Human breast cancer cell line, MDA-MB-231, is highly invasive and aggressive, compared to less invasive cell line, MCF-7. To explore the genes that might influence the malignancy of MDA-MB-231, DNA microarray analysis was performed. The results showed that G0/G1 switch 2 (G0S2) was one of the most highly expressed genes among the genes upregulated in MDA-MB-231. Although G0S2 acts as a direct inhibitor of adipose triglyceride lipase, action of G0S2 in cancer progression is not yet understood. To investigate whether G0S2 affects invasiveness of MDA-MB-231 cells, G0S2 expression was inhibited using siRNA, which led to decreased cell proliferation, migration, and invasion of MDA-MB-231 cells. Consequently, G0S2 inhibition inactivated integrin-regulated FAK-Src signaling, which promoted Hippo signaling and inactivated ERK1/2 signaling. In addition, G0S2 downregulation decreased β-catenin expression, while E-cadherin expression was increased. It was demonstrated for the first time that G0S2 mediates the Hippo pathway and induces epithelial to mesenchymal transition (EMT). Taken together, our results suggest that G0S2 is a major factor contributing to cell survival and metastasis of MDA-MB-231 cells.
Triple-negative breast cancer is a type of breast cancer that lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor (Her2/neu receptor) (Mukherjee and Zhao, 2013; Yadav
The Hippo pathway initially was known as a regulator which controls organ size and proliferation. Recent studies have discovered that the Hippo pathway is strongly involved in cell-cell contraction, cell polarity, apoptosis, and cell cycle. In cancer, the Hippo pathway is a tumor suppressor, and its deregulation is recognized as an important feature in many cancers. The transcription coactivator YAP1, which is a major target of the Hippo pathway, is implicated with induction of EMT as well as proliferation, invasion, metastasis. YAP1 induces expression of genes such as amphiregulin, cysteine-rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) through TEA domain (TEAD) family transcription factors and they are highly activated in metastatic breast cancer cells (Lamar
The G0/G1 switch gene 2 (G0S2) is a protein comprising 103 amino acids (Zandbergen
Besides, there have been several studies related to association between G0S2 and cancers. G0S2 is dramatically hypomethylated in MDA-MB-231 cells compared with MCF-7 cells, which results in overexpression of G0S2 in MDA-MB-231 cells (Cheishvili
Several previous studies have focused on the roles of G0S2 in lipid metabolism. However, there have barely been any studies elucidating the functions and mechanisms of G0S2 in triple-negative breast cancer.
In the current study, we determined a strong correlation between the expression of G0S2 and malignancy of breast cancer cells. Our results demonstrated that knockdown of G0S2 reduced typical tumor progression features, such as cell proliferation, migration, and invasion of MDA-MB-231 cells. Furthermore, these results may be due to activation of the Hippo pathway as well as inactivation of the FAK-Src and ERK1/2 signaling. Thus, we suggested that G0S2 plays critical roles in cell survival and metastasis of MDA-MB-231 cells.
RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Hyclone (Logan, VI, USA). The bicinchoninic acid (BCA) protein assay kit, and goat-anti mouse IgM Alexa Flour 488 were purchased from Thermo Scientific (Waltham, MA, USA). The D-PlusTM CCK cell viability assay kit was purchased from Dongin LS (Seoul, Korea). Mitomycin C was obtained from Sigma (St. Louis, MO, USA). M-MLV reverse transcriptase and RNase inhibitor were purchased from Promega (Madison, WI, USA). dNTP mixture was obtained from TaKaRa Bio (Shiga, Japan). QGreenTM 2X SybrGreen qPCR Master Mix was purchased from CellSafe (Gyeonggi, Korea). Rabbit poly-clonal antibodies for FAK, p-FAK (Tyr925), Src, p-Src (Tyr416), p-ERK1/2 (Thr202/Tyr204), integrin α5, integrin β1, LATS1, p-LATS1 (Ser909), and p-YAP1 (S127) were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibodies for E-cadherin, β-catenin, ERK1, and CYR61, mouse monoclonal antibodies for YAP1 and α-tubulin, and goat anti-rabbit IgG Texas-Red were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody for G0S2 was obtained from CUSABIO (College Park, MD, USA). All other chemicals and reagents were of highest quality commercially available.
