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American Journal of Cancer Research logoLink to American Journal of Cancer Research
. 2018 Oct 1;8(10):1989–2004.

FOXA1 transcriptionally up-regulates cyclin B1 expression to enhance chondrosarcoma progression

Zong-Shin Lin 1, Chiao-Chen Chung 2, Yu-Chia Liu 2, Tsung-Ming Chen 5, Yung-Luen Yu 1,2,3,4, Shao-Chun Wang 1,2,4, Ya-Huey Chen 1,2,3
PMCID: PMC6220138  PMID: 30416851

Abstract

Chondrosarcoma is a malignant and common bone tumor that is highly resistant to radiation and chemotherapy. At this moment, amputation surgery is the only option which unfortunately has serious impact to daily lives of the patients. Thus, there is an urgent need to understand causative molecular mechanisms underlying the disease for more accurate prognosis and more effective targeted treatment. In the current study, we identify the transcription factor FOXA1 through cDNA microarray screening comparing normal versus chondrosarcoma cells and investigate the mechanisms underlying its function in chondrosarcoma development. We show that FOXA1 enhances expression of the cyclin B1 gene, which in turn drives cell cycle progression through G2-M transition thus promotes cell cycle progression of chondrosarcoma cells.

Keywords: Chondrosarcoma, FOXA1, cell cycle, G2/M phase, cyclin B1

Introduction

Chondrosarcoma is the second most malignant and common neoplasm affecting the bones [1]. Long bones such as humerus, femur, and tibia are developed from cartilaginous templates through endochondral ossification, which turns the cartilage into bone. Deregulation of signaling pathways, hedgehog signaling for example, results in enchondromas and osteochondromas, which can be the precursors of central and peripheral chondrosarcoma, respectively [1]. Chondrosarcoma cells have been reported that were highly resistant to chemotherapy with increased interstitial pressure that potentially impends drug delivery [1-4]. Currently surgery is the only option for bone cancer. However, amputation and limb salvage operations affect a patient’s life quality severely. Thus, there is an urgent need of targeted therapies for effective treatment of chondrosarcoma.

Forkhead box (FOX) proteins can both activate and repress gene expression through the recruitment of co-factors or repressors and histone deacetylase (HADACs). Additionally, they can interact extensively with other transcription factors such as p53 and estrogen receptors to modulate gene expression. Consequently deregulation of FOX proteins function directly or indirectly alters their target genes [5]. For example, overexpression of FOXM1 and FOXC2 will promote cell cycle progression in osteosarcoma and angiogenesis for tumor metastasis, respectively. On the other hand, in breast cancer and acute/chronic myeloid leukemia loss of FOXO3A increases the ability to resist apoptosis and promote cell cycle progression [5].

The transcription factor forkhead box protein A1 (FOXA1), also termed hepatocyte nucleus factor 3 alpha (HNF-3α), belongs to the forkhead family and shares the common winged helix DNA-binding domain. Unlike traditional transcription factors, FOXA1 functions as a pioneer factor to shape the chromatin landscape to activate functional regulatory elements, for instance, bookmarking enhancers by reprogramming the chromatin landscape [6], and is required as well as sufficient to trigger enhancer competency [7]. Thus, FOXA1 has intrinsic chromatin remodeling activities to relax condensed chromatin structures in vitro in an ATP-independent manner [7,8].

FOXA1 was first found required for liver development and was the first detectable transcription factor involving in gene enhancer activity required for normal development. In previous studies, FOXA1 has been shown to play an oncogenic role in many types of cancer, including breast, prostate, pancreas, and lung cancer [9-13]. Also, FOXA1 was found interacting with hormone-related transcription factors, such as estrogen receptor (ERα) and androgen receptor (AR). This interaction helps recruit FOXA1 to target genes to regulate hormone-related signal pathway [14]. FOXA1 is known to mediate breast cancer cell progression in ER-positive as well as ER-negative basal like breast cancer [15]. Depression of FOXA1 by RNA interference repressed proliferation of breast cancer cell lines regardless of ErbB2 status [12]. Intriguingly, it has been shown that FOXA1 can increase the transcriptional expression of p27 resulting in tumor suppression, which can be further enhanced in the presence of wild-type BRCA1 [16,17]. FOXA1 has also been associated with favorable prognosis in ER positive breast cancer particularly luminal subtype A breast cancer [16,18]. In Lieu of these prior studies, the role of FOXA1 in the modulation of bone cancer progression remains largely unexplored.

