Estrogen-Related Receptor Alpha Confers Methotrexate Resistance via Attenuation of Reactive Oxygen Species Production and P53 Mediated Apoptosis in Osteosarcoma Cells

Osteosarcoma (OS) is a malignant tumor mainly occurring in children and adolescents. Methotrexate (MTX), a chemotherapy agent, is widely used in treating OS. However, treatment failures are common due to acquired chemoresistance, for which the underlying molecular mechanisms are still unclear. In this study, we report that overexpression of estrogen-related receptor alpha (ERRα), an orphan nuclear receptor, promoted cell survival and blocked MTX-induced cell death in U2OS cells. We showed that MTX induced ROS production in MTX-sensitive U2OS cells while ERRα effectively blocked the ROS production and ROS associated cell apoptosis. Our further studies demonstrated that ERRα suppressed ROS induction of tumor suppressor P53 and its target genes NOXA and XAF1 which are mediators of P53-dependent apoptosis. In conclusion, this study demonstrated that ERRα plays an important role in the development of MTX resistance through blocking MTX-induced ROS production and attenuating the activation of p53 mediated apoptosis signaling pathway, and points to ERRα as a novel target for improving osteosarcoma therapy.


Introduction
Osteosarcoma (OS) is one of the most common forms of childhood and adolescent cancer, comprising approximately 20% of all primary bone cancers, which is the second leading cause of mortality in this age group [1,2]. Currently, the use of neoadjuvant chemotherapy combined with surgical resection is the mainstay therapy in OS [3]. The antifolate, chemotherapeutic agent methotrexate (MTX), is widely used to treat OS. However, approximately 30% of the OS patients suffer the recurrent or metastatic diseases due to the development of drug resistance [4]. Therefore, it is imperative to understand the molecular mechanisms underlying the MTX resistance and identify novel treatment strategies for this disease.
Estrogen-related receptors alpha (ERR ) is an orphan nuclear receptor that plays a key role in regulating metabolic processes. ERR is abundantly expressed in tissues with high-energy demand such as heart, kidney, skeletal muscle, and brown adipose tissues [5,6]. In addition to its role in the control of energy metabolic processes, there is cumulative evidence showing potential functions of ERR in cancer development and progress. ERR overexpression is associated with poor clinical outcomes not only in breast, ovarian, and prostate cancers, but also in nonendocrine related colon cancer [7][8][9][10]. Our previous reports have shown that ERR suppression efficiently induced cancer cell death via induction of reactive oxygen species (ROS) production [11,12]. ROS are chemically reactive molecules and have important roles in cell signaling and homeostasis [13]. ROS level can be dramatically increased as a response to different stress conditions, which can lead to several biological effects, ranging from alterations in cell signaling pathway and gene expression to mutagenesis, mitogenesis, or apoptosis [14,15]. Recent studies demonstrated that chemotherapeutic drugs including MTX can induce cell death by promoting ROS production [16,17]. We thus hypothesized that ERR may be involved in chemotherapy resistance in osteosarcoma.
In this study, we demonstrated that elevated ERR can result in the development of MTX resistance through blocking MTX-induced ROS production and attenuating p53-dependent apoptosis in osteosarcoma cells. Our study suggests ERR is a novel target for improving osteosarcoma therapy. To generate ERR overexpression U2OS stable sublines, full length cDNA of ERR was cloned into the expression vector pLenti4/V5-DEST (Invitrogen). Lentiviruses were produced from these constructs using a three-plasmid packaging system as described [18]. U2OS cells were infected with the lentiviruses and selected with blasticidin (2.5 g/mL, Invivogen, San Diego, CA) for 4-6 weeks. Individual blasticidinresistant clones were isolated and assayed for ERR expression by immunoblotting. Clones homogeneously expressing ERR were maintained in DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum and 1 g/mL blasticidin at 37 ∘ C under 5% CO 2 . All cell lines were used within 20 passages.

Cell Survival Rate
Assay. Cells in logarithmic growth phase were seeded in 6-well plates at a density of 100,000 cells per well and treated with different concentrations of MTX (0-1 M). Cells were harvested after 24, 48, and 72 hours and counted by Beckman Z1 Coulter Counter. Each treatment was performed in triplicate wells at least 3 times.

