A Study On The Role Of GSDME In EGFR-modulated Proliferation In Non-small Cell Lung Cancer Cells

Cancer has been a major disease threatening human survival and health for a long time.We have done a lot of research on the proliferation-promoting characteristics of tumor cells,aiming at finding new targets to treat tumors.The downstream pathway of Epidermal Growth Factor Receptor(EGFR)is very complex,producing a wide range of effects such as promoting proliferation,promoting survival,promoting migration and anti-apoptosis.Activation of the EGFR signaling pathway depends on receptor dimerization and phosphorylation of intracellular tyrosine residues,etc.EGFR signal pathway is down-regulated mainly by EGFR endocytic degradation.In tumor cells,continuous activation of EGFR or its inability to be endocytosed due to excessive ligand production,EGFR gene amplification,EGFR transcriptional up-regulation,EGFR mutation or truncation,thereby causing excessive cell proliferation.Many proteins regulate the EGFR signaling pathway,and an in-depth understanding of these regulations will help us better design drugs targeting the EGFR pathway.GSDME(Gasdermin E)alias DFNA5,was first identified in 1998 as a gene causing autosomal dominant non-integrated hearing loss.GSDME has a self-inhibiting structure consisting of an N-terminal functional domain and a C-terminal regulatory domain.When there is no cutting,the activity of n-terminal functional domain is inhibited.Recent studies have found that GSDME is involved in the process of pyroptosis.When cells are stimulated by chemotherapy drugs,CASP3 is activated,and GSDME is cleaved to release the N-terminal fragment of GSDME,which can perforate the cell membrane,change the osmotic pressure and eventually lead to cell pyroptosis.During the experiment,we found that knockdown of GSDME inhibited cell proliferation by down-regulating CCND1 levels.The expression of CCND1 is regulated by a variety of factors,among which,phosphorylatedERK1/2(Mitogen-Activated Protein Kinase 1)can promote the formation of AP-1(Activator Protein-1)complex and initiate transcription of CCND1.Our results show that phosphorylation of ERX1/2 is inhibited after knockdown of GSDME,and the protein level of CCND1 is also down-regulated.We checked the database and found that GSDME may interact with EGFR.The results of co-immunoprecipitation experiments indicated that the N-terminal fragment of GSDME can bind to the intracellular domain of EGFR.Further studies have found that GSDME promotes endocytic degradation of EGFR via the ubiquitin-proteasome system and the autophagy-lysosomal pathway,leading to down-regulation of its downstream RAS(RAS Proto-Oncogene)-ERK1/2 pathway.We labeled early endosomes with EEA1 and observed that knockdown of GSDME promoted EGFR transport from plasma membrane to early endosomes.Sites that regulate EGFR endocytosis include tyrosine 1045,tyrosine 1068,and tyrosine 1086.Further experimental results showed that GSDME knockdown increased the phosphorylation of EGFR tyrosine at position 1045 to promote c-CBL binding,thereby promoting EGFR degradation and inhibiting cell proliferation.GSDME has a self-inhibiting structure.Further experiments revealed that the N-terminal fragment of GSDME was opposite to the full-length,and the N-terminal fragment of GSDME promoted EGFR degradation.In addition to its effect on endocytic degradation of EGFR,does GSDME directly affect the activity of EGFR pathway?Since GSDME can bind to the EGFR intracellular domain,we hypothesize that this binding may affect EGFR dimerization.The results of co-immunoprecipitation experiments showed that the full length of GSDME could promote EGFR dimerization.Further investigation revealed that GSDME can promote EGFR dimerization to promote phosphorylation of its intracellular tyrosine 1173,and ultimately promote cell proliferation.In summary,this study mainly explored the molecular mechanism of GSDME regulating the proliferation of non-small cell lung cancer cells through EGFR,and found that GSDME full-length and its N-terminal fragment have different regulatory mechanisms on EGFR signaling pathway.When the cells are under normal physiological conditions or under the treatment of chemotherapy drugs,the full-length and N-terminal fragments of GSDME play a role in protecting EGFR from degradation and promoting EGFR degradation,respectively.In addition,the full length of GSDME can also promote EGFR dimerization,thus promoting the activation of its downstream pathway.