FOXC1 Overexpression Research Articles

Forkhead box C1 promotes the pathology of osteoarthritis in subchondral bone osteoblasts via the Piezo1/YAP axis

Subchondral bone sclerosis is a key characteristic of osteoarthritis (OA). Prior research has shown that Forkhead box C1 (FoxC1) plays a role in the synovial inflammation of OA, but its specific role in the subchondral bone of OA has not been explored. Our research revealed elevated expression levels of FoxC1 and Piezo1 in OA subchondral bone tissues. Further experiments on OA subchondral bone osteoblasts with FoxC1 or Piezo1 overexpression showed increased cell proliferation activity, expression of Yes-associated Protein 1 (YAP) and osteogenic markers, and secretion of proinflammatory factors. Mechanistically, the overexpression of FoxC1 through Piezo1 activation, in combination with downstream YAP signaling, led to increased levels of alkaline phosphatase (ALP), collagen type 1 (COL1) A1, RUNX2, Osteocalcin, matrix metalloproteinase (MMP) 3, and MMP9 expression. Notably, inhibition of Piezo1 reversed the regulatory function of FoxC1. The binding of FoxC1 to the targeted area (ATATTTATTTA, residues +612 to +622) and the activation of Piezo1 transcription were verified by the dual luciferase assays. Additionally, Reduced subchondral osteosclerosis and microangiogenesis were observed in knee joints from FoxC1-conditional knockout (CKO) and Piezo1-CKO mice, indicating reduced lesions. Collectively, our study reveals the significant involvement of FoxC1 in the pathologic process of OA subchondral bone via the Piezo1/YAP signaling pathway, potentially establishing a novel therapeutic target.

FOXC1 transcriptionally suppresses ABHD5 to inhibit the progression of renal cell carcinoma through AMPK/mTOR pathway

BackgroundIncreased activity of the transcription factor FOXC1 leads to elevated transcription of target genes, ultimately facilitating the progression of various cancer types. However, there are currently no literature reports on the role of FOXC1 in renal cell carcinoma.MethodsBy using RT-qPCR, immunohistochemistry and Western blotting, FOXC1 mRNA and protein expression was evaluated. Gain of function experiments were utilized to assess the proliferation and metastasis ability of cells. A nude mouse model was created for transplanting tumors and establishing a lung metastasis model to observe cell proliferation and spread in a living organism. Various techniques including biological analysis, CHIP assay, luciferase assay, RT-qRCR and Western blotting experiments were utilized to investigate how FOXC1 contributes to the transcription of ABHD5 on a molecular level. FOXC1 was assessed by Western blot for its impact on AMPK/mTOR signaling pathway.ResultsFOXC1 is down-regulated in RCC, causing unfavorable prognosis of patients with RCC. Further experiments showed that forced FOXC1 expression significantly restrains RCC cell growth and cell metastasis. Mechanically, FOXC1 promotes the transcription of ABHD5 to activate AMPK signal pathway to inhibit mTOR signal pathway. Finally, knockdown of ABHD5 recovered the inhibitory role of FOXC1 overexpression induced cell growth and metastasis suppression.ConclusionIn general, our study demonstrates that FOXC1 exerts its tumor suppressor role by promoting ABHD5 transcription to regulating AMPK/mTOR signal pathway. FOXC1 could serve as both a diagnostic indicator and potential treatment focus for RCC.

IGF-1-mediated FOXC1 overexpression induces stem-like properties through upregulating CBX7 and IGF-1R in esophageal squamous cell carcinoma.

Substantial evidence attests to the pivotal role of cancer stem cells (CSC) in both tumorigenesis and drug resistance. A member of the forkhead box (FOX) family, FOXC1, assumes significance in embryonic development and organogenesis. Furthermore, FOXC1 functions as an overexpressed transcription factor in various tumors, fostering proliferation, enhancing migratory capabilities, and promoting drug resistance, while maintaining stem-cell-like properties. Despite these implications, scant attention has been devoted to its role in esophageal squamous cell carcinoma. Our investigation revealed a pronounced upregulation of FOXC1 expression in ESCC, correlating with a poor prognosis. The downregulation of FOXC1 demonstrated inhibitory effects on ESCC tumorigenesis, proliferation, and tolerance to chemotherapeutic agents, concurrently reducing the levels of stemness-related markers CD133 and CD44. Further studies validated that FOXC1 induces ESCC stemness by transactivating CBX7 and IGF-1R. Additionally, IGF-1 activated the PI3K/AKT/NF-κB and MEK/ERK/NF-κB pathways through its binding to IGF-1R, thereby augmenting FOXC1 expression. Conversely, suppressing FOXC1 impeded ESCC stemness induced by IGF-1. The presence of a positive feedback loop, denoted by IGF-1-FOXC1-IGF-1R, suggests the potential of FOXC1 as a prognostic biomarker for ESCC. Taken together, targeting the IGF-1-FOXC1-IGF-1R axis emerges as a promising approach for anti-CSC therapy in ESCC.

