Forkhead Box C1 Expression Research Articles

Context-Responsive Nanoparticle Derived from Synthetic Zwitterionic Ionizable Phospholipids in Targeted CRISPR/Cas9 Therapy for Basal-like Breast Cancer.

The majority of triple negative breast cancers (TNBCs) are basal-like breast cancers (BLBCs), which tend to be more aggressive, proliferate rapidly, and have poor clinical outcomes. A key prognostic biomarker and regulator of BLBC is the Forkhead box C1 (FOXC1) transcription factor. However, because of its functional placement inside the cell nucleus and its structural similarity with other related proteins, targeting FOXC1 for therapeutic benefit, particularly for BLBC, continues to be difficult. We envision targeted nonviral delivery of CRISPR/Cas9 plasmid toward the efficacious knockdown of FOXC1. Keeping in mind the challenges associated with the use of CRISPR/Cas9 in vivo, including off-targeting modifications, and effective release of the cargo, a nanoparticle with context responsive properties can be designed for efficient targeted delivery of CRISPR/Cas9 plasmid. Consequently, we have designed, synthesized, and characterized a zwitterionic amino phospholipid-derived transfecting nanoparticle for delivery of CRISPR/Cas9. The construct becomes positively charged only at low pH, which encourages membrane instability and makes it easier for nanoparticles to exit endosomes. This has enabled effective in vitro and in vivo downregulation of protein expression and genome editing. Following this, we have used EpCAM aptamer to make the system targeted toward BLBC cell lines and to reduce its off-target toxicity. The in vivo efficacy, biodistribution, preliminary pharmacokinetics, and biosafety of the optimized targeted CRISPR nanoplatform is then validated in a rodent xenograft model. Overall, we have attempted to knockout the proto-oncogenic FOXC1 expression in BLBC cases by efficient delivery of CRISPR effectors via a context-responsive nanoparticle delivery system derived from a designer lipid derivative. We believe that the nonviral approach for in vitro and in vivo delivery of CRISPR/Cas9 targeted toward FOXC1, studied herein, will greatly emphasize the therapeutic regimen for BLBC.

Assessment of FOXC1 expression as a predictor of response to neoadjuvant taxane plus platinum regimens in primary triple-negative breast cancer: Retrospective analysis of three clinical trial cohorts.

569 Background: Taxane and platinum (TP) NAC regimens, e.g. Carboplatin and Docetaxel (CbD), in TNBC are currently of great interest, having good pathologic complete response (pCR) rates but with a significantly more manageable toxicity profile compared to anthracycline-based NAC regimens. Forkhead Box C1 (FOXC1), a transcriptional driver of cell plasticity/partial EMT/metastasis is an established mesenchymal marker diagnostic of basal-like breast cancer having proven prognostic value, but of uncertain predictive value. We sought to evaluate the potential of FOXC1 in predicting pCR to neoadjuvant TP regimens in patients diagnosed with TNBC. Methods: Pre-NAC tumor biopsy FOXC1 mRNA expression status was correlated with rate of pCR in a pooled, ambispective cohort (prospective cohort GEICAM/2006-03, NCT00432172 pooled with multi-institutional retrospective cohort, n = 119). A specific FOXC1 mRNA expression cutoff value was derived to maximize Negative Predictive Value (NPV) and Sensitivity for pCR prediction. The pCR-predictive ability of FOXC1 mRNA expression was then assessed in two validation cohorts of evaluable patients who had been enrolled in prospective clinical trials (UCONN/FIOCRUZ, n = 222, HGUGM, NCT01560663, n = 221). All evaluated patients had been diagnosed with TNBC and had received a Taxane plus Platinum-based NAC regimen. Results: FOXC1 mRNA expression was associated with pCR in CbD/TP treated TNBC patients with pCR rates of 43.48%, 47.89% and 52.73% observed in the discovery and two validation cohorts (two tailed T-test p-values of 0.0005, 0.002, 0.009, respectively). FOXC1 expression above the pre-determined cutoff value was associated with pCR to CbD/TP NAC in patients diagnosed with TNBC in both validation cohorts (OR 4.894, 1.504-15.924; p = 0.004 and OR 2.293, 1.208-4.352; p = 0.006). Conclusions: We report the retrospective validation of pre-NAC breast cancer biopsy FOXC1 mRNA expression for predicting efficacy of CbD/TP NAC in two independent, prospectively accrued TNBC patient cohorts. The described strategy may be acceptable for patient stratification to guide CbD/TP NAC recommendations in TNBC. FOXC1 mRNA or protein expression, assessed using qRT-PCR or routine immunohistochemistry (IHC), respectively, could potentially be utilized in future fixed-arm/adaptive clinical trials to further optimize NAC efficacy, in terms of achieved pCR rates, and to extend disease-free survival in patients diagnosed with TNBC.