In the current study, we identify FOXA1 as the major transcription factor to activate cell cycle-promoting genes in chondrosarcoma and show that cyclin B1 (CCNB1) is the direct target of FOXA1. Inhibition of cyclin B1 in high-level expression of FOXA1 of chondrosarcoma cells significantly suppressed tumor cell growth. Our results suggest that FOXA1 enhances cyclin B1 expression through transcription, subsequently promotes cell cycle progression and reduce apoptosis in chondrosarcoma.

Materials and methods

Cell culture

The human chondrosarcoma cell lines JJ012 and CH2879 were provided by Dr. Joel Block and Dr. Antonio Llombart-Bosch, respectively. The human primary cartilage was obtained from Dr. Teng-Le Huang and the chondrocytes were isolated according to protocol [19]. JJ012 was cultured in 40% Modified Eagle’s medium alpha (MEM-α), 40% high glucose Dulbecco’s Modified Eagle’s medium (HG-DMEM), and 10% F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S) (Gibco). CH2879 was maintained in RPMI1640 containing 10% FBS and 1% P/S. Human primary chondrocytes were maintained in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) containing 10% FBS, 1% antibiotic antimycotic, and 50 μg/ml ascorbic acid 2-phosphate.

Lentiviral infection

Lentiviral shRNA clones were purchased from the National RNAi Core Facility of Academia Sinica (Taiwan). JJ012 and CH2879 were infected with control (pLK0.1 vector alone) or FOXA1 shRNA lentivirus containing polybrene (8 μg/ml) at multiplicity of infection (MOI) of 20. The RNA and protein lysate of cells were extracted after infection for 48 hours and 72 hours, respectively.

Real-time RT-PCR

Total RNA was harvested using TRIzol (Invitrogen) reagent and reverse transcribed into single-stranded cDNA by SuperScriptTM III First Strand Synthesis kit (Invitrogen). Alteration in mRNA expression level were analyzed by real-time PCR (Roche Applied Science, LightCycler 480) using SYBR Green and normalized to β-actin. Primer sequences are listed in Table S1.

Western blot

Cell lysates were extracted by NETN (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, 0.5% Nonidet P-40) lysis buffer containing protease inhibitors on ice. Protein samples (30 μg) were loaded onto SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting. Whole-cell lysates were analyzed with the following antibodies: FOXA1 (GeneTex, GTX100308), p53 (Cell Signaling, #2524), p21 (CALBIOCHEM, OP64), CCNB1 (GeneTex, GTX100911), and β-actin (GeneTex, GTX109639).

Cell proliferation assay

CH2879 and JJ012 were seeded 5,000 and 3,000 cells onto 96-well plates, respectively. At the end of 24, 48, 72 and 96 hr indicated time periods, cell viability was measured by incubation of CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). According to the manufacturer’s instructions, cell viability was determined by OD 490 nm.

Migration assay

JJ012 cells were seeded 2 × 104 cells with serum free culture medium in the upper compartment of a transwell (Corning® 3422 Transwell, Pore Size: 8 µm). Subsequently, the transwell was inset into a 24 well plate which contained 10% serum culture medium. Sixteen or eighteen hours after incubation, the cells were fixed by 100% cold methanol and stained by 0.5% crystal violet. The un-migrated cells on the top of the transwell were removed with a cotton swab and 10 pictures were taken at different high-power fields by a microscope. The migrated cell were counted and plotted.

Wound healing assay

Collagen I (10 μg/ml) was coated onto 24 well plates and incubated at 37°C overnight. 5 × 104 CH2879 cells were suspended in a 70 μl culture medium and seeded into cell inserts in 24 well plates for overnight incubation. A 1 ml medium was added into each well after cell inserts were removed carefully and then photographed after 24 hr incubation.

Soft agar assay (anchorage-independent growth assay)

Anchorage-independent colony formation was assessed using a two-layer soft agar assay. 1 mL of 0.5% agar was added into a 6-well plate. After the agar solidified, 5,000 of JJ012 or CH2879 cells were mixed with 0.3% agar in 1 ml culture medium and plated on top of the 0.5% basis agar layer. Finally, 2 mL of normal culture medium was added on top of the 0.3% agar. After 21 days incubation, colonies were stained with 1% crystal violet staining solution and counted.