FoxC1 activates limbal epithelial stem cells following corneal epithelial debridement

Limbal epithelial stem cells are not only critical for corneal epithelial homeostasis but also have the capacity to change from a relatively quiescent mitotic phenotype to a rapidly proliferating cell in response to population depletion following corneal epithelial wounding. Pax6+/− mice display many abnormalities including corneal vascularization and these aberrations are consistent with a limbal stem cell deficiency (LSCD) phenotype. FoxC1 has an inhibitory effect on corneal avascularity and a positive role in stem cell maintenance in many tissues. However, the role of FoxC1 in limbal epithelial stem cells remains unknown. To unravel FoxC1's role(s) in limbal epithelial stem cell homeostasis, we utilized an adeno-associated virus (AAV) vector to topically deliver human FOXC1 proteins into Pax6+/− mouse limbal epithelium. Under unperturbed conditions, overexpression of FOXC1 in the limbal epithelium had little significant change in differentiation (PAI-2, Krt12) and proliferation (BrdU, Ki67). Conversely, such overexpression resulted in a marked increase in the expression of putative limbal epithelial stem cell markers, N-cadherin and Lrig1. After corneal injuries in Pax6+/− mice, FOXC1 overexpression enhanced the behavior of limbal epithelial stem cells from quiescence to a highly proliferative status. Overall, the treatment of AAV8-FOXC1 may be beneficial to the function of limbal epithelial stem cells in the context of a deficiency of Pax6 function.

FOXC1 Promotes Osteoblastic Differentiation of Bone Marrow Mesenchymal Stem Cells via the Dnmt3b/CXCL12 Axis.

Bone defects have remained a clinical problem in current orthopedics. Bone marrow mesenchymal stem cells (BM-MSCs) with multi-directional differentiation ability have become a research hotspot for repairing bone defects. In vitro and in vivo models were constructed, respectively. Alkaline phosphatase (ALP) staining and alizarin red staining were performed to detect osteogenic differentiation ability. Western blotting (WB) was used to detect the expression of osteogenic differentiation-related proteins. Serum inflammatory cytokine levels were detected by ELISA. Fracture recovery was evaluated by HE staining. The binding relationship between FOXC1 and Dnmt3b was verified by dual-luciferase reporter assay. The relationship between Dnmt3b and CXCL12 was explored by MSP and ChIP assays. FOXC1 overexpression promoted calcium nodule formation, upregulated osteogenic differentiation-related protein expression, promoted osteogenic differentiation, and decreased inflammatory factor levels in BM-MSCs, and promoted callus formation, upregulated osteogenic differentiation-related protein expression, and downregulated CXCL12 expression in the mouse model. Furthermore, FOXC1 targeted Dnmt3b, with Dnmt3b knockdown decreasing calcium nodule formation and downregulating osteogenic differentiation-related protein expression. Additionally, inhibiting Dnmt3b expression upregulated CXCL12 protein expression and inhibited CXCL12 methylation. Dnmt3b could be binded to CXCL12. CXCL12 overexpression attenuated the effects of FOXC1 overexpression and inhibited BM-MSCs osteogenic differentiation. This study confirmed that the FOXC1-mediated regulation of the Dnmt3b/CXCL12 axis had positive effects on the osteogenic differentiation of BM-MSCs.

Exosome-Mediated miR-4792 Transfer Promotes Bladder Cancer Cell Proliferation via Enhanced FOXC1/c-Myc Signaling and Warburg Effect.

