The Role And Mechanism Of FOXF2 In Regulating Basal-like Breast Cancer Lymphatic Metastasis

BackgroudLymphatic metastasis is the chief pathway of breast cancer metastasis,tumor lymphangiogenesis facilitates lymphatic metastasis of breast cancer cells.Forkhead box F2(FOXF2)belongs to Fox transcription factor family members.Our previous study showed that FOXF2 deficiency promotes epithelial-mesenchymal transition(EMT)and metastasis of BLBC.We further found that FOXF2 could bind to the promoter of VEGFR3 gene and inhibit its transcription,which indicates.Vascular endothelial growth factor-C(VEGF-C)and Vascular endothelial growth factor receptor 3(VEGFR3)signal pathway is pivotal for lymphangiogenesis.Therefore,we speculated that FOXF2 could regulate the activity of VEGF-C/VEGFR3 signal and tumor lymphangiogenesis and lymphatic metastasis.PurposesThe purposes of this study are to investigate the role of FOXF2 in tumor lymphangiogenesis and BLBC lymphatic metastasis.Methods1.Analyze the relationship between FOXF2 m RNA expression levels and BLBC lymphatic metastasis on the base of clinical data and the data of The Cancer Genome Atlas(TCGA).Analyze the characteristics of tumor lymphangiogenesis in TNBC with FOXF2 m RNA low expression and lymphatic metastasis by immunohistochemistry and H&E staining.2.231-Luc-sh FOXF2?231-Luc-sh Control cells were injected into the left lower abdominal mammary fat pad of female severe combined immunodeficiency(SCID)mice.Evaluate the role of FOXF2 in lymphatic mimicry and lymphatic metastasis in vivo using bioluminescent imaging,Patent Blue V dye tracing,H&E staining and immunofluorescence.3.Spearman's rank correlation coefficient was used to validate the the correlation of VEGFR3 m RNA and FOXF2 m RNA levels in TNBC primary tumor.RT-q PCR and Western blot were used to detect the VEGFR3 expression in MDA-MB-231and BT549 cells with silenced or overexpressed FOXF2.Chromatin immunoprecipitation(Ch IP),luciferase reporter assay,immunoprecipitation(IP)and Re-Ch IP were used to analye the mechanism of regulating VEGFR3 gene transcription by FOXF2.4.Tube formation assays were used to evaluate the effect of rh VEGF-C on the sprouting and tube formation of MDA-MB-231,BT549 and MCF-7 cells with or without VEGFR3 knockdown.Western blot was used to detect the expression of podoplanin and LYVE-1 and phosphorylation of Akt.Transwell assays were used to evaluate the invasion of MDA-MB-231 and BT549 cells.Tube formation assays were used to evaluate the sprouting and tube formation of MDA-MB-231 cells with or without VEGF-C or VEGFR3 knockdown.5.Tube formation assays,transwell assays and western blot were used to evaluate the effect of rh VEGF-C on the sprouting,tube formation,invasion and the expression of podoplanin and LYVE-1 and phosphorylation of Akt of MDA-MB-231and BT549 cells with or without FOXF2 overexpresison.6.231-Luc,231-Luc-LV-FOXF2 or 231-Luc-LV-vector cells were injected into the left lower abdominal mammary fat pad of SCID mice,the xenograft mice were received rh VEGF-C or saline treatment.To evaluate the role of FOXF2 in lymphatic mimicry and lymphatic metastasis in vivo using bioluminescent imaging,Patent Blue V dye tracing,H&E staining and immunofluorescence.Kaplan-Meier analysis was used to evaluate the clinical value of combined FOXF2 and VEGFR3 m RNA in predicting TNBC prognosis.7.Analyze the relationship between the density of tumor lymphatic vessels in primary tumor of TNBC with lymphatic metastasis.Tube formation assays,MTT assays and transwell assays were used to evaluate the effect of the conditioned medium of MDA-MB-231 on the sprouting,tube formation,proliferation and migration of human dermal lymphatic endothelial cells(HDLECs).8.Tube formation assays,MTT assays and transwell assays were used to evaluate the effect of VEGF-C knockdown in MDA-MB-231 cells,neutralization the role of VEGF-C in MDA-MB-231 cells condition medium by VEGFR3/Fc and VEGFR3 knockdown in HDLECs on the sprouting,tube formation,proliferation and migration of HDLECs.9.RT-q PCR was used to detect the VEGFR3 m RNA expression in HDLECs with silenced or overexpressed FOXF2.Luciferase reporter assays were used to evaluate the regulation VEGFR3 gene transcription by FOXF2.Tube formation assays,MTT assays and transwell assays were used to evaluate the sprouting,tube formation,proliferation and migration of HDLECs with or without FOXF2 overexpression.Results1.The clinical data and the TCGA data showed that BLBC patients with low FOXF2 expression suffered more lymphatic metastasis.We