de-ubiquitinase activity.In term of the regulation of immunology,A20 mainly functions as a negative regulator of NF-κB pathway to restrain immune cells expressing and releasing inflammatory cytokines and chemokines,so that to impede the progression of inflammation.In addition to the effect on immunity and inflammation,the role of A20 in cancer pathogenesis has also been well documented.The dysregulation of A20 is involved in tumor carcinogenesis,tumor growth,invasion and metastasis in many kinds of cancers such as breast cancer,neuroglioma and non-Hodgkin lymphoma,with its ubiquitin-editing function greatly implicated in.Moreover,A20 in tumor-infiltrating CD8~+T cells has been proved as an inhibitor that prevents tumor cells from being killed by CD8~+T cells.However,as a key regulator of immune and inflammatory reaction,the role of tumorous A20 in regulating anti-tumor immunity is rarely studied.Our previous results revealed that A20 expression was prominently increased in melanoma cells compared with melanocytes.The knockdown of A20 significantly impeded the growth and metastasis of melanoma.In addition,tumorous A20expression was in a prominent positive correlation with PD-L1 expression,and was highly correlated with the resistance to anti-PD-1 immunotherapy.However,the detailed underlying mechanism of the role played by A20 in melanoma development and the resistance to anti-PD-1 immunotherapy is still elusive.Based on our previous results,we suppose that A20 may function as an oncogenic factor to facilitate melanoma proliferation and metastasis through the activation of Akt pathway,and meanwhile render the resistance to anti-PD-1/PD-L1 immunotherapy through STAT3-PD-L1 axis.Therefore,A20 is a potential therapeutic target for melanoma therapy via the simultaneous suppression of melanoma growth and immunotherapy resistance.Object:To verify the role of A20 in melanoma development and anti-PD-1/PD-L1immunotherapy resistance in vitro and in vivo and demonstrate the underlying mechanism with a series of molecular biology experiments,the employment of animal model and the analysis of clinical specimens,intending to provide novel therapeutic strategy for melanoma treatment.Methods:1.To detect the expression of A20 in melanomaWestern blot and q RT-PCR assays were used to detect protein and m RNA expression of A20 in human primary melanocytes and melanoma cell lines.Immunofluorescence staining analysis was performed to detect the expression of A20 in xenograft tumors and normal mouse melanocytes in vivo.2.To clarify the role of A20 in melanoma proliferationShort hairpin RNA was used to obtain the knockdown of A20 in melanoma.CCK8and colony formation assays were performed to observe the alteration of cell proliferation and colony formation.Xenograft tumor model was employed to observe the alteration of tumor growth rate and tumor weight.Flow cytometry and western blot assays were performed to detect cell-cycle distribution and the expressions of cell cycle-related proteins.3.To investigate the role of A20 in melanoma metastasisTranswell assay was used to observe the change of melanoma cell migration after the knockdown of A20.Immunofluorescence staining analysis of cell skeleton protein was used to show the cell skeleton arrangement.Western blot was used to detect the expressions of migration-related protein.Lung metastasis model established by tail vein injection of tumor cells was used to observe the number of metastatic lesions in lung after the knockdown of A20.4.To investigate the mechanism underlying the regulatory role of A20 in melanoma developmentWestern blot was used to detect the phosphorylations of Akt and its downstream m TOR after the knockdown of A20.Colony formation and transwell assays were performed to observe melanoma cells proliferation and migration after the treatment with Akt inhibitor in A20-over-expressed cells.5.To clarify the role of A20 in regulating Akt activation-mediated glycolysisThe concentrations of glucose and LDH,as well as PH value in culture medium and intracellular ATP level were detected to observe the glycolysis level of melanoma cells after the knockdown or the overexpression of A20.Bioinformatics analysis in TCGA SKCM database and q RT-PCR was employed to reveal the