Molecular Implications Of DEK/Snail Axis In Gastric Cancer Progression And Chemsensitivity

Background:Gastric cancer(GC)is a common gastrointestinal malignancy.Due to the differences in diet culture,the incidence and mortality of GC in East Asian countries,such as China,South Korea and Japan,are higher than other countries[1].Despite the fact that the great advances have been made in surgical techniques,loss of the best time for surgery remain realistic for GC treatment.To date,the total/subtotal gastrectomy with intravenous chemotherapy is still the dominating choice for patients with advanced GC.The intravenous chemotherapy is related to the quality of survival of patients with GC.Unfortunately,despite the advances have been made in chemotherapy,chemotherapy resistance is still a crucial cause of therapeutic failure in patients with advanced GC[2].Therefore,it is a great significance to identify genes related to tumorigenesis and resistance of chemotherapy in GC.DEK,a transcription factor,is a conserved non-histone nucleoprotein without known paralogs.The functions of DEK involve DNA supercoiling,transcriptional control and cell viability in cell progression and metabolism.As an architectural chromatin oncoprotein,DEK has been detected in numerous human malignancies including breast cancer[3],liver cancer[4],lung cancer[5],cervical cancer[6]and colorectal cancer[7].Our previous study found that The expression levels of DEK protein in GC tissues were significantly higher than that in the adjacent non-tumor tissues.DEK overexpression in GC was correlated with tumor size,histological grade,tumor-node-metastasis(TNM)stage and overall survival(OS)rates,which was consistent with Lee et al[8].However,few researches ever specified the biological functions or the underlying mechanism of DEK in GC cell.Snail,a zinc finger transcription factor,is a member of the SNAIL family which includes Slug,smuc,Scratchl and Scratch2.the Zn2+coordination bonding model in Snail preferentially bound to the CAGGTG regions of DNA near the transcription start sites.Numerous studies have shown that Snail functions as a transcriptional factor,a signaling factor and pro-metastatic factor.It is reported that the overexpression of Snail promoted metastasis in GC.However,few researches ever specified whether Snail affect the other biological functions in GC.Objectives:This study is aimed to investigate the molecular implications of DEK/Snail axis in GC progression and chemosensitivity.(1)to identify the clinicopathological significance of DEK as a potent prognostic biomarker for patients with GC after enlarging the sample size.(2)to investigate the underlying molecular mechanism of DEK implicated in proliferation,chemotherapy resistance and metastasis in GC.(3)To explore the correlation between DEK and Snail,and further understand the effect and mechanism of DEK/Snail axis on GC progression.Materials and Methods:1.GC specimens and database analysis:1)The expression levels of DEK mRNA/protein in pan-cancers including GC were obtained from multiple bioinformatics databases.2)GC samples paired with the adjacent non-tumor tissue samples were selected for immunohistochemical(IHC)staining for DEK.The correlations between DEK overexpression and the clinicopathologica features of gastric cancers were evaluated using the Chi-squared.3)The survival rates were calculated by the Kaplan-Meier plotter bioinformatics database.2.Experiments in vitro:1)The siRNA and DNA plasmid were used to mediate DEK and Snail expression in GC cells.2)The effects of DEK expression on GC cells proliferation were detected by MTT,colony formation assay,soft agar assay and EdU staining.3)The effects of DEK expression on GC cell cycle distribution and cell apoptosis rates were detected by flow cytometry assay.4)The effects of DEK expression on GC cells chemotherapy resistance were analyzed by Cisplatin and 5-FU treatment.5)Wound healing,transwell assay and microtubule formation assay were used to determine the effects of DEK expression on migration,invasion and angiogenesis in GC cells.6)The underlying mechanism between DEK and Snail was measured by immunofluorescence staining and Co-Immunoprecipitation(CoIP)assay.7)The effects of DEK expression on Snail protein stability were detected by CHX(Cycloheximide,a protein synthesis inhibitor)and MG132(a proteasome inhibitor)treatments.8)The expression levels of cell cycle distribution-related proteins,DNA damage response-related proteins and EMT-related proteins were measured by Western Blot.3.Experiments in vivo:GC-chick chorioallantoic membrane(CAM)model were used to determine the effects of DEK/Snail axis on angiogenesis in GC cells.Results:Part 1.DEK is a potent prognostic biomarker for patients with GC:1)DEK overexpression was correlated with poor prognosis in patients with GC:Based on the data from multiple bioinformatics databases,the IHC staining and Western bloting in GC samples paired with the adjacent non-tumor tissue samples,we found that the expression level of DEK in GC tissues was significantly higher than that in the adjacent non-tumor tissues.DEK overexpression in GC was correlated with tumor size,TNM stage,serosal invasion and lymph node metastasis.In the Kaplan-Meier models,overall survival(OS)rates were significantly higher in GC cases with DEK low-expression than in those with DEK overexpression.DEK overexpression influenced OS rates of GC in HER2(+)group and 5-FU treatment group.2)DEK accelerates the proliferation in GC cells:We found that DEK overexpression promoted cell viability,colony formation,anchorage-dependent growth and the abilityof DNA replication by MTT,colony formation assay,soft agar assay and EdU staining in GC cells.3)DEK affects GC cells cycle progression,but apoptosis rates:PI staining showed that DEK expression had an effect on G0/G1 phase transition,regulating the expression levels of CDK,Cyclin D1 and p21WAF1/CIP1.FITC-Annexin V/PI staining showed that DEK expression without impacting the apoptosis rates in GC cells.4)DEK affects chemosensitivity by NHEJ and HR NHEJ repair in GC cells:We found that DEK low-expression promoted GC cells chemosensitivity by DNA damage response(DDR),regulating the expression levels of NHEJ-related protein Ku80 and HR-related protein RAD51.DEK low-expression induced chemosensitivity in Cisplatin/5-FU-treated GC cells.5)DEK enhances the migration,invasion and angiogenesis of GC cells via HIF-la pathway and MMPs:We found that DEK low-expression inhibited the invasion,migration and angiogenesis of GC cells by wound healing,transwell assay,microtubule formation assay and GC-chick chorioallantoic membrane(CAM)model.Furthermore,DEK down-expression blocked HIF-la pathway and MMPs expression.Part 2.DEK contributes to GC progression and chemosensitivity via the regulation of Snail protein stability:1)DEK regulates the nuclear expression level of Snail protein:Positive correlation between DEK and Snail was found by GO/PPI analysis and CoIP assay.And we found that the nuclear expression of DEK protein was down-regulated by DEK low-expression in GC cells.2)DEK regulates Snail protein stability via post-translational protein modification:DEK expression had little influence on Snail transcription.Furthermore,DEK low-expression accelerated the protease degradation in CHX/MG132-treated GC cells,indicating DEK regulated Snail protein stability via post-translational protein modification.3)DEK induced oncogenic activity-involvement of Snail:Silencing Snail led to repressed the proliferation,migration and angiogenesis abilities in DEK overexpression GC cells.4)Snail overexpression was correlated with poor prognosis in patients with GC:Based on the data from multiple bioinformatics databases,we found that the level of Snail expression was elevated in GC samples.Snail overexpression was correlated with poor prognosis in patients with GC.Conclusions:1.DEK and Snail were co-overexpressed in GC tissues,and both correlates with poor prognosis.2.DEK accelerates the proliferation,chemotherapy resistance,metastasis and angiogenesis in GC cells.3.DEK regulates Snail protein stability via post-translational protein modification.Blocking DEK/Snail axis inhibits the proliferation,metastasis and angiogenesis in GC cells.