Forkhead Box C1 Promotes Colorectal Cancer Metastasis Through Trans-activating ITGA7 And FGFR4 Expression

BackgroundColorectal cancer(CRC)is one of the most common malignancies and the second leading cause of cancer mortality worldwide.Recurrence and metastasis are the major cause of death in patients with CRC.Therefore,a better understanding of the molecular mechanisms responsible for CRC metastasis is crucial to identify new predictive biomarkers and facilitate the treatment of CRC.Transcription factors can regulate the expression of many oncogenes and tumor suppressor genes and thus play a role.Forkhead box(FOX)protein family members are a group of transcription factors characterized by a winged-helix forkhead DNA-binding domain.FOX family proteins regulate many biological processes,and deregulation of FOX proteins is involved in the initiation and progression of human cancer.As a member of the FOX family,FOXC1 plays an important role in the different stages throughout embryonic development.In recent years,more and more studies have found that FOXC1 expression is increased in various tumors.FOXC1 promotes tumor metastasis through a variety of mechanisms.However,whether FOXC1 contributes to CRC metastasis remains unknown.Therefore,it is necessary to study the expression of FOXC1 in colorectal cancer and its role in the metastasis of colorectal cancer.AimsTo detect the expression pattern of FOXC1 in colorectal cancer and analyze its relationship with the prognosis of patients.To study the effect of FOXC1 on the metastasis of colorectal cancer cells and search the downstream target genes of FOXC1.To explore the molecular mechanism of FOXC1 in the metastasis of colorectal cancer.MethodsThe expression of FOXC1 in human colorectal cancer tissues and adjacent noncancerous tissues was detected by PCR and immunohistochemistry.The relationship between FOXC1 and clinical pathological parameters and prognosis was analyzed.Western blot was used to detect the expression of FOXC1 in various colorectal cancer cell lines.We selected SW480 and SW620 with high and low metastatic potential for follow-up experiments.We up-regulated FOXC1 expression in low metastatic potential cell line SW480,and down-regulated FOXC1 expression in high metastatic potential cell line SW620.The stable infected cell line was established.Transwell experiments were used to compare changes in cell migration and invasion ability in vitro.Nude mice tail vein injection model was used to compare the ability of cells to undergo lung metastasis in vivo.Human metastasis-associated moleculars PCR microarrays were used in SW480-FOXC1 and SW480-control cells to obtain ITGA7 and FGFR4 that were significantly affected byFOXC1 expression.Serial deletion,site-directed mutagenesis,and chromatin immunoprecipitation assays confirmed the binding sites of FOXC1 in the ITGA7 and FGFR4 promoter regions.Downregulation of ITGA7 and FGFR4 expression in cell lines up-regulated FOXC1 expression,upregulation of ITGA7 and FGFR4 expression in cell lines down-regulating FOXC1 expression,Transwell assay and nude mouse tail vein injection model were used to compare the metastasis capacity of cells.Immunohistochemistry further analyzed the correlation between FOXC1 and ITGA7 and FGFR4 expression,as well as their relationship with clinicopathological parameters and prognosis.ResultsThe m RNA levels of FOXC1 are significantly higher in CRC tissues than in the adjacent non-tumor tissues and normal colon epithelial tissues.Tumors of patients with recurrence of CRC(69 of 120)have higher FOXC1 m RNA expression than the levels in tumors from patients without recurrence(51 of 120).FOXC1 m RNA expression is much higher in primary CRC tissues from patients who developed metastasis than in primary CRC tissues from patients who did not.The m RNA levels of FOXC1 are higher in metastatic versus primary lesions.FOXC1 expression is much higher in CRC tissues than in adjacent non-tumorous tissues,and FOXC1 is primarily localized to the nucleus.Elevated expression of FOXC1 is significantly correlated with poor tumor differentiation,tumor invasion,lymph node metastasis,distant metastasis,and a higher American Joint Committee on Cancer(AJCC)stage.The Kaplan-Meier analysis