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O Publications 2016FOXO1 inhibits the invasion and metastasis of hepatocellular carcinoma by reversing ZEB2-induced epithelial-mesenchymal transitionTianxiu Dong1,Yu Zhang1,Yaodong Chen1,Pengfei Liu2,Tingting An1,Jiuwei Zhang1,Haichao Yang1,Wenjing Zhu1,Xiuhua Yang11Department of Abdominal Ultrasound,The First Affiliated Hospital of Harbin Medical University,Harbin 150001,China2Department of Magnetic Resonance Imaging,The First Affiliated Hospital of Harbin Medical University,Harbin 150001,ChinaCorrespondence to:Xiuhua Yang,email:Keywords:FOXO1,hepatocellular carcinoma,epithelial-mesenchymal transition,ZEB2,TGF-Received:March 24,2016 Accepted:November 02,2016 Published:December 03,2016ABSTRACTThe epithelial-to-mesenchymal transition(EMT)program is critical for epithelial cell cancer progression and fibrotic diseases.FOXO1 influences a broad range of physiological and pathological processes.However,the mechanism by which FOXO1 inhibits EMT is not fully understood.In this study,we demonstrated that FOXO1 overexpression inhibited cell motility and invasiveness in vitro and inhibited lung metastasis in vivo.In addition,we found that FOXO1 couldreverse the EMT program.FOXO1 silencing by siRNA in hepatocellular carcinoma(HCC)cell lines enhanced the expression of mesenchymal markers and decreased the expression of the epithelial markers.Consistent with these findings,FOXO1 overexpression exerted opposite effects.Furthermore,we found that FOXO1 levels were inversely correlated with the levels of EMT inducers,including Snail,Slug,ZEB1,ZEB2 and Twist1 in HCC cells.Co-immunoprecipitation and immunohistochemistry assays revealed that an interaction between FOXO1 and ZEB2.A dual-luciferase reporter assay and a ChIP assay further demonstrated that FOXO1 binds to the ZEB2 promoter.Together,these findings suggest that FOXO1 overexpression or ZEB2 inhibition might be potential therapeutic strategies for treating HCC.INTRODUCTIONHepatocellular carcinoma(HCC)is one of the most common solid malignancies worldwide and is the second leading cause of cancer-related mortality in Asia 1.The prognosis of HCC remains poor due to the lack of effective treatment options and the high probability of metastasis and recurrence after surgical resection 2.In addition,the molecular mechanisms underlying HCC metastasis remain poorly characterized.Thus,identifying the mechanisms that mediate HCC metastasis is imperative to improving outcomes.Epithelial-mesenchymal transition(EMT)plays a vital role in the progression of metastasis in multiple cancers by inducing epithelial cells to adopt mesenchymal attributes 3.During this process,well-organized and tightly connected epithelial cells differentiate into disorganized and motile mesenchymal cells 4.The EMT is regulated by a complex network of interconnected pathways controlled by TGF-signaling 5.EMT-inducing transcription factors(EMT-TFs)include the Snail family protein members Snail1 and Snail2(also referred to as Slug),the two-handed zinc finger factors ZEB1 and ZEB2,and the basic helix-loop-helix(bHLH)protein Twist1 6.ZEB2 plays a key role in the TGF-signaling cascade and promotes tumor cell invasion and metastasis 7.Snail,Slug,ZEB1,ZEB2 and Twist1 are all potent repressors of E-cadherin 811.Determining the structure and functional dynamics of these factors is critical to further characterizing the pathogenesis of EMT in HCC progression.The FoxO family of transcription factors(FoxOs),FOXO1(FKHR),FOXO3(FKHRL1 or FOXO3a),FOXO4(AFX)and FOXO6,integrate multiple cellular signals and translate various environmental stimuli into dynamic patterns of gene expression that influence a O range of physiological and pathological processes,including