relationship between A20 and glycolysis related molecules PKM2,PGAM1,PDK4,HK3,FBP1and ENO2.6.Explore the relationship between A20 expression and the efficacy of PD-1/PD-L1antibodies in melanoma patientsPrimary melanoma tissues were obtained from 11 melanoma patients in our hospital and divided into two groups as drug-sensitive group and drug-resistant group based on the principles in Response Evaluation Criteria In Solid Tumors(RECIST v1.1).Then,Kaplan-Meier survival analysis,western blot assay,histopathology analysis,immunofluorescence staining and flow cytometry assays were employed to analyze the correlation between tumorous A20 expression and the percentage of Ki-67,Perforin and Granzyme B-positive CD8~+T cells in tumor-infiltrating lymphocytes respectively.7.To analyze the role of tumorous A20 in the efficacy of anti-PD-1 immunotherapy in vivoB16F10 cells transfected with CRISPR/Cas9 KO A20 plasmids were injected subcutaneously into the blank of C57/BL6 mice to establish xenograft tumor models with anti-PD-1 antibody treatment.Survival analysis,histopathologic analysis and flow cytometry assays were employed to observe the treatment outcome and the alteration of tumor-infiltrating lymphocytes.In addition,mouse PBMCs were obtained from peripheral blood to establish co-culture system with B16F10 melanoma cells with indicated treatment,and flow cytometry was used to examine the status of tumor cells and CD8~+T cells.8.To investigate the role of tumorous A20 in regulating CD8~+T cells function in vitro PBMCs obtained from above-mentioned xenograft tumors were used to establish co-cultrue system with B16F10 cells with or without the knockdown of A20.Flow cytometry analysis was performed to examine melanoma cells apoptosis and the cytotoxity of CD8~+T cells in co-culture system.9.To demonstrate whether the role played by A20 in regulating anti-PD-1immunotherapy efficacy was dependent on tumor-infiltrating CD8~+T cells in vivoXenograft tumor mice models established by subcutaneous injection with B16F10melanoma cells with or without the transfection with CRISPR/Cas9 KO A20 plasmid were treated by anti-PD-1 and(or)anti-CD8~+T cells antibodies.Tumor growth rates and tumor weights were recorded.Histopathologic study and flow cytometry assay were employed to detect the percentage of CD3~+CD8~+T and PD-1~+CD8~+T cells.10.To analyze the relationship between A20 and PD-L1TCGA SKCM database,histopathological analysis of tissue microarray and western blot analysis of tumor were used to examine the expressions of A20 and PD-L1,and analyze the relationship between these two molecules.Si RNA transfection was used to obtain the knockdown of A20 expression.Western blot,histopathology staining and flow cytometry assays were used to detect the protein level of PD-L1.11.To demonstate that A20 confers the resistance to anti-PD-1 immunotherapy by increasing PD-L1 expressionC57BL/6 mice were used to establish xenograft tumor models by subcutenous injection with B16F10 cells with or without the knockout of A20,followed by anti-PD-1 antibody treatment.The growth rates and weights of tumors were recorded and flow cytometry was performed to detect the number and(or)the cytotoxicity of CD3~+CD8~+T cells and PD-1~+CD8~+T cells.12.To prove that A20 regulates PD-L1 expression and the function of CD8~+T cells in vitroPBMCs derived from xenograft mice were used to establish co-cultrue system with B16F10 cells with the interventions of both A20 and PD-L1 expressions.Flow cytometry analysis was performed to detect melanoma cells apoptosis and CD8~+T cells cytotoxity in co-culture system.13.To uncover the potential singaling pathway linking A20 to PD-L1 expressionKEGG analysis based on TCGA SKCM database,as well as western blot and immunoflourence staining assays were used to screen and confirm the potential pathway linking A20 to PD-L1 expression.Ch IP assay was employed to confirm the transcription factor responsible for PD-L1 up-regulation under the control of A20.14.To screen the proteins that interact directly with A20Co-IP assay combined with MS-based proteomicanalysis were used to verify the molecule that directly interacted with A20 and meanwhile affected STAT3 pathway.Mutant plasmid transinfection,co-IP and protein half-life analysis