The Roles And Mechanisms Of Glycoprotein PTGDS And Its Inhibitor AT56 In Diffuse Large B-Cell Lymphoma

Non-Hodgkin lymphoma(NHL),one of the most common hematologic malignancies,is a group of malignant proliferative disease of lymphoid cells with high heterogeneity.Diffuse large B-cell lymphoma(DLBCL)is the most common aggressive NHL,accounting for 30%-40%of all initial NHL cases.DLBCL has highly heterogeneity in morphology,biology,immunophenotype,genetics and clinical manifestations.With the development and clinical application of new targeted drugs represented by Rituximab,immunomodulatory drugs and cell therapy,the clinical efficacy and prognosis of DLBCL patients have been greatly improved.However,more than one third of of DLBCL patients still cannot be cured because of primary drug resistance or relapse after complete remission.For DLBCL patients with initial treatment failure or short-term recurrence,high-dose salvage chemotherapy and hematopoietic stem cell transplantation could improve the clinical treatment effects,but the vast majority of patients ultimately suffer disease recurrence and even death,resulting in heavy social and economic burden.With the advent of biomedical big data era and the development of multi-omics sequencing,the understanding of molecular mechanisms and genetic characteristics in lymphoma development has been gradually deepened.Precision medicine based on new molecular targets and targeted therapy provides unprecedented opportunities for the treatment of DLBCL patients.Therefore,finding molecular markers for disease recurrence,drug resistance and disease progression,and establishing new targeted therapy and intervention strategies are of great importance for improving the clinical efficacy and prognosis of DLBCL patients.Lipocalin prostaglandin D synthetase(L-PGDS),also known as PTGDS,is located in human chromosome 9 and is one of member of lipocalin family.PTGDS acts as a bifunctional protein that catalyzes prostaglandin metabolism and transports lipophilic substances.It is demonstrated that PTGDS could regulate multiple biological functions of solid tumor such as proliferation,metastasis and stem cell characteristics,and its expression is significantly correlated with the clinical features of tumor patients such as tumor stage,metastasis and prognosis.Glycosylation is an important method of protein modification,and the levels of glycosylation have an influence on the spatial structure,degradation and biological activity of glycoprotein.The 190 amino acids encoded by PTGDS gene can be glycosylated on rough endoplasmic reticulum and Golgi complex,and mature PTGDS protein is with the structure of carbohydrate and complex oligosaccharide.The N-terminal of PTGDS protein contains a signal peptide rich in hydrophobic amino acids,suggesting that it can be secreted out of cells into body fluids such as serum,cerebrospinal fluid and urine.It is found that the concentration of PTGDS in body fluids could reflect the status of several diseases and exogenous addition of PTGDS has been involved in the development of tumors.AT56,an orally active and selective inhibitor of PTGDS,could inhibit the activity of PTGDS in a competitive manner,but the potential role of AT56 in tumor therapy has not been reported.Therefore,further studies on the biological role and molecular mechanism of glycoprotein PTGDS and its inhibitor AT56 in DLBCL development are needed.The purpose of this study is to explore the role and molecular mechanism of PTGDS and its inhibitor AT56 in the development of DLBCL.In this study,we aim to clarify the expression level of PTGDS and its clinical relevance,which could provide potential biomarkers for the early diagnosis and prognosis evaluation of DLBCL.Meanwhile,we aim to reveal the biological role of PTGDS and its inhibitor AT56 in DLBCL,explore the regulatory effects of PTGDS on MYH9 and Wnt-β-catenin-STAT3 signaling pathway,and illuminate the role of glycosylation on the stability,intracellular localization and biological functions of PTGDS protein,which could provide novel molecular targets for targeted therapy in DLBCL patients and lay the theoretical basis and experimental foundation for the application of AT56 in DLBCL treatment.Part Ⅰ.The Expression,Biological Function and Clinical Significance of PTGDS in