Sox12,a Direct Target Of FoxQ1,Promotes Hepatocellular Carcinoma Metastasis Through Up-regulating Twist1 And FGFBP1

Background and Aim Metastasis is the main reason for high recurrence and poor survival of hepatocellular carcinoma(HCC)patients after curative resection.However,the molecular mechanism underlying HCC metastasis remains unclear.The general process of metastasisdissemination of cancer cells from primary tumors and their subsequent seeding of new tumor colonies in distant tissues involves a multi-step process known as the invasion-metastasis cascade.This sequence of events involves the local invasion of primary tumor cells into surrounding tissues;intravasation of these cells into the circulatory system and survival during hematogenous transit;arrest and extravasation through vascular walls into the parenchyma of distant tissues;formation of micrometastatic colonies in this parenchyma and the subsequent proliferation of microscopic colonies into overt,clinically detectable metastatic lesions which is termed colonization.One centrally important process enabling these steps is the cell-biological program termed the epithelial-mesenchymal transition(EMT),which occurs in relatively early stage and is often driven by some classic transcritptional factors including Snail1,Slug,Zeb1 and other non-classical transcriptional factors like Fox C2.Transcprition factors are critical intermediators and central organizers in biological processes including tumor progression and our previous studies have shown the roles of Fox family transcription factors like Fox M1 and Fox Q1 in HCC metastasis or EMT phenotype.In primary analysis of Sox transcription factor family expression Sox12 was identified as one of most up-regulated genes and basic clinical sample and functional analysis indicated Sox12 as HCC metastasis related molecule.Based on the above fact we proposed that Sox12 is essential to HCC metastasis and is a critical factor for HCC progression and prognosis of HCC patients.Methods 1.Western blot was performed to detect the expression of Sox12 in HCC cell lines with different metastatic abilities and validate the over-expression or knockdown efficiency.Real-time PCR was performed to evaluate the expression of Sox12 in HCC tissues compared with adjacent non-tumorous tissues and normal liver tissues.2.Loss-of-function and gain-of-function in vitro and in vivo experiments were carried out to study the roles of Sox12,Twist1 or FGFBP1 in metastasis of HCC.3.Bioinformatics prediction was used to identify the potential binding sites of Fox Q1 or Sox12 in the promotor of Sox12 or Twist1,FGFBP1.A series of experiments including luciferase reporter assay and Ch IP experiment were performed to validate transactivation relationship and the binding sites of Fox Q1 or Sox12.4.Immunohistochemistry was performed to evaluate the clinical significance of Sox12,Twist1,FGFBP1 and Fox Q1.Results 1.The expression of Sox12 in HCC was analyzed in a cohort of 120 paired HCC tissues.The m RNA levels of Sox12 were dramatically up-regulated in HCC tissues compared with adjacent non-tumorous tissues and normal liver tissues.Patients with recurrence of HCC(68 of 120)had higher Sox12 m RNA expression than patients without recurrence(52 of 120).Sox12 m RNA expression was much higher in primary HCC tissues from patients who developed metastasis than in primary HCC tissues from patients who did not develop metastasis.In addition,the m RNA and protein levels of Sox12 were compared in primary and metastatic HCCs in 20 pairs of HCC specimens.Real-time PCR analysis showed that Sox12 m RNA expression was much higher in metastatic HCC tissues than in primary HCC tissues.A higher level of Sox12 expression detected by immunohistochemistry staining was observed in metastatic HCC samples than in primary HCC samples.Sixty percent(12/20)of HCC cases showed higher levels of Sox12 expression in the metastatic tissues than in primary HCC tissues.Next,we analyzed Sox12 protein expression in a tissue microarray of 690 HCC patients(Cohort I)using immunohistochemistry staining.Sox12 expression was dramatically up-regulated in HCC tissues than in adjacent non-tumorous tissues.Sox12 expression was primarily localized to the nucleus.Over-expression of Sox12 was significantly correlated with loss of tumor encapsulation,microvascular invasion,and a higher tumor-nodule-metastasis(TNM)stage.Patients with positive Sox12 expression had shorter overall survival times and higher recurrence rates than patients with negative Sox12 expression.Multivariate analysis showed that Sox12 expression was an independent and significant risk factor for recurrence and reduced survival.The expression and clinical significance of Sox12 was validated in an independent cohort of HCC tissues(Cohort II,n=312)by immunohistochemical staining.Similarly,over-expression of Sox12 was significantly correlated with loss of tumor