Clonogenic assay

Cells were seeded 1000 cells into a 6-well plate for 9-11 days. During colony formation, the plates were washed with PBS once and fixed with a fix solution (75% methanol, 25% acetic acid) at room temperature (RT) for 5 min. After fixation, the plates were stained with 0.5% crystal violet (dissolved in 100% methanol) at RT for 15 min. Following being rinsed with tap water, the colonies were observed under a microscope and the number of the colony was counted for quantification.

Flow cytometric analysis

CH2879 or JJ012 were infected by lentiviral-carried shRNA for 72 hr. For apoptosis assays, cells were harvested and processed for Annexin V staining according to the manufacturer’s instructions (BD Biosciences, cat. 556547). 106 cells of each sample were washed by PBS twice and the cells were re-suspended with 1 × Annexin V buffer. Following being mixed with PI buffer, the samples were incubated in the dark for 15 min before analysis by fluorescence activated cell sorter (FACS). For cell cycle analysis, cells were fixed with 70% ethanol (diluted with PBS) at -30°C overnight and stained with a PI buffer (containing 40 μg/ml PI and 100 μg/ml RNase A) for 30 min at 37°C in the dark. Then, the aggregate cells were filtrated using 70 μm cell strainers and were validated by a FACSVerse laser flow cytometric analysis system (Becton Dickinson).

cDNA microarray

0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and was labeled with Cy3 (CyDye, Agilent Technologies, USA) during the in vitro transcription process. 0.6 μg of Cy3-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with a fragmentation buffer at 60°C for 30 minutes. Correspondingly fragmented labeled cRNA was then pooled and hybridized to Agilent SurePrint G3 Human V2 GE 8 × 60 K Microarray (Agilent Technologies, USA) at 65°C for 17 h. After washing and drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3. Scanned images were analyzed by Feature extraction 10.5.1.1 software (Agilent Technologies, USA), an image analysis and normalization software that was used to quantify signal and background intensity for each feature.

Reporter gene assay

The cyclin B1 (CCNB1) promoter drive luciferase reporter plasmid was amplified from human genomic DNA containing HindIII and XhoI restriction enzyme sites by PCR and then subcloned into pGL3-basic backbone vector. Primer sequences are listed in Table S2. The CCNB1 promoter luciferase reporter plasmid was transfected into 293 cells together with internal control plasmid β-gal or the indicated plasmids using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cell extracts were harvested and luciferase activity was measured by luciferase reporter assay system according to the manufacturer’s instructions (Promega).

Quantitative chromatin immunoprecipitation (qChIP) assay

ChIP assays were performed using the EZ-ChIP kit (Upstate) and according to the manufacturer’s instructions. Specific antibodies against FOXA1 (GeneTex, GTX100308) were used for immunoprecipitation. The immunoprecipitated DNA was subjected to RT-PCR using a SYBR Green system according to the manufacturer’s instructions (Roche Applied Science). Data are shown as the fold enrichment of precipitated DNA relative to 2:100 dilution of input chromatin. Primers are listed in Table S3.

In vivo animal studies

Six-week-old male nude (BALB/cAnN.Cg-Foxn1nu/CrlNarl) mice from the National Laboratory Animal Center (Taipei, Taiwan) were injected subcutaneously in the flank region with 4 × 106 CH2879 cells with or without knockdown of FOXA1. Tumor volume was measured every 3 days using the formula (tumor volume (V) = π/6 × larger diameter × smaller diameter2) for 31 days.

Results

FOXA1 is up-regulated in chondrosarcoma cell lines and enhances their tumorigenicity

