Functional Roles And Mechanisms Of PABPC1 In The Development And Progression Of Natural Killer/T-cell Lymphoma

Background and objectiveNatural killer/T cell lymphoma(NKTCL)is a highly aggressive non-Hodgkin lymphoma,derived from mature NK or cytotoxic T cells,and that has poor prognosis and short survival.The disease has a strong association with Epstein-Barr virus(EBV)and commonly affects adult males,with high prevalence in Asia.Central and South America.NKTCL frequently involves nasal cavity,sinuses,nasopharynx and occasionally occurs primarily in other organs,including the skin,gastrointestinal tract and testis.Patients diagnosed with NKTCL always have a poor sensitivity to anthracycline-based chemotherapy due to high expression level of the drug exporter p-glycoprotein.The current NKTCL standard of care is L-asparaginase or pegaspargase based chemotherapy,with or without radiotherapy.However,due to multiple drug resistance(MDR),the patient's complete response(CR)rate remains only 50-60%and the five-year overall survival(OS)rate is approximatively 50%.Therefore,there is an urgent clinical need to identify NKTCL molecular pathogenesis and develop effective targeted therapy.In the past decade,high-throughput molecular and genomic profiling studies have significantly improved our understanding of NKTCL;however,the pathogenesis of NKTCL remains elusive.Using gene expression profiling,several deregulated signaling pathways,such as the Janus-associated kinase/signal transducer and activator of transcription(JAK/STAT),mitogen-activated protein kinase(MAPK),protein kinase B(Akt),and nuclear factor(NF-?B),have been identified as players in NKTCL development.High-throughput sequencing studies identified JAK3,STAT3,GNAQ,DDX3X and TP53 as the frequently mutated genes in NKTCL.Besides,the involvement of the 6q21-25 deletion and the deregulation of the immune microenvironment have also been demonstrated.Post-transcriptional regulation of genes is important for proteins' expression and functions.Following transcription,RNAs binds to RNA binding proteins(RBPs)to perform biological activities.The dysregulation of RBPs in several human cancers influences every step of cancer development and progression,including sustained cell proliferation,survival,immune surveillance evasion,angiogenesis induction,and metastasis activation.Poly(A)-binding protein.(PABP)is a major component of the messenger RNA-protein complex and that can distinguish and bind the 3' poly(A)tail of the eukaryotic messenger RNA,the eukaryotic initiation factors(eIFs)and the PABP-interacting protein 1(PAIP1).The PABPC family includes at least 3 functional proteins:PABPC1,PABPC3 and PABPC4.PABPC1 plays the most important role in polyadenylation/deadenylation,initiation of mRNA translation and poly(A)tail protection.These functions suggest that PABPC1 may play a role in cancer initiation and progression.Using gene expression profiling,we found that PABPC 1 was highly expressed in NKTCL cell lines.The purpose of this study was to further investigate PABPC 1 expression and functions in NKTCL tumorigenesis.RT-qPCR,western blot and immunohistochemistry(IHC)assays were conducted to determine PABPC 1 expression level in NKTCL cell lines and tissues,and its correlation with NKTCL prognosis.The potential transcription factor binding to PABPC1 was identified using chromatin immunoprecipitation(ChIP)and luciferase reporter assays.Subsequently,PABPC1 gain-or loss-functional assays were used to detect its function in NKTCL cell lines.In addition,a signaling pathway in NKTCL cells overexpressed with PABPC1 was identified by transcriptome sequencing and validated by western blot and a pathway inhibitor.Moreover,gene function and the molecular mechanism were further evaluated using mice xenograft models.The results of this study will provide important evidence for the prediction of prognostic indicators and application of individualized therapy in NKTCL.Part ?:Expression and clinical significance of PABPC1 in natural killer/T-cell lymphomaMethods1.RT-qPCR and western blot were used to evaluate PABPC1,PABPC3 and PABPC4 expression at the mRNA and protein levels in normal NK cells and NKTCL cell lines(YT,NKYS,NK92 and SNK6).2.Tumor tissues from 58 patients diagnosed with NKTCL and normal tissues from 10 patients with lymph nodes' reactive hyperplasia were collected and PABPC1 expression was detected using immunohistochemical(IHC)staining.3.The association between PABPC1 expression and clinicopathological characteristics was analyzed using the Pearson ?2 test or Fisher's two-tailed exact test.4.The