The transcription factor FOXA1 induces epithelial ovarian cancer tumorigenesis and progression

Abstract

FOXA1 (forkhead box A1), a member of the FOXA transcription factor superfamily, plays an important role in tumor occurrence and development. However, the relationship between FOXA1 and ovarian cancer has not been reported. We examined normal ovarian tissue and ovarian cancer tissue and found increased FOXA1 expression in the cancer tissue. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assays demonstrated that transfection with small interfering RNA to silence FOXA1 (si-FOXA1) in ovarian cancer cell lines decreased cell proliferation and induced apoptosis and S-phase arrest. In addition, si-FOXA1 transfection inhibited cell migration and invasion. Western blotting showed that si-FOXA1 transfection decreased the levels of YY1-associated protein 1, cyclin-dependent kinase 1, cyclin D1, phosphatidylinositol-3 kinase, E2F transcription factor 1, B-cell lymphoma 2, and vascular endothelial growth factor A protein. Based on these results, we suggest that FOXA1 plays a catalytic role in ovarian cancer pathogenesis and development by affecting the expression of the above-mentioned proteins.

Introduction

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy; ¹ more than 70% of patients with ovarian cancer are diagnosed with advanced disease because of a lack of sensitive screening tests for early detection. ² Despite advancement in the development of new therapeutic methods, the 5-year survival rate of EOC has remained at approximately 30%. ³ Therefore, understanding the molecular mechanisms of the early events in EOC and searching for novel biomarkers involved in EOC progression are of great value for identifying patients with early-stage disease.

Forkhead box A1/hepatocyte nuclear factor 3α (FOXA1/HNF3α), forkhead box A2/hepatocyte nuclear factor 3β (FOXA2/HNF3β), and forkhead box A3/hepatocyte nuclear factor 3γ (FOXA3/HNF3γ) constitute the FOXA subfamily, which plays an essential role in the development and maintenance of the endoderm-derived organs and are also associated with malignancy. ⁴ As an important transcription factor, FOXA1 plays an important role in metabolism, organ development, and disease. Research on mouse models has found that in adult mice, organs and tissue differentiated from the endoderm, mesoderm, or ectoderm all express FOXA1.^5,6 In organ development, the role of FOXA1 is obvious in hormone receptor–positive organs such as the breast and prostate. The same role has been reported for FOXA1 in kidney, brain, and gastrointestinal tract development.^6,7 The definition of the role of FOXA1 in human malignancy is incomplete, as both pro- and anti-tumorigenic functions have been uncovered.^8–11 There was positive FOXA1 staining in 96.2% of estrogen receptor (ER)-positive breast cancer, and it serves as a good biomarker of breast cancer (except triple-negative breast cancer); ¹² in ER-negative breast cancer, FOXA1 overexpression can resist bisphenol A–induced epithelial–mesenchymal transition. ¹³ In bladder urothelial carcinoma, loss of FOXA1 expression is associated with progression and increased proliferation and metastasis. It is also an independent prognostic factor for decreased survival rates in bladder cancer. ¹⁴ However, little is known of the action of FOXA1 in ovarian cancer. We focused on FOXA1 in the tumorigenesis and development of ovarian cancer and the underlying mechanisms.

Materials and methods

Tissue specimens

EOC and normal ovary tissue specimens were collected from patients who had undergone surgical resection at the Department of Gynecology of the First Affiliated Hospital of China Medical University (Shenyang, China). Two pathologists confirmed the tumor specimens independently. No patient had received preoperative radiotherapy or chemotherapy. Informed consent was obtained from all subjects, and the study was approved by the China Medical University Ethics Committee (No. 2014-27), and all specimens were made anonymous and handled following ethical and legal standards.

Cell culture and transfection

The OVCAR3 and A2780 human ovarian carcinoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (HyClone Laboratories Inc., Logan, UT, USA) and RPMI 1640 (HyClone Laboratories Inc.), respectively, in 5% CO₂ and 37°C, and the culture medium was supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). The si-FOXA1 (sense: CACACAAACCAAACCGUCAdTdT) and the mock (sense: UGACGGUUUGGUUUGUGUGdTdT) transfections were carried out using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Cells were seeded in 96-well plates at a density of 3000 cells per well. At 0, 24, 48, and 72 h after transient transfection, 20 µL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) was added to the wells, which were incubated at 37°C for another 4 h. The medium was then removed and replaced by 150 µL dimethyl sulfoxide. After 10-min shaking, the absorbance at 490 nm was detected using a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA).

Apoptosis assay

The cells were collected, centrifuged for 5 min, and washed with cold phosphate-buffered saline (PBS) twice. Then, the cells were resuspended in the dark in 100 µL 1× buffer plus 5 µL propidium iodide (PI) and 5 µL fluorescein isothiocyanate (FITC)-labeled annexin V (BD Biosciences) per sample. After 400 µL 1× buffer was added, the total apoptotic rate of each sample was determined by flow cytometry within 1 h.