Long Non-Coding RNA ZEB2-AS1 Promotes Hepatocellular Carcinoma Progression by Regulating The miR-582-5p/FOXC1 Axis.

ObjectivesLong non-coding RNAs (lncRNAs) feature prominently in tumors. Reportedly, lncRNA zinc finger E-box-binding homeobox 2 antisense RNA 1 (ZEB2-AS1) is aberrantly expressed in a variety of tumors. The present study was aimed to explore ZEB2-AS1 functions and determine mechanism in hepatocellular carcinoma (HCC) progression. Materials and Methods In this experimental study, expressions of ZEB2-AS1, microRNA (miR)-582-5p and forkhead box C1 (FOXC1) mRNA in HCC tissues and cell lines were detected via quantitative reveres transcription polymerase chain reaction (qRT-PCR). After establishing gain- and loss-of-functions models, cell counting kit-8, 5-bromo-2’-deoxyuridine (BrdU), Transwell assays and flow cytometry analysis were conducted to examine HCC cell multiplication, migration, invasion and apoptosis, respectively. The targeted relationship between miR-582- 5p and ZEB2-AS1 was verified via dual-luciferase reporter gene assay. Western blot was utilized for detecting FOXC1 expression in HCC cells after selectively regulating ZEB2-AS1 and miR-582-5p. Results In HCC tissues and cells, ZEB2-AS1 expression was increased. High ZEB2-AS1 expression was related to relatively large tumor volume, increased tumor-node-metastasis (TNM) stage and positive lymph node metastasis of the patients. ZEB2-AS1 overexpression facilitated HCC cell multiplication, migration, invasion and suppressed apoptosis, while ZEB2-AS1 knock-down caused the opposite effects. It was also confirmed that ZEB2-AS1 could competitively bind with miR-582-5p to repress its expression, and indirectly up-regulate FOXC1 expression level in HCC cells. ConclusionThe current study revealed that ZEB2-AS1 was over-expressed in HCC tissues and cells. It also upregulated FOXC1, through sponging miR-582-5p, to promote HCC progression. This provides new perspectives for elucidating the pathogenesis of HCC.

Evaluation of forkhead-box C1 expression in breast cancer

Background Forkhead box C1 (FOXC1) is considered an important member of forkhead-box transcription factors. Previous studies have shown that FOXC1 is associated with tumor progression and represents a poor prognostic indicator in triple-negative basal-like breast cancer. Method This is a retrospective study conducted on 187 cases of invasive breast carcinoma obtained from Histopathology Laboratory in Oncology Center, Faculty of Medicine, Mansoura University, starting from 2012. Aim and objectives In this study, we aimed to study FOXC1 protein expression in molecular subtypes of breast cancer and to correlate its expression with other prognostic parameters using immunohistochemistry and tissue microarrays. Results FOXC1-positive expression was seen in 26.7% of cases. Positive FOXC1 expression was most common in triple-negative basal-like breast cancer and HER2-enriched subtypes. Positive FOXC1 expression was higher in ER-negative cases than ER-positive cases (39 vs. 21%) with statistical significance (P = 0.01). Similarly, positive FOXC1 was higher in PR-negative cases than PR-positive cases (37.5 vs. 20%), this was statistically significant (P = 0.009). In our series, FOXC1 expression was not associated with survival. Conclusion FOXC1 expression is associated with negative hormonal-receptor status in breast cancer. FOCX1 is not a prognostic indicator of disease-free survival in our patient series.

USP28 promotes aerobic glycolysis of colorectal cancer by increasing stability of FOXC1.

