FOXC1 Enhance The Invasiveness Of Human Glioblastoma By Upregulate β-catenin

Research background:The enhanced invasive ability of tumor cells is related to the process of tumor.Glioblastoma multiforme(GBM),the most common form of primary brain tumor in adults,has the highest mortality rate among all brain malignancies.Extensive invasion is one of the key GBM features,and is often associated with poor prognosis.Forkhead box C1(FOXC1),is characterized by a distinct DNA-binding fork head domain,a member of the forkhead transcription factor family,is over-expressed and plays an important role in tumor progression and metastasis in various cancers.FOXC1 promotes epithelial mesenchymal transition and elicits associated malignant characteristics such as invasion and migration.However,the mechanisms of FOXC1 regulatory network in glioma remain largely unknown.Moreover,β-catenin,an important transcription factor that regulates tumor invasion,was identified as a direct target of FOXC1.The β-catenin protein is a labile protein,and can be rapidly degraded by the proteasome system.In this study,we explored the clinical feature,biological function and potential mechanism of FOXC1 in vitro.We identify FOXC1 as a pivotal regulator of WNT signaling by deubiquitinates and stabilizingβ-catenin through USP4 in glioma.Our findings provide insight into the specific biological role of FOXC1 in tumor invasion,and suggest that targeting FOXC1/USP4/β-catenin axis may be a novel therapeutic approach for blocking glioma invasion.Methods:1.Expression of FOXC1 mRNA and protein was analyzed by qRT-PCR and Western Blot in normal brain tissues and GBM tissues.FOXC1 transcripts was measured with quantitative PCR in a cohort of 81 patient tissues composed of 10 normal brain tissue(NBT)sample and 71 glioma tissue:(11 grade Ⅰ,14 grade Ⅱ,16 grade Ⅲ,and 30 grade Ⅳ).The expression of FOXC1 protein levels were analyzed in different grades of glioma using Western Blot.The levels of FOXC1 in normal human astrocytes(NHAs,control)and 6 GBM cell lines(U87,U251,U118,LN229,T98G and HS683)using qPCR and western blot analysis.2.The FOXC1-siRNA was transfected into U87 and U251 cells.The transfection efficacy of FOXC1-siRNA was examined with qPCR and Western Blot.The wound healing assay and Transwell invasion assay were carried out to investigate the role of FOXC1 on glioma cell invasion.The changes of EMT-related signaling pathway proteins,marker proteins in glioma cells were identified with Western Blot..3.The FOXC1-siRNA was transfected into U87 and U251 cells.Western blot was used to analysis the changes of EMT-inducing transcription factors β-catenin.4.The wound healing assay and Transwell invasion assay were to measure the potential effects of β-catenin overexpression on siRNA-FOXC 1 cellular invasion and migration.Western blot assay were used to analysis the changes of EMT-related signaling pathway proteins,marker proteins.5.RT-PCR and qPCR to measure β-catenin mRNA expression in control cells and cells with FOXC1 knockdown.Western blot assay were used to examine whether FOXC1 affects P-catenin protein stability.6.Western blot assay were used to examine β-catenin after FOXC1-depleted or control U87 and U251 cells treated with the proteasome inhibitor MG132.Ubiquitination level of β-catenin in FOXC1-depleted or control U87 and U251 cells were examined by Western blot assay.7.Ubiquitination level of were used to examine FOXC1 deubiquitinates and stabilizing β-catenin through USP4 in U87、U251 cells and 293Tcells.Results:1.FOXC1 transcripts was markedly increased in patient GBM samples compared with normal brain tissues.Consistent with the increased levels of transcripts,FOXC1 protein was also significantly increased in all the GBM samples.FOXC1 mRNA expression levels were increased in all the glioma groups,and were positively correlated with and patient clinical grading.All the 6 GBM cell lines had significantly higher FOXC1 mRNA and protein expression than that in the NHAs2.Knockdown of FOXC1 significantly reduced the invasion of U87 and U251 in transwell assay and wound healing assay.In both U87 and U251 cells,knockdown of FOXC1 significantly increased the epithelial biomarker E-cadherin,while simultaneously decreased the mesenchymal biomarkers N-cadherin and vimentin.3.β-catenin protein was decreased in both U87 and U251 cell lines with FOXC1 knockdown.4.Downregulation of FOXC1 completely suppressed the invasive growth of U87 and U251 cells by transwell assay and wound healing assay.Overexpression ofβ-catenin could not restore the levels N-cadherin,vimentin and E-cadherin expression in the absence of β-catenin5.No significant differences of β-catenin mRNA expression was observed after FOXC1 depletion.The half-life of β-catenin was significantly shortened in U87 and U251 cells depleted of FOXC1.6.FOXC1 knockdown reduced β-catenin expression,and MG132 treatment restored the expression levels of β-catenin in both cell lines with FOXC1 depletion.Depletion of FOXC1 in U87 and U251 cells significantly decreased the β-catenin protein levels with drastic increased polyubiquitination compared with the control cells.7.FOXC1 stabilizing β-catenin through USP4 and decrease the polyubiquitination of β-catenin protein levelsConclusions:1·FOXC1 is overexpressed in GBM tissues and glioma cell lines.。2.Inhibition ofβ-catenin is involved in the reduced invasive capability of FOXC1 knockdown.3.FOXC1 depletion increased β-catenin polyubiquitination and proteasomal degradation.4.FOXC1 is a regulator of WNT signaling by deubiquitinates and stabilizingβ-catenin through USP4.