Diffuse Large B-cell LymphomaObjective:Diffuse large B-cell lymphoma(DLBCL)is a kind of malignant tumor with high heterogeneity.With the development and clinical application of novel targeted drugs and immunotherapy,most DLBCL patients can obtain clinical cure.However,more than one third of DLBCL patients still suffer drug resistance or relapse,and eventually progress to death.Therefore,there is an urgent requirement to uncover novel molecular markers and therapeutic targets for the establishment of individualized precision treatment in DLBCL patients.Several studies have shown that the expression of PTGDS is disordered in solid tumors,which is closely related to the disease stage and prognosis of tumor patients.However,the expression level and biological role of PTGDS in hematological tumors,especially DLBCL,has not been reported.The aim of this study is to elucidate the expression level of PTGDS in tumor tissues,serum and DLBCL cell lines and analyze the correlation between PTGDS expression and clinical characteristics and prognosis of DLBCL patients,providing new biomarkers for early accurate diagnosis and prognosis assessment in DLBCL patients.Meantime,we also aim to reveal the biological role of PTGDS in the development of DLBCL through exogenous addition of PTGDS,lentivirus-mediated overexpression and knockdown of PTGDS in vitro and in vivo experiments,and explore the potential value of PTGDS as novel therapeutic target in the targeted therapy for DLBCL patients.Materials and Methods:1.The selection of DLBCL and reactive hyperplasia lymphoid(RHL)patients,and the collection of specimen and clinical data;2.Immunohistochemistry(IHC)and Hematoxylin-eosin(HE)staining;3.The collection and processing of peripheral blood from DLBCL patients and healthy control;4.Enzyme linked immunosorbent assay(ELISA);5.The isolation and purity examination of CD19+ B cells from peripheral blood mononuclear cells(PBMCs);6.Protein extraction and Western blotting;7.Bioinformatic analysis based on the GEO database;8.DLBCL cells culture;9.Cell counting kit-8(CCK-8)to detect the proliferation of DLBCL cells;10.Lentivirus-mediated overexpression and knockdown of PTGDS;11.CellTiter-Glo Luminescent assay to detect cell viability;12.The construction of DLBCL xenograft mouse model;13.PI/RNase staining assay to analyze cell cycle;14.Annexin V-PE/7AAD staining to evaluate the level of cell apoptosis;15.Transwell assay to detect cell invasion;16.Statistical analysis.Results:1.The expression of PTGDS was evaluated in 120 newly diagnosed DLBCL patients and 32 reactive hyperplasia patients through IHC staining,and it’s found that PTGDS was upregulated in DLBCL tissues in comparison with control(p<0.001).Although the expression of PTGDS was higher in germinal center of lymphoma tissue,the PTGDS expression was negative in the germinal center of control lymphatic tissue,indicating the specific high expression of PTGDS in lymphoma cells.Similarly,the level of serum PTGDS was higher(p<0.01)in DLBCL patients(n=53)than that in healthy control(n=17).Furthermore,compared with CD19+B cells of PBMCs from healthy donors,the expression of PTGDS protein was increased in DLBCL cell lines(LY1,LY3,LY8,VAL).2.Statistic analysis based on the clinical data of DLBCL patients showed that high expression of PTGDS was correlated with GCB subtype,lower sialic acid(SA)and unfavorable therapeutic efficacy in DLBCL patients(p<0.05).Kaplan-Meier survival curve analysis indicated that DLBCL patients with positive PTGDS displayed reduced progression-free survival(29 months vs 55 months,p=0.003)and overall survival(31 months vs 62 months,p=0.001).Interestingly,positive PTGDS expression was significantly correlated with reduced progression-free survival(p=0.004)and overall survival(p=0.004)in non-GCB subtype,but no significant difference was found in GCB subtype(p>0.05).3.Gene ontology(GO)analysis based on GEO database(GSE31312,GPL570,n=498)revealed that PTGDS was closely related to biological processes involved in tumor progression,such as DNA damage,cell proliferation and death,cell adhesion and migration,apoptosis and so on.The addition of rhPTGDS and PGD2,the catalytic product of PTGDS,obviously promoted the proliferation of DLBCL cells in a dose-dependent manner.Lentivirus mediated PTGDS knockdown(sh-PTGDS#1,sh-PTGDS#2)and