| BackgroudThe mesenchymal transcription factor forkhead boxF2(FOXF2)is a critical regulator of embryogenesis and tissue homeostasis.O ur previous studies demonstrated that FOXF2 is specifically expressed in basal-like breast cancer(BLBC)cells and that FOXF2 deficiency promotes the epithelial-mesenchymal transition(EMT)and aggressiveness of BLBC cells.However,whether or how FOXF2 plays a pivotal role in controlling the aggressive progression and metastasis of BLBC has not been fully elucidated.We found that FOXF2 could transrepresses of TGFB2 and TGFB3 expression in BLBC.Thus,we speculated that FOXF2 functionally represses the TGF-β/SMAD signaling pathway.In turn,we observed that sustained stimulation with TGF-β1,TGF-β2 and TGF-β3 did not change FOXF2 m RNA expression but significantly reduced FOXF2 protein expression in BLBC cells,Thus,we speculated that TGF-β negatively regulates FOXF2 expression at the post-transcriptional level.We speculated that FOXF2 formed a reciprocal repression loop with TGF-β/SMAD signaling pathway in aggressive progression of BLBC.PurposesThis study aims to clarify the mechanism of reciprocal repression loops formed by FOXF2 and the TGF-β/SMAD signaling pathway,To further clarify the role and mechanism of FOXF2 in regulating basal-like breast cancer visceral metastasis.Methods1.We silenced FOXF2 in MDA-MB-231 cells with si FOXF2 sequence and used a FOXF2 plasmid to increase FOXF2 expression in BT549 cells or to ectopically express FOXF2 in MCF7 cells.The SBE reporter assays and Western blot were used to detect the SMAD-driven promoter activity and phosphorylation of SMAD2 and SMAD3.RT-q PCR,Western blot and IHC were used to detect the expression of TGFB2 and TGFB3.C hromatin immunoprecipitation and luciferase reporter assay were used to analye the mechanism of regulating TGFB2 and TGFB3 gene transcription by FOXF2.2.To illustrated TGF-β/SMAD signaling pathway mediates the FOXF2-regulated acquisition of the CAF-like cell state in BLBC cells.The FOXF2-depleted MDA-MB-231 cells were treated with TGF-β signaling inhibitor SB431542,and FOXF2-overexpressing BT549 cells were treated with TGF-β2 and TGF-β3.The CAF-like phenotype were assessed using 3D Matrigel culture,polymerized F-Actin,matrix contraction,wound healing assays and transwell assays in BLBC cel s.3.To illustrated TGF-β/SMAD signaling pathway mediates the FOXF2-regulated viscera metastasis in BLBC cells.MDA-MB-231-Luc cells were infected with recombinant lentiviruses carrying short hairp in RNA(sh RNA)targeting human FOXF2(231-Luc-sh FOXF2)or negative control(231-Luc-sh Control).These cells were selected in 1.0 mg/m L puromycin for 2 weeks to stably silence endogenous FOXF2,and injected into the lower abdominal mammary fat pads of 6-week-old female severe combined immunodeficiency(SCID)mice,and the mice bearing 231-Luc-sh FOXF2 tumors were treated daily with SB431542 b y intraperitoneal injection.To evaluate the metastasis in vivo and survival rates using bioluminescent imaging,H&E staining and Kaplan-Meier analysis.4.To illustrated paracrine TGF-β/SMAD signaling pathway mediates the role of FOXF2-deficient BLBC cells in regulation of biological behavior of neighboring cells.MDA-MB-231 were infected with recombinant lentiviruses carrying EGFP-tagged short hairpin RNA(sh RNA)targeting human FOXF2(231-sh FOXF2-Green)or negative control(231-sh Control-Green).BT549 cells were infected with recombinant lentiviruses carrying EGFP-tagged human full-length FOXF2 c DNA(BT549-FOXF2-Green)or vector(BT549-Vector-Green).Red fluorescent-labeled MDA-MB-231(231-Red)and BT549(BT549-Red)cells were established by infection with lentiviruses expressing m C herry.These cells were selected in 1.0 mg/m L puromycin for 2 weeks to stably silence endogenous FOXF2 or express exogenous FOXF2.MDA-MB-231 and BT549 cells were cultured with CM or cocultured with 231-sh FOXF2-Green or BT549-FOXF2-Green feeder cells,respectively.Western blot were used to detect the expression of TGF-β/SMAD signaling and myofibroblast markers.Wound healing assays and transwell assays were used to detect the cell phenotype of neighboring cel s.5.To further test whether FOXF2-deficient BLBC cells affect the viscera metastatic ability of neighboring cells,equal numbers of 231-Luc and 231-sh FOXF2 or 231-sh Control cells were mixed and then orthotopically inoculated into SCID mice,and the mice bearing 231-Luc plus 231-sh FOXF2 tumors were treated with SB431542 daily by intraperitoneal