PGC-1β cooperating with FOXA2 inhibits proliferation and migration of breast cancer cells

Human breast cancer cell lines (MCF-7 and MDA-MB-231) and normal human breast cell line (MCF-10A) were purchased from the Cell bank of the Chinese Academy of Sciences/American Type Culture Collection (ATCC, VA, USA). Human breast cancer cell line (BT-549) was donated by Dr. Liu from the Affiliated Hospital of Qingdao University. The above cells were maintained and cultured in DMEM HIGH GLUCOSE medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (Gibco, Australia) and 1% penicillin–streptomycin solution in the incubator at 37 °C under 5% CO₂.

A total of 30 paired breast cancer tissues and adjacent non-tumor tissues samples were collected and obtained undergoing breast carcinectomy from the General Hospital of Ningxia Medical University. The fresh samples were frozen and stored in liquid nitrogen until further use. All the tissue samples were signed by patients with informed consent.

The cells were harvested and lysed using RIPA lysis buffer containing configure RIPA, PIC, and PMSF at a ratio of 100:2:1 (Beyotime Biotechnology, Shanghai, China). The total protein was extracted and the protein concentration was detected by BCA protein reagent kit (Thermofisher Scientific, Inc). The samples were electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% defatted milk in TBST for 1 h at room temperature and then incubated with specific primary antibodies overnight at 4 °C (Table 1). Subsequently, the HRP-conjugated secondary antibodies were incubated at room temperature for 1.5 h. The protein bands were detected and scanned by BioImaging Systems (BIO-RAD, CA, USA). Anti-GAPDH antibody was used as an internal control to normalize protein levels.

Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, CA, USA) as per manufacturer’s instructions. cDNA synthesis was performed by using a reverse transcription kit (TaKaRa, ShangHai, China) according to the manufacturer’s protocols. The PCR amplification was performed with specific primers and carried out using the SYBR-Green PCR system (Takara Bio, Inc). GAPDH or β-actin served as internal control. Calculation of the relative expression of each gene was quantified by the 2^−ΔΔCt method.

The plasmids, including shRNA for PGC-1β (sh-PGC-1β), shRNA-NC (NC), pcDNA3.1-PGC-1β (PGC-1β), pcDNA3.1-FOXA2 (FOXA2), shRNA for FOXA2 (sh-FOXA2) were designed and synthesized by Shanghai Genechem Co.Ltd (Shanghai, China). MCF-7 and MDA-MB-231 cells were plated in six-well plates, and the cells were transfected with 2.5 µg plasmid using Lipofectamine 3000 (Invitrogen, CA, USA) when the cell density reaches 70–80% confluence according to the manufacturer’s protocol. Cell transfection efficiency was observed under fluorescence microscope and examined using qRT-PCR and western blot assays.

The cell proliferation ability was assessed with CCK-8 assay and clone formation assay. For the CCK-8 assay, MCF-7 or MDA-MB-231 cells were seeded in 96-well plates and were cultured for 24, 48, 72 h, respectively. The cells were then treated with CCK-8 reagent (KeyGEN BioTECH, Jiangsu, China) and further cultured for 2 h according to the manufacturer’s instructions. The optical density was measured using the spectrophotometer (Glomax Multi Detection Systerm, Promega, USA) at 450 nm. Each group of experiments included five replicates and repeated three times.

For the colony formation assay, 8 × 10³ cells were added into 6-well plate and suspended in DMEM medium. The cells were exchanged every 3 days until the 12 days. Then, these dishes were fixed with 100% methanol for 15 min and stained with crystal violet for 10 min. The number of colonies were observed and calculated from representative areas. All experiments were performed in triplicate.

