Mechanisms Of Annexin A7 On Promoting Apoptosis And Inhibiting Metastasis In Cancer Cells

BACKGROUND AND OBJECTIVEAnnexin A7(ANXA7),a member of annexin superfamily,is a Ca2+-and phospholipid-binding protein and possesses GTPase activity.The ANXA7 gene is located on human chromosome 10q21,where multiple potential tumor suppressor genes exist.Homozygous ANXA7(-/-)knockout mice showed a lethal phenotype at embryonic day 10.Heterozygous ANXA7(+/-)knockout mice have a high frequency of spontaneous tumors,about 20-50%.Spontaneous tumors developed in multiple organs,including liver,prostate,endometrium,salivery gland and thymus.Tumor metastasis was observed in 42%of the mutant mice developing spontaneous tumors.This indicates that ANXA7 plays an important role in inhibiting tumorigenesis and metastasis.Apoptosis is one of the most effective ways against tumors.Studies have shown that ANXA7 promotes apoptosis in prostate cancer,glioblastoma and breast cancer.In our previous study,we identified a small molecule ABO as ANXA7 GTPase inhibitor,which reduced apoptosis in the mouse aortic endothelium and inhibited atherosclerosis in apoE-/-mice.In view of the critical function of ANXA7 in tumor resistance,we speculated that the activation of ANXA7 GTPase might trigger apoptosis in tumor cells.However,so far the regulation of ANXA7 GTPase in tumor cell apoptosis is not clear.In this dissertation,we would identify a new small molecule as ANXA7 GTPase activator,and investigate the mechanism of ANXA7 GTPase in the regulation of cancer cell apoptosis by use of the ANXA7 GTPase activator and inhibitor.Our previous work has demonstrated that ANXA7 could interact with integrin β4(ITGB4)and regulate the phosphorylation of its interactive proteins by exerting GTPase activity.ABO inhibited ANXA7 GTPase activity and decreased ITGB4 Y1494 phosphorylation.ITGB4 is a tumor-related antigen upregulated in multiple tumor cells.The roles of ITGB4 are closely related to its subcellular distribution.When localized on membrane,ITGB4 facilitates tumor growth and metastasis,while ITGB4 in nucleus promotes vascular endothelial cell apoptosis.But the effect and mechanism of ITGB4 nuclear translocation on cancer cell apoptosis are not clear now.The phosphorylation of ITGB4 cytoplasm domain is essential for ITGB4 to be signaling-competent.Y1494 acts as a master regulator of ITGB4 phosphorylation and mutation of Y1494 suppressed overall tyrosine phosphorylation of ITGB4 subunit.Moreover,ANXA7,via its GTPase activity,facilitates membrane fusion,which is important in mediating cell membrane receptor nuclear translocation in vesicle transport manner.Combined with our previous study,we speculated that ANXA7 GTPase activation might increase ITGB4 phosphorylation,induce ITGB4 nuclear translocation and promote cancer cell apoptosis.In the dissertation,we would prove the speculation.Studies have revealed that ANXA7 inhibits metastasis in prostate cancer,glioma and pancreatic carcinoma.However,the mechanism of ANXA7 GTPase in the regulation of tumor metastasis is not clear.Raf kinase inhibitory protein(RKIP)is a tumor metastasis suppressor and downregulated in multiple tumor types.RKIP’s anti-metastasis property was first identified in prostate cancer.Acting as a GTPase,ANXA7 could bind to GTP.It has been reported that RKIP showed affinity to GTP-binding proteins.The binding of RKIP and its target proteins could inhibit the interactive proteins activation by their activators,leading to the inversion of related signaling and the regulation of tumor metastasis.So we wondered whether RKIP might bind to ANXA7 and blocked the activation of ANXA7 GTPase by its activator?In addition,one research found that nonylphenol induced the upregulation of RKIP in testicular Sertoli cells,accompanied with decreased ANXA7 protein level.However,the mechanism of RKIP in the regulation of ANXA7 signal pathway has not been clarified so far.Therefore,in this study,we used metastatic PC3 prostate cancer cell,and constructed RKIP knockout HEK293T cell line(HEK293T RKIP-/-),together with RKIP wild-type HEK293T cell line(HEK293T RKIP+/+)as the cell model,to investigate the mechanism of ANXA7 GTPase on tumor metastasis.In conclusion,in the dissertation,we would use ANXA7 GTPase activator and inhibitor as the tools and the regulation of ANXA7 GTPase on ITGB4 nuclear translocation as the pointcut,to study the mechanisms of ANXA7-mediated ITGB4 nuclear translocation and nuclear ITGB4 on the regulation of apoptosis-related gene expression,and clarify