Human breast cancer cell lines BT-20, HCC-70, T47D, MDA-MB-231, and MCF-7 (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI 1640 medium containing 10% (v/v) heat-inactivated FBS, 100 μg/mL streptomycin, and 100 U/mL penicillin. MCF-10A human breast epithelial cells were obtained by Dr. Aree Moon (Duksung Woman’s University, Seoul, Korea) and cultured in DMEM/F12 medium (Welgene, Seoul, Korea) containing 5% horse serum, 0.5 mg/mL hydrocortisone, 10 μg/mL insulin, 100 ng/mL cholera toxin, 20 ng/ mL EGF, 100 μg/mL streptomycin, and 100 U/mL penicillin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Cells were transfected with G0S2-specific siRNA (37.5 nM, target sequence: 5′-TGCACTAGGGAGGAAGGATAA-3′, QIAGEN) with Neon® Transfection System (Invitrogen, Carlsbad, CA, USA) and cultured in RPMI 1640 medium supplemented with 10% FBS without antibiotics overnight. The medium was replaced with RPMI 1640 medium containing 10% FBS with antibiotics and cultured for 24 h.
G0S2 siRNA-transfected cells (7×103 cells/well) were seeded in 96-well cell culture plates. After stabilization for 48 h, 10 μL of D-PlusTM CCK solution was treated to each well, followed by incubation at 37°C for 2 h. The formed formazan dyes were quantified by measuring the absorbance at 450 nm with a SunriseTM microplate reader (Tecan, Männedorf, Switzerland).
Cells were transfected with G0S2 siRNA for 48 h, and then, the cells were harvested with 0.05% trypsin-EDTA and washed with PBS. The obtained cell pellets were fixed in 70% ethanol overnight at −20°C. After washed with PBS to remove ethanol, the cells were incubated in 200 μL of MUSE® cell cycle reagent (Merck Millipore, Darmstadt, Germany) for 30 min at room temperature in the dark. Cell cycle was measured by a MUSE® Cell Analyzer (Merck Millipore).
After culturing for 48 h with G0S2 siRNA, the cells were harvested with 0.05% trypsin-EDTA and washed with PBS. The cell pellets were treated with caspase-3/7 working solution containing a DNA-binding dye at 37°C for 30 min, and then, treated with 7-AAD working solution for 5 min at room temperature. Apoptotic cells were measured by fluorescence-activated cell sorting using a MUSE® Cell Analyzer (Merck Millipore).
Cells were transfected with G0S2 siRNAs, and then, 200 cells were seeded in a well of a 6-well culture plate and cultured for 10 days. The culture medium was changed every two days. Subsequently, 4% formaldehyde solution in PBS was treated for 10 min at room temperature to fix the cells. After that, the fixed cells were washed with PBS several times and stained with 0.2% crystal violet for 10 min. After drying enough, the number of colonies containing over 50 cells was measured using a microscope (OLYMPUS, Tokyo, Japan).
Transfected cells (7×105 cells/well) were seeded in 6-well culture plates. After stabilization for 48 h, cells with over 90% confluence were washed with PBS and incubated in mitomycin C solution (10 μg/mL) at 37°C for 2 h. After washing several times, a scratch into cell monolayer was created using a 200 μL pipette tip. Scratch images were captured using a microscope at the indicated time points.
Measurement of cell invasion was performed using the QCMTM 24 well Invasion Assay Kit (Merck Millipore) in accordance with the manufacturer’s instructions. In brief, cells transfected with G0S2 siRNA were seeded in cell culture inserts with 8-μm pore size polycarbonate membrane coated with reconstituted basement membrane extract. Invasive cells through the membrane were stained with 4′,6-diamidino-2-phenylindole (DAPI). Five fields per membrane in a cell culture insert were photographed using the ZEISS LSM700 Confocal Laser Scanning Microscope (Carl Zeiss, Jena, Germany) and the number of invasive cells was counted.
Total RNA was extracted using RibospinTM kit (GeneALL, Seoul, Korea). 0.5 μg of total RNA was reverse-transcribed as follows: initially at 37°C for 1 h, followed by at 95°C for 5 min in 20 μL total volume containing 5× RT buffer, 10 mM dNTPs, 100 pmol oligo-dT primer, 200 units of Moloney murine leukemia virus reverse transcriptase (M-MLV), and 40 units of RNase inhibitor. qPCR was conducted using the QGreenTM 2X SybrGreen qPCR Master Mix. Each reaction contained 10 μL of qPCR Master Mix, 2 μL of cDNA, 1 μM oligonucleotide primers in a final volume of 20 μL. Amplification was carried out as follows: one cycle of pre-denaturation 95°C for 5 min, 40 cycles of denaturation at 95°C for 15 s, followed by a combined annealing and extension step at each primer’s melting temperature (Tm) for 30 s. Data were analyzed using QIAGEN Rotor-Gene Q Series software (QIAGEN, Venlo, Netherlands).