Aerobic glycolysis is essential for cancer cell metabolism and growth. Deubiquitinase, USP28 (ubiquitin specific peptidase 28), could maintain stability of proteins involved in tumor progression. This study was performed to investigate the role of USP28 in aerobic glycolysis of colorectal cancer. Our data showed that USP28 mRNA and protein expressions were enhanced in colorectal cancer tissues and cells. Functional assays demonstrated that overexpression of USP28 promoted cell proliferation and aerobic glycolysis of colorectal cancer, while USP28 inhibition could reverse these effects. Protein expression of Forkhead Box C1 (FOXC1) was increased by USP28 over-expression, whereas knockdown of USP28 aggravated cycloheximide (CHX; protein synthesis inhibitor) stimulated decrease of FOXC1. Moreover, proteasome inhibitor, MG132, could rescue USP28 silence-induced degradation of FOXC1. Overexpression of FOXC1 counteracted the suppressive effects of USP28 interference on colorectal cancer cell viability and aerobic glycolysis. In conclusion, USP28 enhanced cell viability and aerobic glycolysis of colorectal cancer by stabilizing FOXC1, suggesting that USP28-FOXC1 might be a novel therapeutic avenue for colorectal cancer.

Knockdown of HCG18 Inhibits Cell Viability, Migration and Invasion in Pediatric Osteosarcoma by Targeting miR-188-5p/FOXC1 Axis.

Understanding the underlying mechanisms of pediatric osteosarcoma (OS) migration and invasion is important for prognosis and treatment. We tried to measure the expression of long non-coding RNA HLA complex group 18 (HCG18) in OS and reveal its function in the malignant behaviors of OS cells. This study detected the expression of HCG18, miR-188-5p and forkhead box C1 (FOXC1) in OS tissues and cell lines by quantitative real-time PCR (qRT-PCR). The relevance between miR-188-5p and HCG18 or FOXC1 was affirmed by dual-luciferase reporter (DLR) assay. Cell viability was analyzed by MTT assay. Transwell assay was utilized to test cell invasion and migration. FOXC1 protein expression was detected by western blot. HCG18 expression was elevated in OS tissues, and enhanced HCG18 expression was related to metastasis. HCG18 silencing repressed the viability, migration and invasion of OS cells. Moreover, HCG18 interacted with miR-188-5p. MiR-188-5p up-regulation repressed cell viability, invasion and migration in OS cells. FOXC1, a known target of miR-188-5p, was negatively modulated by miR-188-5p. Furthermore, miR-188-5p inhibition or FOXC1 over-expression partially abolished the reduced of cell viability, invasion and migration mediated by HCG18 silencing in OS cell lines. This study revealed that HCG18 knockdown repressed the viability, invasion and migration of OS cells by targeting miR-188-5p and regulating FOXC1 expression. Thus, HCG18/ miR-188-5p/FOX may be a hopeful target for OS therapy.

Long Non-Coding RNA LINC01929 Accelerates Progression of Oral Squamous Cell Carcinoma by Targeting the miR-137-3p/FOXC1 Axis.

Recently, additional long noncoding RNAs (lncRNAs) have been identified and their possible roles were investigated in a variety of human tumors. One of these lncRNAs, LINC01929, promoted the progression of some cancers, whereas its expression and biological function in human oral squamous cell carcinoma (OSCC) remains still mostly uncertain. The LINC01929 expression in OSCC tissues or cell lines was identified via quantitative real-time polymerase chain reaction. The cell counting kit-8, transwell migration, wound-healing, and flow cytometry assays were utilized to characterize the functions of LINC01929 in OSCC cells. The interactive relationships between LINC01929 and miR-137-3p, miR-137-3p and Forkhead box C1 (FOXC1) were investigated by the dual-luciferase activity assay. Our findings demonstrated that LINC01929 was highly expressed in OSCC tissue samples and cell lines, whereas miR-137-3p expression was downregulated. LINC01929 acted as a carcinogenic lncRNA with accelerated OSCC cell proliferation, migration and invasion, and suppression of apoptosis. We further indicated that LINC01929 facilitated tumor growth in xenograft mouse models. Mechanistically, LINC01929 acted as a sponge for miR-137-3p to elevate FOXC1 expression, which is the target of miR-137-3p. In addition, downregulated miR-137-3p expression rescued the suppressive behaviors of LINC01929 knockdown on the biological behaviors of OSCC cells. Taken together, LINC01929 functioned as a tumor-promoting lncRNA via the miR-137-3p/FOXC1 axis in OSCC, suggesting novel targets for OSCC therapy.

The miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of human adipose-derived stem cells via the AKT and p38 signalling pathways

BackgroundHuman adipose-derived stem cells (hADSCs) are stem cells with the potential to differentiate in multiple directions. miR-204-5p is expressed at low levels during the osteogenic differentiation of hADSCs, and its specific regulatory mechanism remains unclear. Here, we aimed to explore the function and possible molecular mechanism of miR-204-5p in the osteogenic differentiation of hADSCs.MethodsThe expression patterns of miR-204-5p, Runx2, alkaline phosphatase (ALP), osteocalcin (OCN), forkhead box C1 (FOXC1) and growth differentiation factor 7 (GDF7) in hADSCs during osteogenesis were detected by qRT-PCR. Then, ALP and alizarin red staining (ARS) were used to detect osteoblast activities and mineral deposition. Western blotting was conducted to confirm the protein levels. The regulatory relationship among miR-204-5p, FOXC1 and GDF7 was verified by dual-luciferase activity and chromatin immunoprecipitation (ChIP) assays.ResultsmiR-204-5p expression was downregulated in hADSC osteogenesis, and overexpression of miR-204-5p suppressed osteogenic differentiation. Furthermore, the levels of FOXC1 and GDF7 were decreased in the miR-204-5p mimics group, which indicates that miR-204-5p overexpression suppresses the expression of FOXC1 and GDF7 by binding to their 3′-untranslated regions (UTRs). Overexpression of FOXC1 or GDF7 improved the inhibition of osteogenic differentiation of hADSCs induced by the miR-204-5p mimics. Moreover, FOXC1 was found to bind to the promoter of miR-204-5p and GDF7, promote the deacetylation of miR-204-5p and reduce the expression of miR-204-5p, thus promoting the expression of GDF7 during osteogenic differentiation. GDF7 induced hADSC osteogenesis differentiation by activating the AKT and P38 signalling pathways.ConclusionsOur results demonstrated that the miR-204-5p/FOXC1/GDF7 axis regulates the osteogenic differentiation of hADSCs via the AKT and p38 signalling pathways. This study further revealed the regulatory mechanism of hADSC differentiation from the perspective of miRNA regulation.

MiR-138-5p knockdown promotes osteogenic differentiation through FOXC1 up-regulation in human bone mesenchymal stem cells.

This study examined the hypothesis that the microRNA miR-138-5p reduces the osteodifferentiation of human bone mesenchymal stem cells (hBMSCs) by downregulating the expression of forkhead box C1 (FOXC1). For this, hBMSCs were separated from bone marrow and osteogenic induction medium was added to stimulate osteogenic differentiation. Flow cytometric analysis was applied to evaluate the expression of cell-surface antigens associated with hBMSCs, including CD29, CD44, CD90, CD45, and CD34. qRT-PCR assays and Western blot assays were used to measure the mRNA and protein expression of miR-138-5p, osteocalcin, runt-related transcription factor 2, bone sialoprotein, alkaline phosphatase (ALP), and FOXC1. ALP staining assays and Alizarin Red staining (ARS) assays were used to confirm osteogenic differentiation. We used a luciferase assay to test the interaction between miR-138-5p and FOXC1. We demonstrated that miR-138-5p is downregulated in osteogenic differentiated hBMSCs. Further, overexpression of miR-138-5p reduced the expression of markers for osteodifferentiation, ALP activity, and ARS activity. Furthermore, we showed that FOXC1 is a downstream target gene of miR-138-5p, and that knockdown of miR-138-5p improves the osteogenesis differentiation of hBMSCs by upregulating FOXC1. The results from this study indicate miR-138-5p as a new target for osteogenic differentiation of hBMSCs and the treatment of bone defects.

Circ_0004913 sponges miR-1290 and regulates FOXC1 to inhibit the proliferation of hepatocellular carcinoma

