Study Of Antitumor Effects Of KLF4 And ATO In Mantle Cell Lymphoma Through Targeting Wnt Signaling Pathway

Mantle cell lymphoma is an aggressive mature B-cells derived non-Hodgkin lymphoma(NHL)with genetic hallmark of translocation t(11;14)(q13;q32),resulting in overexpression of cyclin D1.The median survival time is between 3-5 years for MCL patients.The cytogenetic abnormalities contribute to high frequency of relapse after conventional chemotherapy.Therefore,it is important to explore mechanisms for the pathogenesis of the diseases,and develop target therapy.Kruppel-like factors(KLFs)consist of 17 widespread expressed human transcription factors which are characterized by 3 highly conserved zinc fingers of C2H2 type and a transactivating domain in the N-terminus.The KLF protein control essential cellular processes such as proliferation,differentiation and migration.There is a transcriptional activation domain and a transcriptional repression domain in KLF4 protein so that allows KLF4 alter its positive or negative transcriptional effect after binding DNA sequences with the downstream promoter elements in the nucleus.KLF4 functions as an oncogene or tumor suppressor depending on the type of tissue involved.Overexpression of KLF4 was observed in head and neck squamous cell carcinoma and ductal carcinoma of the breast,and was considered as an oncogene.The oncogenic properties might be explained via negative transcriptional effect by binding P53 with the transcriptional repression domain.In contrast,in many epithelial cancers such as colon,esophageal,gastric,bladder,pancreatic and lung cancer,KLF4 functions as a tumor suppressor.Studies on anticancer effects of KLF4 reveal that KLF4 induces p27kipl expression in cervical cancers,represses Wnt and Notch signaling pathway in the gastrointestinal diseases.In hematologic malignancies,KLF4 was found to act as a tumor suppressor and was epigenetic silenced in B-cell lymphomas such as DLBCL,follicular lymphoma,Burkitt lymphoma,and cHL cases.But it remains unknown whether and,if so,how KLF4 contributes to the development and progression of CLL and MCL.The Wnt/?-catenin pathway is responsible for growth and development in normal cells,constitutively activation of Wnt/?-catenin pathway was demonstrated in CLL and MCL and appeared to promote tumorigenesis.In normal status,?-catenin phosphorylated and degraded by GSK-3? complex so that kept a low expression.With the dysregulation of Wnt inhibitory factors and inactivation of GSK3(3,?-catenin accumulated in the cytoplasm and translocated to the nucleus,in which it promotes transcription of downstream target genes including cyclin D1 that could contribute to oncogenesis.Furthermore,in colon cancer and gastric cancer,KLF4 was reported to inhibit ?-catenin and regulate the biological behavior of cancer cells.The synergism of KLF4 and P-catenin can induce the expression of hTERT in the pluripotent stem(iPS)cells.Arsenic derivatives were first used as therapeutic compounds or poison in ancient Greek and China.It is noteworthy that ATO and oral trans-retinoic acid(ATRA)combination has been the standard of care for patients with non-high-risk APL that were newly diagnosed.The therapeutic potential of ATO has also been developed in many other hematologic malignancies.However,there are rare reports on the effects and mechanism of ATO used in mantle cell lymphoma.There is a growing body of evidences that abnormal epigenetic regulations occur in the hematological diseases.DNA methyltransferase-1(DNMT-1)has become a highlight of the methylation study.KLF4 was reported to be a tumor suppressor and epigenetically silenced in gastrointestinal diseases and lymphomas as DLBCL,follicular lymphoma,cHL cases and T cell acute lymphoblastic leukemia,indicating the methylation of KLF4 promoter is closely associated with the tumorigenesis.All these promote us hypothesize that KLF4 is in close relationship with Wnt/?-catenin pathway in CLL and MCL,and the epigenetic silencing of KLF4 may contribute to the pathogenesis of diseases.Our findings highlight the significance of KLF4 in carcinogenesis and implicate KLF4 as a novel potential therapeutic target for MCL.Part ? Krtippel-like factor 4 contributes to the pathogenesis of mantle cell lymphomaObjective:Kruppel-like factor 4(KLF4)is a Zinc finger structural contained transcription factor,which also participated in the induction of iPS cells with other three factors.KLF4 functions as an oncogene or tumor suppressor depending on the type of tissues involved.The exact co-relationship of KLF4 with hematological malignancies was still in controversial.Mantle cell lymphoma(MCL)is an aggressive subtype of B-cell non-Hodgkin lymphoma(NHL)with poor prognosis.It is urgent to introduce new detecting techniques and clinical trials on novel target agents to provide chances to explore more bio-targets and test their effects as tumor suppressors for therapeutic intervention.In this study,we aim to illuminate the role of KLF4 in MCL and the mechanism of how KLF4 functions in MCL pathogenesis.Material and Methods:1.Cell culture and MCL Specimen collection;2.RNA extraction and real-time quantitative PCR;3.Immunohistochemistry(IHC);4.Protein extraction and western blot analyses;5.GFP-KLF4 lentivirally transfection in Jeko-1;6.The apoptosis of transduced Jeko-1 cells was detected by flow cytometry.The proliferation was analyzed using Cell Counting Kit-8(CCK8)method;7.Immunoprecipitation assay was used for detection of the binding of KLF4 and?