injection for 6 weeks from the day 7 after initial inoculation.To evaluate the metastasis in vivo and survival rates using bioluminescent imaging,H&E staining and Kaplan-Meier analysis.6.To illustrated the role of TGF-β/SMAD signaling in regulation of the expression of FOXF2 and miR-182-5p.MDA-MB-231 cells were stimulated with TGF-β1,TGF-β2 and TGF-β3 during diffenrent time.RT-q PCR,Western blot and dual-luciferase reporter assays were performed to demonstrate the post-transcriptional regulation of miR-182-5p on FOXF2.7.Ch IP and dual-luciferase reporter assays were performed to demonstrate the transcriptional regulation of SMAD3 and FOXF2 on miR-182.Then the miR-182-5p expression were detected by RT-q PCR in MDA-MB-231 and BT549 cells with silenced or overexpressed FOXF2.Results1.The SBE reporter assays revealed that FOXF2 negatively regulated SMAD-driven promoter activity in MDA-MB-231 and BT549 cells but had no effect in MCF-7 cells.Consistently,FOXF2 negatively regulated the phosphorylation of SMAD2 and SMAD3 but had no effect in MCF-7 cells.FOXF2 negatively regulated the transcription of genes coding all three TGF-β forms,TGFB1,TGFB2 and TGFB3,in MDA-MB-231 and BT549 cells and did not affect the expression of any of these genes in MCF-7 cells.Consistently,TGF-β1,TGF-β2 and TGF-β3 protein levels were also negatively regulated by FOXF2 in the CM of MDA-MB-231 and BT549 cells.Ch IP-PCR and Ch IP-q PCR assays confirmed that FOXF2 was recruited to the TGFB2 and TGFB3 promoters containing FOXF2 binding sites in MDA-MB-231 cells transfected with FOXF2-FLAG.Luciferase reporter assays showed that FOXF2 negatively regulated TGFB2 and TGFB3 promoter activity only in the presence of the FOXF2 binding motif in MDA-MB-231 and BT549 BLBC cells.These results indicate that FOXF2 controls TGF-β/SMAD signaling pathway activation through direct transrepression of TGFB2 and TGFB3 expression.2.FOXF2-depleted MDA-MB-231 cells adopted a more mesenchymal/fibroblast-like morphology,with a spindle-like cell shape and cell scattering in two-dimensional(2D)culture,and invasive branching in 3D Matrigel culture.Consistently,FOXF2 depletion in MDA-MB-231 cells led to increased stress fiber formation revealed by immunofluorescence staining of polymerized F-Actin,enhanced matrix contraction when the cells were embedded in type I collagen gel,increased migration in wound healing assays,increased migration and invasion in transwell assays,and upregulated expression of the myofibroblast/C AF markers α-SMA,FAP,fibronectin,and PDGFRβ.As expected,the CAF morphology and phenotype of FOXF2-depleted MDA-MB-231 cells were reversed by the TGF-β signaling inhibitor SB431542.Conversely,FOXF2 overexpression and treatment with TGF-β2 or TGF-β3 elicited the opposite effects in BT549 cells.These results indicate that FOXF2-deficient BLBC cells adopt a CAF-like phenotype through autocrine TGF-β signaling.3.231-Luc-sh FOXF2 and 231-Luc-sh Control cells were injected into the left lower abdominal mammary fat pad of female severe combined immunodeficiency(SCID)mice.Bioluminescence imaging and metastatic photon flux analysis at day 50 after injection revealed that the number and extent of metastases were greater in mice injected with FOXF2-depleted cells than in control mice,and SB431542 treatment abolished FOXF2 depletion-enhanced metastases.The survival rates of the mice bearing 231-Luc-sh FOXF2 tumors were significantly lower than the control mice,and extended by SB431542 treatment.Histological examination by H&E staining identified that FOXF2 depletion significantly increased metastases in visceral organs,including the lungs,livers,pancreases and kidneys,and SB431542 treatment abolished this effect.Immunohistochemical staining also confirmed that the expression of the myofibroblast/CAF markers α-SMA,FAP,PDGFRβ and p-SMAD3 was increased in sh FOXF2 tumors compared with sh Control tumors,and the FOXF2 depletion-increased expression of these proteins was significantly repressed by SB431542 treatment.These results indicate that FOXF2 deficiency promotes BLBC visceral metastasis through increasing autocrine TGF-β signaling that induces the cel s to transdifferentiate into the myofibroblast/CAF phenotype.4.MDA-MB-231 and BT549 cells were cultured with CM from 231-sh FOXF2 and BT549-FOXF2 cells,p-SMAD2 and p-SMAD3 levels were upregulated in MDA-MB-231 cells exposed to 231-sh FOXF2 CM and downregulated in BT549 cells exposed to BT549-FOXF2 CM,and these alterations in