The Study On The Roles And The Mechanisms Of Wnt5a/JNK And γ-catenin In CML Cells

Aim:The relations between Wnt and tumors or stem cells always are the focus of domesticand foreign studies. Previously, we showed Wnt5a acted as a tumor suppressor in leukemia;Wnt5a binded Ror2receptor, then activated non-canonical Wnt signaling, and inhibitedcanonical Wnt signaling and tumor promoting of β-catenin. Which signaling protein doesWnt5a inhibit β-catenin through? Whether Wnt5a can influence the effects of ImatinibMesylate on the CML cells? These questions have not yet answered.It has been reported that in some cells Wnt5a can bind and activate Ror2, Fz receptor andthen activates JNK——activating Wnt5a/JNK pathway. In the CML, the studies on the JNKare limited, and consist of diverse viewpoint: a much earlier report showed that BCR-ABLactivated JNK, and JNK promoted the transformation of BCR-ABL positive cells; on theother hand, JNK was involved in the apoptosis or autophagy induced by a serials of medicinesin the CML cells. Whether JNK can be activated by Wnt5a in CML cells? Whether JNKparticipates in Wnt5a affecting Imatinib Mesyltate and what is the mechanism for that?These questions need answers.γ-catenin is a member of Armadillo protein family, and is involved in the signalingtransduction and cell adhesion. As a member of Wnt family，whether γ-catenin can beregulated by Wnt5a and participate in Wnt5a’ roles in CML? There are no answers for thesequestions, and it worth our studing. It has been shown that the fused proteins ofAML(AML1-ETO, RARα, PLZF-RARα) can promoted the expression of γ-catenin, butwhether the fused protein of CML(BCR-ABL) can influence γ-catenin is unknown. In thestudy of Kim et al, the CML patient at the stages of Accelerated Phase or Blast Crisisshowed a elevated level of γ-catenin compared with patients at the stage of CP, suggestingγ-catenin play pivotal roles in the CML, but roles of γ-catenin in CML and the mechanism has not been well understood yet.On the basis of our previous studies, this research aims to answer the followingquestions:1) Whether can Wnt5a influence JNK and the response of CML cells to ImatinibMesylate?2) Whether can γ-catenin be regulated by Wnt5a?3) Whether can γ-catenin beregulated by BCR-ABL? what the roles does γ-catenin play in CML? Whether can γ-catenininfluence the response of CML cells to Imatinib Mesylate? The research is beneficial fordeeper exploring the roles of Wnt5a/JNK and γ-catenin in leukemia, which provides a newstrategy for CML treatment.Method:1. The study on the influences of Wnt5a/JNK signaling pathway on the ImatinibMesylate inhibiting CML cells and its mechanism.1.1Construct KU812cells overexpressing Wnt5a; Measure the effects of Wnt5a onthe Imatinib Mesylate inhibiting CML cell proliferation， inducing apoptosis, andsuppressing BCR-ABL; Detect the influence of Wnt5a on the β-catenin and its target geneSurvivin，and γ-catenin in CML cells.1.2Detect the influence of Wnt5a on the expression and the activity(phosphation level)of JNK; After inhibiting JNK with SP600125, measure the influence of inhibiting JNK onthe effects on Wnt5a on the Imatinib Mesylate, and the influences of inhibiting JNK on theeffects of Wnt5a inhibiting β-catenin and its target gene Survivin.1.3Construct the CML cells overexpressing JNK1/JNK2; Measure the influences ofJNK on the effects of Imatinib Mesylate, and the effects of JNK on β-catenin and its targetgene Survivin，and γ-catenin.1.4Construct xenograft tumor animal model (NOD/SCID mouse), and observe theinfluences of Wnt5a on the effects of Imatinib Mesylate in vivo.2. The study on the roles of γ-catenin the CML2.1Measure the expressions of γ-catenin in7leukemia cells; Construct CML cellssuppressing BCR-ABL with siRNA; Detect the influence of