To identify the genes that mediate chondrosarcoma progression using an unbiased approach, we utilized a genome-wide human cDNA microarray to compare gene expression profiles in normal chondrocytes versus chondrosarcoma cell lines. The results indicated that RNA expression of the FOXA1 gene was over 4-fold higher in chondrosarcoma cell lines compared with normal chondrocytes (Figure 1A). To confirm the results of the cDNA microarray data, we investigated the expression of FOXA1 mRNA and protein in chondrosarcoma cell lines by q-RT-PCR and western blot analysis, respectively. In contrast to the normal chondrocytes tested, FOXA1 expression was significantly increased in chondrosarcoma cell lines (Figure 1B and 1C). FOXA1 has been reported to be aberrantly expressed in many cancer types, and plays an oncogenic or a tumor suppressing role in different cancer types or even in the same cancer types [9-12,18,20-23]. To test the role of FOXA1 in chondrosarcoma tumorigenicity, chondrosarcoma cell lines with or without FOXA1 short hairpin RNA (shRNA) delivered by lentiviral transduction were assessed for their proliferative, migratory, and anchorage-independent colony-forming abilities. The knockdown efficacy of FOXA1 shRNA was validated in which FOXA1 mRNA and protein expression in chondrosarcoma cell lines comparing the viral vector control and two different FOXA1 shRNAs (Figure 2A, 2B). Knockdown of the endogenous FOXA1 gene attenuated proliferation, migration, and anchorage-independent colony formation of chondrosarcoma cells (Figure 2C-H). Conversely, ectopical expression of FOXA1 resulted in higher colony forming activity than that of the vector control (Figure 3). Collectively, these results suggest that FOXA1 positively regulate chondrosarcoma progression.

Figure 1.

Figure 1

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The expression of FOXA1 in chondrosarcoma cell lines and normal primary chondrocyte. A. Normal primary chondrocyte and JJ012 chondrosarcoma cell line were analyzed by cDNA microarray. Compared to normal primary chondrocyte, four-fold change genes in chondrocarcoma cell line were shown in heat map. B. Relative FOXA1 mRNA expression of chondrosarcoma cell lines and normal primary chondrocyte cells were measured by qRT-PCR. C. The FOXA1 protein expression level of chondrosarcoma and normal primary chondrocyte were determined by western blot.

Figure 2.

Figure 2

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The tumorogenesis of chondrosarcoma cell lines is inhibited with knockdown of FOXA1. A and B. The expression of FOXA1 in chondrosarcoma cell lines was knocked down via lentivirus system, and the knockdown efficiency was examined by qRT-PCR and western blot. C and D. Chondrosarcoma cells were infected with lentivirus expressing control or FOXA1 shRNA. MTS assay was used to clarify cell proliferation at different time points including 24, 48, 72, and 96 hr. E. Migration of cells was assessed by wound healing assay. CH2879 cells were infected by control or FOXA1 shRNA and seeded into wound creating inserts for 24 hr. Images and quantification of cell migration is shown. F. After infection, images and quantification of cell migration of JJ012 was determined by transwell assay. G and H. Cells infected with lentivirus encoding control or FOXA1 shRNA. The anchorage-independent growth was analyzed through soft agar assay. Data are means ± SD, Student’s t-test. *P < 0.05, **P < 0.01. ***P < 0.001.

Figure 3.

Figure 3

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Ectopic-expressed FOXA1 enhances colony forming activity in chondrosarcoma cell lines. A and B. qRT-PCR and immunoblot of stable CH2879 and JJ012 transfectants, established by the transfection of cells with control vector or plasmid-expressing FOXA1. C and D. Images and quantification of colony forming ability assay of CH2879 and JJ012 stable cell lines. Data are means ± SD, Student’s t-test. *P < 0.05, **P < 0.01. ***P < 0.001.

FOXA1 promotes cell cycle progression and reduces apoptosis in chondrosarcoma cell lines

To further understand the tumor-promoting function of FOXA1, a gene expression profiling analysis of chondrosarcoma cell lines harboring the control shRNA or two FOXA1 shRNA was carried out. In comparison with the control shRNA, affected genes in the FOXA1-depleted cells were analyzed by KEGG gene ontology analysis (Figure 4A). The top three gene categories regulated by FOXA1 were DNA replication, cell cycle, and the p53 signaling pathways (Figure 4A). Dysregulated genes of the cell cycle group are shown in a heat map (Figure 4B). To confirm the gene expression from these categories, expression of cyclin B1 (CCNB1), cyclin A1 (CCNA1), hepatocyte growth factor (HGF), neurogenic locus notch homolog protein 4 (NOTCH4), and tumor protein P53 inducible nuclear protein 1 (TP53INP1) were assessed by qRT-PCR. Depletion of FOXA1 expression by two different shRNA consistently showed reduction of cell cycle activator genes including cyclin B1 and cyclin A1, and cell cycle inhibitor genes upregulated by p53 (Figure 4C). Conversely, growth factors including HGF and NOTCH4 were down-regulated in FOXA1 depleted cells (Figure 4C). We next examined whether knockdown of FOXA1 affects cell cycle distribution by FACS analysis. We found that the subG0/G1 and G2/M phases were increased when FOXA1 was inhibited (Figure 5A, 5B), indicating that depletion of FOXA1 may promote cell death and mitotic arrest. Apoptosis induction by depletion of FOXA1 in chondrosarcoma cells was further verified by flowcytometry which showed that repression of FOXA1 significantly increased annexin V-positive cells (Figure 5C). These results suggest that FOXA1 downregulation leads to G2/M arrest which in turn induces apoptosis.