Whole cells were harvested and solubilized with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA, 50 mM NaF, and 1 mM sodium orthovanadate. The total amount of protein in the lysates was measured by the BCA method in accordance with the supplier’s recommendations. Proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8 to 15% polyacrylamide gels and transferred onto 0.45 μm PVDF membranes. After that, membranes were blocked with 5% (w/v) bovine serum albumin or 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) at 4°C for 2 h, and then, incubated overnight with primary antibodies at 4°C. After washed in TBS-T 3 times 10 min at room temperature, membranes were incubated with secondary antibody for 2 h at 4°C. All these steps related to membranes were conducted on a shaker. Each target protein band was visualized by the enhanced chemiluminescence (ECL) (Bionote, Gyeonggi, Korea), and the band intensities were analyzed using a Chemi-Doc XRS densitometer and quantified using Quantity One software (Bio-Rad, CA, USA).
Cells were transfected with G0S2 siRNA and seeded on poly D-lysine-coated coverslips on a 6-well cell culture plate. After stabilization for 48 h, the cells were fixed with 4% formaldehyde solution in PBS at room temperature for 10 min. After washed with cold PBS, the fixed cells were incubated in 0.5% Triton X-100 at room temperature for 15 min to permeabilize the cells, followed by washing with cold PBS three times. The cells were blocked with 3% bovine serum albumin in PBS at room temperature for 1 h. Incubation steps of antibodies were conducted as follows: primary antibody (1:250) incubation at 4°C overnight, washing with cold PBS three times for 3 min, subsequently, incubation with goat anti-rabbit IgG-Texas Red (1:250) or goat anti-mouse IgM-Alexa Fluor 488 (1:250) at room temperature for 4 h. After washed with cold PBS several times for 3 min, the coverslips were mounted on glass slides using Ultra CruzTM mounting medium containing DAPI (Santa Cruz Biotechnology). Fluorescence signals were measured and captured using a ZEISS LSM 800 confocal laser scanning microscope (Carl Zeiss).
All data are shown as mean ± standard errors of the means (SEM). Comparison of two groups with one characteristic was conducted using unpaired Student’s
To explore which genes might affect the invasiveness of MDA-MB-231 cells, differential gene expressions were evaluated and compared between highly invasive MDA-MB-231 cells and less invasive MCF-7 cells by DNA microarray analysis. Table 1 shows the ten highly expressed genes in MDA-MB-231 cells compared with MCF-7 cells. The difference in
To investigate whether G0S2 overexpression is a characteristic of triple-negative breast cancer lines, mRNA levels of
To determine the association between G0S2 expression and relapse-free survival (RFS) of triple-negative breast cancer patients, Kaplan-Meier analysis from the KM plotter database (http://kmplot.com) was used. As shown in Fig. 2C, higher
To explore the role of G0S2 in tumorigenesis in MDA-MB-231 cells, cell viability and colony formation were determined. When cells were treated with G0S2 siRNA, approximately 20% of cell viability was decreased (Fig. 3A, 3B). In addition, G0S2 knockdown remarkably reduced the number of colonies (Fig. 3C). These results demonstrated that G0S2 induced cell proliferation and colony formation related to tumorigenesis in MDA-MB-231 cells.
To identify whether G0S2 affected apoptosis, cells were transfected with G0S2 siRNA, and then, cell cycle was analyzed by flow cytometry. As shown in Fig. 3D, the proportion of G0S2 knockdown cells in the G1 phase was higher in comparison to the control, whereas the proportion of cells in the S and G2/M phases was decreased relative to control. In addition, apoptosis was analyzed using caspase-3/7 staining and flow cytometry. Increase in the percentage of apoptotic cells (∼7%) was observed in cells treated with G0S2 siRNA compared to the control (Fig. 3E). These results were consistent with the increased percentage of sub-G1 in the cell cycle assay, which indicated increase in apoptosis (Fig. 3D). Together, these results showed that G0S2 knockdown prevented MDA-MB-231 cells from entering the S phase and induced apoptosis.
To further determine whether G0S2 is involved in the metastatic progression of MDA-MB-231 cells, wound healing and transwell invasion assays were performed. In wound healing assay, the results showed that the wound closure of the cells after treatment with G0S2 siRNA was inhibited by about 50% compared to the control at 24 h after scratching, while the wound of control cells was closed completely at 24 h after scratching (Fig. 4A). In the transwell invasion assay, the number of invaded cells across pores was significantly decreased in the group treated with G0S2 siRNA compared to the control (Fig. 4B). These results demonstrated that G0S2 promoted metastasis by inducing migration and invasion of MDA-MB-231 cells.