The migration ability of the tumor cells were examined by transwell and wound healing assays. The cells were seeded in twelve-well plate and transfected with interfering or overexpressing plasmids for 48 h. The cell monolayer was wounded by scraping with 200 µl pipet tip and the exfoliated cells were washed with PBS. Subsequently, the speed of wound closure was observed and photographed under a microscope. Cell mobility was assessed by measuring three randomly perpendicular wound width. For the transwell assay, Corning Incorporated transwell Chambers (Corning, 8 μm, NY, USA) were used to detect the cells of migration capacity. The chambers were placed into a 24-well plate containing culture medium supplemented with 20% FBS as a chemo-attractant. After transfection with 48 h, MCF-7 and MDA-MB-231 cells were collected and suspended in serum-free medium and loaded onto the upper chamber, incubated and allowed to migrate at for 24 h and 48 h, respectively. The cells were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet for 10 min. The non-invading cells were removed using swab, whereas total numbers of cells were imaged and counted from representative areas under a microscope (Olympus, Tokyo, Japan).

MCF-7 and MDA-MB-231 cells were planted in six-well plate and treated with transfection reagent for 48 h, and the cells were digested with trypsin without EDTA and counted 1 × 10⁶ cells for analysis of each sample. After being washed twice with cold PBS, the cells were collected and prepared to form a single cell suspension in a binding buffer, and stained with AnnexinV-APC for 10 min and propidium iodide (PI) for 10 min according to instructions of the AnnexinV-APC/PI apoptosis detection kit (BestBio, Shanghai, China). Samples were examined using BD flow cytometer and the data were analyzed with FlowJo software (TreeStar Corporation, Ashland, OR, USA).

Cells were grown in 24-well chamber slides (NEST, USA) for 48 h, and washed with cold PBS and fixed in 4% paraformaldehyde for 15 min. Subsequently, the coverslips were permeabilized with 0.3% Triton X-100 for 6 min and blocked with 5% normal goat serum for 30 min at room temperature. Then, the cells were incubated with anti-PGC-1β (ab176328, Abcam) and anti-FOXA2 (ab117542, Abcam) overnight at 4 °C, and followed by incubation with fluorescent secondary antibody for 1 h at room temperature. The nucleus was visualized with DAPI solution (dilution: 1:1000, Sigma, USA) for 6 min in the dark. Confocal laser scanning microscope was utilized for image analysis.

The paraffin-embedded tissues were deparaffinized using xylene and rehydrated through graded concentrations of alcohol solution. The slides were incubated with 0.3% hydrogen peroxide for 30 min to inactivate endogenous peroxidase prior to the infiltration of the citrate buffer with the tissue parts to repair the antigen in a microwave. The slides were rinsed with PBS for 15 min, blocked with 5% goat serum for 1 h at room temperature and incubated with the primary antibodies at 4 °C overnight. Subsequently, the slides were incubated with biotinylated secondary antibody at room temperature for 1 h. The sections were visualized with 3, 3′-diaminobenzidine (DAB) chromogenic fluid and counterstained with hematoxylin (CWBIO, Beijing, China).

293T and MCF7 cells were seeded in 10 cm dish, and were co-transfected with PGC-1β and FOXA2 overexpression plasmids by Lipofectamine 3000. The cells were lysed using RIPA lysis buffer containing protease inhibitor cocktails (Thermo, USA). The cell lysates were used in immunoprecipitation after centrifuged at 12,000 rpm for 20 min. The proteins extracted from the cells were incubated the primary antibody or normal mouse/rabbit IgG at 4 °C for 4–6 h and then incubated with 30 μl Protein A/G Plus-Agarose beads (GE Healthcare Bio-Sciences, Sweden) for overnight. The immune complexes were precipitated by centrifuged for 5 min, and the precipitates were washed three times with corresponding washing buffer. The precipitates were resuspended in 5× SDS-PAGE sample loading buffer to boiled (100 °C) for 10 min for western blot assay.

