Study Of Klotho And IGF-1R Signaling Pathway In Non-hodgkin Lymphoma

Non-Hodgkin lymphoma(NHL),originated from lymphoid system,is one of the most common type of hematological malignancies,with the morbidity and mortality in the top 10 of human malignancies.NHL is divided into different types according to the diagnostic criteria of WHO(World health organization),including B-NHL and T-NHL,and a small number of NK-cell lymphoma.NHL is characterized by high heterogeneity in molecular pathology,diagnosis,treatment and personalized prognostic stratification.Therefore,development of accurate prognostic stratification and specific targeted therapy will improve the survival of NHL patients.With the development of biological targeted therapy and cellular immunotherapy,the 5 year overall survival rate has been significantly improved,however,there are still approximately 30%?40%of them presented reftactory or replased process.Rescue therapies including allogeneic hematopoietic stem cell transplantation are difficult to achieve long-term survival.Therefore,it is urgently needed to investigate the specific biomarkers and cellular signaling pathways.Several studies have demonstrated that the pathogenesis and progression of NHL were closely related to the aberrant expression of multifactors and dysregulation of signaling pathways.Further investigation on the molecular pathogenesis of NHL will provide more effective treatment strategies.Klotho is an anti-aging gene which was recently identified.It plays significant role in aging and aging-related disease through exerting modulatory effects on phosphate homeostasis,energy metabolism,oxidative stress and ion channels.With the deepening of experimental research,Klotho was demonstrated to be closely related to the development and progression of human malignancies.The tumor suppressive activity of Klotho was first identified in breast cancer in 2008.Recent investigations have indicated that Klotho is extensively down-regulated in several solid tumors,including cervical cancer,pancreatic cancer,melanoma,gastric cancer and hepatocellular carcinoma.Among these malignancies,Klotho was elucidated to be a modulator of several signaling pathways,including the fibroblast growth factor(FGF)signaling,insulin-like growth factor-1 receptor(IGF-1R)and Wnt/?-catenin signaling pathways,which are also involved in the pathogenesis of hematological malignancies.Deregualtion of these signaling pathways were also involved in the development of T-cell lymphoma.However,the role and functional mechanism of Klotho in NHL have not been reported.In the present study,we aimed to investigate the effect and functional mechanism of Klotho in NHL.The expression level of Klotho in NHL was evaluated.We further explored the biological function and the relevant mechanism of Klotho in NHL.Our results will provid the experimental and theoretical basis for the diagnosis,prognostic evaluation and novel targeted therapy of NHL.Part ?.Klotho Acts as a Tumor Suppressor and Modulator of IGF-1R Signaling Pathway in Diffuse Large B-cell LymphomaObjective:Diffuse large B-cell lymphoma(DLBCL)is the most common form of adult NHL,accounts for nearly 40%of all newly diagnosed cases.This disease presents as an aggressive process and exhibits high heterogeneity in gene expression,clinical presentation and prognosis.Although majority of DLBCL patients could be cured by anthracycline-based chemotherapies combined with rituximab,one-third of them present refractory or relapsed process.The definite etiology and molecular mechanisms of DLBCL is still not clear.Mice with Klotho knockout exhibited obvious impairment in B-cell development.Evolving evidence indicates that Klotho functions as a tumor suppressor in human malignancies by regulating several signaling pathways.However,the role of Klotho in DLBCL is still not clear.The aim of this study is to investigate the biological function and the related mechanism of Klotho in DLBCL.Material and Methods:1.Patients selection and specimen collection,2.Immunohistochemistry(IHC),3.Cell culture,4.Isolation of peripheral blood mononuclear cells(PBMCs),5.Purification of CD19+ B cells,6.Real-time quantitative polymerase chain reaction(RT-PCR),7.Protein isolation and Western blotting,8.Cell transfection by lentivirus vectors either encoding Klotho(LV-KL)or an empty lentiviral vector(LV-Con),9.Cell proliferation assay by Cell Counting Kit-8(CCK-8),10.Detection of cell apoptosis by Annexin V-PE/7AAD staining,11.Assessment of serum Klotho levels by enzyme-linked immune sorbent assay(ELISA),12.Effect of Klotho on DLBCL growth were evaluated by DLBCL xenograft mouse model,13.Statistical analysis.Results:1.Compared with reactive hyperplasia,expression level of Klotho was significantly decreased in DLBCL tissues.Klotho positive rate was 14%(7 of 50)in DLBCL whereas 80%(16 of 20)in reactive hyperplasia.Patients with stage ? or ?presented markedly lower Klotho expression level than those with stage ? or ?