the mechanism of ANXA7 GTPase on promoting tumor cell apoptosis.We used metastatic PC3 prostate cancer cell,HEK293T RKIP+/+ and RKIP-/-cells as the cell model,to study the mechanism of ANXA7 GTPase on inhibiting prostate cancer metastasis.These efforts provide theoretical and physical foundation to the finding of effective target and novel drugs in tumor therapy.STUDY CONTENTS1.To investigate the mechanism of ANXA7 on ITGB4 nuclear translocation and pro-apoptotic effect in cancer cells.1.1 The identification of ANXA7 GTPase activator.1.2 The mechanism of ANXA7 in the regulation of ITGB4 nuclear translocation.1.3 The regulation of nuclear ITGB4 in apoptosis-related gene expression.1.4 The in vivo confirmation in human xenograft model on the chick embryo chorioallantoic membrane(CAM).2.To study the mechanism of ANXA7 on AMPK activation and anti-metastatic effect in cancer cells.2.1 The effect of ANXA7 inhibited PC3 prostate cancer cell migration.2.2 The mechanism of ANXA7 in activating AMPK and inhibiting pro-metastatic gene expression.2.3 The inhibitory effect of the binding of RKIP to ANXA7 on signal pathways mediated by ANXA7 GTPase activation.2.4 The in vivo confirmation in PC3-3M-Luc orthotopic prostate tumor growth and metastasis model.METHODS1.We used chemical small molecules SEC(ANXA7 GTPase activator)and ABO(ANXA7 GTPase inhibitor)as tools to do the related study in the dissertation.2.ANXA7 GTPase activity assay:Purification of wild-type ANXA7,T275A and T286A ANXA7 mutant proteins.Purified ANXA7 was incubated with SEC and the GTPase activity of ANXA7 was measured by use of the ATPase/GTPase Activity Assay Kit.3.Western blot analysis is used to detect relevant protein levels and phosphorylation levels.qPCR and RT-PCR are used to detect the mRNA levels of relevant genes.4.Cell viability is estimated by SRB assay.Cell apoptosis is estimated by Hoechst 33258 staining.5.Detection of the ITGB4 location in cytoplasm and nucleus:5.1 Immunofluorescence assay to determine subcellular distribution of ITGB4 in cytoplasm and nucleus.5.2NE-PERTM Nuclear and Cytoplasmic Extraction Reagents Kit used to extract nuclear and cytoplasmic proteins.Western blot to detect ITGB4 protein level in nuclear and cytoplasmic extracts.6.Chromatin immunoprecipitation(ChIP)is used to determine the binding of nuclear ITGB4 to ATF3 promoter.7.Detection of the interactions between proteins:7.1 Co-immunoprecipitation assay to analyze the binding of proteins.7.2 Immunofluorescence assay to evaluate the co-localization between proteins.8.Investigation of the roles of relevant proteins in signal pathways:8.1 RNA interference and gene knockout technology to inhibit specific protein expression.8.2 Plasmid transient transfection to overexpress or restore the expression of certain proteins.8.3 Site-directed mutagenesis to construct ANXA7 and ITGB4 point mutation plasmids:mCherry-ANXA7-wt,mCherry-ANXA7-mtl(T275A),-mt2(T286A),-mt3(T286D)and pEGFP-ITGB4-wt,pEGFP-ITGB4-mt(Y1494A).And to characterize the roles of corresponding sites in the regulation of ANXA7 GTPase activity and function and ITGB4 nuclear translocation.9.Confirmation of pro-apoptotic effect of SEC in vivo:9.1 Establishing human xenograft model on CAM to detect tumor volume with SEC treatment for 7 days.9.2 The frozen tumor tissue sections were used in TUNEL assay to detect apoptosis.10.Detection of the effect of SEC on angiogenesis:10.1 In vivo the CAM model was used to measure angiogenic action of SEC on CAM with gelatin sponge as deposition carrier.10.2 In vitro,Matrigel angiogenesis assay was used to detect the effect of SEC on vascular endothelial cell angiogenesis.11.Detection of cell migration:11.1 Wound healing assay to evaluate cell migration ability.11.2 Western blot to detect the protein levels of epithelial marker E-Cadherin and mesenchymal marker Vimentin,and to evaluating epithelial-mesenchymal transition(EMT).12.Confirmation of anti-metastatic effect of SEC in vivo:12.1 Establishing PC3-3M-Luc orthotopic prostate tumor growth and metastasis model,followed by bioluminescence in vivo imaging with SEC injection for 3 weeks.12.2 Isolation of aortic lymph node,followed by bioluminescence and count lymph node with prostate cancer metastasis.RESULTS1.SEC increased ANXA7 GTPase activity.1.1 SEC dose-and time-dependently increased the activity of ANXA7 GTPase.The point mutation to alanine at Thr275 had no effect on SEC-increased ANXA7 GTPase activity.In contrast,with mutation of Thr286 to alanine,SEC could not increase ANXA7 GTPase