CGA Promotes Chemoresistance In Gastric Cancer Through EGFR-GATA2 Positive Feedback Circuit

【Background】Chemotherapy is widely applied for clinical therapy of gastric cancer,and it can remarkably improve the overall survival（OS）and progression-free survival（FPS）of patients with stage II-III gastric cancer,and can make improvement the life-quality of advanced gastric cancer patients.However,chemoresistance often occurs along with chemotherapy and is an important cause of clinical oncology therapy failure.Tumor drug resistance biomarkers play an important role in predicting chemotherapeutic resistance.However,currently known drug-resistance biomarkers are mostly located in cell membrane or cytoplasm,which determines their lack of accessibility and non-invasiveness.Therefore,it is particularly important to find more representative and easier to detect tumor drug resistance markers.Secreted proteins are a group of proteins that are synthesized inside the cell and secreted outside the cell,including many types:hormones,cytokines,antibodies,and complement.The secreted proteins of tumor cells are important sources of potential diagnostic markers and therapeutic targets due to their strong specificity and ease of detection.However,little is known about the secreted proteome of gastric cancer chemotherapy-resistant cells.This project will screen drug-resistance-related secreted proteins from gastric cancer chemotherapy-resistant cell models,and explore its role in the diagnosis and treatment of gastric cancer chemoresistance.【Objectives】To explore the mechanism of glycoprotein hormone alpha subunit（CGA）elevation in gastric cancer chemotherapy-resistant cells and tissues,and its molecular mechanism to promote gastric cancer multidrug resistance,providing new theoretical and experimental basis for solving clinical chemotherapy resistance.1.To identify the relationship between CGA and chemotherapy resistance in gastric cancer;2.To clarify the function of CGA in chemoresistance and prognosis of patients with gastric cancer;3.To reveal the molecular biological mechanism of CGA function in gastric cancer;4.To explore the causes of the dysregulation of CGA expression in gastric cancer;5.To explore the clinical value of CGA in predicting chemotherapy resistance of gastric cancer and the significance of targeting CGA in the treatment of chemoresistance.【Methods】1.Differences in IC50 of gastric cancer cells and gastric cancer chemoresistant cells to multiple chemotherapeutic agents were tested by Cell Counting Kit-8;Mass spectrometry techniques were employed to profile secreted proteins distinctly expressed in gastric cancer parental cells and drug-resistant cells,and the candidate drug-resistant related genes were screened by combined analysis with previous transcriptome data.The expression differences of CGA in gastric epithelium and gastric cancer cells,specimens of gastric cancer treated with chemotherapy and PDX tissues were examined by Western blot,immunofluorescence and immunohistochemistry.The Kaplan-Meier Plotter database was analyzed for the predictive value of target molecules for gastric cancer.2.CGA was knocked down or knocked out in gastric cancer cells using small interfering RNA or CRISPR/Cas9 gene editing techniques.The CGA plasmid was used to construct an overexpression lentiviral vector to establish a CGA gain-of-function model.The effects of CGA on tumor malignant phenotypes such as proliferation ability and chemotherapy resistance were investigated by flow cytometry,cell survival assay,cell proliferation curve assay and nude mice xenograft assay.3.Phosphorylated receptor tyrosine kinase（RTK）antibody array was performed for high-throughput screening of phosphorylated receptors activated by addition of CGA protein;The effect of overexpression or deletion of CGA on EGFR phosphorylation was studied by Western blot.The binding of CGA and EGFR were predicted by Clus Pro website and measured by Surface plasmon resonance（SPR）.Immunofluorescence staining was adopted to explore the localization and degradation of CGA and EGFR in cells.Co-IP assay was employed to study the binding ability of CGA to EGFR.and its extracellular domain（ECD）.Transcriptome sequencing was applied to profile distinctly expressed genes and altered signaling pathways in response to knockdown of CGA.4.The glycosylation sites of CGA were detected by Mass spectrometry.The effect of CGA glycosylation site mutations on chemo-resistance was analyzed via cell survival assay.Western blot and immunofluorescence assays were conducted to investigate effects of CGA glycosylation site mutations on the ability to bind to EGFR.5.Bioinformatic analyses were used to predict the candidate transcription factors of CGA.The effect of up-regulation of GATA2 on CGA expression was detected by real-time quantitative PCR and Western blot.Dual luciferase reporter gene assay,chromatin immunoprecipitation（Ch IP）and electrophoretic mobility shift（EMSA）were deployed to examine the binding of GATA2 to the CGA promoter.The influence of CGA overexpression or deletion and EGFR signaling pathway on the expression of GATA2 was analyzed via real-time quantitative PCR and Western blot.Real-time quantitative PCR and Ch IP assay were used to investigate the inducing of sublethal dose chemotherapy drugs on GATA2 self-regulation.6.Expression of GATA2 and p-EGFR in gastric cancer chemotherapy and PDX tissue specimens examined by immunohistochemical staining.The correlation between CGA,GATA2 and EGFR was analyzed using the Gene Expression Omnibus（GEO）database.Enzyme-linked immunosorbent assay（ELISA）was taken to determine the concentration of CGA in healthy donors,non-chemotherapy and chemotherapy patients with gastric cancer.7.Database analyses were used to predict the candidate mi RNAs that regulate CGA,and these mi RNAs were verified by PCR and Western blot.CGA plasmids with or without3’-UTR were constructed and co-transfected with mi R-708-3p and mi R-761,and the change of chemotherapy resistance was detected using cell survival assay.Nude mouse tumorigenicity assay was adopted to evaluate the role of mi R-708-3p and mi R-761 in overcoming chemotherapy resistance in gastric cancer.