Integrins are well-known ECM receptors and crucial for cell migration and invasion (Hood and Cheresh, 2002). Of them, integrin α5β1 is highly expressed in invasive breast cancer cell lines. Focal adhesion kinase (FAK) is a key mediator of intracellular signaling through integrin α5β1, and FAK-Src signaling in cancer promotes cell migration, invasion, and metastasis through cytoskeleton remodeling and MMP-mediated extracellular matrix proteolysis (Guan, 2010; Mierke
To further investigate the mechanism of action of G0S2 on proliferation, migration, and invasion, the expression of proteins involved in integrin α5β1-mediated FAK-Src and ERK1/2 signaling was evaluated in MDA-MB-231 cells. First, decreased levels of G0S2 protein were confirmed using G0S2 siRNA (Fig. 5A). Interestingly, we found that the knockdown of G0S2 decreased expression of integrin α5β1 protein in MDA-MB-231 cells (Fig. 5B). Furthermore, phosphorylation of FAK at Tyr915, induced by activation of integrin α5β1, and phosphorylation of Src at Tyr416, induced by FAK activation, was also reduced. In addition, phosphorylation of ERK1/2 was decreased by G0S2 inhibition (Fig. 5C). These results indicated that G0S2 activated FAK-Src and ERK1/2 signaling by inducing integrin α5β1 expression to promote proliferation, migration, and invasion in MDA-MB-231 cells.
Recent studies have revealed that Hippo signaling pathway may contribute to tumorigenesis and cancer development (Zygulska
To further elucidate whether the Hippo signaling pathway is activated by G0S2 knockdown, the degrees of phosphorylation of LATS1 and YAP1 were evaluated. As a result, phosphorylation of LATS1 at Ser909 and YAP1 at Ser127 were significantly increased, and YAP1 and CYR61 protein levels were decreased after G0S2 inhibition (Fig. 6B). Furthermore, confocal microscopy analysis revealed that knockdown of G0S2 expression might inhibit YAP nuclear localization by increasing the accumulation of phosphorylated YAP1 protein in the cytoplasm or inducing degradation of phosphorylated YAP1, because 14-3-3 proteins binds to the phosphorylation site, Ser127, of YAP1, or phosphorylation of YAP1 induces proteasome-mediated degradation (Fig. 6C) (Freeman and Morrison, 2011; Lamar
EMT is a complicated process that epithelial cells gain the properties of migratory and invasive mesenchymal cells. EMT affects cancer progression and metastasis through lose cell-cell adhesion and polarity, and reorganizes their cytoskeletons (Son and Moon, 2010). E-cadherin which is one of the typical epithelial cell markers in EMT, and its downregulated expression is associated with highly invasive and metastatic cancers (Elisha
MMPs are well-known markers of cell invasion. To determine whether the inhibition of invasion is associated with MMPs when cells were treated with G0S2 siRNA, the levels of different MMPs (1, 2, 3, 7, 9, 12, 13, and 14) were measured by qPCR. Our results showed that the knockdown of G0S2 expression reduced mRNA levels of
Inflammatory cytokines, such as
The first step of metastasis is migration of cancer cells away from the primary tumor site, a process called tumor invasion (Glentis
The CpG site in the G0S2 gene has been demonstrated to be overwhelmingly hypomethylated in MDA-MB-231 cells compared with MCF-7 cells, and the mRNA expression of
Our results demonstrated that G0S2 tended to be over-expressed in triple-negative breast cancer cell lines. Consistently, overexpression of G0S2 increases accumulation of LDs (Antalis
Our studies showed that G0S2 promoted cell proliferation, migration, and invasion in MDA-MB-231 cells by activating integrin α5β1-mediated FAK-Src and ERK1/2 signaling through phosphorylation of Tyr925, Tyr416, or Thr202, and Tyr204. This is consistent with previous results that revealed that FAKSrc signaling activation promotes cell survival, adhesion, angiogenesis, migration, and metastasis by upregulating integrin α5β1 expressions (Mitra and Schlaepfer, 2006; Mierke
Previous studies have demonstrated that integrins directly bind to fibronectin and then stimulate FAK-Src signaling. After activation of FAK-Src signaling, YAP1 is activated in a PI3K-PDK1-dependent manner (Kim and Gumbiner, 2015). Subsequently, dephosphorylated LATS1/2 activates YAP1, resulting in YAP1 accumulation in the nucleus (Fan
In addition, our results showed that G0S2 knockdown suppressed β-catenin protein levels and mRNA levels of
G0S2 is highly expressed in MDA-MB-231 cells. However, Heckmann
In summary, inhibition of G0S2 suppressed proliferation, migration, and invasiveness of invasive breast cancer MDA-MB-231 cells. The scheme in Fig. 9 summarizes novel findings about the mechanisms induced by G0S2. Taken together, overexpression of G0S2 in MDA-MB-231 cells plays a key role in malignant tumor progression. Thus, G0S2 might be an effective therapeutic target for triple-negative breast cancers.
The authors declare that they have no conflicts of interest.
This research was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (grant numbers. NRF-2015R1A5A1008958 and NRF-2017R1E1A1A01074032).