GSDME Regulate Malignant Behavior Of Non-small Cell Lung Cancer Through JAK/STAT3 Signaling Pathway And MGA Mutation As A Biomarker For Immunotherapy For Patients With Lung Adenocarcinoma

Background:The gasdermins(GSDMs)family has attracted much attention for its role in mediating proinflammatory programmed cell death-pyroptosis.At present,most of the studies on GSDMs family focus on their role in antitumor immunity,but little attention has been paid to the effect of GSDMs family on the characteristics of tumor cells.The role and mechanism of GSDME in non-small cell lung cancer(NSCLC)are unclear.Methods:Tumor tissue specimens and clinical information of NSCLC patients were collected in this study.The expression level of GSDME in tumor tissues was evaluated by immunohistochemical staining,and the correlation between GSDME expression level and chemotherapy efficacy and prognosis was analyzed.In the cell model,the effect of GSDME on the malignant behavior and chemosensitivity of NSCLC was explored,and the underlying mechanism was explored by the next-generation sequencing analysis.The pathway inhibitors were used to verify the effect of GSDME on NSCLC at the cell level and subcutaneous tumor model level in nude mice.Finally,the effects of GSDME on tumor immune microenvironment and sensitivity to chemotherapy and anti-PD-1 monoclonal antibody treatment were explored in the subcutaneous tumor models of immunocompetent mouse.Results:Among the 43 NSCLC patients,GSDME was highly expressed in 9 patients and low or no expression in 34 patients.Correlation analysis showed that patients with high expression of GSDME had a better response to chemotherapy than those with low expression of GSDME(p value=0.023).Kaplan-Meier survival analysis showed that patients with high expression of GSDME had a longer disease-free survival(median survival times:33.3 months vs.21.5 months,p value=0.035).In the cell model,overexpression of GSDME significantly inhibited the proliferation,migration,invasion,angiogenesis and other malignant biological behaviors of NSCLC cells,and increased the sensitivity of tumor cells to cisplatin.Knockdown of GSDME expression can significantly promote the malignant biological behavior of NSCLC and reduce its sensitivity to cisplatin.In the nude mice experiment,compared with the control group(shNC),the tumor in the stable knockdown of GSDME group(shGSDME)grew faster,the tumor cells injected into the tail vein were more likely to develop lung metastasis,and the sensitivity of the tumor to cisplatin was decreased,and the response to chemotherapy was worse.The results of next-generation sequencing showed that the JAK-STAT pathway was enriched by functional enrichment analysis of differential genes in the four cell lines of GSDME intervention group and control group.Western blot showed that overexpression of GSDME inhibited the activation of JAK-STAT3 pathway in NSCLC cells,while knockdown of GSDME activated the JAK-STAT3 pathway in NSCLC cells.The p-STAT3 inhibitor SH-4-54 could significantly reverse the promoting effect of GSDME knockdown in tumor cells and subcutaneous model of nude mice.In the subcutaneous model of immunocompetent mice,the tumors expressed in the shGSDME group grew faster and were less sensitive to cisplatin and anti-PD-1 monoclonal antibody than those in the shNC group.The next-generation sequencing results showed that functional enrichment analysis of the differential genes in the shNC group and the shGSDME group showed that JAK-STAT3 pathway,PD-L1 expression and PD-1 checkpoint pathway were significantly enriched.The ImmuCellAI-mouse platform was used to calculate the abundance of immune cells in tumor tissues.The results showed that the infiltrating CD8+T cells in tumor tissues of shGSDME group were significantly decreased compared with those of shNC group(p value=0.01).The enrichment and abundance of immune-negative regulation cells,such as mast cells,Treg cells and neutrophils in the shGSDME group were higher than those in the shNC group,but the difference was not statistically significant(p value=0.116,0.375,0.073,respectively).Conclusions:In NSCLC,GSDME may inhibit the proliferation,migration,invasion,angiogenesis and other malignant biological behaviors of tumor cells by inhibiting the JAK-STAT3 pathway,and it could improve the sensitivity of the tumor to chemotherapy.In terms of tumor microenvironment,GSDME may mediate active immune microenvironment by inhibiting the JAK-STAT3 pathway,thereby inhibiting tumor progression and improving tumor response to chemotherapy and immunotherapy.GSDME is expected to be a biomarker for the prognosis and efficacy prediction of patients with NSCLC.Background:With the continuous revolutionary breakthrough of immune checkpoint inhibitors(ICIs)therapy represented by anti-PD-1/PD-L1 monoclonal antibody in tumor treatment,which significantly prolongs the survival of tumor patients,how to find better predictive biomarkers to identify and screen patients who are effective to ICIs therapy has become the current problem to improve the quality of life of cancer patients.It is one of the most important research hotspots to prolong the survival of cancer patients.Methods:The predictive effect of Max’s giant associated protein(MGA)mutations on the response and prognosis of ICIs treatment in multiple tumor types was analyzed in the MSKCC cohort and validated in a validation cohort.In addition,gene set enrichment analysis(GSEA)and immune cell infiltration analysis were performed on lung adenocarcinoma transcriptome sequencing data of the TCGA database to explore the potential mechanism of MGA mutation as a potential biomarker for ICIs efficacy and prognosis.Results:In the MSKCC discovery cohort,MGA mutation was a marker of ICIs efficacy and prognostic in patients with lung adenocarcinoma(LUAD),while was not in patients with other tumor types.MGA mutation was positively correlated with TMB score.The results of the validation cohort were consistent with those of the discovery cohort in LUAD patients.Moreover,patients with MGA mutations had longer survival in the low TMB or high PD-L1 expression subgroups.Multivariate analysis showed that MGA mutation was an independent prognostic marker in LUAD patients treated with ICIs.Mechanistically,we found that MGA’s co-mutated genes(PTRPD et al.)did not affect the predictive role of MGA mutation on the prognosis of immune checkpoint inhibitors.In addition,GSEA showed that primary immunodeficiency pathway was enriched in the MGA wild-type group.Immune cell infiltration analysis showed that activated NK cells were more abundant in MGA-mutant LUAD patients.Conclusion:Tumor tissue MGA mutation could be a predictive biomarker for the response and prognosis of ICIs therapy in patients with LUAD,independent of PD-L1 expression and TMB.These results may provide a new insight for identifying patients with LUAD who may potentially benefit from ICIs.