based on western blot assay were used to observe the ubiquitin-editing function of A20 and the ubiquitin binding site of PHB.15.To denmonstrate the translational potential of A20-PHB-PD-L1 axis in melanoma immunotherapyTCGA SKCM database was used to analyze the relationship among PHB,PD-L1 and A20.PBMCs derived from melanoma patients were used to observe the functional status of PD-1~+CD8~+T cells.The relationship between tumorous A20 expression and melanoma patients’prognosis was ultimately investigated.Result1.A20 expression is significantly up-regulated in melanomaCompared with human primary melanocytes,both m RNA and protein levels of A20 were significantly up-regulated in melanoma cells.Additionally,A20 expressions in both B16and B16F10 mouse melanoma cells in xenograft tumors were significantly higher than that of melanocytes in the tail of C57BL/6.Cg-Tg(KRT14-Kitl*)4XTG2Bjl/J mice.2.Up-regulated A20 promotes melanoma cell proliferationThe knockdown of A20 expression prominently impeded the proliferation and colony formation of melanoma cells.In addition,the knockdown of A20 induced lower tumor growth rate as well as decreased tumor weight and volume.Further,cell-cycle progression was prominently restrained and the expressions of cell-cycle related proteins were accordingly altered after the knockdown of A20.These results demonstrated A20 as an important facilitator of melanoma proliferation via the regulation of cell-cycle progression.3.Up-regulated A20 facilitates melanoma metastasisThe knockdown of A20 induced the disarrangement of cell skeleton and impaired the expressions of migration-related proteins,as well as the migratory capacity of melanoma cells.Moreover,the knockdown of A20 significantly decreased the number of metastatic lesions in lung in mice.These results demonstrated that up-regulated A20 expression contributed to cell migration and tumor metastasis in melanoma.4.A20 promotes Akt activation to facilitate melanoma developmentThe phosphorylations of Akt and its downstream molecules m TOR,4E-BP1 and 70S6K were inhibited after the knockdown of A20,whereas increased after the over-expression of A20.Moreover,the phosphorylation of Akt was decreased after the knockdown of A20 in xenograft tumors in vivo.In addition,Akt inhibitor could partially reverse the increase of melanoma cell proliferation and migration induced by the overexpression of A20.These results demonstrated that up-regulated A20 promoted melanoma cell proliferation and migration via the activation of Akt pathway.5.A20 promotes glycolysis via the activation of Akt pathwayThe overexpression of A20 significantly induced the increase of extracellular lactic acid level and intracellular ATP level,as well as the decrease of PH value and glucose concentration in medium,while Akt inhibitor could partially reverse these alterations.Moreover,the m RNA level of A20 was positively correlated with PKM2,PGAM1,PDK4,HK3,FBP1 and ENO2 respectively,and the knockdown of A20 could impede the m RNA levels of these glycolytic molecules.6.The up-regulation of tumorous A20 expression is related with poor clinical response to anti-PD-1 immunotherapy in melanoma.Melanoma patients who were relatively resistant to anti-PD-1 immunotherapy had higher tumorous A20 expression.In addition,there are less lymphocytes,especially CD8~+T cells infiltrating in A20-high-expressed tumors compared with A20-low-expressed tumors.The expression of tumorous A20 was negative correlated with Ki67,granzyme B and perforin expression in tumor-infiltrated CD8~+T cells.These results demonstrated that tumorous A20 expression was positively correlated with the resistance to anti-PD-1 immunotherapy in melanoma patients.7.A20 impairs the efficacy of anti-PD-1 immunotherapy in vivoTumor weight and growth rate were decreased in xenograft tumor model after the knockout of A20 under the condition of anti-PD-1 immunotherapy.In addition,mice beard tumors with A20 deficiency revealed better prognosis after anti-PD-1 immunotherapy.Moreover,the knockout of A20 expression induced the increase of tumor-infiltrating lymphocytes,as well as Ki67 and granzyme B expression in tumor-infiltrating CD8~+T cells.In line with the results in vivo,the results in co-cultured system also revealed higher