The Roles And Mechanisms Of CUL4B In Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma(DLBCL)is the most common histologic subtype of non-Hodgkin lymphoma(NHL),accounts for approximately forty percent of most newly diagnosed NHL cases.DLBCL is an aggressive lymphoma which is extremely heterogeneous in morphology,genetics,biology,and clinical manifestations.Recently,with the application of new targeted drugs represented by CD20 monoclonal antibody and immunomodulatory drugs,the prognosis of DLBCL patients has been significantly improved.Nevertheless,40%～50%of DLBCL patients still have primary drug resistance or relapse after complete remission.Although high-dose rescue chemotherapy and hematopoietic stem cell transplantation can improve the treatment efficiency of refractory/relapsed DLBCL patients,the majority of patients still suffer from disease recurrence and even eventually to death.Therefore,more accurate and effective therapeutic measures are urgently needed to improve the efficacy of DLBCL patients in clinical practice.Cullin4B(CUL4B)is a member of the cullin family.As a scaffold protein of the CUL4B-RING E3 ubiquitin ligase complex(CRL4B),CUL4B plays a key regulatory role in DNA replication,gene transcription,cell cycle,signal transduction,and various other biological processes.There are eight cullin family members in mammals,including CUL1,CUL2,CUL3,CUL4A,CUL4B,CUL5,CUL7 and PARC,among which CUL4A and CUL4B have the highest homology and the most similar structure.Though similar,CUL4A and CUL4B are not identical copies of one another in functions.Unlike CUL4A,CUL4B has a unique N-terminal nuclear localization sequence.It can play biological roles not only in the cytoplasm but also in the nucleus.Recent studies have revealed that CUL4B was highly expressed in many solid cancers and contributed to the development of various tumors,in many solid cancers,including esophageal cancer,gastric cancer,lung cancer,liver cancer,colon cancer,cervical cancer,and osteosarcoma.Mechanistically,studies have shown that CUL4B can cooperate with PRC2 complex and SUV39H1/HP1/DNMT3A complex to participate in histone modification and DNA methylation modification,playing a transcriptional inhibitory role,and inhibiting the expression of multiple tumor suppressor genes.CUL4B functions as an oncogenic role in cancer.CUL4B can upregulate CDK2 by transcriptionally repressing the expression of miR-372 and miR-373 to regulate cell cycle and DNA replication.It can promote tumor invasion and metastasis through positively upregulating HER2 via the transcriptional repression of miR-125a.CUL4B can also form a dual negative feedback loop with mutual inhibition of mir-194 to participate in the regulation of tumor progression.CRL4B can establish a double-negative feedback loop with miR-194 to participate in the the regulation of tumorigenesis.In addition,the abnormal expression of CUL4B is also closely related to the abnormal activation of multiple signaling pathways,such as Wnt/β-catenin,AKT/GSK3β,p53-ROS pathway,etc.,which play driving roles in the process of tumor occurrence and progression,invasion and migration.Studies have shown that abnormal activation of the above signaling pathway also exists in DLBCL.Although CUL4B has been demonstrated to regulate the survival of several solid neoplasms cells,its expression and biological functions in DLBCL have not yet been validated.In the present study,we aimed to explore the expression and function of CUL4B in DLBCL,including the demonstration of molecular mechanisms through loss-of-function and gain-of-function assays in DLBCL.The exploration of the mechanisms of CUL4B in the tumorigenesis and progression of DLBCL may help us find new therapeutic targets.This study provided a theoretical basis and experimental basis for the new approach to DLBCL therapy.Part I The Expression of CUL4B and its Clinicopathological Significance in Diffuse Large B-cell LymphomaObjective:Diffuse Large B-cell Lymphoma(DLBCL)is a highly heterogeneous and aggressive non-Hodgkin lymphoma(NHL).With the recent advancement of novel targeted therapy,such as monoclonal anti-CD20 antibody,the outcome of DLBCL patients have been significantly improved.However,there are still more than 30%of DLBCL patients suffering from a relapse.Therefore,it is of great significance to explore the mechanism and root cause of DLBCL in relapse,resistance and progression.Searching for more effective chemotherapeutic regimens for therapeutic management of DLBCL is warranted.Aberrant expression of CUL4B was identified in various types of solid cancers.CUL4B overexpression was significantly correlated with differentiation and undesirable outcomes of tumors.However,the expression of CUL4B in DLBCL is still unknown.This study aims to explore the expression of CUL4B in DLBCL,and analyze the relationship between CUL4B expression and clinical characteristics of DLBCL patients to preliminarily explore its clinical significance.Material and Methods:1.Use the Oncomine platform data to analyze the expression of CUL4B in DLBCL2.Case selection and specimen collection;3.Immunohistochemistry(IHC);4.Public data platform TCGA database data bioinformatics analysis;5.Cell