p-SMAD2 and p-SMAD3 levels were abolished by SB431542 and TGF-β2/3,respectively.The mesenchymal cell and myofibroblast markers fibronectin,vimentin and α-SMA and an EMT-TF Slug changed in a consistent manner.Cell migratory and invasive capabilities assessed by wound healing and transwell assays also changed consistently when 231-Red or BT549-Red cells were cocultured with 231-sh FOXF2-Green or BT549-FOXF2-Green feeder cells,respectively,or with CM derived from their feeder cells.These results indicate that FOXF2 expression in BLBC cells controls TGF-β/SMAD signaling pathway activation and the transition toward the mesenchymal cell/myofibroblast phenotype in neighboring cells through negatively regulating paracrine TGF-β signaling.5.Bioluminescence imaging and metastatic photon flux analyses at day 60 after injection revealed that 231-sh FO XF2 cells encouraged 231-Luc cells to form more metastases than did 231-sh Control cells,and SB431542 treatment abolished this effect.Consistently,the mice bearing 231-Luc plus 231-sh FOXF2 tumors had worse survival than the mice bearing 231-Luc plus 231-sh Control tumors,and SB431542 treatment extends the overall survival of the 231-Luc plus 231-sh FOXF2 xenograft group.231-sh FOXF2 cells enhances the metastasis in the lungs,livers,pancreases and kidneys of 231-Luc cells that were identified by H&E staining and immunohistochemistry staining for luciferase and SB431542 treatment reversed this effect.Immunohistochemical staining confirmed that the expression of the mesenchymal cell and myofibroblast/CAF markers α-SMA,fibronectin,vimentin and p-SMAD3 was increased in tumors derived from 231-Luc cells mixed with 231-sh FOXF2 cells compared with those derived from 231-Luc cells mixed with 231-sh Control cells,and SB431542 treatment inhibited the increased expression of these markers in 231-Luc cells mixed with 231-sh FOXF2 cells.These results confirm that FOXF2-deficient BLBC enhances the metastasis of neighboring cells through increasing paracrine TGF-β signaling that induces cells to adopt a myofibroblast/CAF phenotype.6.We observed that sustained stimulation with TGF-β1,TGF-β2 and TGF-β3 did not change FOXF2 m RN A expression but significantly reduced FOXF2 protein expression in MDA-MB-231 cells,indicating that TGF-β negatively regulates FOXF2 expression at the post-transcriptional level.Luciferase reporter assays showed that FOXF2 3’UTR reporter activity was significantly reduced by miR-182-5p mimic transfection and induced by miR-182-5p inhibitor treatment in MDA-MB-231 and BT549 cells,and these effects were abrogated by a 7-bp mutation at the miR-182-5p binding site in the FOXF2 3’-UTR reporter.Consistently,the levels of FOXF2 protein,not m RNA,were increased by miR-182-5p mimic transfection and decreased by miR-182-5p inhibitor treatment.Furthermore,TGF-β-induced silencing of FOXF2 expression was restored by miR-182-5p inhibitor treatmen.These data indicate that FOXF2 and TGF-β undergo reciprocal repression in BLBC cells and that miR-182-5p mediates the silencing of FOXF2 expression in response to TGF-β.7.Ch IP-qPCR assays confirmed that both SMAD3 and FOXF2 bound the miR-182 promoter regions containing the predicted binding elements in MDA-MB-231 and BT549 cells.And the recruitment of SMAD3 to the miR-182 promoter was significant increased by TGF-β stimulation and decreased by SB431542 treatment in MDA-MB-231 and BT549 cells,respectively.Consistently,the activity of the miR-182 promoter containing the SMAD3 binding element was enhanced by TGF-β treatment and inhibited by SB431542 treatment in MDA-MB-231 and BT549 cells,respectively.FOXF2 also negatively regulated the activity of the miR-182 promoter containing the FOXF2 binding element and miR-182-5p expression in MDA-MB-231 and BT549 cells.These results demonstrate that miR-182 is a target of FOXF2 and SMAD3.Therefore,in addition to mediating the reciprocal repression loop between FOXF2 and TGF-β in BLBC,miR-182-5p also forms a direct reciprocal repression loop with FOXF2.Conclusions1.FOXF2-deficient BLBC cells adopt a myofibroblast/cancer-associated fibroblast-like phenotype and tend to metastasize to visceral organs through increasing autocrine TGF-β signaling.2.FOXF2-deficient BLBC cells confers aggressiveness on neighboring cells through increasing paracrine TGF-β signaling.3.FOXF2 forms a reciprocal repression loop with miR-182-5p and TGF-β/SMAD signaling pathway... |
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