BackgroundCircular RNA (circRNA), an novel type of non-coding RNA, could interact with miRNA and protein molecules to regulate the occurrence and progression of hepatocellular carcinoma (HCC). However, little is known about the pathogenesis of circ_0004913 in HCC.MaterialsThrough the GEO (Gene Expression Omnibus database) to find dysfunctional circRNAs in HCC, and circ_0004913 was selected as the research object. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression level of circ_0067934 in HCC tissues and cells. CCK-8, Edu and flow cytometry assays were used to determine the malignant behavior of transfected HCC cells. Mechanistically, RNA immunoprecipitation and dual-luciferase reporter gene assay were performed to explore the relation between circ_0067934, miR-1290 and FOXC1 (Forkhead box C1) in HCC.ResultsThe expression of circ_0004913 was down-regulated in HCC tissues and cell lines, while the overexpression of circ_0004913 attenuates the malignant behavior of HCC cells. Bioinformatics predicted that circ_0004913 interacts with miR-1290, which targeted FOXC1 mRNA. In fact, miR-1290 promoted the malignant behavior of HCC cells, while FOXC1 had the opposite effect. In addition, circ_0004913 overexpression enhanced FOXC1 expression by reducing miR-1290 expression, thereby inhibiting the proliferation of HCC cells.ConclusionsCirc_0004913 / miR-1290 / FOXC1 regulatory axis could inhibit the progress of HCC. Our findings may provide potential new targets for the diagnosis and treatment of HCC.

Abstract 3148: FOXC1 expression is associated with a triple-negative basal-like phenotype in breast cancer

Abstract Introduction: Triple-negative breast carcinoma (TNBC) is a group of aggressive tumors that are resistant to hormonal therapy. Forkhead box C1 (FOXC1) has been shown to play a crucial role in cancer pathogenesis by cancer stem cell regulation, epithelial to mesenchymal transition and tumor progression in basal-like breast cancer (BLBC). This study examined the expression of FOXC1 in basal-like TNBC, which may potential be exploited as a treatment target for this group of tumors. Materials and Methods: Immunohistochemical Peroxidase-DAB system and Opal immunofluorescence staining system were employed to detect FOXC1 and cytokeratin 14 (CK14) expressions in 44 formalin-fixed paraffin-embedded (FFPE) invasive ductal breast carcinoma (IDC) and 150 IDC samples on tissue microarray. CK14 was used to identify the BLBC subtype. The staining was scored semi-quantitatively according to the intensity with a mean score from 0-4. Results: CK14 identified 15 (34%) BLBC in the FFPE tissues and 24 (16%) BLBC in the samples on tissue microarray. Of the 44 FFPE tissues, FOXC1 expression was observed in 100% of the BLBC and 83% of TNBC, while it was only found in 15% of non-TNBC. The mean staining intensity of FOXC1 was lower in non-TNBC (0.30) compared to TNBC (2.16) and basal-like TNBC (2.60). These findings were confirmed in the tissue microarray, which also revealed a strong positive correlation of FOXC1 with basal-like TNBC. In addition, our results showed that FOXC1 was more frequently expressed in ER-negative (22%), PR-negative (21%) and HER2-negative (19%) breast cancers than in ER-positive (3%), PR-positive (3%) and HER2-positive (5%) breast cancers. Moreover, FOXC1 expression was significantly associated with high tumor grade (p = 0.000041) and high tumor-infiltrating lymphocytes (TILs) (p = 0.000001). Conclusion: FOXC1 is highly expressed in TNBC, particularly among the basal-like subgroup, as opposed to other breast cancers. It accurately classifies the BLBC subtype which may be used as a surrogate marker for this molecular subtype. We find that FOXC1 has an inverse correlation with ER, PR and HER2, as well as a positive correlation with tumor grade and TILs. Our finding suggests that FOXC1 may serve as a potential treatment target for basal-like TNBC. Citation Format: William C. Cho, Victor W. Ma, Wah Cheuk, Grace Y. So, Rebecca Y. Chin. FOXC1 expression is associated with a triple-negative basal-like phenotype in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3148.

LncRNA MCM3AP-AS1 promotes proliferation, migration and invasion of oral squamous cell carcinoma cells via regulating miR-204-5p/FOXC1

Oral squamous cell carcinoma (OSCC) is a lethal malignancy. It is reportedly demonstrated that long non-coding RNA (lncRNA) participates in the development of OSCC. The purpose of this study was...

Forkhead Box C1 (FOXC1) Expression in Stromal Cells within the Microenvironment of T and NK Cell Lymphomas: Association with Tumor Dormancy and Activation.