Bladder cancer is the second-most common malignancy in the urogenital system and the most common in men. However, our understanding of the driving mechanisms of bladder cancer remains incomplete. The forkhead box (FOX) family of transcription factors is implicated in urogenital development and bladder malignancies. Many exosomal microRNAs have been identified as regulators and mediators of the expression of FOX, including the expression of FOXC1. miR-4792 has been known as a tumor miRNA suppressor. However, the function of miR-4792/FOXC1 signaling in bladder cancer development remains unknown. Here, we studied the role of miR-4792/FOXC1 signaling in bladder cancer by using multiple bladder cancer cell lines and bladder cancer mouse models through in vitro and in vivo approaches. We showed that FOXC1 is highly expressed in multiple bladder cancer cell lines and bladder tumor tissues. The knockdown of FOXC1 expression in bladder cancer cell lines decreases c-Myc expression levels, retards cell growth, and reduces aerobic glycolysis (also known as the Warburg effect) and lactic acid content. By contrast, the overexpression of FOXC1 elicits the opposite effects. FOXC1-downregulated bladder cancer cells form significantly smaller tumors in vivo. The inhibition of c-Myc reverses the effects of FOXC1 overexpression and leads to reduced cell proliferation, aerobic glycolysis, and lactic acid content. miR-4792 expression is downregulated in bladder tumor tissues. miR-4792 exposure to bladder cancer cells reduces the expression levels of FOXC1 and c-Myc, slows down cell growth, and decreases aerobic glycolysis and lactic acid content. However, the enhanced miR-4792 expression elicits opposite effects. These findings provided the first evidence that the exosome-mediated delivery of miR-4792 could play an important role in bladder cancer development through the downregulation of FOXC1 and c-Myc, which further inhibited aerobic glycolysis and lactic acid content.

RETRACTED ARTICLE: Forkhead-box C1 attenuates high glucose-induced trophoblast cell injury during gestational diabetes mellitus via activating adenosine monophosphate-activated protein kinase through regulating fibroblast growth factor 19

Statement of Retraction Shan Cao, Shuxuan Zhang- 2022, Forkhead-box C1 attenuates high glucose-induced trophoblast cell injury during gestational diabetes mellitus via activating adenosine monophosphate-activated protein kinase through regulating fibroblast growth factor 19, Bioengineered 13(1), doi: 10.1080/21655979.2021.2018094 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

FOXC1 promotes HCC proliferation and metastasis by Upregulating DNMT3B to induce DNA Hypermethylation of CTH promoter

BackgroundForkhead box C1 (FOXC1), as a member of the FOX family, is important for promote HCC invasion and metastasis. FOX family protein lays a pivotal role in metabolism. ROS is involved in tumor progression and is associated with the expression of lots of transcription factors. We next explored the mechanism underlying FOXC1 modulating the metabolism and ROS hemostasis in HCC.MethodsWe used amino acids arrays to verify which metabolism is involved in FOXC1-induced HCC. The kits were used to detect the ROS levels in HCC cells with over-expression or down-expression of FOXC1. After identified the downstream target genes and candidate pathway which regulated by FOXC1 during HCC progression in vitro and in vivo, we used western blot, immunohistochemistry, bisulfite genomic sequencing, methylation-specific PCR, chromatin immunoprecipitation analysis and luciferase reporter assays to explore the relationship of FOXC1 and downstream genes. Moreover, the correlation between FOXC1 and target genes and the correlation between target genes and the recurrence and overall survival were analyzed in two independent human HCC cohorts.ResultsHere, we reported that FOXC1 could inhibit the cysteine metabolism and increase reactive oxygen species (ROS) levels by regulating cysteine metabolism-related genes, cystathionine γ-lyase (CTH). Overexpression of CTH significantly suppressed FOXC1-induced HCC proliferation, invasion and metastasis, while the reduction in cell proliferation, invasion and metastasis caused by the inhibition of FOXC1 could be reversed by knockdown of CTH. Meanwhile, FOXC1 upregulated de novo DNA methylase 3B (DNMT3B) expression to induce DNA hypermethylation of CTH promoter, which resulted in low expression of CTH in HCC cells. Moreover, low levels of ROS induced by N-acetylcysteine (NAC) which is an antioxidant inhibited the cell proliferation, migration, and invasion abilities mediated by FOXC1 overexpression, whereas high levels of ROS induced by L-Buthionine-sulfoximine (BSO) rescued the suppression results mediated by FOXC1 knockdown. Our study demonstrated that the overexpression of FOXC1 that was induced by the ROS dependent on the extracellular regulated protein kinases 1 and 2 (ERK1/2)- phospho-ETS Transcription Factor 1 (p-ELK1) pathway. In human HCC tissues, FOXC1 expression was positively correlated with oxidative damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG), p-ELK1 and DNMT3B expression, but negatively correlated with CTH expression. HCC patients with positive co-expression of 8-OHdG/FOXC1 or p-ELK1/FOXC1 or FOXC1/DNMT3B had the worst prognosis, whereas HCC patients who had positive FOXC1 and negative CTH expression exhibited the worst prognosis.ConclusionIn a word, we clarify that the positive feedback loop of ROS-FOXC1-cysteine metabolism-ROS is important for promoting liver cancer proliferation and metastasis, and this pathway may provide a prospective clinical treatment approach for HCC.