culture;6.Isolation of human’s peripheral blood mononuclear cells(PBMCs);7.RNA extraction,reverse transcription and real-time quantitative polymerase chain reaction(qRT-PCR);8.Protein extraction and western blot assay;9.Statistical analysis.Results:1.Firstly,we analyzed the microarray datasets obtained from the Oncomine database to explore CUL4B expression in B-lymphocyte,germinal center B-lymphocyte and DLBCL samples.It was observed that DLBCL tissues exhibited higher expression of CUL4B compared to B-Lymphocyte and Germinal Center B-Lymphocyte tissues(P<0.01).We next validated the expression of CUL4B in DLBCL cell lines(LY1 and LY8)by qRT-PCR and western blot.As expected,the expression of CUL4B was upregulated in DLBCL cell lines compared to the normal PBMCs.2.To investigate the protein expression of CUL4B in DLBCL,IHC staining was performed in 48 DLBCL tissues and 30 reactive hyperplasia lymphoid(RHL)tissues.CUL4B protein was located in the nucleus of DLBCL cells.The number of CUL4B-positive cells was higher in DBLCL tissues than in RHL tissues.Positive expression of CUL4B was observed in 70.8%(34/48)of DLBCL samples and in 3.3%(1/30)of control tissues.3.We further analyzed the relationship between CUL4B expression and clinical characteristics of DLBCL patients.CUL4B overexpression was found to be positively associated with higher Ann Arbor stage(P=0.049),the presence of B symptoms(P=0.043),elevated serum lactate dehydrogenase(LDH,P=0.019),and high international prognostic index(IPI)score(P=0.041)of DLBCL patients.By analyzing previously published gene expression data of 48 DLBCL patients from the Cancer Genome Atlas(TCGA),Kaplan-Meier survival analysis indicated that patients with the high expression level of CUL4B presented a shorter overall survival(OS)than those without CUL4B overexpression(P<0.05,n=45).Conclusions:1.CUL4B expression was increased in human DLBCL tissues and associated with tumor progression2.CUL4B overexpression was positively associated with an advanced Ann Arbor stage,the presence of B symptoms,elevated serum LDH,and high IPI scores of DLBCL patients.3.Patients with the high expression level of CUL4B presented a shorter overall survival(OS)than those without CUL4B overexpression.Above data suggested that CUL4B may serve as a biomarker of DLBCL progression.Part Ⅱ The Biological Functions of CUL4B in Diffuse Large B-Cell LymphomaObjective:The relationship between CUL4B and tumor has become a focus in recent years.The function of CUL4B in a variety of tumors has been reported,while the biological function of CUL4B in DLBCL remains unclear.The functions of CUL4B in many solid malignancies have been reported,including regulation of cell proliferation,cell cycle,cell apoptosis,invasion,migration,epithelial-mesenchymal transformation by regulating the signal transduction.However,the role of CUL4B in DLBCL is still unclear.In order to explore the biological functions of CUL4B in DLBCL,this study aimed to establish CUL4B low-and overexpression cell model and DLBCL xenograft mice model,and investigate the effects of CUL4B on cell proliferation,cell cycle,invasion,migration,and cell apoptosis.Material and Methods:1.Cell culture;2.Cell transfection by lentivirus vectors either encoding CUL4B(shCUL4B,lvCUL4B)or empty lentivirus vectors(shcontrol,lvcontrol);3.The expression of GFP protein in lentivirus transfection cells by flow cytometry;4.RNA extraction,reverse transcription,and the real-time quantitative polymerase chain reaction;5.Protein extraction and western blot assay;6.Cell proliferation using the Cell Counting Kit-8(CCK-8)method;7.Analyses cell cycle by PI staining assay;8.Analyses cell apoptosis by annexin V PE/7-aminoactinomycin D(7-AAD)assay;9.Cell invasion and migration by transwell assay;10.Effect of CUL4B on DLBCL cell growth evaluated by DLBCL xenograft mice models;11.Immunohistochemistry(IHC);12.Statistical analysis.Results:1.To investigate the role of CUL4B in DLBCL cells,stable knockdown(designated as shCUL4B#1,shCUL4B#2,shCUL4B#3)and overexpression of CUL4B(designated lvCUL4B)in LY1 and LY8 cells were established.Effective silencing or overexpression of CUL4B in LY1 and LY8 cells were confirmed by qRT-PCR and western blo assay,and shCUL4B#3,one of the three lentivirus-mediated RNA interference vectors against CUL4B,exhibited highest efficacy of CUL4B-depletion.Compared to the lvcontrol group,the mRNA and protein CUL4B expression of CUL4B was higher in lvCUL4B group.2.CCK8 assay was used to investigate cell proliferation.Silencing of CUL4B resulted in growth suppression of LY1 and LY8 cells,whereas CUL4B overexpression promoted the growth of LY1 and LY8 cells.3.Furthermore,cell cycle analysis was performed with PI staining by flow cytomertry assay.The result revealed that shCUL4B increased the accumulation of cells in the G1 phase of cell cycle.It is suggested that CUL4B could promote DLBCL cells pass through G0/G1 phase to accelerate cell cycle progression,and then promote cell proliferation.4.Regulatory effect of CUL4B on cell apoptosis was also investigated.However,no significant difference of cell apoptosis was observed in either shCUL4B groups or lvCUL4B groups compared to control groups.5.To determine the