cancer and aging 12.FoxOs are associated with the insulin/IGF and growth factor-activated receptor tyrosine kinase(RTK)-phosphoinostide-3-kinase(PI3K)signaling pathway 13.FOXO1 dysregulation has been observed in several human cancers,and this aberration influences multiple cellular functions,including apoptosis,cell cycle control,DNA damage repair,carcinogenesis,glucose metabolism and tumor immunity 1416.Furthermore,cancer stem cells(CSCs)in pancreatic ductal carcinoma are FOXO1-negative,and the loss of FOXO1 might be a more accurate CSC marker than the expression of CD133 or ALDH1 17.A recent report demonstrated that FOXO1 regulates the vascular growth that couples metabolic and proliferative activities in endothelial cells 18.However,the impact of FOXO1 on invasion and metastasis in HCC is poorly understood.In addition,the underlying mechanisms that promote HCC invasion and metastasis remain elusive.Thus,we sought to investigate the mechanisms by which FOXO1 inhibits HCC migration and invasion.In this study,we provide the first evidence that FOXO1 can reverse EMT in HCC via the transcription inducers Snail,Slug,ZEB1,ZEB2 and Twist1,with ZEB2 playing a particularly critical role in this process.Furthermore,we demonstrated that FOXO1 disrupts TGF-induced EMT.RESULTSFOXO1 inhibits HCC cells migration and invasionTo explore the effects of FOXO1 on the invasiveness of HCC cells,we examined the expression of FOXO1 in five HCC cell lines(SMMC-7721,Huh7,HCCLM3,MHCC97H and SK-HEP-1),which exhibit different invasive behaviors.The expression of FOXO1 in these HCC cell lines was compared with that in a normal hepatic cell line,L02.FOXO1 protein levels were highest in normal hepatic L02 cells and were decreased in SMMC7721 and Huh7 cells(low metastatic potential),and finally,were lowest in HCCLM3,MHCC97H and SK-HEP-1 cells(high metastatic potential)(Figure 1A).To further evaluate the role of FOXO1 in HCC cells migration and invasion,we established stable cell lines that were infected with the LV-NC lentivirus(referred to as HCCLM3-Control and SK-HEP-1-Control)or with the LV-FOXO1 lentivirus(referred to as HCCLM3-FOXO1 and SK-HEP-1-FOXO1).In addition,the SMMC7721 and Huh7 cell lines were transfected with siRNA-NC or siRNA-FOXO1.A western blot analysis indicated that FOXO1 expression was up-regulated by FOXO1 overexpression and silenced by siRNA-FOXO1.FOXO1 silencing increased the wound-healing capability of SMMC7721 and Huh7 cells,while FOXO1 overexpression reduced the wound-healing capability of HCCLM3 and SK-HEP-1 cells(Figure 1B and 1E).Consistent with these findings,a Transwell assay demonstrated that FOXO1 silencing significantly enhanced the migration of SMMC7721 and Huh7 cells by 2.57-and 1.39-fold,respectively,and enhanced the invasion of these cells by 6.91-and 3.36-fold,respectively(Figure 1C and 1D).Conversely,FOXO1 overexpression markedly inhibited the migration of HCCLM3 and SK-HEP-1cells by 3.34-and 1.33-fold,respectively,and inhibited the invasion of these cells by 3.73-and 2.46-fold,respectively(Figure 1F and 1G).Together,these data indicate that FOXO1 inhibits the motility and invasiveness of HCC cells.FOXO1 represses epithelial-to-mesenchymal transition in HCC cellsEMT is a developmental program that converts adherent epithelial cells into mesenchymal migratory cells capable of promoting tumor metastasis.To investigate whether FOXO1 suppresses HCC invasion and migration by reversing EMT,we evaluated epithelial and mesenchymal markers in HCC cells using western blot and immunofluorescence assays.A western blot analysis confirmed that FOXO1 protein levels were down-regulated in both SMMC-7721 and Huh7 cells that were transfected with FOXO1 siRNA and were up-regulated in HCCLM3 and