PurposeForkhead box C1 (FOXC1) is critical for maintaining bone marrow microenvironments during hematopoiesis, but its role in hematological malignancies remains obscure. Here, we investigated whether FOXC1 regulates tumor dormancy and activation in the microenvironments of T and natural killer (NK) cell lymphomas.Materials and MethodsOne hundred and twenty cases of T and NK cell lymphomas were included; the immunohistochemical expression of FOXC1 was investigated in stromal cells, and numbers of FOXC1+ stromal cells were counted. Furthermore, the expression of phosphorylated p38 (p-p38) and phosphorylated ERK1/2 (p-ERK1/2) in tumor cells was investigated using immunohistochemistry.ResultsFOXC1 was variably expressed in C-X-C motif chemokine 12–associated reticular stromal cells, histiocytes, (myo)fibroblasts, and endothelial cells. The phenotypes of cases were categorized as dormant (high p-p38/low p-ERK1/2; n=30, 25.0%), active (high p-ERK1/2/low p-p38; n=25, 20.8%), or intermediate (others; n=65, 54.2%). Lower FOXC1+ stromal cell infiltration was associated with the dormant phenotype, the precursor T lymphoblastic leukemia/lymphoma subtype, and inferior overall survival rates, whereas higher FOXC1+ stromal cell infiltration was associated with the active phenotype and favorable patient prognosis (p < 0.05 for all).ConclusionThese results suggested that FOXC1+ stromal cells within the microenvironments of T and NK cell lymphomas might be related to tumor phenotypes.

C/EBPα Regulates FOXC1 to Modulate Tumor Growth by Interacting with PPARγ in Hepatocellular Carcinoma.

Forkhead box C1 (FOXC1) is an important cancer-associated gene in tumor. PPAR-γ and C/EBPα are both transcriptional regulators involved in tumor development. We aimed to clarify the function of PPAR-γ, C/EBPα in hepatocellular carcinoma (HCC) and the relationship of PPAR-γ, C/EBPα and FOXC1 in HCC. Western blotting, immunofluorescent staining, and immunohistochemistry were used to evaluate protein expression. qRT-PCR was used to assess mRNA expression. Co-IP was performed to detect the protein interaction. And ChIP and fluorescent reporter detection were used to determine the binding between protein and FOXC1 promoter. C/EBPα could bind to FOXC1 promoter and PPAR-γ could strengthen C/EBPα's function. Expressions of C/EBPα and PPAR-γ were both negatively related to FOXC1 in human HCC tissue. Confocal displayed that C/EBPα was co-located with FOXC1 in HepG2 cells. C/EBPα could bind to FOXC1 promoter by ChIP. Luciferase activity detection exhibited that C/EBPα could inhibit FOXC1 promoter activity, especially FOXC1 promoter from -600 to -300 was the critical binding site. Only PPAR-γ could not influence luciferase activity but strengthen inhibited effect of C/EBPα. Further, the Co-IP displayed that PPAR-γ could bind to C/EBPα. When C/EBPα and PPAR-γ were both high expressed, cell proliferation, migration, invasion, and colony information were inhibited enormously. C/EBPα plasmid combined with or without PPAR-γ agonist MDG548 treatment exhibited a strong tumor inhibition and FOXC1 suppression in mice. Our data establish C/EBPα targeting FOXC1 as a potential determinant in the HCC, which supplies a new pathway to treat HCC. However, PPAR-γ has no effect on FOXC1 expression.

FOXC1 negatively regulates BMP-SMAD activity and Id1 expression during osteoblast differentiation.

Bone morphogenetic proteins regulate a diverse range of biological processes through their activation of SMAD1, SMAD5, or SMAD8 proteins that, in turn, regulate gene expression. These SMAD transcription factors achieve a layer of functional specificity in different cell types largely through actions with additional transcriptional regulatory molecules. In this study, we demonstrate that the forkhead box C1 (FOXC1) transcription factor can modulate bone morphogenetic protein (BMP) signaling to impair the expression of BMP4-responsive genes and prevent the efficient osteoblast differentiation. We demonstrate that repression occurs downstream of BMP signaling and impacts the ability SMAD1 or SMAD5 to activate gene expression. Repression of SMAD activity requires FOXC1 DNA-binding capacity and the transcriptional inhibitory domain of FOXC1. We report that FOXC1 inhibits BMP4 induction of Id1 expression and identify a motif in the regulatory region of mouse Id1 gene that FOXC1 binds. We determine that this inhibition by FOXC1 binding does not affect SMAD1, SMAD5, or SMAD8 binding to its target sequence in the Id1 gene. Finally, we determine that the elevated expression of FOXC1 can reduces expression osteogenic differentiation genes in mouse embryonic stems directed to the osteoblast lineage through BMP4 treatment. Together, these findings indicate that FOXC1 can negatively regulate certain aspects of BMP4 signaling required for osteoblast differentiation. We propose that FOXC1 acts to attenuate the initial BMP-activated pathways that establish osteoblast differentiation and allow for terminal osteoblast differentiation to conclude.