MicroRNA miR-106a-5p targets forkhead box transcription factor FOXC1 to suppress the cell proliferation, migration, and invasion of ectopic endometrial stromal cells via the PI3K/Akt/mTOR signaling pathway

ABSTRACT Emerging evidence has exhibited an obvious decreased expression of miR-106a-5p in the ectopic endometrial tissue of endometriosis (EMS) patients. Thus far, the pathophysiological function of miR-106a-5p in EMS is unknown. A previous study showed an increased FOXC1 expression in the ectopic endometrial tissue of patients with EMS. Moreover, we found that there was a binding site of miR-106a-5p on the 3ʹUTR of FOXC1 through bioinformatics predictions. Hence, we speculated that miR-106a-5p might affect the development of EMS via targeting FOXC1. We first showed a decreased level of miR-106a-5p and an increased level of FOXC1 mRNA in ectopic endometrial tissues compared with normal tissues. Functionally, we transfected ectopic endometrial stromal cells (ESCs) with miR-106a-5p mimics or NC mimics and indicated an inhibitory role of miR-106a-5p on ESC proliferation, invasion, and migration. Mechanistically, FOXC1 was found to be a target gene of miR-106a-5p. To confirm whether miR-106a-5p exerted an inhibitory activity in ESCs via targeting FOXC1, miR-106a-5p mimic was co-transfected into ESCs with the FOXC1-plasmid or vector. We found that FOXC1 overexpression evidently reversed the results caused by a miR-106a-5p mimic in ESCs. Additionally, our results demonstrated that miR-106a-5p mimic inhibited the expression of p-Akt and p-PI3K. Collectively, these results revealed that miR-106a-5p inhibited the proliferative, migratory, and invasive ability of ESCs via directly binding to FOXC1, likely through the suppression of the PI3K and its downstream signaling pathway, which offered a potential and novel therapeutic strategy for EMS treatment.

Activation of the LINC00242/miR-141/FOXC1 axis underpins the development of gastric cancer

BackgroundLong non-coding RNAs (LncRNAs) are a class of newly identified transcripts recognized as critical governors of gene expression during human carcinogenesis, whereas their tumor-suppressive or tumor-promoting effects on gastric cancer (GC) are required for further investigation. In the study, we identify the expression pattern of a novel lncRNA LINC00242 in GC and its possible permissive role in the development of GC.MethodsThe study included 68 pairs of GC and adjacent normal gastric tissue samples. The viability, migration, and invasion of cultured human GC cells HGC27 were evaluated by CCK-8 and Transwell chamber assays. In vitro tube formation of human brain microvascular endothelial cells (HBMVECs) in HGC27 cell coculture was detected. The regulatory network of LINC00242/miR-141/FOXC1 was verified using dual luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay. Subcutaneous xenografts of HGC27 cells were performed in nude mice.ResultsLINC00242 was highly expressed in GC tissues and cells and contributed to poor prognosis. LINC00242 knockdown inhibited HGC27 cell viability, migration and invasion, and tube formation of HBMVECs. LINC00242 interacted with miR-141 and positively regulated FOXC1, a target gene of miR-141. LINC00242 knockdown was partially lost in HGC27 cells upon miR-141 inhibition or FOXC1 overexpression. The tumor-promoting effect of LINC00242 on GC was demonstrated in nude mice.ConclusionTaken together, the present study demonstrates the oncogenic role of the LINC00242/miR-141/FOXC1 axis in GC, highlighting a theoretical basis for GC treatment.

Overexpression of Foxc1 regenerates crushed rat facial nerves by promoting Schwann cells migration via the Wnt/β-catenin signaling pathway.