relationship between PABPC1 expression and survival outcomes was calculated using the Kaplan-Meier method and compared by the log-rank test.Prognostic factors of OS and PFS were detected using the Cox proportional hazard regression models.Results1.PABPC1 mRNA expression in YT(P=0.011),NKYS(P=0.003),NK92(P=0.000)and SNK6(P=0.005)was higher than that in normal NK cells.There were no significant changes in PABPC3 and PABPC4 mRNA expression levels.PABPC1 protein expression was consistent with the mRNA results.2.PABPC1 had a cytoplasmic staining and was highly expressed in NKTCL.A total number of 30(51.7%)patients had a high PABPC1 expression and 28(48.3%)patients had a low expression.The expression of the reactive hyperplasia of lymph nodes was lower than that in tumor tissues.The PABPC1 expression in NKTCL was higher than that in reactive hyperplasia of lymph nodes(P=0.002).3.PABPC1 expression level had no significant correlation with age,sex,B symptoms,ECOG score,LDH EBV-DNA,lymphoma node,NKPI,therapy and treatment response;However,the Ann Arbor stage significantly correlated with PABPC1 expression(P=0.031).4.The high PABPC1 expression was associated with poor OS(median,27 months compared with not reached;HR 3.9,P=0.008)and PFS(median,21 months compared with not reached;HR 2.8,P=0.009).PABPC1(P=0.049)and NKPI(P=0.035)were independent prognostic factors for overall survival of NKTCL patients.Summary1.PABPC1 expression was upregulated in NKTCL cell lines(YT,NKYS,NK92 and SNK6)compared to normal NK cells,whereas,the expression of PABPC3 and PABPC4 showed little change.2.PABPC1 was highly expressed in NKTCL tumor tissues.3,The expression of PABPC1 was significantly associated with Ann Arbor stage,but not with the other clinical characteristics.4.PABPC1 was an independent prognostic factor for OS in NKTCL patients.Part ?:Regulation of PABPC1 expression,function and mechanism in natural killer/T-cell lymphomaMethods1.Bioinformatic analysis was performed using JASPAR and TRANSFAC to predict binding transcription factors.2.RT-qPCR and western blot were used to evaluate mRNA and protein expression of the potential transcription factor in normal NK cells and NKTCL cell lines(YT,NKYS,NK92 and SNK6).PABPC1 mRNA and protein levels were evaluated in YT and NK92 cells that were transduced with an ATF4 overexpressing lentiviral vector.3.ChIP assay was performed to identify the direct interaction between the transcription factor and PABPC1 promoter.A dual-luciferase reporter assay was used to detect the expression regulation of PABPC1 expression.4.A PABPC1-overexpressing model(YT-Lv-PABPC1)was constructed by infecting YT cells with Lv-PABPC1 lentivirus.A PABPC1-silenced model(NK92-sh-PABPC1)was generated by infecting NK92 cells with an sh-PABPC1 lentivirus.The transfected clones were selected by puromycin.RT-qPCR and western blot were used to evaluate expression levels of PABPC1 mRNA and protein.5.Gain or loss of functional assays were performed to detect the role of PABPC1 in NKTCL,including Cell Counting Kit-8(CCK-8)assay,flow cytometry,soft ager and transwell assays.6.To investigate activity changes of potential signaling pathways following PABPC1 overexpression,transcriptome sequencing and bioinformatic analysis were used.Key proteins expression was determined using western blot.7.Western blot and loss of function assays were performed to evaluate the impact of a PI3K/mTOR inhibitor on YT-Lv-PABPC 1.Results1.The activating transcription factor 4(ATF4)was predicted to bind PABPC1 promoter region at two potential binding sites.2.ATF4 mRNA was upregulated in YT(P=0.026),NKYS(P=0.008),NK92(P=0.000)and SNK6(P=0.000),when compared to the normal NK cells.A significant increase in PABPC1 mRNA level was observed in ATF4-overexperssing YT(P=0.001)and NK92(P=0.001)cell lines.PABPC1 protein expression was consistent with the mRNA results.3.ATF4 was enriched at two binding sites on the PABPC1 promoter region in NKTCL cell lines.Enhanced ATF4 expression dramatically elevated luciferase intensity in wild type cells(P=0.013);however,luciferase activity was not altered by ATF4 in mutant type cells.4.PABPC1 mRNA was upregulated in YT cells following transduction with Lv-PABPC1(P=0.000);whereas,it was downregulated in NK92 cells transduced with the sh-PABPC1 lentivirus(P=0.000).PABPC1 protein expression was consistent with the mRNA results.5.Enhanced PABPC1 expression promoted cell growth of YT cells(P<0.05),whereas its silencing in NK92 cells induced the suppression of cell proliferation(P<0.05).PABPC1 induced S phase blockage(P=0.016),while its knockdown induced a decrease