Proteomic Analysis Of Relapsed/Refractory NK/T Cell Lymphoma And Functional Study Of Key Proteins

Background and objective:The incidence of malignant lymphoma increases year by year,which becomes one of the major cancers threatening human health and life.NK/T cell lymphoma(NKTCL)is one distinct subtype of aggressive non-Hodgkin lymphoma,which is characterized by poor survival and dismal prognosis.Epstein-Barr virus(EBV)infection plays a crucial role in the pathogenesis of NKTCL,and the EBV encoded latent membrane protein 1(LMP1)and microRNAs promote tumorgenesis and immune escape.Advanced NKTCL patients experience more progressive disease and usually suffer from hemophagocytic syndrome,which lead to high mortality rate.Currently,standard therapy for advanced NKTCL patients has not been established.DDGP regimen(cisplatin,dexamethasone,gemcitabine and pegaspargase)have show remarkable efficacy in the treatment of advanced NKTCL with overall response rates of 87.5%,while primary or acquired drug-resistance and high relapse rate frequently result in treatment failure.Moreover,for high risk or relapsed/refractory patients,if we could predict the efficacy of chemotherapy prior to treatment,modified therapy strategy would prolong their survival and improve quality of life.Currently,few parameters are available to predict chemosensitivity in advanced NKTCL.Only pretreatment EBV-DNA copy number in whole blood was correlated with tumor response and disease recurrence.Proteomic analysis of the differentially expressed proteins between responders and relapsed/refractory patients is expected to screen biomarkers associated with therapeutic response and discover precise targets to overcome drug-resistance,which provide scientific basis for target therapy and individual therapy in advanced NKTCL.The mechanism network of antitumor response by the host is rather complicated,in which certain proteins with biological effect are secreted into blood by tumor cells,immune cells and the inflammatory cells infiltrating in tumor microenvironment.Serums are precious samples from patients which contain much valuable information reflecting the prognosis of disease.With the development of high-throughput proteomic technology,it is available to screen protein profilings of complex samples such as serum and tissue.Isobaric tags for relative and absolute quantitation(iTRAQ)coupled with liquid chromatography tandem mass spectrometry(LC-MS/MS)has several advantages: be more sensitive to detect low abundance protein,possessing higher ability to separate proteins and wider analytical range,the qualitative and quantitative result be more accurate.In the present study,we conducted proteomic analysis to screen and identify differentially expressed proteins between responders and relapsed/refractory patients.We selected S100A9 and ORM1 as candidate biomarkers and further confirmed them in serum from a larger cohort of patients by ELISA and examine their reliability.In addition,serum levels of candidate biomarkers were correlated with clinicopathologic characteristics,response rate,relapse rate and survival.Expression of S100A9 were examined in tumor tissues and normal tissues.We constructed recombinant expression plasmid pET-16b-S100A9 and obtained recombinant S100A9 protein after induction of expression.Finally,we investigated the effects of S100A9 recombinant protein on biological characteristics of NKTCL cell and tumor growth,and further uncovered the molecular mechanisms underlying the effects.Part 1 Proteomic analysis of relapsed/refractory NK/T celllymphoma and validation of candidate proteins Methods:(1)Pretreatment serum samples from chemosensitive and relapsed-refractory advanced NKTCL patients were collected;high abundance proteins in serum were removed using affinity chromatography columns.(2)Serum proteins were digested into peptides and labeled with iTRAQ reagents;the complexity of peptides sample was firstly reduced utilizing strong cation exchange(SCX)chromatography.(3)High performance liquid chromatography connected with mass spectrometer were utilized to separate peptides,do chromatographic analysis and quantification.