encapsulation,microvascular invasion,and higher TNM stage.Sox12 over-expression was significantly correlated with poor prognosis.Multivariate analysis showed that Sox12 over-expression was an independent predictor for postoperative recurrence and overall survival.2.Western blot showed that Sox12 expression in highly metastatic HCC cell lines was much higher than in HCC cell lines with low metastatic potential.To determine the role of Sox12 in the invasion and metastasis of HCC cells,firstly we established two stable cell lines Huh7-Sox12 and HCCLM3-sh Sox12.In vitro transwell assay showed that up-regulation of Sox12 expression significantly increased the migration and invasion potential of the Huh7 cells,whereas down-regulation of Sox12 decreased the migration and invasion potential of HCCLM3 cells.In vivo metastatic assay also confirmed that the up-regulation of Sox12 increased the incidence of lung metastasis and the number of metastatic lung nodules while decreasing the overall survival time of the Huh7-Sox12 group.However,the down-regulation of Sox12 decreased incidence of lung metastasis and the number of metastatic lung nodules while prolonging the overall survival time of the HCCLM3-sh Sox12 group.Next,we determined whether Sox12 has any effect on HCC proliferation and apoptosis.MTT and colony formation assays showed that up-regulation of Sox12 increased cell proliferation of Huh7 and SMMC7721 cells,whereas down-regulation of Sox12 decreased cell proliferation HCCLM3 and MHCC97 H cells.Soft agar colony formation assay showed that over-expression of Sox12 significantly enhanced the anchorage-independent growth of Huh7 and SMMC7721 cells in soft agar,whereas knockdown of Sox12 decreased the anchorage-independent growth of HCCLM3 and MHCC97 H cells in soft agar.In vivo tumorigenicity assays showed that up-regulation of Sox12 increased tumor growth of Huh7 cells,whereas down-regulation of Sox12 significantly decreased tumor growth of HCCLM3 cells.However,Annexin V/PI FACS analysis showed that knockdown of Sox12 had no significant effect on apoptosis of HCC cells.3.Immunofluorescence showed that up-regulation of Sox12 decreased E-cadherin expression and increased Vimentin expression in Huh7 cells,whereas down-regulation of Sox12 increased E-cadherin and decreased Vimentin expression in HCCLM3 cells.Western blot assay showed that up-regulation of Sox12 also induced decreased expression of epithelial markers(E-cadherin and ZO-1)and the increased expression of mesenchymal markers(Vimentin and Fibronectin).In contrast,down-regulation of Sox12 significantly increased the expression of epithelial markers and decreased the expression of mesenchymal markers.Then m RNA expression profiles between Huh7-Sox12 and Huh7-control cells were compared using an EMT RT-PCR Array.Up-regulation of Sox12 increased the expression of several metastatic-related genes,including FGFBP1,TWIST1,JAG1,MST1 R,ZEB2,VIM,MMP9,ERBB3,FN1,ITGA5 and ZEB1.Of particular interest were FGFBP1 and Twist1,which were the top two up-regulated genes in response to Sox12 over-expression.Considering the critical role of Twist1 in EMT and metastasis we sought out to determine if Twist1 was regulated by Sox12 in HCC and involved in Sox12-mediated HCC EMT and metastasis.Western blot showed that down-regulation of Twist1 expression attenuated the loss of E-cadherin expression induced by Sox12 over-expression,whereas up-regulation of Twist1 significantly inhibited the increase in E-cadherin expression induced by Sox12 knockdown.Luciferase reporter assay showed that Sox12 trans-activated Twist1 promoter activity.Sequence analysis revealed three putative Sox12 binding sites in the Twist1 promoter.Serial deletion and site-directed mutagenesis showed that the second and third Sox12 binding sites were critical for Sox12-induced Twist1 trans-activation.A Ch IP assay also confirmed the direct binding of Sox12 to the Twist1 promoter in HCC cells and human HCC tissues.Considering the important role of Twist1 in invasion and metastasis,we wondered whether Twist1 is involved in Sox12-mediated HCC metastasis.Down-regulation of Twist1 significantly reduced Sox12-enhanced cell migration and invasion,whereas the up-regulation of Twist1 rescued the decreased migration and invasion induced by Sox12 knockdown.In vivo metastatic assays showed that the down-regulation of Twist1 decreased the incidence of lung metastasis and the number of metastatic lung nodules while increasing the overall survival time of the Huh7-Sox12 group.In contrast,the up-regulation of Twist1 rescued the decreased incidence of lung metastasis and the number of metastatic lung nodules while decreasing the overall survival time of the HCCLM3-sh Sox12 group.4.We also confirmed that up-regulation of Sox12 increased FGFBP1 expression,whereas down-regulation of Sox12 decreased FGFBP1 expression in HCC cells.A luciferase reporter assay