Wound-healing assay

Cells (1 × 10⁶ per well) were seeded in a six-well plate and cultured to 80% confluence. The monolayers were scratched with a 200-µL pipette tip, and the cells were washed with PBS and cultured in FBS-free medium. The wounds were observed under microscope and photographed at 0, 24, and 48 h. The nude areas were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The wound-healing rate was represented by: (area of original wound − area of wound at different time points)/area of original wound × 100%.

Invasion assay

Matrigel-coated Transwell cell culture chambers (BD Bioscience, San Jose, CA, USA) were used for the invasion assay. Filters were coated with 30 µL basement membrane Matrigel (1:10). Cells (5 × 104 L⁻¹) resuspended in 200 µL serum-free medium were layered in the upper compartment of the Transwell inserts. The bottom chambers contained 600 µL complete medium serving as the chemoattractant. After 48 h of incubation at 37°C, cells on the upper chamber were removed using a cotton swab, and the invaded cells at the bottom of the top chamber were fixed with formaldehyde, stained with crystal violet, and counted under an Olympus fluorescence microscope (Tokyo, Japan).

Western blotting

Total cell proteins were harvested and lysed in radioimmunoprecipitation assay buffer containing protease inhibitors. Protein lysates were separated on 10% sodium dodecyl sulfate–polyacrylamide gels and electrotransferred to Hybond membranes (Amersham, Munich, Germany). Albumin Bovine V (BSA, 3%) was used to block the membranes for 2 h at room temperature. Subsequently, primary antibodies against cyclin-dependent kinase 1 (CDK1), cyclin D1 (CCND1), E2F transcription factor 1 (E2F1), B-cell lymphoma 2 (Bcl-2), phosphatidylinositol-3 kinase (PI3K), vascular endothelial growth factor A (VEGFA; Proteintech, Chicago, IL, USA) and YY1-associated protein 1 (YAP; ABclonal, Woburn, MA, USA) were incubated with the blots overnight at 4°C. The membranes were washed three times with Tris-buffered saline with Tween 20 (TBST) before they were incubated with the secondary antibody for 2 h at room temperature. Finally the proteins were visualized using an enhanced chemiluminescence system according to the manufacturer’s protocol (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Cell-cycle analysis

EOC cells were trypsinized, collected, washed, and fixed in 70% ice-cold ethanol and then washed again, and PI containing RNase A (BD Biosciences, San Jose, CA, USA) was added in the dark for 30 min. The cell-cycle profiles were determined by flow cytometry.

Immunohistochemistry

Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a different concentration of ethanol solutions and then incubated for 20 min in 3% H₂O₂ followed by antigen repair. Non-specific binding was blocked by incubation with 10% goat serum for 2 h at room temperature. Slides were then incubated overnight at 4°C with anti-FOXA1 primary antibody. An appropriate secondary antibody was added and incubated for 20 min at 37°C, and binding was visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB). After each treatment, slides were washed three times with TBST for 5 min. Positive expression was graded as follows: negative = 0; 1%–50% = 1; 51%–74% = 2; and more than 75% = 3. The staining intensity was graded as follows: weak = 1; intermediate = 2; and strong = 3. The two grades were mutiplied to obtain a final score: 0 = − ; 1–2 = +; 3–4 = ++; 6–9 = +++).

Statistical analysis

Data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) and are presented as mean ± standard deviation (SD). Two groups were compared using an unpaired, two-tailed Student’s t-test. All p values are two-sided, and p < 0.05 was considered to be statistically significant.

Results

FOXA1 expression was associated with EOC tumorigenesis

Immunohistochemistry (IHC) showed significantly higher FOXA1 protein expression in ovarian carcinoma tissue than in normal ovarian tissues (Figure 1; p < 0.05). Details can be found in Table 1.

Figure 1.

Correlation of FOXA1 expression with tumorigenesis of ovarian carcinoma. Our immunohistochemistry assay showed that FOXA1 was upregulated in primary ovarian cancer tissues than normal ovarian tissues (Table 1; p < 0.05).

Table 1.

FOXA1 expression in ovarian tissues.

Groups	FOXA1 (− = 0, + = 1, ++ = 2, and +++ = 3)				Total	PR (%)	p value
	0	1	2	3
Normal ovarian tissues	19	2	0	0	21	9.52	0.034
Ovarian carcinoma	104	42	5	2	153	32.03

PR: positive rate; FOXA1: forkhead box A1.

The value given in boldface represents p < 0.05.

Effect of FOXA1 on EOC cell proliferation

The tetrazolium (MTT) proliferation assay was used to observe EOC cell viability. EOC cell proliferative ability was significantly decreased after FOXA1 expression had been downregulated (Figure 2; p < 0.05).

Figure 2.