Figure 4.

Figure 4

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FOXA1 mediates chondrosarcoma tumorogenesis through regulation of cell cycle. A. cDNA microarray analysis of FOXA1 knockdown in chondrosarcoma cells. Top, western blot analysis of FOXA1 expression in chondrosarcoma cells infected with lentivirals contain control or FOXA1 shRNA. Actin served as the control. Bottom, the genes numbers were plotted based on KEGG classification and P value of each category. B. A list of top candidates includes message-encoding proteins of cell cycle. C. Verifying the gene expression from cDNA microarray, which is involved in cell cycle, growth factor, p53 signal pathway by qRT-PCR. Data are means ± SD.

Figure 5.

Figure 5

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Depletion of FOXA1 by shRNA induces G2/M arrest and apoptosis of chondrosarcoma cells. A. Lysates from CH2879 cells infected with lentiviruses expressing control or four different FOXA1 shRNAs were immunoblotted with antibodies against the indicated protein. B. After infection, chondrosarcoma cells were stained with PI and analyzed for the distribution of cell cycle phases by flow cytometry. C. Flow cytometric analysis of CH2879 with or without FOXA1 shRNA cells stained with annexin V and PI. Data are shown as means ± SD; n = 3 sets of cells. Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

FOXA1 transcriptionally up-regulates cyclin B1 expression to enhance cell cycle progression in chondrosarcoma

Mitotic entry is triggered by cyclin B1 expression and activation of cyclin B1-cyclin-dependent kinase 1 (Cdk1) complex [24]. Mounting evidence exhibited that the cyclin B1 level was minimal in G1, began to rise in S phase, and peaked at the G2/M transition [25,26]. As depletion of FOXA1 arrests chondrosarcoma cells in the G2/M phase, we investigated whether FOXA1 alters the expression of cyclin B1. Using two specific FOXA1 shRNA to knockdown FOXA1 expression and ectopic stable transfectants of FOXA1, we found the RNA expression level of cyclin B1 was significantly reduced and increased, respectively (Figure 6A and 6B). These results are consistent with our cDNA microarray data that knockdown of FOXA1 transcriptionally repressed cyclin B1 expression in chondrosarcoma cells (Figure 4C). To determine whether cyclin B1 is critical for FOXA1-medicated chondrosarcoma tumorigenicity, cyclin B1 expression was depleted using two different shRNAs that specifically target cyclin B1. Western blotting analysis indicated that cyclin B1 was highly expressed in CH2879 chondrosarcoma cells compared to normal primary chondrocytes (Figure 6C) and the two cyclin B1 shRNAs effectively attenuated cyclin B1 expression without affecting the expression of FOXA1 (Figure 6D). Reducing cyclin B1 expression inhibited proliferation of CH2879 cells as determined by MTT assay (Figure 6E). Compared to control shRNA, treatment by cyclin B1-specific shRNA decreased the colony forming activity (Figure 6F). Moreover, depletion of cyclin B1 expression by treated with cyclin B1 shRNA of FOXA1-overexpressed CH2879 cells significantly attenuated cell proliferation in compared to shRNA control (Figure 6G and 6H). These results were consistent to those in the FOXA1 knockdown experiments (Figure 2) and suggest that cyclin B1is likely the downstream target and upregulated by FOXA1 to promote cell growth.

Figure 6.

