Downregulation Of GSDMD Attenuates Tumor Proliferation Through Caspase-3-Mediated Apoptosis And Inhibition Of EGFR/Akt Signaling In NSCLC

BackgroundLung cancer is a leading cause of cancer-related mortality worldwide,which comprises non-small cell lung cancer(NSCLC,approximately 85%)and small cell lung cancer.Our study focused on the main histological subtype NSCLC,which can be further classified as lung adenocarcinoma(LUAD),lung squamous cell carcinoma(LUSC)and large cell carcinoma.Pyroptosis is an inflammatory form of programed cell death.Gasdermin D(GSDMD)is a newly described pyroptosis executive protein.It can be cleaved by inflammatory caspases into a GSDMD-N fragment.GSDMD-N fragments can form functional pores in the plasma membrane,leading to cell swelling and lysis.Pyroptotic cells can release large amounts of damage associated molecular patterns(DAMPs),such as IL-1?,and has a critical role in promoting inflammation As we known,inflammation has a distinct role in carcinogenesis and molecules involving in pyroptosis signaling,such as NLRP3,are reported to regulate tumor proliferation,invasion and metastasis.However,the precise role of GSDMD in carcinogenesis remains nearly unknown.Our study investigated the biological function of GSDMD in NSCLCMethods1.Two commercial tissue microarrays of LUAD and LUSC were used to analyze the protein expression profile of GSDMD in NSCLC and paired adjacent normal specimens.Correlation between GSDMD expression,and clinicopathological characteristics,and prognosis in NSCLC were analyzed as well.2.PC9 and H1703 cell lines with high levels of GSDMD were used for si-RNAs knockdown experiments.The MTT assay and colony formation assay were performed for detecting cell growth and proliferation.3.In GSDMD knockdown NSCLC cell lines,flow cytometry of Annexin V/PI staining labeled cells were used to detect cell apoptosis,flow cytometry of JC-1 staining labeled cells were used to detect mitochondrial membrane potential(MMP),apoptosis associated proteins were analyzed by Western Blot and release of lactate dehydrogenase(LDH)were detected by LDH cytotoxicity assay.4.GSDMD knockdown PC9 cell lines were pretreated with lipopolysaccharide(LPS)plus adenosine 5'-triphosphate(ATP)or not.Inflammasome associated proteins were detected using Western Blot,release of IL-1? were detected using ELISA,Annexin V/PI staining labeled cells were analyzed by flow cytometry and morphology of treated cells were observed with the light microscope.5.A normalized NSCLC GEO array was downloaded at MERAV database and co-expression genes of GSDMD were identified using Morpheus tools.KEGG enrichment analysis was performed using the OmicShare tools.Identified pathways were further confirmed by Western blot.6.PC9 cells stably transduced with lentiviral vectors encoding GSDMD or negative shRNAs were subcutaneously injected into the nude mice.The tumor volume and weight were measured.Immunohistochemistry of Ki-67 staining and TUNEL staining were used for cell proliferation and apoptosis detection,respectively.Results1.The LUAD tissue array showed that the GSDMD protein level was significantly up-regulated in LUAD compared with matched adjacent specimens.Higher GSDMD expression correlated with larger tumor size and more advanced TNM stages.Moreover,higher GSDMD protein expression indicated a poor prognosis in LUAD.2.The LUSC tissue array showed that the GSDMD protein level was significantly up-regulated in LUSC compared with matched adjacent specimens.Higher GSDMD expression correlated with more advanced TNM stages.However,GSDMD protein expression revealed no correlation with prognosis in LUSC.3.GSDMD knockdown in PC9 and H1703 cell lines limited tumor growth and colony formation.4.NSCLC cell lines with GSDMD knockdown revealed more early and late apoptosis cells,decreased MMP,increased PARP-1 cleavage and capsase-3 cleavage proteins.LDH cytotoxicity assay showed no obvious increase in LDH release by GSDMD knockdown cells.Moreover,z-VAD-FMK,a general caspase inhibitor,partially alleviated the decreased cell proliferation induced by GSDMD knockdown.5.There was intrinsic activation of pyroptotic signaling in NSCLC cell lines,confirmed by NLRP3,caspase-1 and cleaved capsase-1 expression,along with the spontaneous release of IL-1?.Moreover,PC9 cells pretreated with LPS/ATP stimuli showed only increased Annexin V/PI double positive cells with swelling cellular morphology,while GSDMD knockdown PC9 cells pretreated with LPS/ATP stimuli showed increased Annexin V single positive and Annexin V/PI double positive cells with shriveled cellular morphology.6.KEGG enrichment analysis showed that the co-expressed genes were enriched in several classical signal transduction pathways,including Rapl,ErbB,PI3K-Akt signaling.We chose the PI3K-Akt signaling which was vitally involved in regulation of cell survival and apoptosis for further investigation.Western Blot revealed obvious down-regulation of phosphorylated Akt,phosphorylated EGFR and phosphorylated mTOR in GSDMD knockdown cells.7.The xenograft experiment showed that GSDMD knockdown limited tumor growth in vivo.Both tumor volume and weight were significantly lower in GSDMD depletion group.Moreover,there were lower Ki-67 staining and higher TUNEL positive staining in GSDMD depletion group.ConclusionsTogether,our results revealed that the GSDMD level was up-regulated in NSCLC and GSDMD was an independent prognostic biomarker for LUAD,but not LUSC.GSDMD knockdown limited tumor growth in vitro and in vivo,and promoted cell apoptosis though activating caspase-3.Moreover,there may be a crosstalk between pyroptotic signaling and apoptosis in tumor cells.Intrinsic and extrinsic activation of NLRP3/caspase-1 signaling in GSDMD knockdown cells may contribute to regulation of cell apoptosis.Additionally,co-expression analysis indicated a correlation between GSDMD and EGFR/Akt signaling.