showed that patients with positive FOXC1 expression have shorter overall survival and higher recurrence rates than patients with negative FOXC1 expression.A multivariate Cox proportional hazards model indicated that FOXC1 expression is an independent and significant factor for recurrence and reduced survival.The FOXC1 expression is higher in metastatic CRC cell lines than in primary CRC cell lines.Transwell assay showed that up-regulation of FOXC1 expression significantlyincreases the migration and invasion abilities of SW480 cells,whereas the inhibition of FOXC1 expression in SW620 cells dramatically decreases cell migration and invasion.An in vivo metastatic colonization assay showed that the down-regulation of FOXC1 decreases the incidence of metastatic colonization and the number of metastatic lung nodules while increasing the overall survival time.In contrast,the up-regulation of FOXC1 increases the incidence of metastatic colonization and the number of metastatic lung nodules while decreasing the overall survival time.Over-expression of FOXC1 up-regulates ITGA7 and FGFR4 expression in SW480 cells,whereas knockdown of FOXC1 decreases ITGA7 and FGFR4 expression in SW620 cells.The luciferase reporter assays show that FOXC1 up-regulates ITGA7 and FGFR4 promoter activities.Chromatin immunoprecipitation(Ch IP)assay results confirm the direct binding of FOXC1 protein to the FOXC1 binding sites within both the ITGA7 and FGFR4 promoters in CRC cells and human CRC tissues.Transwell assay showed that the depletion of both ITGA7 and FGFR4 significantly decreases the migration and invasion abilities induced by FOXC1 over-expression,whereas the up-regulation of ITGA7 and FGFR4 rescues the decreased migration and invasion abilities induced by FOXC1 knockdown.An in vivo metastatic colonization assay showed that the down-regulation of ITGA7 and FGFR4 decreases the incidence of metastatic colonization and the number of metastatic lung nodules while increasing the overall survival time of the SW480-FOXC1 group.In contrast,the up-regulation of ITGA7 and FGFR4 rescues the decreased incidence of metastatic colonization and the number of metastatic lung nodules while decreasing the overall survival time of the SW620-sh FOXC1 group.FOXC1 expression is positively correlated with both ITGA7 and FGFR4 expression.Elevated expression of both ITGA7 and FGFR4 are significantly correlated with poorer tumor differentiation,lymph node metastasis,distant metastasis and higher AJCC stage.CRC patients with positive expression of ITGA7 and FGFR4 have shorter overall survival and higher recurrence rates than patients with negative expression.Patients with positive co-expression of both FOXC1/ITGA7 and FOXC1/FGFR4 have the highest recurrencerate and shortest overall survival.ConclusionsFOXC1 expression is much higher in CRC tissues compared with adjacent non-tumorous tissues.Elevated expression of FOXC1 is correlated with the patient’s clinicopathological parameters.CRC patients with positive FOXC1 expression have poorer prognosis than patients with negative FOXC1 expression.FOXC1 expression is an independent and significant risk factor for disease recurrence and reduced survival.In vitro and in vivo experiments showed that down-regulation of FOXC1 expression inhibited the metastasis ability of cells and up-regulated FOXC1 expression enhanced the metastasis ability of cells.Up-regulation of FOXC1 promoted the expression of ITGA7 and FGFR4 and down-regulated FOXC1 inhibited the expression of ITGA7 and FGFR4.ITGA7 and FGFR4 are direct transcriptional targets of FOXC1.Knockdown of ITGA7 and FGFR4 decreases FOXC1-mediated CRC migration,invasion and metastasis,whereas ectopic over-expression of ITGA7 and FGFR4 rescue the decreased CRC migration,invasion and metastasis induced by FOXC1 knockdown.The high expression of ITGA7 and FGFR4 was significantly correlated with the clinicopathological parameters and poor prognosis of patients.The patients with positive co-expression of FOXC1 and ITGA7 or FGFR4 indicates poorer prognosis.Therefore,FOXC1 is a prognostic biomarker in patients with CRC and selective targeting this signaling pathway may provide a potential effective therapeutic approach for the treatment of CRC.