Figure 6

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Knockdwon of cyclin B1 inhibits tumorigenicity of chondrosarcoma. A. CH2879 cells were infected with lentivirus expressing control or FOXA1 shRNA. qRT-PCR of CCNB1 gene expression in CH2879 with control or FOXA1 shRNA. B. CH2879 cell lines stably expressing vector control or FOXA1 were used in this assay. qRT-PCR of CCNB1 gene expression in CH2879 stable cell lines. C and D. Lysates of CH2879 cells were immunoblotted as indicated antibodies. Actin served as the control. E. MTT assay was performed to assess cell proliferation at different time points including 24, 48, 72 and 96 hr. F. Images and quantification of colony forming ability assay of CH2879 cells. G. CH2879 cell lines stably expressing vector control or FOXA1 were infected with control or two specific cyclin B1 shRNA. Lysates were harvested and immunoblotted. H. MTT assay of indicated cells was determined for cell proliferation at different time points including 24, 48, 72, and 96 hr. Data are means ± SD, Student’s t-test. *P < 0.05, **P < 0.01. ***P < 0.001.

To further investigate the growth-promoting mechanisms of FOXA1, FOXA1 depletion and ectopic expression was conducted in distinct chondrosarcoma cell lines. Western blot analysis revealed that depletion of FOXA1 decreased the expression of cyclin B1 (CCNB1) while increased expression of p21 and p53. Given that CDK1 functioned as a cell-cycle kinase during G2/M phase and also bound to cyclin B1 to form an active cyclin B1-CDK1 complex [27], we next detected whether phosphorylation of CDK1 was regulated by FOXA1 in chondrosarcoma cell line. Indeed, depletion FOXA1 suppressed phosphorylation of CDK1 compared to cells treated with the control shRNA (Figure 7A). Furthermore, knockdown of FOXA1 enhanced caspase 3 cleavage, a hallmark of apoptosis (Figure 7B). In contrast, ectopic expression of FOXA1 reduced the expression of p53 and p21 as well as induction of phosphorylation of CDK1 (Figure 7C). These results suggest that FOXA1 mediates cell cycle progression by promoting cyclin B1 expression and attenuating expression of p53 and p21, subsequently activates cyclin B1-CDK1 complex to promote cell cycle and prevents from cell death.

Figure 7.

Figure 7

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FOXA1 regulates chondrosarcoma progression through cell cycle and apoptosis-dependent pathway. A, B. CH2879 cells were infected with lentivirus expressing control or FOXA1 shRNA. Lysates of CH2879 cells were harvested. Actin served as the control. C. CH2879 cell lines stably expressing vector control or FOXA1 were collected. Immunoblots of experiment performed as indicated antibodies in CH2879 stable cell lines.

To understand how FOXA1 upregulates cyclin B1, we examined the proximal promoter region 1000 bases upstream from the start site of the cyclin B1 gene and identified four conserved FOXA1 binding sites (Figure 8A) [28]. To validate whether FOXA1 directly activates cyclin B1 promoter, reporter genes fused with two different promoter lengths of the cyclin B1 promoter were constructed (Figure 8A). The results indicate the cyclin B1 promoter region at -400 bp upstream of the transcriptional start site was stimulated by FOXA1 to the highest extent among the constructs (Figure 8B). To test whether FOXA1 stimulates the reporter through its genuine transcriptional activity, the FOXA1 binding site in the -400 bp region of cyclin B1 promoter region was mutated. Luciferase reporter assay demonstrated that disruption of FOXA1 binding site abolished the FOXA1-mediated gene activation of the reporter (Figure 8C). These findings suggest that the FOXA1 binding site located at 400 bp upstream of the cyclin B1 promoter is critical for FOXA1-mediated cyclin B1 promoter activation. We then accessed the binding activity of FOXA1 at the cyclin B1 promoter in chondrosarcoma cells by a quantitative chromatin immunoprecipitation (ChIP) assay by immunoprecipitation of fragmented chromatin with a FOXA1 antibody followed by quantitative PCR of the corresponding genomic region surrounding the FOXA1-binding site. The data showed that FOXA1 binding to the cyclin B1 promoter was strongly enhanced in chondrosarcoma cells expressing ectopic FOXA1 compared with the control (Figure 8D). Given the potent activation of chondrosarcoma tumorigenicity by FOXA1 in vitro, we clarified the role of FOXA1 in vivo by employing a mice tumor model. Male nude BALB/cAnN.Cg-Foxn1nu/CrlNarl mice with subcutaneous xenograft tumors from CH2879 cells with FOXA1-knockdown or CH2879 cells harboring control shRNA were generated. The tumors from FOXA1-knockdown cells were smaller than the tumors from the control cells by thirty-one days after implantation (Figure 9A, 9B). The average weight of FOXA1-knockdown tumors was significantly less than that of the control tumors. These results together suggest a model in which FOXA1 acts as a transcriptional activator to enhance the cyclin B1 promoter activity through the key binding site in the promoter to increase its expression, which is required for the activation of mitosis during cell cycle progression (Figure 9C).