Facial paralysis can result in severe implications for patients. A good prognosis depends on the degree of nerve regeneration. Schwann cells (SCs) play an important role in facial nerve development and regeneration through migration. Forkhead box C1 (Foxc1), a member of the forkhead transcription factor family, is implicated in cell migration. However, the role of Foxc1 in the progression after facial nerve crush remains unknown. Our aim was to evaluate the effect of Foxc1 overexpression on SC migration and recovery of facial nerves after crush injury. The rat facial nerve crush injury model was established through the use of unilateral surgery. The results showed that the expression of Foxc1 was increased in the surgery group compared to that of the control group. SCs were isolated from the sciatic nerves and cultured. Foxc1, delivered by an adeno‐associated virus in vivo, or adenovirus in vitro, both induced overexpression of Foxc1, and increased the expression of CXCL12 and β‐catenin. After the transfection of Foxc1, the migration of SC was increased both in vitro and in vivo, was reduced by the inhibition of CXCL12 or β‐catenin. The facial nerve function and the nerve axon remyelination of the rats transfected with Foxc1 were significantly improved after nerve crush injury. Overall, the results demonstrated that overexpression of Foxc1 promoted SC migration by regulating CXCL12 via the Wnt/β‐catenin pathway, thus contributing to improved facial nerve function after crush injury.

Identification of a novel microRNA-141-3p/Forkhead box C1/β-catenin axis associated with rheumatoid arthritis synovial fibroblast function in vivo and in vitro.

Rationale: Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis in which synovial fibroblasts (SFs) play key roles in cartilage and bone destruction through tumor-like proliferation, migration, invasion and inflammation. This study aimed to research forkhead box protein C1 (FoxC1) and microRNA (miR)-141-3p, which modulate pathological changes in the synovial membrane, to find possible strategies for treating RA.Methods: FoxC1, β-catenin and miR-141-3p gene expression in synovial tissues and SFs was quantified by real-time PCR; FoxC1 and β-catenin protein levels were evaluated by immunohistochemistry, immunofluorescence, and Western blotting. We transiently transfected human SFs with FoxC1 and β-catenin overexpression and silencing vectors and assessed proliferation, migration, invasion and inflammation by cell function and enzyme-linked immunosorbent assays. We also assessed downstream signaling activation using immunofluorescence, real-time PCR and Western blotting. Double luciferase, coimmunoprecipitation and chromatin immunoprecipitation assays were used to verify miR-141-3p, FoxC1 and β-catenin gene and protein combinations. Finally, the therapeutic effects of FoxC1 silencing and miR-141-3p overexpression were evaluated in type II collagen-induced arthritis (CIA) rats.Results: We found that FoxC1 expression was significantly upregulated in synovium and SFs in both RA patients and rats with collagen-induced arthritis (CIA). FoxC1 overexpression increased β-catenin messenger RNA (mRNA) and protein levels and upregulated cyclin D1, c-Myc, fibronectin and matrix metalloproteinase 3 (MMP3) mRNA and protein expression in RA SFs (RASFs). In contrast, FoxC1 knockdown reduced β-catenin mRNA and protein levels as well as cyclin D1, c-Myc, and fibronectin mRNA and protein levels in RASFs. Furthermore, altering FoxC1 expression did not significantly change GSK3β and pGSK3β levels. FoxC1 overexpression promoted proliferation, migration, invasion and proinflammatory cytokine (interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)) production and reduced anti-inflammatory cytokine (IL-10) levels in RASFs. FoxC1 bound to the β-catenin promoter, and β-catenin mediated the FoxC1-induced pathological changes. We also observed downregulated microRNA (miR)-141-3p expression in SFs from both RA patients and CIA rats and further found that miR-141-3p bound to the FoxC1 3′UTR and suppressed FoxC1 expression. Intra-ankle miR-141-3p agomir or FoxC1-specific siRNA injection hindered CIA development in rats.Conclusions: FoxC1 and miR-141-3p participate in RA pathogenesis by mediating inflammation and SF proliferation, migration, and invasion and thus could be novel targets for RA therapy as a nonimmunosuppressive approach.

Forkhead box C1 promotes the pathology of osteoarthritis by upregulating β-catenin in synovial fibroblasts.