SK-HEP-1 cells that were infected with the LV-FOXO1 lentivirus.E-cadherin,-catenin and ZO-1 were down-regulated in FOXO1-silenced HCC cells compared with the negative control,whereas the mesenchymal markers Vimentin and N-cadherin were strongly up-regulated(Figure 2A and 2B).FOXO1 overexpression exerted the opposite effects(Figure 2C and 2D).FOXO1 inhibits TGF-induced epithelial-to-mesenchymal transitionTGF-signaling pathways are associated with the development of liver cancer 19.We next investigated whether TGF-signaling-induced EMT was repressed by FOXO1.A western blot analysis was used to determine the level of FOXO1 protein in response to different doses of TGF-1 in HCCLM3 and SK-HEP-1 cells.The analysis showed that,FOXO1 expression decreased in response to low doses of TGF-1 and that FOXO1 expression was lowest at a concentration of 10 ng/ml TGF-1(Figure 3A).We next evaluated the FOXO1 levels in HCCLM3 and SK-HEP-1 cells treated with 10 ng/ml TGF-1 for 24 h,48 h or 72 h,and observed the greatest decrease at 72 h(Figure 3B).Real-time PCR and western blotting revealed that TGF-1 expression was increased in FOXO1-silenced HCC cell lines.In contrast,TGF-1 expression was decreased in HCC cell lines overexpressing FOXO1(Figure 3C and 3D).Moreover,O 1:FOXO1 inhibits the migration and invasion of HCC cells.A.Western blotting analysis of FOXO1 expression in different HCC cell lines and the normal hepatic cell line L02.B.Wound-healing C.Transwell migration assays.D.Matrigel invasion assays in FOXO1-silenced SMMC-7721 and Huh7 cells.E and F.Cell migration and G.Invasion assays in HCCLM3 and SK-HEP-1 cells overexpressing FOXO1.Cells were counted in 3 randomized fields at a magnification of 100.The error bar represents the meanSD of triplicate assays(*p0.05,*p0.01,*p0.001;p-values were calculated using Students t-test).O silencing enhanced Smad3 phosphorylation,while FOXO1 overexpression inhibited Smad3 activation(Figure 3D).These data indicate that TGF-1 and FOXO1 each repress the expression of the other.HCC cells treated with TGF-1 led to a promotion of cell migration and invasion,but this promotion was greatly attenuated by FOXO1 overexpression(Figure 3E and 3F).FOXO1 overexpression inhibited N-cadherin expression,and this inhibition persisted even after TGF-1 treatment.In addition,E-cadherin expression was enhanced in the FOXO1-overexpressing cells.The FOXO1-induced increase in E-cadherin expression was inhibited by treatment with TGF-1(Figure 3G).These results suggest that the effects of FOXO1 associated with TGF-1 more potently affect N-cadherin expression compared with E-cadherin expression and indicate that FOXO1 expression in HCC cells inhibits TGF-signaling.FOXO1 overexpression in luciferase-labeled HCCLM3 and SK-HEP-1 cells inhibits lung metastasis in nude miceTo investigate the role of FOXO1 in vivo,we injected stably transfected cell lines(HCCLM3-Control,SK-HEP-1-Control,HCCLM3-FOXO1 and SK-HEP-1-FOXO1)into the tail vein of nude mice and examined the mice for the presence of lung metastatic nodules.The expression of FOXO1 was verified by western blotting,which showed that FOXO1 expression in HCCLM3-FOXO1 and SK-HEP-1-FOXO1 cells was increased approximately 5.25-and 6.93-fold,respectively,compared with the control(Figure 4A).Bioluminescent imaging of nude mice demonstrated that the incidence of lung metastasis was lower in mice injected with HCCLM3-FOXO1 or SK-HEP-1-FOXO1 cells compared with mice injected with HCCLM3-Control or SK-HEP-1-Control cells(Figure 4B).Moreover,mice injected with HCCLM3-FOXO1 or SK-HEP-1-FOXO1 cells exhibited fewer and smaller lung metastases compared with the control group(Figure 4C-4E).Histologic analyses of the dissected lungs further confirmed the presence of