found that TNBC cells could form tube structures with positive expression of lymphatic vessel markers which were connected to tumor lymphatic vessels.2.The bioluminescent imaging and H&E staining revealed that the231-Luc-sh FOXF2 mice suffered more sentinel lymph nodes(SLNs)metastasis than the 231-Luc-sh Control mice.The Patent Blue V dye was drained to the collecting lymphatic vessels(CLVs)and SLNs in 50.0%(3/6)231-Luc-sh FOXF2 mice but not in the 231-Luc-sh Control mice.Immunofluorescence found tube structures which were podoplanin and luciferase double positive in the primary tumor of231-Luc-sh FOXF2 mice but not in that of 231-Luc-sh Control mice.3.VEGFR3 m RNA levels inversely correlate with the FOXF2 m RNA levels in34 cases of TNBC primary tumor tissues.FOXF2 knockdown significantly increased the expression of VEGFR3 and enforced expression of FOXF2 decreased the expression of VEGFR3 at both m RNA and protein levels in BLBC cells.Ch IP assay and luciferase reporter assay showed that FOXF2 suppress the transcriptional activity of the VEGFR3 promoter.IP assays revealed TATA box binding protein(TBP)could interact with Pol II,and overexpression of FOXF2 in MDA-MB-231 and BT549 cells resulted in decreased binding activity of TBP and Pol II.Ch IP and Re-Ch IP assays showed overexpression of FOXF2 in MDA-MB-231 cells resulted in recruitment of TBP/FOXF2 cemplex at the(Initiator element,Inr)of VEGFR3 gene and led to reduced recruitment of Pol II and TBP/Pol II complex.4.Tube formation assay and tranwell assay showed that rh VEGF-C could promoted formation tube and invasion of MDA-MB-231 and BT549 cells,VEGFR3knockdown reversed the effect of rh VEGF-C.Western blot showed that treatment with rh VEGF-C activated Akt phosphorylation and increased the expression of LYVE-1 and podoplanin.rh VEGF-C could not induce MCF-7 cells to form tube on the matrigel and the expression of lymphatic vessel marker.5.FOXF2 overexpression could reversed the promoting role of rh VEGF-C in tube formation and invasion of MDA-MB-231 and BT549 cells.Western blot showed that FOXF2 overexpression reduced the expression of VEGFR3,LYVE-1,podoplanin and the phosphorylation level of Akt in MDA-MB-231 and BT549 cells with rh VEGF-C treatment.FOXF2 knockdown enhanced the tube formation and invasion of MDA-MB-231 cells with rh VEGF-C treatment and the expression of LYVE-1,podoplanin and the phosphorylation level of Akt.VEGFR3 knockdown could reversed the effect of FOXF2 knockdown.6.The bioluminescent imaging analysis and H&E staining revealed that the mice with rh VEGF-C treatment suffered more SLNs,lung and liver metastasis than the control mice.The drainage of Patent Blue V dye from the primary tumor to the CLVs and SLNs in the mice with rh VEGF-C treatment was significantly more than control mice.The mice with rh VEGF-C treatment have worse disease free survival DFS than the control mice.FOXF2 overexpression in 231-Luc cells could reverse the effect of rh VEGF-C treatment.Immunofluorescence found tube structure with podoplanin and luciferase double positive expression in the primary tumor of the mice with rh VEGF-C treatment and 231-Luc-sh FOXF2 mice,FOXF2 overexpression could eliminate lymphatic mimicry.Kaplan–Meier analysis revealed that BLBC patients in the FOXF2_low/VEGFR3_high groups exhibited the worst DFS.7.The condition medium of MDA-MB-231 cells could promote the tube formation,proliferation and migration of HDLEC cells.8.VEGF-C knockdown in MDA-MB-231 cells,neutralization the role of VEGF-C in MDA-MB-231 cells condition medium by VEGFR3/Fc,VEGFR3knockdown in HDLEC cells could reduced the promoting role of MDA-MB-231 cells condition medium in tube formation,proliferation and migration of HDLEC cells.9.Overexpression of FOXF2 in HDLEC cells decreased the expression of VEGFR3 and also reduced the promoting role of MDA-MB-231 cells condition medium in tube formation,proliferation and migration of HDLEC cells.Conclusions1.Our study demonstrates that lymphatic mimicry provides channels for lymphatic metastasis in BLBC.2.FOXF2 binds to the promoter of VEGFR3 and regulates its transcription.3.BLBC cells with activated VEGF-C/VEGFR3 signal could transdifferentiate into lymphatic endothelial cells phenotype and form lymphatic mimicry.4.BLBC cells secrete VEGF-C to stimulate VEGF-C/VEGFR3 signal in lymphatic endothelial cells to induce tumor lymphangiogenesis.5.FOXF2 inhibits the activity of VEGF-C/VEGFR3 signaling,eliminates both tumor lymphangiogenesis formed by lymphatic endothelial cells and lymphatic mimicry.