activity.1.2 SEC could bind to ANXA7.The point mutation to alanine at Thr275 had no effect on the binding,while the mutation of Thr286 to alanine blocked the binding of SEC to ANXA7.2.SEC induced apoptosis in tumor cells with high expression of ITGB4 by promoting ITGB4 nuclear translocation.2.1 SEC significantly decreased the cell viability and increased apoptosis in tumor cells expressing a high level of ITGB4—prostatic cancer(PC3),lung carcinoma(A549),colorectal cancer(HCT116)and breast cancer(MCF-7).The viability of hepatic cells(L-02)and human embryonic kidney cells(HEK293),with low ITGB4 level,was not affected by SEC.However,SEC significantly decreased the viability of HEK293 cells with overexpression of ITGB4.2.2 SEC induced nuclear translocation of endogenous ITGB4 in tumor cells and exogenous overexpressed ITGB4 in HEK293 cells.3.SEC facilitated the binding of ANXA7 to ITGB4 and promoted ITGB4 nuclear translocation by exerting GTPase activity.3.1 SEC dose-dependently promoted the binding of ANXA7 to ITGB4.ANXA7 knockdown blocked the capacity of SEC to induce nuclear localization of ITGB4.3.2 ABO inhibited the SEC-promoted binding of ANXA7 and ITGB4,which was concomitant with minor levels of nuclear ITGB4.3.3 Cells transfected with mCherry-ANXA7-mt2(T286A)expressed extreme low levels of nuclear ITGB4 as compared with cells transfected with mCherry-ANXA7-wt and-mtl(T275A).4.ANXA7 GTPase activity enhanced ITGB4 Y1494 phosphorylation and its subsequent nuclear translocation.4.1 The mutation of ITGB4 Y1494 to alanine blocked SEC-induced ITGB4 nuclear translocation.4.2 SEC increased Y1494 phosphorylation of ITGB4,which was markedly inhibited by knockdown ANXA7,treatment with ANXA7 GTPase inhibitor ABO or overexpression of mCherry-ANXA7-mt2(T286A).5.Nuclear ITGB4 induced the transcription of target genes.5.1 SEC increased the mRNA levels of ATF3,PPP1R15A,IL8 and TR1B3.After RNAi-mediated knockdown of ITGB4,SEC stimulation had no effect on the expression of target genes.5.2 Nuclear ITGB4 could bind to the promoter of ATF3.6.SEC inhibited the growth of human tumor xenografts in an avian embryo model.6.1 SEC promoted apoptosis and inhibited the growth of tumor xenografts in CAM.6.2 SEC had no effect on CAM normal angiogenesis and did not interfere with vascular endothelial cell angiogenesis.7.SEC inhibited the cell migration of PC3 cells and HEK293T RKIP-/-cells.7.1 SEC inhibited the cell migration and EMT in PC3 cells expressing a low RKIP level and RKIP-null HEK293T cells,while had no effect on RKIP-carrying HEK293T cells.7.2 In PC3 and HEK293T RKIP-/-cells SEC reduced the mRNA levels of the pro-metastatic genes,including CCL2,APLN and IL6ST,while SEC had little effect on HEK293T RKIP+/+cell.Restoration of RKIP expression in HEK293T RKIP-/-cells resulted in SEC ineffective8.ANXA7 activated AMPK and inhibited the expression of pro-migration genes.8.1 SEC enhanced the binding of ANXA7 to AMPK and increased AMPK phosphorylation,which were inhibited by ANXA7 GTPase inhibitor ABO.8.2 Activated AMPK blocked mTORC1 activity and decreased STAT3 phosphorylation,leading to the inhibition of STAT3 nuclear translocation and the expression of pro-metastatic genes9.RKIP could bind to ANXA7 and inhibit the signal pathway mediated by ANXA7 GTPase activation.9.1 In HEK293T RKIP+/+cells,RKIP could bind to ANXA7.9.2 In HEK293T RKIP+/+cells,RKIP,via binding to ANXA7,inhibited the interaction between ANXA7 and AMPK induced by SEC10.SEC suppressed tumor metastasis in PC-3M-Luc orthotopic implantation nude mice model.10.1 SEC decreased the bioluminescent signals expressed as photon counts.10.2 SEC suppressed the lymph node metastatic capacities of the nude mice model.CONCLUSIONS1.SEC increased ANXA7 GTPase activity and activated ANXA7 promoted ITGB4 nuclear translocation by triggering ITGB4 phosphorylation at Y1494.2.Nuclear ITGB4 can bind to the ATF3 promoter region and activate the expression of ATF3,then upregulate the downstream pro-apoptosis genes including PPP1R15AA IL8 and TRIB3,leading to apoptosis.3.In PC3 cells expressing a low RKIP level and RKIP-null HEK293T cell,SEC increased AMPK phosphorylation via activating ANXA7 GTPase.Activated AMPK inhibited STAT3 nuclear translocation via mTORCl/STAT3 pathway.In RKIP-carrying HEK293T cells RKIP bound to ANXA7 and inhibited the signal pathway mediated by ANXA7 GTPase activation.4.The inhibition of STAT3 nuclear translocation could lead to the downregulation of pro-metastatic genes including CCL2,APLN and IL6ST,and inhibit metastasis.