metastases(Figure 4F).These results demonstrate that FOXO1 plays a critical role in inhibiting HCC invasion and metastasis.FOXO1 negatively regulates EMT-inducing transcription factors and interacts with ZEB2 in HCC cellsSnail,Slug,ZEB1,ZEB2,and Twist1 are key regulators of the EMT program.We examined the expression of these transcription factors to further investigate their potential interactions with FOXO1 during EMT.We found that endogenous mRNA levels of Snail1,Slug,ZEB1,ZEB2 and Twist1 were increased in FOXO1-silenced cells and were decreased in FOXO1-overexpressing cells(Figure 5A and 5C).A western blot analysis demonstrated similar results;however,Slug expression was not noticeably affected in the Huh7 cell Figure 2:FOXO1 reverses EMT in HCC cells.A.Western blot and B.Immunofluorescence staining assays revealed a down-regulation of the expression of epithelial markers(E-cadherin,-catenin and ZO-1)and an up-regulation of the expression of mesenchymal markers(Vimentin and N-cadherin)in SMMC7721 and Huh7 cells transfected with FOXO1 siRNA.In contrast,C.Western blotting and D.Immunofluorescence staining revealed that FOXO1 overexpression up-regulated the expression of epithelial markers and decreased the expression of mesenchymal markers in HCCLM3 and SK-HEP-1 cells.Scale bar indicates 25m.O 3:FOXO1 reverses TGF-induced EMT.A.FOXO1 expression levels in HCCLM3 and SK-HEP-1 cells treated with different doses of TGF-1.B.FOXO1 expression levels in HCCLM3 and SK-HEP-1 cells treated with activated TGF-1(10 ng/ml)at 3 time points were evaluated by western blotting.C.TGF-1 mRNA levels were determined using real-time PCR in HCC cells treated with si-NC,siRNA-FOXO1,empty vector or LV-FOXO1.The error bar represents the meanSD of triplicate assays.(*p0.05,*p0.01,*p0.001;p-values were calculated using Students t-test).D.TGF-1,p-Smad3,and Smad3 expression in HCC cell lines was analyzed by western blotting.Down-regulation of FOXO1 using siRNA markedly increased the expression of TGF-1 and p-Smad3 in SMMC7721 and Huh7 cells.In contrast,the overexpression of FOXO1 significantly attenuated the expression of TGF-1 and inhibited Smad3 phosphorylation.Relative changes of the number of HCCLM3 and SK-HEP-1 cells that migrated E.and invaded F.after TGF-1 treatment or FOXO1 overexpression.(*p0.05,*p0.01,*p0.001;p-values were calculated using Students t-test).G.Western blot analysis of FOXO1,E-cadherin and N-cadherin expression after treatment with TGF-1(10 ng/ml)in HCCLM3 and SK-HEP-1 cells that were infected with the LV-NC lentivirus or LV-FOXO1 lentivirus.O 5B and 5D).These findings suggest that FOXO1 might reverse EMT by interacting with EMT-associated transcription factors in HCC cells.According to our results,ZEB2 can promote EMT.We found that ZEB2 silencing enhanced the expression of E-cadherin and decreased the expression of N-cadherin(Figure 5E).In addition,ZEB2 silencing led to morphological changes in HCCLM3 and SK-HEP-1 cells(Figure 5F).We also found that FOXO1 and ZEB2 levels were inversely correlated and that ZEB2 was regulated by FOXO1 transcription.In addition,an immunohistochemistry analysis of metastatic nodules obtained from the nude mice revealed that FOXO1 overexpression inhibited ZEB2 expression(Figure 5G).Co-immunoprecipitation assays confirmed the interaction between FOXO1 and ZEB2(Figure 5H).Based on the results that EMT transcriptional factors(including ZEB2)were decreased in FOXO1-overexpressing cells(Figure 5C and 5D),we hypothesized that FOXO1 might suppress ZEB2 promoter activity.ZEB2 promoter region was cloned into pGL3-Basic plasmid and the transcriptional regulation of FOXO1 was investigated through a dual-luciferase reporter assay.We first co-transfected pEGFP-FOXO1 or a negative control