Figure 8.

Figure 8

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FOXA1 binds specifically to the CCNB1 promoter in chondrosarcoma cells. A. The FOXA1 binding motif on CCNB1 promoter was predicted by PROMO 3.0 and is visually represented in WebLogo (http://weblogo.berkeley.edu). A schematic of the FOXA1 binding site located in CCNB1 promoter region within -1000 and -400 bp to transcription starting site. B. Luciferase reporter assay of the promoter activities of CCNB1 promoter-deletion mutants with or without ectopic expression of FOXA1 in 293 cells. C. One FOXA1 binding site located in the CCNB1 promoter within -400 bp to transcription starting site was mutated by site direct mutagenesis. Reporter assay of CCNB1 promoter activity with FOXA1-binding site mutations in 293 cells ectopically expressing vector control or FOXA1. D. CH2879 cells stably expressing vector control or FOXA1 were subjected to ChIP assay. Cross-linked chromatin was immunoprecipitated by utilizing FOXA1 or IgG antibodies. The input and immunoprecipitated DNA were subjected to q-PCR primers corresponding to the promoter region of CCNB1 (Table S1). P values were determined by Student’s t-test, error bars represent ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9.

Figure 9

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Knockdown of FOXA1 attenuates chondrosarcoma tumor growth in vivo. A. Representative images of tumors harvested from subcutaneous mice xenografted with CH2879-control or FOXA1-knockdown cells. B. Tumor volume was monitored every 3 days and plotted. n = 4 per group. C. A proposed model of FOXA1 transcriptionally mediated chondrosarcoma cell cycle progress. In chondrosarcoma cells, FOXA1 binds specifically to the indicated region of CCNB1 promoter to facilitate its expression and thereby promote G2/M transition via cycllinB1-CDK1 complex activation in the cell cycle. Data are shown as means ± SD; Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Discussion

In this study, we have demonstrated that chondrosarcoma cells highly express FOXA1 protein compared with normal chondrocytes and further revealed that depletion of FOXA1 reduceed the proliferation, migration, and colony forming activities in chondrosarcoma. We have found that shRNA-mediated silencing of FOXA1 significantly suppresses proliferation through G2/M arrest, which likely results in apoptosis. Conversely, ectopic expression of FOXA1 promoted colony forming activity of chondrosarcoma. We showed that increased FOXA1 expression directly targeted to the cell cycle activator cyclin B1 promoter and activated cyclin B1 transcription. In chondrosarcoma, induction of cyclin B1 expression leads to CDK1 activation, consequently promoted of G2/M transition. Also, this FOXA1-cyclin B1 mediated cell cycle progression negatively correlates to p53 and p21 dependent pathway. This suggests that FOXA1 may exert an oncogenic role in chondrosarcoma progression.

In prior studies, FOXA1 has been shown to be amplified and overexpressed in esophageal, lung adenocarcinomas, anaplastic thyroid carcinoma (ATC), and ovarian cancer [29-31]. In ATC, it has been reported that knockdown of FOXA1 substantially inhibits cell proliferation by blocking G1-S transition and enhancing expression of the cell cycle inhibitor p27kip1 [30]. Mounting evidence supports that FOXA1 functions as a pioneer factor for steroid hormone receptors (SR), including the androgen receptor (AR) in prostate cancer cells and the estrogen receptor (ER) in breast cancer cells, controlling AR- and ER-regulated hormone responsive genes [32-34]. In androgen-independent prostate cancer such as castration-resistant prostate cancer, FOXA1 binding is essential for AR recruitment to regulate G2/M phase progression by directed transcriptional regulation of G2/M phase-specific genes, including ubiquitin-conjugating enzyme E2C (UBE2C) and CDK1 [35]. Additionally, FOXA1 regulates the cell cycle by driving G1 to S transition in AIPC through a steroid receptor (SR)-independent mechanism. It has been shown that FOXA1 acts as a master cell cycle regulator not only via direct regulation of cyclin E2 (CCNE2) but also indirect regulation of cyclin A2 (CCNA2) and E2F1 in AIPC [36]. Consistent with the critical role of FOXA1 in modulating the cell cycle, we found a previously undescribed yet pivotal role for FOXA1: direct targeting cyclin B1 (CCNB1) for G2/M progression in chondrosarcoma. Hence, in chondrosarcoma, FOXA1 seems to mainly function as a transcription factor to directly mediate G2/M cell-cycle related genes rather than as a co-factor. This transcriptional regulation mechanism is also similar to other forkhead box (Fox) transcription factors such as FoxM, which has been shown acts as a critical effector of cell cycle progression driving expression of cyclin B and cyclin A in lung cancer [37] and prostate cancer [38], respectively. FOXA1 overexpression is capable of increasing p27kip1 expression in breast cancer cell models [16], suggesting that FOXA1 promotes cell cycle progression hence cell growth in different cell types.

