| ObjectiveImmunocheckpoint inhibitors improved the ORR,PFS and OS of melanoma patients.However,many patients still cannot benefit from it.Although studies have addressed the latent mechanisms involved in the blockade of PD-1,the mechanisms of PD-L1 upregulation and reliable biomarkers for anti-PD-1 therapy are unknown.This study was to investigate the potential role of combined IGFBP2 and PD-L1 in predicting the efficacy of immunotherapy in melanoma.Determine whether IGFBP2 regulates PD-L1 expression and clarify the signaling pathway and mechanism.Methods1.IHC was used to detect IGFBP2 and PD-L1 protein expression in 127 melanoma patients who had not received anti-PD-1 therapy.Based on the clinical and follow-up data,survival analysis was performed to verify the clinical significance.2.The efficacy of anti-PD-1 therapy in 13 melanoma patients who failed chemotherapy at Tianjin Medical University of cancer Institute and Hospital was evaluated.Tissue samples were collected for IHC staining of IGFBP2 and PD-L1.3.Bioinformatics analysis was used to annalyse the RNA-seq data(GSE78220)of 28 melanoma patients who have received anti-PD-1 therapy.4.According to the m RNA levels of PD-L1 and IGFBP2,443 melanoma patients in TCGA were divided into three groups via quartile method.CIBERSORT was used to analyze the differences of infiltrating immune cells in different groups.5.The expression of IGFBP2 was altered by adding exogenous IGFBP2 protein,constructing IGFBP2 stably overexpression cell lines and using si RNAs targeting IGFBP2 in melanoma cell lines A875 and A375.Western blot was used to determine the signaling pathway of IGFBP2 regulating PD-L1 expression.6.In the melanoma cell lines A875 and A375,Co-IP,IF,ICC and IHC were used to determine the molecular mechanism of IGFBP2 regulates PD-L1 expression.Results1.IHC results showed the high expression of IGFBP2 and PD-L1 in melanoma tissues.IGFBP2 is positively correlated with PD-L1 expression.Patients with high IGFBP2 or PD-L1 expression had worse OS(P < 0.05).When patients were divided into four groups based on the expression of IGFBP2 and PD-L1,patients with both high IGFBP2 and PD-L1 expression had worst OS(P < 0.05).2.Among the 13 melanoma patients receiving anti-PD-1 therapy,seven petients had enough data to do efficacy evaluation.One patient achieved PR,three patients achieved SD and three patients suffered from PD.In addition,IHC results showed high expression of both IGFBP2 and PD-L1 in PR patient.3.At the m RNA level,patients with both high PD-L1 and IGFBP2 expression response better than the other groups.And the AUC was also the largest.4.CIBERSORT results showed among 443 melanoma patients in TCGA,patients with both high IGFBP2 and PD-L1 had increased CD8 T cells,CD4 memory activated T cells,activated NK cells and M0 macrophages;Patients with low IGFBP2 and PDL1 had increased CD4 memory resting T cells and resting NK cells.5.By altering the expression level of IGFBP2 in A875 and A375,we verified the increased IGFBP2 led to the activation of EGFR/STAT3 and upregulation of PD-L1.Using si RNA to inhibit IGFBP2 expression reduced the activation of EGFR/STAT3 and down-regulated PD-L1 expression.6.Western blot results showed EGFR was a key factor in IGFBP2 regulated PDL1 expression.Co-IP demonstrated IGFBP2 could bind to EGFR;IGFBP2 and EGFR co-localized,which was obvious in the nucleus.The subsequent Western blot and IHC results showed nuclear IGFBP2 promoted EGFR nuclear accumulation as well as the activation of EGFR/STAT3 signaling pathway,inducing PD-L1 expression.ConclusionOur study confirmd the high expression of IGFBP2 and PD-L1 is related to poor OS;revealed that IGFBP2 combined with PD-L1 may have potential predictive role of anti-PD-1 therapy.Furthermore,this study also confirmed that IGFBP2 facilitates EGFR nuclear accumulation as well as the activation of EGFR/STAT3 signaling pathway to regulate PD-L1 protein expression.The results may help to develop a new therapeutic strategy to potentiate PD-L1-targeted immunotherapy in melanoma. |
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