Roles And Mechanisms Of 14-3-3? In Regulating Cisplatin Resistance Of Gastric Cancer

Gastric cancer(GC)is one of the most common digestive tract malignant tumors.China is a country with a high incidence of gastric cancer,and its mortality rate ranks third among malignant tumors.The overall prognosis of gastric cancer is poor.On the one hand,due to its strong heterogeneity,easy metastasis and recurrence,on the other hand,owing to insidious onset,most patients are already in the advanced stage when the diagnosis is confirmed.Platinum drugs are used as first-line chemotherapy drugs for patients with advanced gastric cancer.The disadvantage is that drug resistance induced by tumor heterogeneity during long-term treatment often leads to tumor recurrence,and the patient has a short benefit time and poor prognosis.14-3-3?isoform is a member of the 14-3-3 protein family,which is a serine/threonine binding protein of about 28 to 33 k Da acidic polypeptides,including seven highly homologous isoforms(?,?,?,?,?,?and?).By binding to phosphorylation-dependent proteins,they may affect subcellular localization and protein complex stability,thereby involved in multiple cellular functions,such as cell proliferation,cell cycle progression,regulation of apoptosis and adhesion.Substantial evidence indicated that 14-3-3 family are involved in tumor development,progression and drug resistance by transduction of signaling pathways.However,the mechanism of action of 14?3?3 members in the CDDP resistance of GC remains to be elucidated.In this study,we systemically analyzed the expression and prognostic value of 14-3-3family,and found that high 14-3-3?level was associated with the poor response to cisplatin chemotherapy in GC.Phosphoproteomic analysis revealed that 14-3-3?behaved as a phosphorylation-dependent molecular switcher to alter phosphorylation expression profile and interacted with phosphorylated Hsp90B as a critical pathway conferring acquired cisplatin resistance.Thus,14-3-3?might serve as a potential novel target in GC cisplatin resistance.Methods1.Patients and tissue microarrayIn total,127 pairs of gastric cancer tissues and paired non-tumor tissues were obtained from the Second Affiliated Hospital of Nanjing Medical University from 2010 to 2015.At the same time,the tissue microarray was constructed to study the relationship between protein expression and clinicopathological characteristics of gastric cancer.2.Construction of CDDP-resistant cellsTo obtain CDDP-resistant cells,the AGS cells were cultured in RPMI-1640 medium with 0.1?M of CDDP for 7 days.After an exponential growth was again reached in these treated cells,RPMI-1640 medium with 1?M of CDDP was added and cells was cultured for another period of time.Likewise,the cells were gradually exposed to stepwise increasing concentrations for a harsh selection.After a continuous induction of approximately 5 months,AGS/CDDP cells were successfully established after a sustained growth and steady passage in the RPMI-1640 medium contained 5?M of CDDP.3.RNA extraction and quantitative real-time PCR(q RT-PCR)Total RNA was extracted from GC cells and the concentration was measured.Then,RNA was reverse transcribed into complementary DNA(c DNA).q RT-PCR was performed with Light Cycler 96 SYBR Green I Master Mix machine and Universal SYBR Green Master Mix.The CT value was measured during the exponential growth phase and the 2^-??CT method was used to detect and evaluate gene amplification efficiency and relative expression.4.Western blottingTotal protein was extracted,the BCA kit was applied to measure the concentrations.Then,the target proteins were separated by SDS–PAGE gel electrophoresis and transferred to PVDF membrane.After blocking,samples were incubated overnight 4?with the primary antibodies.The next day each membrane was washed with TBST and reacted with secondary antibodies for 1 hour.Finally,target protein expression was detected by ECL luminescent liquid.5.Cell viabilities and calculation of the 50%inhibitory concentrations(IC₅₀)Using CCK-8 kit to measure cell viabilities.The gastric cancer cells were treated with different concentrations of cisplatin for 24h.The ratio of absorbance between the CDDP treatment group and the control group is the relative cell viability.The IC50s were calculated via Graph Pad Prism 8.0 software.Using the three-parameter dose-response equation and non-linear regression to calculate the the inhibition ratio of each test.The ordinate and abscissa,respectively,represented the inhibition ratio and the log(concentrations),results were exhibited as“best-fit values”±“standard errors”.6.Measurement of apoptosis and cell growthAfter transfection,the cells were treated with 5?M cisplatin for 24 hours:one part of the adherent cells was collected and counted under a microscope with a cell counter,and the other part was used to detect apoptosis by caspase 3 Activity Assay Kit.According to the Caspase-3 activity detection reaction system,reagent was added and incubated at room temperature for 4 hours in the dark,then use enzyme labeling.The meter detects the absorbance value at 405 nm.7.Phospho-specific protein microarray analysisThe phospho explorer antibody array(PEX100)was used to screen for downstream key protein.Protein phosphorylation level was measured as following formula:phosphorylation ratio=phosphorylated value/un-phosphorylated value.8.Bioformatics analysisTCGA-STAD gene expression data was downloaded from TCGA database.Kaplan-Meier Plotter database was used to analyze the correlation between the 14-3-3 family members expression with overall survival(OS)of GC.DAVID database was used to perform the gene ontology(GO)and Kyoto Encyclopaedia of Genes and Genomes(KEGG)pathway analysis.The p<0.05 was set as the cut-off criterion for significant enrichment.9.Xenograft and treatmentsAGS^CRcells(approximately 2×10⁶ cells/m L)were