MDA-MB-231 cells were infected with lentivirus vector and subcutaneously implanted into 4–6-week-old female BalB/C nude mice, which purchased from the Beijing Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). The tumor nodules were assessed with electronic calipers by recording tumor length and width for 15 days. Subsequently, the mice were euthanized and tumor mass were collected, weighed and photographed. All animal experiments were performed in accordance with principles and guidelines approved by the Ethics Committee of Animal Research.

Statistical testing was performed with the SPSS 17.0, GraphPad Prism version 6.0 and Microsoft Office Excel 2013 software. All assays were conducted three times and results were presented as mean ± SD. Statistical differences among groups were determined by two-tailed Student t test or one-way analysis of variance (one-way ANOVA). Results were considered significant when P values < 0.05.

To detect expression of PGC-1β and FOXA2 in breast cancer, qRT-PCR was performed on 30 pairs of breast cancer tissues and adjacent normal tissues. Elevated levels of PGC-1β and low levels of FOXA2 were observed in breast cancer tissues (Fig. 1a). Moreover, we examined the expression of both genes in human breast cancer cell lines (MCF-7, MDA-MB-231 and BT-549) and normal breast cell line (MCF-10A). Consistent with the findings in the clinical breast cancer specimens, the PGC-1β mRNA and protein expression levels were significantly upregulated in the breast cancer cell lines. Whereas, the expression of FOXA2 was negatively correlated with PGC-1β (Fig. 1b, c). Results from human breast cancer tissues indicated that abnormal expression of PGC-1β was closely related to the clinicopathological characteristics of patients, such as Lymph node metastasis (P = 0.026) and Progesterone receptor (P = 0.026) (Table 2). Immunohistochemical assay showed strong staining of PGC-1β and weak staining of FOXA2 were observed in breast tumor tissues, which was consistent with the above results (Fig. 1d). Moreover, we detected the localization of PGC-1β and FOXA2 through immunofluorescence assay. It was found that PGC-1β localized mainly in the cytoplasm, and FOXA2 localized mostly in the nucleus (Fig. 1e). Collectively, these data clearly demonstrated that abnormal expression of PGC-1β and FOXA2 were frequent event in breast cancer, and they might play vital roles in the development and progression of breast cancer.

To confirm the interaction of PGC-1β and FOXA2, we detected the binding of two proteins in MCF-7 cell by Co-immunoprecipitation (Co-IP) assay. The FLAG-tagged PGC-1β and HA-tagged FOXA2 were co-expressed into MCF-7 cells. As shown in Fig. 2a, anti-FLAG antibody was co-precipitated with HA-FOXA2. However, HA-FOXA2 did not pull down FLAG-PGC-1β. Co-IP assay indicated that endogenous PGC-1β protein might target to FOXA2 in breast cancer. The subcellular colocalization of PGC-1β and FOXA2 were examined to further determine whether PGC-1β can combine with FOXA2 physically through immunofluorescence (IF) staining. The overexpression plasmids of PGC-1β and FOXA2 were transfected into HEK293T and MCF-7 cell lines. The cells were fixed and incubated with anti-PGC-1β and anti-FOXA2 antibodies. PGC-1β was found to be mainly expressed in the cytoplasm in the cells transfected with PGC-1β plasmid alone, and FOXA2 was expressed in the nucleus in the cells transfected with FOXA2 plasmid only. Interestingly, PGC-1β and FOXA2 were co-localized in the nucleus when the cells were co-transfected with PGC-1β and FOXA2 overexpression plasmids (Fig. 2b, c). These findings suggested that PGC-1β and FOXA2 can physically interact with each other and form complex.