Osteoarthritis (OA) is a joint disease characterized by articular cartilage degeneration, and no effective treatment is available. The OA classification has shifted from a cartilage-only disease to a whole-joint disease, and the synovial membrane plays an important role. Therefore, studies are needed to identify additional genes that regulate the pathologicalchanges in the synovial membrane to develop a promising therapeutic strategy for OA. Here, we validated that the expression of forkhead box protein C1 (FoxC1) and β-catenin was upregulated in OA synovial membranes and synovial fibroblasts (SFs). Gain- and loss-of-function studies revealed that FoxC1 overexpression promoted, whilst silencing inhibited OA synovial fibroblast (OASF) proliferation and pro-inflammatory cytokine [interleukin 6 (IL-6), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α)] production. FoxC1 overexpression increased β-catenin mRNA, total and nuclear protein expression in OASFs and upregulated a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS-5), fibronectin, matrix metalloproteinase 3 (MMP3) and matrix metalloproteinase 13 (MMP13) mRNA and total protein expression in OASFs. Conversely, FoxC1 knockdown reduced β-catenin mRNA, total and nuclear protein expression in OASFs and reduced ADAMTS-5, fibronectin, MMP3 and MMP13 mRNA and total protein expression in OASFs. β-catenin mediates FoxC1-induced pathologicalchanges (proliferation, catabolic regulation and inflammation) in OASFs. MicroRNA-200a-3p (miR-200a-3p) binds to the 3'-UTR of FoxC1 and mediates FoxC1 expression. Intra-articular FoxC1-specific siRNA transfection hindered OA development in mice. Therefore, our results demonstrate the key role FoxC1 plays invivo and invitro in OA synovial pathology, possibly identifying a potential novel therapeutic target for OA.

Overexpression of Forkhead box C1 attenuates oxidative stress, inflammation and apoptosis in chronic obstructive pulmonary disease

AimChronic obstructive pulmonary disease (COPD) is a disease caused by cigarette smoke, which has been emerging as a serious health problem worldwide. The aim of this study is to explore the mRNA expression profile of lung tissues from the COPD rats and to characterize the role of Forkhead box C1 (Foxc1) in COPD. Main methodsWistar rats were exposed to cigarette smoke during 16 weeks for COPD model establishment. The microarray was used to identify the differential gene expression in the lung of rats. Adenovirus carrying Foxc1 was administered to rats by intratracheally instillation once a week for 16 weeks. Human bronchial epithelial cell line (16HBE) cells were transfected with Foxc1 siRNA followed by incubation in the presence of CSE (10%) for 24 h. Subsequently, the pathological changes, fibrosis, apoptosis, inflammatory cytokines and oxidative stress were detected. Key findingsMicroarray results showed an upregulation of Foxc1 in lung tissues in COPD rats. Overexpression of Foxc1 mitigated the lung injury, as evidenced by reducing alveolar fusion, inflammatory cell infiltration and oxidative stress. Additionally, the apoptosis was remarkably increased in the lung in rats exposed to cigarette smoke, which was suppressed by Foxc1 overexpression. Furthermore, downregulation of Foxc1 aggravated the inflammation, oxidative stress and apoptosis in 16HBE cells with CSE treatment. SignificanceOverexpression of Foxc1 could prevent oxidative stress, inflammation responses and cell apoptosis and knockdown of Foxc1 has the opposite effect, suggesting that Foxc1 may be available for lung protection during COPD.

The Forkhead Box C1, a Novel Negative Regulator of Osteogenesis, Plays a Crucial Role in Odontogenic Differentiation of Dental Pulp Stem Cells.

The forkhead box C1 (Foxc1) protein, a member of the forkhead/winged helix transcription factor family, is required in stem cell developmental processes. Recently, multiple studies have indicated the crucial role of Foxc1 in mesenchymal stem cell differentiation, but the precise effects and mechanisms on dental pulp stem cells (DPSCs) remain unclear. In this study, we evaluate the role of Foxc1 on the odontogenic differentiation and proliferation of DPSCs. Our results show that Foxc1 decreases time dependently in odontogenic differentiation of DPSCs. Meanwhile, overexpression of Foxc1 could significantly inhibit the mineralization of DPSCs and the expression of odontogenic-related genes, such as runt-related transcription factor 2 (Runx2), dentin sialophosphoprote (DSPP), and dentin matrix acidic phosphoprotein 1 (DMP-1). Foxc1 overexpression does not significantly alter the proliferation of DPSCs. In addition, Foxc1 reduces the expression of p-Smad1/5, an important modulator of bone morphogenetic protein (BMP)/Smad signaling pathway, inhibiting BMP/Smad signaling pathway. In conclusion, our data demonstrated that Foxc1 inhibits odontogenic differentiation of DPSCs and odontogenic-related gene expression through the BMP/Smad signaling pathway which may be useful for the dental regeneration and repair.