FOXA1 contains a helix-turn-helix motif and is flanked on either side by a “wing” domain that affects the stability of DNA binding [39]. This DNA binding domain directly binds to the consensus sequence AAR(C/T)AA in the major groove [40]. Since the DNA recognition element for FOXA1 lacks a GC-rich sequence, DNA binding by FOXA1 may not be subject to status of DNA methylation within chromatin. There is profound evidence supporting that epigenetic modification of histones, including the methylation of histone H3 lysine 4 (H3K4), plays an important role in FOXA1-mediated transcription [32]. Through genome-wide survey of FOXA1-binding sites analysis in MCF-7 breast cancer cells, 12,904 FOXA1-binding motifs were found, which represents only 3.7% of the sites comprising FOXA1-recognition elements. The active transcription events such as mono- and di-methylation of H3K4 correlate with FOXA1 recruitment. Thus it appears that chromatin-modifying enzymes may indirectly manipulate FOXA1 activity via redirecting FOXA1 to specific regions of the chromatin harboring histone H3K4 methylation [32]. However, the FOXA1-targeted genes and their epigenetic signatures mediated by FOXA1 in chondrosarcoma are poorly understood.

FOX proteins may become therapeutic targets by utilizing both chemical and biological strategies. However, many of these strategies are in early stages, and the clinical effectiveness in tumor suppression awaits further investigation. The antibiotic thiazole compound siomycin A has been shown to reduce the expression and transcriptional activity of FOXM1, leading to suppression of FOXM1 target genes and promotion of apoptosis in FOXM1-expressing osteosarcoma cells [41]. The same research group also generated a cell-penetrating alternative reading frame (ARF)26-44 peptide inhibitor of FOXM1 to effectively and selectively increase apoptosis in human hepatocellular carcinoma cell lines and mouse models [42]. Recently, the small molecule inhibitor, MC-1-F2, was shown to be able to upregulate cadherin switching and reverse epithelium mesenchymal transition (EMT) by degrading FOXC2 and inhibiting its unclear localization in breast cancer [43]. In light of these findings, direct inhibition of FOXA1 entailing peptides or small molecule inhibitors may be a promising therapeutic strategy of chondorsarcoma.

In summary, here we show that a) increased expression of FOXA1 is a frequent event in human chondrosarcoma cells, b) inhibition of FOXA1 in chondrosarcoma cells suppresses cell proliferation, migration, and cologenic activities, c) knockdown of cyclin B1 in FOXA1 overexpression CH2879 cells attenuates cell proliferation and d) FOXA1 may substantially modulate chondrosarcoma progression by regulating expression of the cell cycle activator cyclin B1.

Acknowledgements

We thank Ian Crews, Drs. Yung-Luen Yu and Shao-Chun Wang for the critical editing of the paper. This work was supported in part by the following grants: Ministry of Science and Technology, Taiwan (MOST104-2320-B-039-047-MY3, MOST106-2320-B-039-047, and MOST107-2320-B-039-003 to Y.-H.C.; MOST106-2320-B-039-051-MY3 and MOST107-2320-B-039-004 to Y.-L.Y.; MOST-107-2320-B-039-002, MOST-106-2320-B-039-049-MY3 to S.-C.W.; the Ministry of Health and Welfare, Taiwan (MOHW107-TDU-B-212-112-015). And the work was also financially supported by the “Drug Development Center, China Medical University” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan, ROC.

Disclosure of conflict of interest

None.

Supporting Information

ajcr0008-1989-f10.pdf (150.4KB, pdf)

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