(4)Gene ontology(GO)annotation and KEGG pathway mapping were performed to analyze the differentially expressed proteins.(5)In larger cohort of patients,ELISA assay was performed to examine expression of candidate protein S100A9 and ORM1;the sensitivity and specificity of candidate proteins for predicting therapeutic response were calculated.(6)The expression of serum S100A9 and ORM1 were correlated with clinical characteristics,response rate,early relapse rate and survival.multivariate analysis using Cox regression model was conducted to explore independent prognostic factors for advanced ENKL patients.(7)Expression of S100A9 in serum healthy individuals and NKTCL patients were detected by ELISA assay;expression of S100A9 in NKTCL tumor tissues and normal tissues were detected by immunohistochemistry;expression of S100A9 in NKTCL cell lines were detected by western blot.Results:(1)Serum samples were qualified for further study and high abundance proteins were removed effectively,and twelve fractions were obtained after strong cation exchange(SCX)chromatography.(2)Compared with responders,a total of 83 differentially expressed proteins were identified in relapsed/refractory patients which include 61 upregulated proteins and 22 downregulated proteins(3)These differentially expressed proteins were mainly involved in the process of inflammatory/acute phase response,DNA damge and enriched in chemokine signaling pathway,hippo signaling pathway.(4)In relapsed/refractory NKTCL patients,mean levels of serum S100A9 and ORM1 were much higher than that in responders with(p?0.0001).Serum levels of S100A9 at the concentration of 62.0ng/ml possessed a sensitivity of 81.5% and a specificity of 71.4%.Serum levels of ORM1 at the concentration of 1436ng/ml possessed a sensitivity of 85.2% and a specificity of 77.1%.When S100A9 in combined with ORM1,the sensitivity increased to 100%.(5)The response rate in high-S100A9 group was lower than that in low-S100A9 group(63.6% vs 93.1%,p=0.006)and relapse rate was higher in high-S100A9 group(82.8% vs 33.3%,p<0.001).The response rate in high-ORM1 group was lower than that in low-ORM1 group(64.5% vs 90.3%,p=0.007)and relapse rate was higher in high-ORM1 group(51.6% vs 35.5%,p?0.001).(6)High S100A9 and ORM1 in serum were correlated with inferior overall survival and poor progression free survival.Serum S100A9,ORM1 and EBV copy number were independent prognostic factors in advanced NKTCL patients.(7)The expression of S100A9 in serums from NKTCL patients were much higher than that in healthy individuals.The expression of S100A9 in tumor tissues was much higher than that in normal nasal mucous,and it was expressed mainly in tumor stroma infiltrating inflammatory cells and were not expressed by tumor cells.The NKTCL cell lines and normal NK did not express S100A9 preotein.SummarySeveral differentially expressed proteins with biological function were identified in serums from responders and relapsed/refractory NKTCL patients.Serum S100A9 in combination with ORM1 could serve as reliable biomarkers for predicting therapeutic response of advanced NKTCL patients,and S100A9 and ORM1 were independent prognostic factors for advanced NKTCL patients.Elevated expression of S100A9 in serum and tumor microenvironment in NKTCL patients suggest that it may play a key role in the pathogenesis of NKTCL.Part 2 Construction of S100A9 recombinant expression plasmid and induction,purification and identification of recombinant protein Methods:(1)The whole coding sequence of human S100A9 gene was synthesized,and RT-PCR was performed to amplify and introduce two restriction sites Ndel and BamHI.(2)The PCR products and expression vector pET16 b were digested,pufified and then connected with T4 DNA ligase to construct recombinant expression plasmid.(3)The recombinant expression plasmid was transformed into E.coli DH5? cells,and the positive transforms were selected by PCR,and further validated by enzyme digestion and DNA sequence.