The FOXC1/FBP1 signaling axis promotes colorectal cancer proliferation by enhancing the Warburg effect.

Aberrant expression of Forkhead box (FOX) transcription factors plays vital roles in carcinogenesis. However, the function of the FOX family member FOXC1 in maintenance of colorectal cancer (CRC) malignancy is unknown. Herein, FOXC1 expression in CRC specimens in The Cancer Genome Atlas (TCGA) cohort was analyzed and validated using immunohistochemistry with a tissue microarray. The effect of FOXC1 expression on proliferation of and glycolysis in CRC cells was assessed by altering its expression in vitro and in vivo. Mechanistic investigation was carried out using cell and molecular biological approaches. Our results showed that FOXC1 expression was higher in CRC specimens than in adjacent benign tissue specimens. Univariate survival analyses of the patients from whom the study specimens were obtained, and validated cohorts indicated that ectopic FOXC1 expression was significantly correlated with shortened survival. Silencing FOXC1 expression in CRC cells inhibited their proliferation and colony formation and decreased their glucose consumption and lactate production. In contrast, FOXC1 overexpression had the opposite effect. Furthermore, increased expression of FOXC1 downregulated that of a key glycolytic enzyme, fructose-1,6-bisphosphatase 1 (FBP1). Mechanistically, FOXC1 bound directly to the promoter regions of the FBP1 gene and negatively regulated its transcriptional activity. Collectively, aberrant FBP1 expression contributed to CRC tumorigenicity, and decreased FBP1 expression coupled with increased FOXC1 expression provided better prognostic information than did FOXC1 expression alone. Therefore, the FOXC1/FBP1 axis induces CRC cell proliferation, reprograms metabolism in CRCs, and constitutes potential prognostic predictors and therapeutic targets for CRC.

Inhibition of lobuloalveolar development by FOXC1 overexpression in the mouse mammary gland

The forkhead box transcription factor FOXC1 plays a critical role in embryogenesis and the development of many organs. Its mutations and high expression are associated with many human diseases including breast cancer. Although FOXC1 knockout mouse studies showed that it is not required for mammary gland development during puberty, it is not clear whether its overexpression alters normal mammary development in vivo. To address this question, we generated transgenic mice with mammary-specific FOXC1 overexpression. We report that transgenic FOXC1 overexpression suppresses lobuloalveologenesis and lactation in mice. This phenotype is associated with higher percentages of estrogen receptor-, progesterone receptor-, or ki67-positive mammary epithelial cells in the transgenic mice at the lactation stage. We also show that expression of the Elf5 transcription factor, a master regulator of mammary alveologenesis and luminal cell differentiation, is markedly reduced in mammary epithelial cells of transgenic mice. Likewise, levels of activated Stat5, another inducer of alveolar expansion and a known mediator of the Elf5 effect, are also lowered in those cells. In contrast, the cytokeratin 8-positive mammary cell population with progenitor properties is elevated in the transgenic mice at the lactation stage, suggesting inhibition of mammary cell differentiation. These results may implicate FOXC1 as a new important regulator of mammary gland development.

FOXC1 overexpression is a marker of poor response to anthracycline-based adjuvant chemotherapy in sporadic triple-negative breast cancer

PurposeBecause of its aggressive characteristics and poor prognosis, triple-negative breast cancer (TNBC) has become a hot topic in cancer research. Chemotherapy is currently the only treatment for patients with TNBC. The transcription factor FOXC1 has been associated with TNBC prognosis, but little is known about its effect on chemosensitivity. The aim of this study was to investigate the effects of FOXC1 on chemosensitivity.MethodsA case–control study was performed on 25 TNBC patients who experienced relapse and/or metastasis. Another 25 patients without relapse or metastasis were randomly selected as controls. Medical records were reviewed for relevant information, and immunohistochemistry was performed to measure FOXC1 levels. The Kaplan–Meier method and Cox analysis were used to analyze differences in disease-free survival (DFS) and overall survival (OS). The correlation of FOXC1 expression with chemosensitivity was analyzed. Data were analyzed using SPSS 21.0 software, and a P value <0.05 was considered to be statistically significant.ResultsIn 15 of 22 case patients, FOXC1 was overexpressed, whereas only 8 control patients exhibited FOXC1 overexpression (P < 0.05). FOXC1 expression had no correlation with pathological indicators. An anthracycline-based regimen was administered to 21 study patients and 23 control patients. FOXC1 expression was significantly associated with a worse DFS (HR 2.62, 95% CI 1.05–6.50, P = 0.038) but presented no correlation with OS (HR 2.53, 95% CI 0.76–8.40, P = 0.131) among these 44 patients.ConclusionsThis study shows that FOXC1 is correlated with chemosensitivity to anthracycline and could be used as an indicator of chemosensitivity in sporadic TNBC.