Comparative Study of FOXC1 Expression in Selective Odontogenic Cysts and Tumours

Introduction: Many studies have indicated that Forkhead Box C1 (FOXC1) is highly expressed in a various malignant neoplasms and its over expression is associated with tumour development, progression and metastasis. However, the role of FOXC1 in odontogenic lesions remains to be elucidated. Aim: This study aimed to investigate FOXC1 expression in selective Odontogenic Cysts (OC) and Odontogenic Tumours (OT). Materials and Methods: A descriptive study on OC and OT was performed on oral biopsy specimens at Faculty of Dentistry, Naresuan University, Phitsanulok, Thailand, between January 2009 and December 2016. Institute of Cancer Research (ICR) mouse were used as study animal. Immunohistochemical reaction was performed using antibody against FOXC1 in 23 formalin-fixed and paraffin-embedded tissues of eight Ameloblastoma (AM), one Adenomatoid Odontogenic Tumour (AOT), seven Odontogenic Keratocyst (OKC), five Dentigerous Cyst (DC), two Calcifying Odontogenic Cyst (COC) and three mouse embryonic head at Embryonic Day (E)14. The expression level of FOXC1 was evaluated using semi-quantitative analysis. Results: Immunoreactivity of FOXC1 was not detected in the epithelial lining of OKC, DC and COC but it was identified in the epithelial compartment of AM and AOT. Overall, semi-quantitative analysis demonstrated moderate staining of FOXC1 and staining score was 4.13 in AM and 5 in AOT. In addition, FOXC1 was not detected in normal tooth bud of mouse including both enamel organ and condensing ectomesenchyme. Conclusion: Expression of FOXC1 may contribute in tumouriogenesis of OT whereas it is may not be related with normal odontogenesis.

COUP-TFII Knock-down Promotes Proliferation and Invasion in Colorectal Cancer Cells via Activation of Akt Pathway and Up-regulation of FOXC1.

The chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) regulates cancer cell proliferation and invasion via complex molecular mechanisms. We aimed to investigate whether COUP-TFII modulates proliferation and invasion of the colorectal adenocarcinoma cell line HT-29. HT-29 cells were stably tranfected with COUP-TFII shRNA plasmid to knock-down COUP-TFII (COUP-TFII shRNA-HT-29 cells). Cell proliferation, colony formation assay, invasion assay, microarray assays and western blot analyses were performed. Cell proliferation and invasion were significantly enhanced in COUP-TFII shRNA-HT-29 cells. The protein levels of forkhead box C1 (FOXC1), p-Akt, p-glycogen synthase kinase-3β (p-GSK-3β), and β-catenin, which are known to be involved in cell proliferation and invasion, were significantly increased in COUP-TFII shRNA-HT-29 cells. Akt inhibitor IV and dominant negative (DN)-Akt expression vector transfection reversed the increased proliferation and invasion, which was accompanied by decreased protein levels of p-Akt, p-GSK-3β, β-catenin and FOXC1. COUP-TFII knock-down promoted proliferation and invasion via activation of Akt/GSK-3β/β-catenin and up-regulation of FOXC1. Further studies on the molecular mechanism of interaction between β-catenin and FOXC1 expression may reveal novel target molecules for metastatic colorectal cancer therapy.

P38-regulated FOXC1 stability is required for colorectal cancer metastasis.