showed that Sox12 trans-activated FGFBP1 promoter activity.Sequence analysis revealed two putative Sox12 binding sites in the FGFBP1 promoter.Serial deletion and site-directed mutagenesis showed that both Sox12 binding sites were critical for Sox12-induced FGFBP1 trans-activation.A Ch IP assay further confirmed that Sox12 directly binds to the FGFBP1 promoter in both HCC cells and human HCC tissues.To test whether FGFBP1 is involved in Sox12-mediated metastasis,we stably down-regulated FGFBP1 expression in Huh7-Sox12 cells and up-regulated FGFBP1 expression in HCCLM3-sh Sox12 cells.In vitro migration and invasion assay showed that down-regulation of FGFBP1 significantly decreased Sox12-enhanced migration and invasion,whereas up-regulation of FGFBP1 rescued the decreased migration and invasion induced by Sox12 knockdown.The in vivo metastatic assay also confirmed that the down-regulation of FGFBP1 decreased the incidence of lung metastasis and the number of metastatic lung nodules while increasing the overall survival time of the Huh7-Sox12 group.In contrast,the up-regulation of FGFBP1 rescued the decreased incidence of lung metastasis and the number of metastatic lung nodules while decreasing the overall survival time of the HCCLM3-sh Sox12 group.5.We further evaluated the possible association between Sox12 and either Twist1 or FGFBP1 expression in human HCC tissues(Cohort I).FGFBP1 expression was found to be significantly up-regulated in HCC tissues as compared with adjacent non-tumorous tissues.Over-expression of FGFBP1 was significantly correlated with poorer tumor differentiation and higher TNM stage.HCC patients with positive expression of FGFBP1 had shorter overall survival and higher recurrence rates than patients with negative expression of FGFBP1.Over-expression of Twist1 was also associated with poor prognosis and aggressive tumor behavior.Immunohistochemistry showed that Sox12 expression was positively correlated with both FGFBP1 and Twist1 expression in Cohort I HCC tissues.Kaplan-Meier analysis showed that patients with positive co-expression of both Sox12/FGFBP1 and Sox12/Twist1 had the highest recurrence rates and shortest overall survival times.The expression correlation and prognostic value of Sox12,Twist1,and FGFBP1 were validated in another independent cohort of HCC patients(Cohort II)by immunohistochemical staining.Similarly,over-expression of both FGFBP1 and Twist1 were associated with poor prognosis and aggressive tumor behavior.Sox12 expression was positively correlated with Twist1 and FGFBP1 expression in Cohort II HCC tissues.Patients with positive co-expression of both Sox12/Twist1 and Sox12/FGFBP1 had the highest recurrence rates and shortest overall survival times.6.Four potential Fox Q1 binding sites were identified in the Sox12 promoter.Western blot showed that up-regulation of Fox Q1 increased Sox12 expression,whereas down-regulation of Fox Q1 decreased Sox12 expression in HCC cells.Luciferase reporter assay showed that Fox Q1 trans-activated Sox12 promoter activity.Serial deletion and site-directed mutagenesis showed that the third and fourth Fox Q1 binding sites were critical for Fox Q1-induced Sox12 trans-activation.Ch IP assay further confirmed that Fox Q1 binds directly to the Sox12 promoter in HCC cells and human HCC tissues.In vitro transwell experiment showed that the down-regulation of Sox12 decreased Fox Q1-enhanced migration and invasion,whereas the up-regulation of Sox12 rescued the decreased migration and invasion induced by knocking down Fox Q1.An in vivo metastatic assay also showed that the down-regulation of Sox12 decreased the incidence of lung metastasis and the number of metastatic lung nodules while increasing the overall survival time of the SMMC7721-Fox Q1 group.In contrast,the up-regulation of Sox12 rescued the decreased incidence of lung metastasis and the number of metastatic lung nodules while decreasing the overall survival time of the HCCLM3-sh Fox Q1 group.Furthermore in HCC tissues of Cohort I,Fox Q1 expression was found to be positively correlated with Sox12 expression detected by immunnohistochemisty.Kaplan-Meier analysis showed that patients with positive co-expression of Fox Q1 and Sox12 had the highest recurrence rates and shortest overall survival times.Similarly,Fox Q1 expression was positively correlated with Sox12 expression in Cohort II HCC tissues and patients with positive co-expression of Fox Q1 and Sox12 had poorer prognosis.Conclusion 1.Sox12 was significantly up-regulated in HCC tissues compared with adjacent normal tissues and correlated with poor prognosis and clinical invasive behaviors.2.Sox12 through transactivating Twist1 and FGFBP1 promoted HCC metastasis.3.Sox12 was critical for Fox Q1 mediated HCC metastasis.4.Expression of Twist1,FGFBP1 or Fox Q1 with or without co-expression of Sox12 is correlated with poor HCC prognosis and clinical invasive behaviors.