【Results】1.Mass spectrometry identified 102 proteins specifically secreted in chemotherapy-resistant gastric cancer cells,and they were combined with gene expression profile data to finally screen out 9 secreted drug resistance-related genes.Western blotting and immunofluorescence staining demonstrated that there was a noticeably greater expression of CGA in gastric cancer chemo-resistant cells than in parental cells.Immunohistochemical staining indicated that CGA expression in gastric cancer after chemotherapy was dramatically increased compared to pre-chemotherapy,and the staining of CGA was apparently more intense in fluorouracil-treated PDX tissues.Further analysis of the database revealed that CGA was closely related to the prognosis of patients with gastric cancer and patients treated with fluorouracil chemotherapy.2.Western blot assay confirmed that CGA was successfully knocked out in SGC7901^ADR and SGC7901^VCR cells.Flow cytometry,cell survival assay and cell proliferation curve assay showed that knocking out CGA reduced gastric cancer cell drug resistance and replenishing human recombinant CGA protein（r CGA）could block the reduction of drug resistance caused by CGA knockout.In MKN45 and BGC823 cells,it was also found that CGA deletion caused a decrease in resistance to chemotherapeutics,while r CGA supplementation markedly reversed the decrease.Nude mouse tumorigenicity assay revealed that knockdown of CGA suppressed tumor growth and declined chemotherapy resistance in gastric cancer cells.3.Cell survival assay showed that CGA mutant plasmids could not restore chemotherapy resistance of CGA knockout cells.Western blot and RTK antibody array analysis showed that CGA depletion reduced EGFR phosphorylation and r CGA promoted EGFR phosphorylation.Co-IP experiment showed that CGA could bind to EGFR and EGFR ECD.The Clus Pro website predicted that 19 amino acid residues in the CGA structure were bound to residues in EGFR ECD through hydrogen bonding.CGA showed rapid association with the chip-immobilized ECD of EGFR and a dose-dependent resonance signal using SPR analysis.Immunofluorescence staining displayed that CGA and EGFR co-located in the cell membrane and subsequently entered the endosome.Transcriptome sequencing analysis and pathway enrichment showed that there was a significant change in“ERBB signaling pathway”after CGA downregulation.Cell survival assay and nude mouse tumorigenicity assay further demonstrated that targeted EGFR therapy could enhance chemotherapy cytotoxicity.4.Mass spectrometry analysis revealed that CGA was modified by N-glycosylation at Asn52 and Asn78 sites.Cell survival assay showed that CGA glycosylation site mutant plasmids could not restore chemoresistance of CGA knockout cells.Western blot analysis showed that the glycosylation site mutation affected the stability and secretion of CGA protein.Western blot,Co-IP and immunofluorescence assay indicated that CGA proteins with mutations in glycosylation sites could not induce EGFR phosphorylation,nor could they bind to EGFR.5.Through bioinformatic analyses,GATA2 was predicted as a possible CGA transcription factor.Real-time PCR and Western blot indicated that GATA2 affected CGA expression.Promoter binding experiments showed that GATA2 was capable of binding to two sites in the CGA promoter region.PCR and Western blot assays displayed that up-regulation or deletion of CGA and inhibition of EGFR signal pathway could affect the expression of GATA2.Real-time quantitative PCR and Ch IP assays indicated that GATA2could be triggered by chemotherapy and then initiated self-regulation.6.Immunohistochemical staining displayed a significant positive relationship between the CGA,GATA2 and p-EGFR expressing in gastric cancer chemotherapy specimens.The staining intensities of CGA,GATA2 and P-EGFR were consistent in PDX tissues.Analysis of the GEO database disclosed a positive correspondence between CGA,EGFR and GATA2.ELISA analysis demonstrated that CGA expression was remarkably elevated in patients undergoing chemotherapy compared to nonchemotherapy patients and healthy donors,and that CGA expression in gastric cancer patients was correlated to adverse outcome.7.Four candidate mi RNAs that regulated CGA were screened via bioinformatic analyses.Real-time quantitative PCR,Western blot and double luciferase reporter assays were applied for confirming that mi R-708-3p and mi R-761 regulate CGA expression by targeting CGA 3’-UTR.CGA with or without 3’-UTR plasmids were co-transfected with mi RNA mimics,and mi R-708-3p and mi R-761 were found to suppress drug resistance of gastric cancer cells by directly binding to CGA 3’-UTR.Nude mouse tumorigenicity assay showed that mi R-708-3p and mi R-761 could significantly enhance fluorouracil cytotoxicity.【Conclusion】This current study clarified the expression pattern of CGA in gastric cancer drug-resistant cells and tissues,revealing the important role of CGA in gastric cancer chemoresistance and the value of CGA in forecasting chemotherapy resistance.The mechanism of interaction between CGA and EGFR was revealed,and the effect of glycosylation modification on CGA promoting chemotherapy resistance was clarified.Through transcriptional regulation studies,we illuminated the interactive regulation of GATA2 and CGA and uncovered the mechanism of CGA-EGFR-GATA2 positive feedback loop promoting gastric cancer chemoresistance.The clinical value of mi RNA targeting CGA in the treatment of chemotherapy resistance in gastric cancer was also explored.This study clarified the role and mechanism of CGA in promoting gastric cancer chemoresistance,deepened the understanding of tumor drug resistance mechanism,and provided a new clinical diagnosis and treatment strategy for gastric cancer chemoresistance.