inoculated into the right armpit of mice to construct a subcutaneous tumor model.The cells were grouped as follows:(1)MOCK group,(2)14-3-3?knockdown(KD)group,(3)CDDP treatment group,(4)14-3-3?KD+CDDP treatment group.After 5 weeks,the mice were sacrificed and tumor tissues were collected for further investigation.10.Immunohistochemistry(IHC)For IHC,sections mounted on slides were dewaxed using xylene and rehydrated in ethanol.Then,sections were incubated in 10%normal bovine serum albumin for 5 min,followed by incubation with primary antibody at 4?C overnight.The slides were then incubated with secondary antibody for 30 min at room temperature.Samples were then visualized using diaminobenzadine,dehydrated,cleared,mounted,and photographed under a panoramic-scan digital slice scanning system.The graphs were analyzed using Image-Pro-Plus 6.0 software as described previously.11.Statistical analysisData sets were compared using Graph Pad 8.0 Software.The t test is used to compare the differences between the two groups,and the Kaplan-Meier survival curve and log-rank test are used for the OS and PFS analysis,Chi-square test is used to analyze clinicopathological characteristics,and Cox regression model is used to analyze independent prognostic factors related to survival.p<0.05 was considered statistically significant.Results14–3-3?is a key molecule for cisplatin resistance in GCWe observed that high expression of?,?,?and?isoforms were significantly related with poor overall survival(OS)and progression free survival(PFS)in GC by bioinformatic analysis.Then,we found only high expression of 14-3-3?turned to predict a poor OS in patients with GC after meta-analysis.As well,we investigated that?and?isoforms were increased in recurrence patients than those in patients without recurrence.Finally,we constructed resistant cell models to examine the expression of14-3-3 protein family in GC cells.The results suggested that the expressions of 14-3-3?were significantly higher in AGS^CR than those in parental AGS cells by q RT-PCR.Collectively,these results revealed that 14-3-3?plays an important role in cisplatin resistance in gastric cells.14-3-3?regulated phosphorylated modification of cancer associated proteome in cisplatin-resistant GC cellsBy performing functional experiments,we found that 14-3-3?plays an important role in cisplatin resistance in GC cells.So,to efficiently clarify the profile protein phosphorylation status mediated by 14-3-3?on cisplatin resistance in gastric cancer cells,a phospho-antibody microarray was used in MOCK and 14-3-3?-knockdown AGS^CR cells.Using this approach,we detected and analyzed phosphorylation events at1318 specific sites using the PEX100 array to explore the potential downstream effectors of 14-3-3?.Based on PEX100 results,after knockdown of 14-3-3?,the phosphorylation of VEGFR,IGFR,FGFR and EGFR,the receptors with extracellular signal transduction function declined obviously.Collectively,these results indicated that,14-3-3?mediated downstream protein could form a special phosphorylation modification system in cisplatin resistance in gastric cells.Identification of p-Hsp90B as an important downstream factor in regulating CDDP resistance in GC cellsIn the above-mentioned PEX100 array,multiple phosphorylation sites have been significantly changed,and the phosphorylation level of Hsp90B(Ser226)has the most dramatic variation.We also found that the expression of Hsp90B was associated with malignant progression and poor prognosis by bioinformatics tools.Further functional studies confirmed that the expression of p-Hsp90B was regulated by 14-3-3?.It also showed that 14-3-3?knockdown enhanced the caspase-3 activity,the phenomenon in14-3-3?-overexpression group were opposite,while significantly elevated when treated with Hsp90 inhibitor AUY922.Furthermore,the IC50s of CDDP were also decreased in both AGS and HGC-27 cells in the presence of AUY922.Therefore,we hypothesized that 14-3-3?/Hsp90B pathway may form a vicious circle to stimulate resistance to cisplatin in gastric cancer.14-3-3?promotes CDDP resistance of GC cells in vivoWe performed in vivo experiments to further confirm our results.Treating the xenografts with CDDP alone mildly inhibited tumor growth.However,combining with knockdown of 14-3-3?facilitated the CDDP-caused inhibition of tumor growth,and enhanced the apoptosis.Collectively,these results suggested that,by phosphorylation of Hsp90B,binding sites for 14-3-3?are created,which in-turn activated the anti-apoptosis/pro-survival process,leading to the increase of CDDP resistance in GC cells.Prognostic value of the combination of 14-3-3?and Hsp90B in GCBased on the mechanism above,we proceeded to explore the clinical relevance of 14-3-3?and Hsp90B in our study.We found that the expressions of 14-3-3?and Hsp90B were higher in GC tissues than those in normal tissues.Then we investigated that a significant positive correlation was found between 14-3-3?and Hsp90B levels.Moreover,Kaplan-Meier survival analysis showed that“14-3-3?and Hsp90B both high”group had the worst OS and PFS.A univariate analysis showed that OS and PFS were obviously related to clinical stage,pathologic differention,14-3-3?and Hsp90B expression level.Subsequently,multivariate analysis indicated that 14-3-3?expression,along with clinical stage,was an independent risk factor for OS and PFS.These results indicated that,14-3-3?might be regarded as a potential risk factor of GC,predicting poor prognosis and CDDP resistance.Conclusions1.Among 14-3-3 family,14-3-3?isoform in GC can result in cisplatin resistance2.14-3-3?induced/maintained cisplatin resistance in GC Cells by proteomics phosphorylation modification3.Phosphorylated Hsp90B is a key downstream factor regulated by 14-3-3?in cisplatin resistance in GC4.14-3-3?could be a potential therapeutic target for reversing cisplatin resistance in GC patients...