(4)The recombinant plasmid was transformed into Rosetta2 cells,and expression of recombinant protein was induced by IPTG,purified with Ni-NTA agrose and identified by SDS-PAGE and western blot.Results:(1)The recombinant expression plasmid pET16b-S100A9 was successfully constructed;PCR of bacteria liquor,enzyme digestion and DNA sequence all indicated that the recombinant plasmid pET16b-S100A9 contained the whole coding sequence of human S100A9 gene.(2)After transformation of recombinant plasmid,the recombinant protein was obtained by induction,purification and concentration.Recombinant proteins was characterized as human S100A9 proteins through SDS-PAGE and western blot.SummaryRecombinant expression plasmid pET-16b-S100A9 was successfully constructed which contained coding sequence of human S100A9 gene;The S100A9 protein was obtained after induction,purification and concentration,which provide abudndant materical for further study.Part 3 The effects of S100A9 recombinant protein on biologicalcharacteristics of NKTCL cells and studies of mechanisms Methods:(1)CCK-8 assay were performed to examine the effect of S100A9 recombinant protein on cell proliferation of NKTCL cells.(2)flow cytometry were performed to examine the effect of S100A9 recombinant protein on cell apoptosis and cell cycle distribution of NKTCL cells.(3)Changes in expression of PD-L1 were detected upon treatment with S100A9 recombinant protein through western blot.(4)Investigate the targeted receptor on tumor cell surface by S100A9 protein:The experiment include six groups: blank,S100A9 protein,RAGE inhibitor,TLR 4 inhibitor,S100A9 protein in combination with RAGE inhibitor S100A9 protein in combination with TLR4 inhibitor.(5)Changes in expression of p65,ERK1/2,JNK1/2,p38 MAPK and their phosphorylated protein were detected upon treatment with S100A9 recombinant protein through western blot.(6)Cotreatment of NKTCL cells with S100A9 protein and specific ERK1/2 inhibitor,cell proliferation and expression of PD-L1 were detected.The experiment include four groups: blank,S100A9 protein,ERK1/2 inhibitor,S100A9 protein in combination with ERK1/2 inhibitor.(7)Observe the effect of Tasquinimod(S100A9 inhibitor)on tumor growth in vivo.Results:(1)Low and mediate concentration of S100A9 protein promoted cell proliferation of NKTCL cells,while high concentration of S100A9 did not enhance the growth promotion.(2)Cell apoptosis were not affected upon treatment with S100A9 protein;the cells in S phase were increased and G1 phase were decreased upon S100A9 treatment.(3)The expression of PD-L1 in tumor cells were increased when exposed to S100A9 protein.(4)Blockade of RAGE receptor reduced the effect of S100A9 on tumor cells while blockade of TLR4 did not exert any effect.(5)Upon treatment with S100A9 protein,expression of phosphorylated ERK1/2 was increased,while the levels of p65,ERK1/2,JNK1/2,pJNK1/2,p38 MAPK and p-p38 MAPK were not changed;RAGE inhibitor decreased the activation of ERK1/2 signaling by S100A9 protein.(6)ERK1/2 inhibitor decreased the proliferation promotion and upregulation of PD-L1 expression induced by S100A9 protein.(7)Tasquinimod treatment significantly reduced tumor growth compared with control group.SummaryS100A9 protein promotes cell proliferation of NKTCL cells and upreglates PD-L1 expression via interaction with RAGE receptor and activation of ERK1/2 signaling pathway.Blockade of the interaction between RAGE receptor reduced proliferation promotion and upregulation of PD-L1 expression induced by S100A9 protein,and interruption of S100A9/RAGE interaction decreases tumor growth in vivo.Conclusion1.Serum S100A9 in combination with ORM1 could serve as reliable biomarkers for predicting therapeutic response of advanced NKTCL patients,and S100A9 and ORM1 were independent prognostic factors for advanced NKTCL patients.2.Recombinant expression plasmid pET-16b-S100A9 was successfully constructed,and His-tag labeled recombinant S100A9 protein was obtained.3.S100A9 promoted proliferation of NK/T cell lymphoma cells and upregulated PD-L1 expression via RAGE-mediated activation of ERK1/2 pathway.Targeting S100A9 protein decreased tumor growth in mice.