Foxc1 Regulates Early Cardiomyogenesis and Functional Properties of Embryonic Stem Cell Derived Cardiomyocytes.

Embryonic Stem Cells (ESCs) hold great potential for regeneration of damaged myocardium, however the molecular circuitry that guides ESC differentiation into cardiomyocytes remains poorly understood. This is exemplified by the elusive role of the transcription factor, Foxc1, during cardiac development. The only known Foxc1 target during heart development is Tbx1. Because Foxc1 null mice contain heart mutations that are far more severe than Tbx1 null mice, it is likely that Foxc1 has additional regulatory roles during heart development. The goal of our study was to test whether Foxc1 is critical for ESC differentiation into functional cardiomyocytes through proper regulation of specific downstream gene networks. Converging evidence from Foxc1 deficient and overexpression ESC models reveals a close relationship between Foxc1 levels and early cardiomyogenic factors Isl1, Mef2c, and Nkx2.5 and also the production of functional cardiomyocytes. We show Foxc1 regulates early cardiomyogenesis during a specific window of differentiation, D4-D6. Through whole transcriptome RNA-sequencing analysis, we report pathways regulated by Foxc1 involved in cardiac function including actin cytoskeleton, cell adhesion, tight and gap junctions, and calcium signaling. Our data indicate a novel Foxc1 direct gene target, Myh7, which encodes the predominant myosin heavy chain isoform, MHCβ, expressed during cardiac development. These data lead us to conclude that Foxc1 regulates both early cardiomyogenesis and the functional properties of ESC-derived cardiomyocytes. Our findings shed light on the molecular circuitry governing cardiomyogenesis that may lead to the development of better translational strategies for the use of pluripotent stem cells in regenerative medicine towards repairing damaged myocardium. Stem Cells 2016;34:1487-1500.

Abstract 14304: The Role of Foxc1 During the Differentiation of Embryonic Stem Cells Into Functional Cardiomyocytes

Background: Embryonic Stem Cells (ESCs) hold great potential for regeneration of damaged myocardium, however the molecular circuitry that guides ESC differentiation into cardiac lineages remains poorly understood. This is exemplified by the elusive role of the developmental transcription factor, Foxc1, during cardiac development. When one human FoxC1 allele is mutated it causes Axenfeld-Rieger syndrome characterized by ocular defects and often heart defects. Foxc1 homozygous null mice are either embryonic lethal or die soon after birth with the majority of these mice containing severe heart malformations. The only known Foxc1 target during heart development is Tbx1, a transcription factor necessary for the proper alignment of the outflow tract. Because Foxc1 null mice contain heart defects that are far more severe than Tbx1 null mice, it is likely that foxc1 has additional regulatory roles in heart development. It is unknown in which specific cell types of the heart Foxc1 functions. Hypothesis: Foxc1 is critical for ESC differentiation into functional cardiomyocytes through the regulation of a downstream regulatory transcription network. Methods and Results: Using a murine Nkx2.5-GFP reporter ESC line, we show that Foxc1 knockdown (KD) reduces the number of Nkx2.5+ cells upon differentiation. Foxc1 KD also reduces a panel of cardiomyocyte marker transcripts as well as inhibits the beating of embryoid bodies (EBs). On the other hand, using an inducible Foxc1 overexpression (OE) ESC line, we show that Foxc1 OE increases a panel of cardiomyocyte transcripts and enhances the number beating of EBs. Additionally, Foxc1 OE beating EBs have an enhanced spontaneous beat rate and possess functional electrophysiological properties, beating in synchrony with surrounding cardiomyocytes upon electrical stimulation. RNA-sequencing results identify over 3,000 differentially regulated transcripts after Foxc1 OE during cardiomyogenesis. Conclusion: Both molecular and functional analyses reveal a positive correlation between Foxc1 expression level and cardiomyogenesis. Currently chromatin-immunoprecipitation sequencing is being performed to determine the direct downstream regulatory network of Foxc1 during cardiomyogenesis.