(p=0.002).Kaplan-Meier analysis revealed that Klotho protein expression was negatively correlated with the median overall survival(OS)of DLBCL patients,suggesting that lower Klotho expression was associated with poorer OS in DLBCL(p<0.05).Reduction of Klotho mRNA and protein expression levels were also confirmed in DLBCL cell lines(LY1 and LY8).2.To further establish the biological function of Klotho,human DLBCL cells were stably transfected with either negative control lentivirus vectors(LV-Con)or Klotho-overexpression lentivirus vectors(LV-KL).Significant reduction of cell proliferation was observed in DLBCL cells transfected with LV-KL,compared with those transfected with empty-vector(p<0.01).Consistent with the in vitro results,mice treated with Klotho overexpression cells revealed remarkable reduction in tumor volume compared to that transfected with empty-vector(p<0.01).In addition,relative higher expression of Klotho protein and lower positive rate of Ki67 were identified in mice treated with LV-KL-transfected LY1 cells compared to the control group.3.Flow cytometry analysis indicated that Klotho overexpression resulted in enforced apoptosis rates in both LY1(6.56±0.71%in LV-Con vs.16.41±2.16%in LV-KL group,p<0.01)and LY8 cells(7.23±0.65%in LV-Con vs.16.03±1.85%in LV-KL group,p<0.01).Dramatically reduction of anti-apoptotic protein Mcl-1 and increased Cleaved-Caspase-3 expression were observed in DLBCL cells transfected with LV-KL.4.Cell proliferation was less restored by IGF-1 in DLBCL cells transfected with LV-KL compared to that transfected with LV-Con(p<0.05).Decreased phosphorylation levels of IGF-1R and its downstream targets,including AKT and ERK1/2,were observed in cells transfected with LV-KL.Decreased phosphorylation levels of IGF1-R and its downstream targets were also observed in DLBCL xenograft mice treated with LV-KL compared to the control group.5.The rhKL was found to be active in vitro and significantly reduced the proliferation of DLBCL cells by 30%?40%(p<0.05).Moreover,rhKL increased the sensitivity of DLBCL cells to adriamycin(ADR).The in vivo activity of rhKL in DLBCL xenograft mouse model was also detected.Significantly decreased tumor volumes were noted in mice treated with rhKL compared with those treated with vehicle control(p<0.05).Moreover,reductive expression level of Ki67 was observed in rhKL-treated group.Lower serum Klotho levels were noted in DLBCL patients than the control subjects(p<0.05).Conclusions:Our observations identified for the first time that reduced expression of Klotho in DLBCL tissues and cell lines.Loss of Klotho expression contributed to the development and poor prognosis of DLBCL via modulating IGF-1R signaling pathway.Given the in vivo tumor suppressive activity of secreted Klotho protein,it may serve as a potential strategy for the development of novel therapeutic interventions for DLBCL.Part II.Study of the Role and Mechanism of Klotho in T-cell LymphomaObjective:T-cell lymphoma is the aggressive malignancy originated from T lymphocytes.This disease is more common in our country compared to Western countries.T-cell lymphoma is characterized by highly heterogeneous in both molecular and clinical characteristics.Due to the lack of definitive results focusing on the chemotherapy and transplantation of randomize multicenter clinical trails,none standard treatment strategy is used in clinical therapy at present.Patients with T-cell lymphoma revealed lower sensitivity to chemotherapy and presented worse prognosis than B-cell lymphoma.It was reported that Klotho,an anti-aging gene,could inhibit the growth of cancer cells by modulating IGF-1R signaling in several solid tumors.Aberrant activation of IGF-1R signaling was elucidated to be involved in the development and progression of T-cell lymphoma.However,the potential role of Klotho in T-cell lymphoma has not been reported.In the present study,we aimed to explore the expression and the molecular mechanism of Klotho in T-cell lymphoma.Material and Methods:1.Patients selection and specimen collection,2.Immunohistochemistry(IHC),3.Cell culture,4.Isolation of peripheral blood mononuclear cells(PBMCs),5.Real-time quantitative polymerase chain reaction(RT-PCR),6.Protein isolation and Western blotting,7.Cell transfection with lentivirus vectors either encoding Klotho or empty lentiviral vector,8.Cell proliferation assay by Cell Counting Kit-8(CCK-8),9.Detection of cell apoptosis by Annexin-V PE/7AAD staining,10.Immunofluoresence(IF)analysis,11.Statistical analysis.Results:1.We observed markedly decreased level of Klotho protein in the tissues of T-cell lymphoma.Klotho positive rate was 14.3%(5 of 35)in T-cell lymphoma tissues whereas 75%(15 of 20)in the reactive hyperplasia.Reduction of Klotho mRNA and protein expression levels were also confirmed in T-cell lymphoma cell lines(Jurkat,Molt-3,Karpas299 and MyLa3676).2.To explore the function of Klotho in the progression of T-cell lymphoma,T-cell lymphoma cell lines(Jurkat,Molt-3,Karpas299 and MyLa3676)were stably transfected with either Klotho-overexpression lentivirus vectors(LV-KL)or empty vector control(LV-Con).Significant reduction of cell proliferation was noted in T-cell lymphoma cells transfected with LV-KL,compared with those transfected with LV-Con(p<0.05).3.T-cell lymphoma cells transfected with LV-KL presented markdely increase of apoptosis rates compared with the LV-Con group(p<0.05).In addition,apoptosis promotive effect of Klotho was further confirmed by Western blotting analysis.Remarkable decreased expression levels of Mcl-1 and Pro-Caspase-3 protein were observed in Jurkat and Karpas299 cell lines.4.Cell proliferation was less restored by IGF-1 in T-cell lymphoma cells transfected with LV-KL compared to the control group.Jurkat and Karpas299 cells transfected with LV-KL revealed significantly decreased phosphorylation level of IGF-1R.Moreover,the downstream targets of IGF-1R signaling,including AKT and ERK1/2,were also inhibited by Klotho overexpression.Conclusions:This study indicated that Klotho acts as a tumor suppressor in T-cell lymphoma by suppressing cell-proliferation and promoting cell apoptosis.Activation of IGF-1 R signaling could be inhibited by Klotho in T-cell lymphoma.Taken together,Klotho may serve as a hopeful target for development of novel therapeutic strategy of T-cell lymphoma.