expression of Ki67 and granzyme B in CD8~+T cells after the knockout of A20 in melanoma cells.These results demonstrated that A20 conferred the resistance to anti-PD-1immunotherapy in melanoma.8.A20 expression is positively correlated with PD-L1 expression in melanomaA20 expression is positively correlated with PD-L1 in both m RNA and protein levels in melanoma according to the results in TCGA SKCM database and our immunohistochemical staining analysis of TMA.A20 expression deficiency could lead to the down-regulation of PD-L1 expression both in vitro and in vivo.These results proved that A20 promoted PD-L1 expression in melanoma.9.A20 mediates the resistance to anti-PD-1 immunotherapy via the regulation of PD-L1expressionAfter the treatment with anti-PD-1 antibody,the overexpression of PD-L1 could partially reverse the decrease of tumor weight and growth rate after the knockout of A20.In line with this,the numbers of tumor-infiltrating CD8~+T cells and PD-1~+CD8~+T cells were also partially reversed.In in vitro co-cultured system,the overexpression of PD-L1 could also reverse the increase of Ki67 and granzyme B expression in CD8~+T cells that caused by A20 expression deficiency.These results demonstrated that A20 mediated the resistance to anti-PD-1 immunotherapy via the up-regulation of PD-L1 expression.10.A20 promotes PD-L1 expression via the activation of STAT3JAK-STAT3 pathway was highly correlated with A20 expression in TCGA SKCM database according to KEGG pathway analysis.The knockdown of A20 significantly down-regulated the phosphorylation of STAT3.Moreover,the knockdown of A20 also impeded the binding of STAT3 to the promoter region of PD-L1.In addition,WP1066 that is a STAT3 phosphorylation inhibitor,could partially impair the increase of PD-L1 m RNA and protein levels induced by A20 overexpression.These results demonstrate that A20promoted PD-L1 transcription and expression via the activation of STAT3 pathway.11.A20 ubiquitinates PHB to promote its degradation and thereby facilitates STAT3phosphorylation.Our co-IP and MS-based proteomic analysis revealed that A20 directly interacted with PHB and mediated the linkage of K48-specific ubiquitin chain to K186 and K202 residues in PHB protein,which subsequently promoted PHB protein degradation and alleviated the suppression on STAT3 phosphorylation.Meanwhile,the knockdown of PHB expression could prominently promote STAT3 activation and PD-L1 expression.Collectively,these results demonstrated that A20 promoted STAT3 phosphorylation and PD-L1 expression via the ubiquitination and degradation of PHB.12.The involvement of A20-PHB-STAT3 axis in anti-PD-1 immunotherapy in melanoma patients.Our analysis in TCGA SKCM database revealed that the m RNA level of PHB was negatively correlated with A20 and PD-L1 expression,respectively.After anti-PD-1antibody treatment,patients with higher tumorous A20 expression presented worse prognosis than that with lower tumorous A20 expression.More importantly,tumorous A20expression was positively correlated with the ratio of Ki-67 percentage in PD-1~+CD8~+T cells to tumor burden,and negatively correlated with granzyme B and perforin expression in PD-1~+CD8~+T cells.These results revealed the involvement of A20-PHB-STAT3 axis in anti-PD-1 immunotherapy in melanoma patients,and tumorous A20 expression could be a potential biomarker for predicting the efficacy of anti-PD-1 immunotherapy in melanoma patients.ConclusionIn the present study,we firstly uncovered that A20 expression was prominently increased in melanoma cells compared with that in normal human melanocytes.Then,we proved A20 as a promoter in melanoma growth and metastasis via activating Akt pathway and enhancing glycolysis.Further,we demonstrated A20 as a contributor to the resistance to anti-PD-1 immunotherapy in melanoma.To be specific,A20 added K48 ubiquitin on PHB to facilitate its degradation,and then promoted STAT3 phosphorylation to facilitate PD-L1transcription and expression,thus rendering the resistance to anti-PD-1 immunotherapy.Taken together,A20 could be regarded as a promising therapeutic target via the simultaneous suppression of tumor growth and anti-PD-1 immunotherapy resistance in melanoma. |
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