in S phase(P=0.007).Enhanced PABPC1 expression decreased cell apoptosis(P=0.011)and increased the ability of colony formation(P=0.004)and cell mobility(P=0.000)in YT cells.Whereas PABPC1 silencing in NK92 cells increased cell apoptosis(P=0.017)and decreased the ability of colony formation(P=0.005)and cell mobility(P=0.006).6.Transcriptome sequencing showed that 92 genes were upregulated and 243 genes were downregulated in YT-Lv-PABPC1 cells when compared with YT-Lv-NC cells.Subsequently,KEGG and GSEA analysis were performed and we found that the phosphatidylinositiol 3-kinase(PI3K)/Akt/mammalian target of rapamycin(mTOR)signaling pathway was activated by PABPC1.Western blot also showed that PABPC1 directly affected expression levels of proteins involved in the PI3K/Akt/mTOR pathway(P<0.05).7.p-Akt and p-mTOR were downregulated following treatment with a PI3K/mTOR inhibitor in YT-Lv-PABPC1 cells(P=0.0009 P=0.000).The elevated S phase,cell proliferation,and the decreased cell apoptosis caused by PABPC1 overexpression were also significantly reversed by PF-502(P=0.001,P=0.000,P=0.000).Summary1.ATF4 was upregulated in NKTCL cell lines when compared to normal NK cells.PABPC1 was specifically activated by ATF4 transcription factor in NKTCL.2.PABPC1 promoted cell growth,colony formation ability and cell mobility.3.The silencing of the PI3K/Akt/mTOR signaling pathway,using an inhibitor,reversed PABPC1-induced cell proliferation.Part ?:In vivo validation of PABPC1 function and mechanism in natural killer/T-cell lymphomaMethods1.YT cells that were stably transduced with Lv-PABPC1 or Lv-NC were injected into the flanks of BALB/c nude mice.2.When the average tumor volume reached about 100 mm3,the YT-Lv-PABPC1 group was divided into YT-Lv-PABPCI/Vehicle and YT-Lv-PABPC1/PF-502.The YT-Lv-PABPCI/PF-502 group was treated by oral gavage with 5 mg/kg/d PF-502.The YT-Lv-NC/Vehicle and YT-Lv-PABPC1/Vehicle were placebo treated using the same delivery method.3.Tumors volume was measured every 4 days.The primary tumors were surgically removed and tumors wight was measured.4.The tumors were fixed,paraffin-embedded and sectioned.The sections were observed under a microscope after hematoxylin and eosin(H&E)staining.The protein levels of CD5 6,perforin and Granzyme B were evaluated using IHC staining.5.IHC staining was used to detect Ki-67 expression.Terminal deoxynucleoitidyl transferase-mediated nick end labeling(TUNEL)was performed to evaluate the level of apoptosis.6.p-Akt and p-mTOR expression levels in each group were determined using IHC.Results1.Eight days after injection,YT-Lv-NC and YT-Lv-PABPCl animal models were successfully conducted and the tumor formation rate was 100%.2.Seventeen days after injection,compared with the YT-Lv-NC/Vehicle group,the tumor volume of YT-Lv-PABPC1/Vehicle group significantly increased(P=0.013).Twenty-one days after injection,the.tumor volume of YT-Lv-PABPC1/PF-502 group was smaller than that in the vehicle-treated group(P=0.000).3.The YT-Lv-PABPC1/Vehicle group exhibited an increase in tumor mass(P=0.000);whereas PF-502 showed a significant inhibition of the YT-Lv-PABPC1 group(P=0.000).4.The results of H&E staining of tumor tissues were consisted with the cellular morphology of malignant tumor cells.IHC staining indicated that CD56,perforin and GrB were present in each group of tumor tissues.5.Enhanced PABPC1 expression promoted Ki-67 expression(P=0.003)and decreased cell apoptosis(P=0.049);whereas,the elevated Ki-67 and decreased cell apoptosis were significantly reversed by PF-502(P=0.000,P=0.000).6.PABPC1,p-Akt and p-mTOR were upregulated in YT-Lv-PABPC1 tumor tissues.p-Akt and p-mTOR were significantly downregulated by treatment with PF-502 in YT-Lv-PABPC1 cells.Summary1.An NKTCL animal model was successfully generated.2.PABPC1 promoted NKTCL tumor growth via activating PI3K/Akt/mTOR signaling pathway.3.The PI3K/mTOR inhibitor reversed PABPC1-induced NKTCL tumor growth.Conclusions1.PABPC1 was upregulated in NKTCL cell lines and tumor tissues and PABPC1 was an independent prognostic factor for NKTCL patients' OS.2.ATF4 was upregulated in NKTCL cell lines and PABPC1 was specifically activated by the ATF4 transcription factor in NKTCL.3.PABPC1 promoted cell growth,colony formation ability and cell mobility via activating the PI3K/Akt/mTOR signaling pathway.The silencing of the PI3K/Akt/mTOR signaling pathway using an inhibitor reversed PABPC1-induced cell proliferation.4.PABPC1 promoted tumor growth via activating the PI3K/Akt/mTOR signaling pathway in vivo.The PI3K/mTOR inhibitor reversed PABPC1-induced tumor growth.