-catenin;8.Statistical analysis.Result:1.Compared with peripheral blood mononuclear cells(PBMCs)from healthy donors,both mRNA and protein levels of KLF4 were significantly decreased in human MCL cell lines(Jeko-1,Mino and SP53).KLF4 was significant down-regulated in 65%(13/20)MCL tissue samples analyzed by immunohistochemical staining.Down-regulation of KLF4 is associated with MCL clinical staging(P<0.05).2.The expression of critical molecules of Wnt/?-catenin pathway such as ?-catenin,c-Myc and cyclinDl in MCL cell lines Jeko-1,Mino,SP53 and MCL patients'lymphoma tissues were extremely upregulated compared with PBMCs and reactive hyperplasia of lymphnode tissues manifested by western blot analysis and immunohistochemical staining.The statistical analyses showed that the downregulation of KLF4 was associated with overexpression of ?-catenin in MCL lymph node tissue samples(P<0.05).3.Overexpression of KLF4 in Jeko-1 cells induced significant cellular apoptosis(P<0.05).CCK8 assay showed that the cell proliferation was distinctly decreased after lentivirally transfected with KLF4 in Jeko-1 cells.Significant binding of KLF4 with?-catenin was observed after overexpression by lentivirus-mediated transfection.4.The expression of methyltransferase-1(DNMT-1)protein was up-regulated in MCL cell lines Jeko-1,Mino,SP53 and MCL patients' lymphoma tissues compared to PBMCs and reactive hyperplasia of lymph node tissues manifested by western blot analysis and immunohistochemical staining.After cultured with demethylating agent 5-azaC in gradient concentrations(0,0.5,1.0,2?M)for 48 hours,the expression of DNMT-1 decreased and KLF4 increased in a dose dependent manner in Jeko-1 and Mino cells.Nuclear ?-catenin,c-myc and cyclin D1 all decreased gradiently with increased 5-azaC concentrations.Conclusion:Our study suggested that the downregulated expression of KLF4 was associated with MCL clinical progression and prognosis.The overexpression of KLF4 suppressed the the expression of ?-catenin,cyclinD1 and c-myc,and the binding of KLF4 and P-catenin stopped the activation of the downstream molecules of Wnt/?-catenin signaling.DNMT-1 was strongly expressed in MCL,the demethylated drug could restore KLF4 expression and inhibit the Wnt/?-catenin pathway.The downregulation of KLF4 is supposed to be in a relationship with hypermethylation and KLF4 may be a novel potential demethylated therapeutic target for MCL.Part ?.The antitumor effects of arsenic trioxide in mantle cell lymphoma via targeting Wnt/?-catenin pathway and methyltransferase-1Objective:It is noteworthy that ATO and oral trans retinoic acid(ATRA)combination has been the standard of care for patients with nonhigh-risk APL that were newly diagnosed.Most of MCL patients have an aggressive clinical behavior and poor survival rates.In this study,we investigated the mechanism of ATO especially the effect on Wnt/?-catenin pathway and DNMT-1 in MCL and aim to provide better treatment options for MCL patients.Material and Methods:1.Cell culture and MCL Specimen collection;2.Immunohistochemistry(IHC);3.Protein extraction and western blot analyses;4.RNA extraction and real-time quantitative PCR;5.Cell culture with needed drug(ATO,5-azaC,LiCl);6.The apoptosis of cells treated with ATO were detected by flow cytometry;7.Statistical analysis.Result:1.The apoptosis rate of MCL cell lines(Jeko-1 and Mino)treated with ATO(0,0.5,2.0,5.0 ?,M)for 24 hours was measured by flow cytometry.Significant increase of apoptosis rates were detected in Jeko-1 and Mino cells with higher concentrations of ATO(P<0.05)compared with 0.5 ?M ATO treated group.Early apoptosis rates in both two cell lines increased slightly with higher concentrations of ATO,however,the increasement of late apoptotic cells was more remarkable.The viability of cells was notably inhibited by ATO with increased concentrations.2.In MCL cell lines Jeko-1,Grant519,SP53 and Mino,the expression of DNMT-1,?-catenin and c-Myc in all cell lines were notably higher compared with PBMCs from healthy volunteers.Furthermore,the conclusion was also confirmed in MCL tissue samples by immunohistochemical staining.In our follow up and survival analysis,we found that the survival rate of MCL patients with negative DNMT-1 expression was higher than the positive expression group before 39 months according to the survival curve.3.The expression of DNMT-1,?-catenin and the downstream molecules of Wnt/?-catenin pathway such as c-myc,cyclin D1 and MMP7 were all decreased in a dose dependent manner with ATO.4.ATO also attenuated upregulation of ?-catenin after LiCl stimulation and provided synergistic effect with 5-azaC on the DNMT-1 inhibition.Conclusions:Our study indicated that ATO has an anticancer effect on MCL by targeting Wnt/?-catenin pathway and DNMT-1 in a dose dependent manner.ATO also attenuated upregulation of P-catenin after LiCl stimulation and provided synergistic effect with 5-azaC on the DNMT-1 inhibition However,the mechanism and unique metabolism of ATO still need further research to explore its potential.