role of CUL4B in the invasion and migration of DLBCL cells,transwell assays were performed.Significant reduction in cell invasion,more than half decrease were observed in shCUL4B group compared with control group.Conversely,CUL4B overexpression displayed the opposite effect in LY1 and LY8 cells.6.To further explore the role of CUL4B on tumor growth in vivo,we established a xenograft mice model with human DLBCL cells.ShCUL4B or control cells were injected subcutaneously into the right axilla of SCID beige mice.Consistent with results in vitro,CUL4B knockdown greatly decreased tumor growth in DLBCL xenograft model.IHC assay confirmed that CUL4B expression in the xenograft tumor sections derived from shCUL4B cells was significantly lower than that in control cells.In addition,the Ki67 and c-MYC percentage score of tumor cells in the shCUL4B group were relatively reduced compared with those in the control group.In all,these results supported CUL4B as an important regulator of tumor proliferation in DLBCL.Conclusions:1.CUL4B played a stimulus role in the proliferation of DLBCL cells2.Knockdown of CUL4B could induce G0/G1 arrest.3.No significant difference of cell apoptosis was observed in either shCUL4B groups or lvCUL4B groups.4.CUL4B promoted DLBCL invasion and migration in vitro5.Knockdown of CUL4B suppressed tumour growth in DLBCL xenograft modelsPart Ⅲ CUL4B Regulates Autophagy via JNK Signaling in Diffuse Large B-cell LymphomaObj ective:Study of autophagy in the medical field has maken rapid development.Autophagy plays an important role in various physiological and pathological human processes,including cell survival,apoptosis,and senescence.The roles of autophagy on the occurrence and progression of tumors have two sides.On the one hand,autophagy can inhibit the proliferation of tumor cells;on the other hand,it is conducive to the high metabolism of tumor cells and thus promotes the occurrence of tumors.Studies have found that gene expression,signaling pathways,and drugs are involved in the regulation of autophagy in tumor cells.We found that CUL4B is highly expressed in DLBCL and can be involved in regulating multiple biological functions of DLBCL cells.However,the effect of CUL4B on autophagy of DLBCL cells has not been reported in literature.In the study,we aim to observe the effect of CUL4B on DLBCL cell autophagy and to discuss its theory and mechanism.Our study may lay a foundation for further study on its biological function and molecular mechanism.Material and Methods:1.Cell culture;2.Silence and overexpression the expression of CUL4B in DLBCL cells by lentivirus vectors;3.RNA extraction,reverse transcription and real-time quantitative polymerase chain reaction(qRT-PCR);4.Protein extraction and western blot assay;5.Autophagosome observation by transmission electron microscopy;6.Statistical analysis.Results:1.We detected the expression of LC3 and Beclinl in DLBCL cells transfected with shCUL4B and lvCUL4B.Downregulated CUL4B resulted in a significant decrease in the expression of LC3 and Beclinl proteins in DLBCL cells.Whereas,the expression level of LC3 protein was upregulated when CUL4B was overexpressed.2.Furthermore,the autophagic vesicles and double-membrane autophagosomes in the cytoplasm of shCUL4B,lvCUL4B,and control cells were visualized by transmission electron microscopy.The number of autophagosomes(another golden hallmark of autophagy)with engulfed bulk cytoplasm and cytoplasmic organelles in shCUL4B cells was lower than that in control cells.Contrarily,CUL4B overexpression exhibited the opposite effect.These results indicated that CUL4B contributed to the autophagy induction in DLBCL cells.3.Phosphorylation of AKT,mTOR,and JNK was assessed upon lentiviral vectors mediated inhibition or elevation of CUL4B expression in LY1 and LY8 cells.The phosphorylation of AKT protein was significantly inhibited in DLBCL cells with CUL4B knockdown but increased with CUL4B overexpression.However,neither CUL4B depletion nor overexpression exerted a substantial effect on mTOR phosphorylation.Despite all this,western blot assay further revealed that phosphorylation within JNK and c-JUN was downregulated in shCUL4B cells but elevated in lvCUL4B cells.4.We then investigated whether JNK signaling had a functional impact on autophagy induced by the changed expression of CUL4B.We combined a pan-JNK activator(Anisomycin)with CUL4B knockdown and evaluated the autophagy.As shown in Figure 8A,treatment of shCUL4B cells with Anisomycin increased the LC3 protein expression.Meanwhile,more autophagosomes were observed by transmission electron microscopy.CUL4B-knockdown cells within Anisomycin treatment showed more autophagosomes than control cells.Combination of SP600125(an inhibitor of JNK signalling)with lvCUL4B cells reduced the expression of LC3.Autophagosomes were fewer in CUL4B-overexpression cells within SP600125 treatment than in cells without SP600125 treatment.Conclusions:1.CUL4B could upregulate the elevation of autophagy in DLBCL cells.2.CUL4B activated the JNK signaling pathway and promoted JNK phosphorylation in DLBCL.3.CUL4B activated the protective autophagy of DLBCL cells via JNK signaling pathway to promote cell proliferation.