overexpression(LV-PTGDS)was constructed and transfected into DLBCL cells(LY1,LY3).The effective regulation of PTGDS expression through lentivirus transfection was demonstrated by Western blotting,with sh-PTGDS#2 exhibiting higher efficacy of PTGDS knockdown.PTGDS knockdown decreased cell proliferation、viability and the expression of pro-proliferation protein c-myc while PTGDS overexpression enhanced cell proliferation.In DLBCL xenograft model,mice bearing LV-PTGDS cells displayed increased growth rate,higher tumor volume and weight,bioluminescence and Ki-67 expression.Conversely,significant reduction in tumor growth was found in mice bearing sh-PTGDS cells.4.In flow cytometry experiments,PTGDS knockdown was observed to induce obvious cell cycle arrest in G0/G1 phase(p<0.01)and inhibit the expression of Cyclin D1 and CDK2 in DLBCL cells,which promoted the transformation of cell cycle from G1 phase to S phase.DLBCL cells with PTGDS knockdown displayed increased cell apoptosis rates(p<0.05),increased expression of pro-apoptotic proteins(Bax,and the cleaved forms of caspase-3,caspase-9 and PARP)and decreased expression of anti-apoptotic protein Bcl-xl.In addition,Transwell assays showed the significant reduction of cell invasion and decreased expression of zeb1 and vimentin in sh-PTGDS cells(p<0.01).These results indicated that PTGDS is involved in the development of DLBCL.Conclusions:1.The expression of PTGDS is significantly upregulated in DLBCL,and it is closely related to clinical features,treatment efficacy and worse prognosis in DLBCL patients,suggesting that PTGDS can serve as biomarkers for DLBCL disease progression and prognosis assessment.2.In vitro and in vivo studies indicates that PTGDS participates the development of DLBCL through regulating cell viability,proliferation,apoptosis,cell cycle,and invasion,indicating PTGDS as promising therapy target for DLBCL therapy.Part Ⅱ.The Anti-tumor Effects of PTGDS Inhibitor AT56 in Diffuse Large B-cell LymphomaObjective:In recent years,with the proposal of precision medicine and the development of targeted intervention,several novel small-molecule targeted drugs have achieved great clinical benefits in tumor treatment,including malignant hematological diseases.The purpose of this study is to clarify the effects of PTGDS inhibitor AT56 on the biological functions of DLBLC cells such as cell viability,proliferation,cycle,apoptosis and invasion in vitro and in vivo experiments,and explore the effects of AT56 on the drug sensitivity of DLBCL cells to common chemotherapeutic drugs.Therefore,we could reveal the potential utility of AT56 in the precise targeted therapy and combined chemotherapy for DLBCL patients and provide novel therapeutic strategies to improve the prognosis and clinical benefit of DLBCL patients.Materials and Methods:1.DLBCL cell culture;2.ELISA;3.CellTiter-Glo Luminescent assay to detect cell viability;4.CCK-8 to detect the proliferation of DLBCL cells;5.Protein extraction and Western blotting;6.PI/RNase staining assay to analyze cell cycle;7.Annexin V-PE/7AAD staining to evaluate the level of cell apoptosis;8.Transwell assay to detect cell invasion;9.The construction of DLBCL xenograft mouse model;10.IHC and HE staining;11.RNA extraction,reverse transcription and real-time fluorescent quantitative polymerase chain reaction(qRT-PCR);12.Comet assay to detect the level of DNA damage;13.Immunofluorescence assay(IF)and confocal microscopy;14.Statistical analysis.Results:1.ELISA assay showed that the concentration of PGD2 in cell culture supernatant was decreased by AT56(p<0.05).AT56 inhibited cell viability in sh-Con cells,but not in sh-PTGDS cells,supporting the specificity of AT56 on PTGDS in DLBCL cells.The cell proliferation was reduced by the incubation with AT56 in a dose-and time-dependent manner.AT56 also inhibited the expression of c-myc in DLBCL cells.2.AT56 induced the elevation of G0/G1 phase cells and decreased the expression of Cyclin D1 and CDK2 in a dose-dependent manner.Flow cytometry demonstrated that AT56 dose-dependently increased the early apoptotic cell populations in DLBCL cells.Notably,the expression of pro-apoptotic proteins(Bax,and the cleaved forms of caspase-3,caspase-9 and PARP)was upregulated while the expression of anti-apoptotic protein Bcl-xl was decreased