Si-FOXA1 inhibited ovarian carcinoma cells proliferation. Silence of FOXA1 showed decline of cell viability in MTT (*p < 0.05) compared with control and mock-transfected cells.

Effect of FOXA1 on EOC cell cycle

Flow cytometry showed that si-FOXA1 induced significant S-phase arrest as compared to the control and mock-transfected cells (Figure 3; p < 0.05).

Figure 3.

Si-FOXA1 induced S-phase arrest in ovarian carcinoma cells. Si-FOXA1 induced significant S-phase arrest as compared to the control and mock-transfected cells by flow cytometry (p < 0.05).

Effect of FOXA1 on EOC cell apoptosis

Flow cytometry demonstrated that si-FOXA1 transfection increased the percentage of cells in early-stage apoptosis at 48 h after transfection (Figure 4; p < 0.05).

Figure 4.

Si-FOXA1 induced apoptosis in ovarian carcinoma cells. Flow cytometry demonstrated that si-FOXA1 transfection promotes apoptosis of ovarian carcinoma cells at 48 h after transfection (p < 0.05).

Changes in EOC cell migration and invasive ability

The wound-healing experiment showed that FOXA1 silencing decreased EOC cell migration (Figure 5(a); p < 0.05). Transwell assay evaluation showed significantly suppressed invasive ability of si-FOXA1-transfected EOC cells (Figure 5(b); p < 0.05) as compared with the mock-transfected group.

Figure 5.

Si-FOXA1 inhibited EOC cell migration and invasive ability. (a) Si-FOXA1 decreased EOC cell migration ability (p < 0.05). (b) si-FOXA1-transfected decrease of the invasion ability of EOC cells (p < 0.05).

Effects of si-FOXA1 transfection on EOC cell genotype in vitro

Downregulated FOXA1 reduced the expression of YAP, CDK1, CCND1, PI3K, E2F1, Bcl-2, and VEGFA protein (Figure 6; p < 0.05).

Figure 6.

Si-FOXA1 transfection changes the genotype of on EOC cell. Si-FOXA1 reduced the expression of YAP, CDK1, cyclin D1 (CCND1), PI3K, E2F E2F1, Bcl-2, and VEGFA protein (p < 0.05).

Discussion

To explore the role of FOXA1 in ovarian cancer pathogenesis and development, we examined FOXA1 expression in tissue from normal ovaries and ovarian carcinoma using IHC and found increased FOXA1 protein expression. The results suggest that FOXA1 can promote ovarian cancer.

In FOXA1-silenced ovarian cancer cell lines, cell proliferation, migration, and invasion were decreased; early apoptosis was increased significantly; and S-phase arrest was induced, which indicate the potential oncogenic role of FOXA1 in ovarian cancer pathogenesis and development. Silencing FOXA1 decreased the expression of the YAP, CDK1, CCND1, PI3K, E2F1, Bcl-2, and VEGFA proteins.

FOXA1 opening of the compacted chromatin facilitates cyclic adenosine monophosphate (AMP) response element–binding protein (CREB)-mediated YAP transcription. Metallothionein 1D, pseudogene (MT1DP)-inhibited liver cancer cell growth in vivo was rescued by a combination of FOXA1, runt-related transcriptionfactor 2 (RUNX2), and YAP over expression. ¹⁵ Excessive YAP activation increased ovarian cancer cell proliferation and migration, resistance to chemotherapeutic drugs, and anchorage-independent growth. ¹⁶ In castration-resistant prostate cancer (CRPC), FOXA1 acts as a pivotal driver of the cell cycle, promoting G₁–S transition and G₂–M transition. Specific FOXA1 binding sites directly upregulate CCNE2 in CRPC cells and upregulate CCNA2 via E2F1. ¹⁷ CDKs, including three interphase CDKs (CDK2, CDK4, and CDK6) and a mitotic CDK (CDK1), are vital regulators of cell-cycle progression and are likely to play irreplaceable roles in cell-cycle regulation. The cyclin family consists of CCNA–Y, and CCNA2, CCNB1, CCND1, and CCNE play major roles in regulating the cell cycle.^18,19 CCND1 plays an important and positive role during the key rate-limiting point of G₁–S transition. Activation of the PI3K/AKT and/or extracellular signal–regulated kinase (ERK) pathways can lead to cell proliferation and growth by regulating CCND1 levels. ²⁰ However, VEGF directly induces tumor angiogenesis by increasing microvessel permeability, thereby creating a favorable environment for endothelial cell proliferation. ²¹ Bcl-2 is a member of the Bcl-2 family of anti-apoptotic proteins. ²²

Based on this study results, we suggest that FOXA1 acts as an oncogene in the pathogenesis and development of ovarian cancer by influencing the expression of multiple proteins. This may provide new ideas for diagnosing and treating ovarian cancer, and further in-depth exploration of the molecular mechanisms involved in ovarian cancer is warranted.