The specific shRNA against PGC-1β (sh-PGC-1β) and overexpression against FOXA2 (FOXA2) were transfected with MCF-7 and MDA-MB-231 cells. The results indicated that compared with the corresponding control group (NC), transfection of sh-PGC-1β reduced the expression of endogenous PGC-1β (Fig. 3a) and transfection of FOXA2 overexpression significantly increased FOXA2 expression (Fig. 3d) by qRT-PCR assay. At the same time, the MCF-7 and MDA-MB-231 cells were also transfected with the overexpression plasmid of PGC-1β and the interference plasmid of FOXA2. The results showed that there were no significant difference between the two groups (P > 0.05, Fig. 3b, c). Western Blotting results showed that transfected with PGC-1β interference and FOXA2 overexpression vector, the proteins expression of PGC-1β and FOXA2 were decreased and increased, respectively (Fig. 3e, f). Therefore, the PGC-1β interference and FOXA2 overexpression vectors were used for the subsequent experimental studies.

To explore the biological functions of PGC-1β and FOXA2 in breast cancer progression, we examined the effects of both genes on cell proliferation and clonogenicity by CCK-8 and colony formation assays. The results demonstrated that the cell viability and proliferation were inhibited in MCF-7 and MDA-MB-231 cells after knockdown of PGC-1β or overexpression of FOXA2. It is worth noting that co-transfection with plasmids of sh-PGC-1β and FOXA2 dramatically reduced cell proliferation ability compared with individual transfection (Fig. 4a, b). Afterwards, we assessed whether PGC-1β and FOXA2 were involved in regulating the cells migration. Consistent with the above results, the cell migration was more significantly inhibited when co-transfected with both of plasmids (Fig. 4c, d). Overall, knockdown of PGC-1β combined with overexpression of FOXA2 apparently inhibited proliferation and migration of breast cancer cells in vitro.

With AnnexinV-APC/PI staining and flow cytometry analysis, we evaluated the effects of PGC-1β and FOXA2 on cell apoptosis. The results indicated that downregulation of PGC-1β or upregulation of FOXA2 promoted apoptosis of MCF-7 and MDA-MB-231 cells compared to the control groups. Of note, co-transfection with plasmids of sh-PGC-1β and FOXA2 significantly increased cell apoptosis in comparison to that in cells with individual transfection (Fig. 5a, b). Subsequently, mouse subcutaneous xenograft experiments were performed to detect the effects of PGC-1β and FOXA2 on tumor growth in vivo. The tumor weight of xenografts derived from LV-sh-PGC-1β or LV-FOXA2 were significantly lighter compared with LV-NC. However, there was no significant difference in tumor weight when co-transplanted with LV-sh-PGC-1β and LV-FOXA2 (P > 0.05, Fig. 5c). Additionally, immunohistochemistry assay proved that PGC-1β inhibition or FOXA2 overexpression effectively reduced Ki67 expression compared with the NC group (Fig. 5d). The above results indicated that the combination of sh-PGC-1β and FOXA2 further suppressed xenograft tumor growth and promoted apoptosis in breast cancer.

PI3K-AKT-mTOR pathway has been reported that frequently involves in variety of human cancers and regulates numerous cellular processes, such as cell growth, proliferation and migration [34]. To investigate the molecular mechanisms of PGC-1β and FOXA2 in breast cancer progression, we detected the expression of related proteins of PI3K-AKT-mTOR pathway. The results showed that the expression of PGC-1β, p-PI3K, p-AKT, PDK1, mTOR and Raptor were downregulated in MCF-7 cells when transfected with sh-PGC-1β. However, the expression of FOXA2 was increased (Fig. 6a). Then, we found that the expression of p-PI3K, p-AKT, PDK1, p-mTOR, p-S6K1 and Raptor were significantly decreased when co-transfected with sh-PGC-1β and FOXA2 (Fig. 6b). To further prove the role of PI3K-AKT-mTOR pathway in breast cancer growth and metastasis, we use rapamycin, an inhibitor of mTOR protein, to suppress the PI3K-AKT-mTOR pathway. We found the downregulation of p-PI3K, p-AKT and mTOR being induced (Fig. 6c). All the above results confirmed that the combination of PGC-1β and FOXA2 inhibited breast cancer cells proliferation, migration and induced apoptosis through regulating the PI3K-AKT-mTOR signaling pathway.