with elevated concentration of AT56.Besides,the negative effect of AT56 on cell invasion was demonstrated in Transwell assay and AT56 was observed to dose-dependently decrease the expression of zeb1 and vimentin in DLBCL cells.These results indicated that AT56 exerted significant anti-tumor effects in DLBCL cells.3.In DLBCL xenograft model,mice receiving AT56(n=5)displayed reduced growth rate,tumor weight and volume.Besides,AT56 treatment decreased the expression of Ki67,c-myc and Cyclin D1,and increased the expression of Bax and cleaved PARP in DLBCL tissue.4.qRT-PCR experiments showed that adriamycin and bendamustine decreased the expression of PTGDS mRNA in DLBCL cells.Notably,the addition of AT56 to adriamycin and bendamustine showed enhanced cytotoxicity in terms of cell proliferation and apoptosis in DLBCL cells.As adriamycin and bendamustine exerted anti-tumor effects through promoting DNA damage,further comet assay showed that DLBCL cells with AT56 treatment displayed longer tail moment,indicating enhanced DNA damage.Moreover,in Western blotting and confocal immunofluorescence experiments,AT56 induced elevated expression of DNA damage marker(p-H2AX)in DLBCL cells.These results provided evidence that AT56 could sensitize DLBCL cells to chemotherapeutic drugs through promoting DNA damage.Conclusions:These studies have confirmed that PTGDS specific inhibitor AT56 could exert significant anti-tumor effects in DLBCL through inhibiting cell viability and proliferation,inducing cell cycle arrest,promoting cell apoptosis and inhibiting cell invasion.Furthermore,AT56 enhances the sensitivity of DLBCL cells to adriamycin and bendamustine by promoting DNA damage.These results provide theoretical basis and experimental foundation to establish novel targeted therapy and combination regimen for DLBCL treatment.Part Ⅲ.Glycoprotein PTGDS Promotes the Development of Diffuse Large B-cell Lymphoma by MYH9-mediated regulation of Wnt-β-catenin-STAT3 signalingObjective:Our previous study found that the high expression of PTGDS is significantly correlated with the clinical efficacy and prognosis in DLBCL patients,and PTGDS knockdown and its specific inhibitor AT56 could exert antitumor effects through regulating the biological functions of DLBCL cells,such as cell proliferation,cycle,apoptosis and invasion.However,the specific underlying molecular mechanism remains incompletely understood.The present study aims to explore the molecular mechanism of PTGDS in regulating the development of DLBCL,and elucidate the effects of glycosylation on the degradation,intracellular localization and biological functions of PTGDS protein,providing experimental and theoretical basis for the application of PTGDS and AT56 in the individualized precision therapy for DLBCL patients.Materials and Methods:1.DLBCL cell culture;2.Co-immunoprecipitation(CoIP)and mass spectrometry;3.IF and confocal microscopy;4.Lentivirus-mediated knockdown of MYH9;5.Protein extraction and Western blotting;6.CellTiter-Glo Luminescent assay to detect cell viability;7.CCK-8 to detect the proliferation of DLBCL cells;8.PI/RNase staining assay to analyze cell cycle;9.Annexin V-PE/7AAD staining to evaluate the level of cell apoptosis;10.Transwell assay to detect cell invasion;11.RNA extraction,reverse transcription and qRT-PCR;12.Chromatin immunoprecipitation(ChIP);13.The extraction of nuclear and cytosolic proteins;14.Statistical analysis.Results:1.We screened PTGDS interactive proteins through CoIP and mass spectrometry,and analyzed the protein-protein interactions by STRING website,among which MYH9 ranked first.Bioinformatic analysis based on GSE31312 indicated the correlation between the expression level of PTGDS and MYH9(p<0.001).Confocal immunofluorescent images and CoIP verified the colocalization and interaction between endogenous PTGDS and MYH9 in DLBCL cells.2.Lentivirus mediated RNA interference vectors against MYH9 displayed effective knockdown in DLBCL cells,of which sh-MYH9#1 exhibited the highest efficacy.MYH9 knockdown and MYH9 inhibitor,Blebbistatin,displayed anti-tumor effects in DLBCL through inhibiting cell proliferation and viability,inducing cell cycle arrest,promoting cell apoptosis,and decreasing cell invasion.In DLBCL cells with MYH9 knockdown,no effect of Blebbistatin on cell