BCR-ABL on the expression ofγ-catenin; Observe the interaction of BCR-ABL and γ-catenin using Co-IP(Co-Immunoprecipitation).2.2Construct the CML cells suppressing γ-catenin with siRNA; Detect the influence ofinhibiting γ-catenin on the proliferation and transformation of CML cells and the genes related to proliferation(cycliD1and c-Myc).2.3Detect the influences of inhibiting γ-catenin on the effects of Imatinib Mesylate(inhibiting proliferation and inducing apoptosis) and the expression of Suvivin and Bcl-xL.2.4Detect the influence of inhibiting γ-catenin on the activity of transcription factorSTAT5and β-catenin. Detect the influence of inhibiting γ-catenin on the GSK3β which is onthe key proteins regulating β-catenin.Results1. Wnt5a/JNK signaling pathway enhanced the effects of Imatinib Mesylate on CMLcells through inhibiting β-catenin/Survivin, and Wnt5a suppressed γ-catenin not throughJNK.1.1In the Wnt5a-overexpressing CML cells, we found: Wnt5a potentiated the effectsof Imatinib Mesylate on CML cell (inhibiting proliferation and inducing apoptosis, andsuppressing BCR-ABL); Wnt5a promoted the phosphation of β-catenin in two CML cells,and supprsessed the expression of β-catenin in KU812cells; Wnt5a suppressed the expressionof Survivin and γ-catenin.1.2Wnt5a increased the the activity(phosphation level) of JNK but had no influenceon the expression of total JNK; Inhibiting JNK with SP600125relieved the effects onWnt5a on the Imatinib Mesylate. It also was relived by inhibiting JNK that Wnt5ainhibiting β-catenin and its target gene Survivin.1.3In the JNK-overexpressing CML cells, we found: JNK also potentiated the effectsof Imatinib Mesylate on CML cell; JNK suppressed the expression of β-catenin andpromoted its phosphation; JNK suppressed the expression of Survivin but had no effect onγ-catenin.1.4The xenograft tumor animal model (NOD/SCID mouse) was constructed, andWnt5a potentiated the effects of Imatinib Mesylate in vivo.2. γ-catenin downregulation inhibited CML cell growth and enhanced the response ofCML cells to Imatinib Mesylate through inhibiting β-catenin.2.1Among7leukemia cells, two CML cells(K562, KU812) expressed γ-catenin; TheCML cells suppressing BCR-ABL with siRNA were constructed; Suppressing BCR-ABLreduced the expression of γ-catenin; Co-IP(Co-Immunoprecipitation) assay showedBCR-ABL protein and γ-catenin protein cannot bind each other. 2.2The CML cells suppressing γ-catenin with siRNA were constructed; inhibitingγ-catenin reduced proliferation and transformation of CML cells and the genes related toproliferation(cycliD1and c-Myc).2.3Inhibiting γ-catenin potentiated the effects of Imatinib Mesylate and suppressed theexpression of Suvivin and Bcl-xL.2.4Inhibiting γ-catenin suppressed the activity of transcription factor STAT5and thenucleus translocation of β-catenin and promoted the phosphation of β-catenin but had noinfluence on total β-catenin. Inhibiting γ-catenin promoted the expression of GSK3β andreduced phosphation of GSK3β.Conclusion:1. In CML, Wnt5a promotes the activity of JNK, and Wnt5a/JNK pathway enhancesthe effects of Imatinib Mesylate, the mechanism of which is suppressing β-catenin and itstarget gene Survivin. Wnt5a can suppress γ-catenin but not through JNK, suggestingγ-catenin also plays important roles in the effects of Wnt5a on Imatinib Mesylate inhibitingCML cells.2. γ-catenin is over-expressed in CML cells and can be regulated by BCR-ABL in anindirect pattern. Downregulation of γ-catenin suppresses the proliferation and transformationof CML cells, enhances the effects of Imatinib Mesylate on CML cells, which is related tosuppressing the expressions of cycliD1, c-Myc, Suvivin and Bcl-xL through regulatingGSK3β/β-catenin and STAT5. These suggest that γ-catenin functions as a leukemia promoterin CML.