Aberrant expression of forkhead box C1 (FOXC1) promotes tumor metastasis in multiple human malignant tumors. However, the upstream modulating mode and downstream molecular mechanism of FOXC1 in metastasis of colorectal cancer (CRC) remain unclear. Herein we describe a systematic analysis of FOXC1 expression and prognosis in CRC performed on our clinical data and public databases, which indicated that FOXC1 upregulation in CRC samples was significantly associated with poor prognosis. FOXC1 knockdown inhibited migration and invasion, whereas FOXC1 overexpression caused the opposite phenotype in vitro and in vivo. Furthermore, MMP10, SOX4 and SOX13 were verified as the target genes of FOXC1 for promoting CRC metastasis. MMP10 was demonstrated as the direct target and mediator of FOXC1. Interestingly, Ser241 and Ser272 of FOXC1 were identified as the key sites to interact with p38 and phosphorylation, which were critically required for maintaining the stability of FOXC1 protein. Moreover, FOXC1 was dephosphorylated by protein phosphatase 2A and phosphorylated by p38, which maintained FOXC1 protein stability through inhibiting ubiquitination. Expression of p38 was correlated with FOXC1 and MMP10 expression, indirectly indicating that FOXC1 was regulated by p38 MAPK. Therefore, FOXC1 is strongly suggested as a pro-metastatic gene in CRC by transcriptionally activating MMP10, SOX4 and SOX13; p38 interacts with and phosphorylates the Ser241 and ser272 sites of FOXC1 to maintain its stability by inhibiting ubiquitination and degradation. In conclusion, the protein stability of FOXC1 mediated by p38 contributes to the metastatic effect in CRC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Forkhead box C1 promotes metastasis and invasion of non-small cell lung cancer by binding directly to the lysyl oxidase promoter.

Increasing evidence indicates that human forkhead box C1 (FOXC1) plays important roles in tumor development and metastasis. However, the underlying molecular mechanism of FOXC1 in non–small cell lung cancer (NSCLC) metastasis remains unclear. Here, we identified FOXC1 as an independent prognostic factor in NSCLC and showed clear biological implications in invasion and metastasis. FOXC1 overexpression enhanced the proliferation, migration and invasion of NSCLC cells, whereas FOXC1 silencing impaired the effects both in vitro and in vivo. Importantly, we found a positive correlation between FOXC1 expression and lysyl oxidase (LOX) expression in NSCLC cells and patient samples. Downregulation of LOX or LOX activity inhibition in NSCLC cells inhibited the FOXC1‐driven effects on cellular migration and invasion. Xenograft models showed that inhibition of LOX activity by β‐aminopropionitrile monofumarate decreased the number of lung metastases. Mechanistically, we demonstrated a novel FOXC1‐LOX mechanism that was involved in the invasion and metastasis of NSCLC. Dual‐luciferase assay and ChIP identified that FOXC1 bound directly in the LOX promoter region and activated its transcription. Collectively, the present study offered new insight into FOXC1 in the mediation of NSCLC metastasis through interaction with the LOX promoter and further revealed that targeted inhibition of LOX protein activity could prevent lung metastasis in murine xenograft models. These data implicated FOXC1 as a potential therapeutic strategy for the treatment of NSCLC metastasis.

Prognostic Significance of FOXC1 in Various Cancers: A Systematic Review and Meta-Analysis.

Forkhead box C1 (FOXC1), a member of the Forkhead box (Fox) transcription factor family, plays an essential role in lymphatic vessel formation, angiogenesis and metastasis. Observational studies examining the relationship between the protein biomarker FOXC1 and breast cancer prognosis have reported conflicting findings. This systematic review and meta-analysis evaluates the prognostic value of the FOXC1 expression in association with patient survival in breast cancer and other types of cancers in order to identify the overall prognostic effectiveness of FOXC1. This study followed the guidelines established in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). We conducted a broad search on the online bibliographic databases EMBASE, PubMed, Science Direct and Scopus, limiting search to publications from 2010 to 2018. The prognostic value was demonstrated by a random effects model meta-analysis using the hazard ratio (HR) with 95% confidence interval (CI) for overall survival (OS) in various cancer patients. The heterogeneity was measured by the I2 statistic. Publication bias and quality assessment for the selected articles was performed. Subgroup analysis was conducted based on the data available from the selected articles. A total of 16 studies met the predefined selection criteria established for our systematic review and meta-analysis, with multiple studies using diverse methodologies and reported on differing clinical outcomes, falling under a common banner of FOXC1 expression and survival in cancer. Overall, we observed a statistically non-significant association between FOXC1 protein expression and patients survival (HR: 1.186 and 95% CI 1.122-1.255, p=0.000, I2=88.83%). In summary, FOXC1 protein expression indicated poor survival outcome in various carcinomas, especially in patients with breast cancer, suggesting it as a possible biomarker for the prognosis in multiple carcinomas. Further clinical evaluation and large-scale cohort studies are required to accurately identify its possible clinical utility.