proliferation,viability,or cell cycle was found,supporting the specificity of Blebbistatin on MYH9 in DLBCL.3.Analysis based on CoIP and mass spectrometry indicated that several PTGDS-associated proteins influenced the activation of Wnt pathway.In vitro and in vivo studies showed that AT56 and PTGDS knockdown decreased the expression of MYH9 and STAT3,and suppressed the activation of Wnt pathway.MYH9 knockdown could also inhibit the activation of Wnt pathway and the expression of STAT3.Interestingly,it was found that PTGDS and MYH9 inhibition decreased the protein level of β-catenin,the key molecule of Wnt pathway,but not its mRNA expression,which indicated enhanced degradation ofβ-catenin protein.Confocal immunofluorescent images and CoIP verified the colocalization and interaction between endogenous MYH9 and β-catenin in DLBCL cells.The knockdown of PTGDS and MYH9 decreased the ubiquitination level of GSK3-β and prolonged its half-life in DLBCL,which could induce the degradation of β-catenin and inhibit the activation of downstream molecules in Wnt pathway.Besides,PTGDS and MYH9 inhibition decreased both the mRNA and protein levels of STAT3 in DLBCL cells,and ChIP assay showed the binding of TCF4,a transcription factor in Wnt pathway,and STAT3 promoter,indicating STAT3 as the downstream of Wnt pathway in DLBCL cells.4.When Wnt signaling was activated by Wnt3a treatment,the proliferation inhibition,cell cycle arrest and enhanced apoptosis by AT56 was significantly reversed.Besides,the regulatory effect of AT56 on the expression of important molecules in Wnt pathway could be partly reversed by Wnt3a,indicating that AT56 exerted anti-DLBCL effects through inhibiting Wnt pathway.Moreover,the decreased expression of MYH9 after AT56 treatment could not be reversed by Wnt3a,suggesting that MYH9 might not act as the downstream of Wnt pathway in DLBCL.STAT3 inhibitor WP1066 reversed the proliferation promotion induced by PTGDS overexpression,and enhanced the proliferation inhibition and apoptosis promotion caused by AT56,indicating the involvement of STAT3 in the oncogenic role of PTGDS in DLBCL.Moreover,MYH9 inhibitor Blebbistatin reversed the enhanced cell proliferation and the activation of Wnt-β-catenin-STAT3 pathway caused by PTGDS overexpression,indicating that MYH9 mediated the regulatory role of PTGDS on Wnt-β-catenin-STAT3 pathway and DLBCL development.5.Based on Universal Protein Resource(UniProt)database,it’s found that PTGDS was with three specific glycosylation sites,including two N-glycosylation sites(Asn51 and Asn78)and an O-glycosylation site(Ser29).The removal of glycan chains using PNGase F and tunicamycin increased the ratio of low molecular weight PTGDS protein,confirming the glycosylation of PTGDS protein in DLBCL.Besides,glycosylation inhibition by tunicamycin was found to promote the nuclear translocation of PTGDS protein in DLBCL cells.Compared with CD 19+B cells,DLBCL cells displayed increased PTGDS protein level and decreased PTGDS mRNA expression,and the glycosylation level of PTGDS in DLBCL cells was found to be lower than normal B cells in Western blotting experiments.In cycloheximide chase assay,deglycosylation led to degradation inhibition and longer half-life of PTGDS in DLBCL,which might partly explain the discrepancy between PTGDS protein and mRNA expression level.6.Point mutations were created in two potential N-glycosylation sites(Asn51 and Asn78)and it’s found that mutated PTGDS protein displayed lower molecule weight than wild type PTGDS protein,which verified the glycosylation and its sites of PTGDS in DLBCL.Point mutation promoted the translocation of PTGDS protein to nucleus in confocal immunofluorescent images,and increased cell proliferation and the expression of c-myc in DLBCL cells.Conclusions:In summary,our study demonstrates that PTGDS might promote DLBCL progression through MYH9-mediated regulation of Wnt-β-catenin-STAT3 signaling,and the abnormal glycosylation in Asn51 and Asn78 can lead to prolonged half-life,nuclear location and enhanced oncogenic role of PTGDS protein,revealing the molecule mechanism by which glycoprotein PTGDS participates the development of DLBCL.These results provide a rationale for PTGDS as a novel biomarker and molecule target to establish the individualized precision therapy for DLBCL patients.