Trypan Blue Exclusion
Assay. Cells in logarithmic growth phase were seeded in 6-well plates at a density of 100,000 cells per well and treated with different concentrations of MTX (0-1 M). Cells were harvested after 24, 48, and 72 hours and counted under a light microscope after trypan blue exclusion (0.4% trypan blue, Life Technologies, USA) according to instructions of manufacturer. Each treatment was performed in triplicate wells per experiment.

ROS Level.
The production of intracellular ROS was measured using a 6-corboxy-2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe (Invitrogen, USA). The DCFH-DA passively enters the cell where it reacts with ROS to form the highly fluorescent compound dichlorofluorescein (DCF). In brief, cells were plated in 6-well plates and allowed to attach overnight. After treatment with the indicated concentrations of MTX for 24 h, the cells were washed 2 times with DMEM without FBS and then loaded with 10 M DCFHDA at 37 ∘ C, 5% CO 2 for 20 min. Cells were washed another three times by PBS and then observed under a fluorescene microscope. ROS was also measured as previously described [19]. The cells were lysed using 1% SDS and sonicated. After 5 min centrifuge (14,000 rpm), supernatant was aliquoted (100 mL) into 96-well plate and measured by Victor 3 microplate reader. Signal intensity was normalized to protein concentration.

Western Blotting Analysis.
Cell lysates were prepared with lysis buffer. The lysates were cleared by centrifugation and total protein concentration was measured using bicinchoninic acid assay kit (Bio-Rad Laboratories). The protein samples were separated on SDS-PAGE at 10-15% and transferred to a polyvinylidene difluoride membrane (Immobilon-P transfer membrane; Millipore) with a Bio-Rad Trans-Blot semidry transfer cell apparatus. The membranes were blocked by 5% nonfat milk in Tris-buffered saline Tween-20 (TBST, pH 7.6) for 90 min at room temperature.
Primary antibodies were diluted in 5% BSA of TBST and incubated for 2 h at room temperature or overnight at 4 ∘ C. After washing 3 times with TBST, the membranes were incubated with anti-rabbit, anti-mouse, or anti-goat (1/2,000; R&D Systems) secondary antibodies coupled to horseradish peroxidase for 1 h at room temperature. The membranes were washed 3 times with TBST; then the bands were detected using electrochemiluminescence (ECL) (GE Healthcare UK Ltd.).

Statistical
Analysis. Data were expressed as means ± SD. All experiments were independently repeated at least three times. The statistical significance of differences was determined by Student's two-tailed -test.
< 0.05 was considered statistically significant. Asterisks indicate the level of significance. The data were analyzed using SPSS 17.0.

ERR Overexpression Blocks MTX-Induced Osteosarcoma
Cell Death. To investigate the potential function of ERR in osteosarcoma (OS) progression, we analyzed ERR gene expression in Gene Expression Omnibus (GEO) datasets. We found that ERR expression was significantly upregulated in the metastatic osteosarcomas compared with nonmetastatic tumors in GSE21257 dataset (Figure 1(a)). Metastatic osteosarcomas that are resistant to conventional chemotherapy are the major cause of death. To explore the potential role of ERR in chemotherapeutic resistance, we first established U2OS stable sublines that overexpress ERR (U2OS-ERR number 1 and U2OS-ERR number 2) (Figure 1(b)). We then examined the effect of ERR overexpression on the sensitivities of U2OS cells to chemotherapeutic agent MTX. Results in Figures 1(c) and 1(d) demonstrated that overexpression of ERR strongly protected the cells from MTX-induced growth inhibition. By trypan blue exclusion assay, we found that the elevated ERR can efficiently block MTX-induced cell death (Figure 1(e)). As shown in Figure 1(f), MTX at a low concentration (0.125 M) effectively induced apoptosis in the parental U2OS cells, as detected by cleaved PARR and cleaved caspase 7. However, in U2OS-ERR cells, MTX even at 8-fold higher concentration (1 M) did not efficiently cause cell apoptosis. These results suggested that ERR conferred resistance to MTX in human osteosarcoma cells through inhibition of apoptosis.

MTX Induces Cell Death by Increasing ROS Production in Osteosarcoma Cells.
A number of studies indicated that reactive oxygen species (ROS) are involved in a variety of different cellular processes ranging from apoptosis and necrosis to cell proliferation and carcinogenesis [20,21]. To determine whether ROS is involved in MTX-induced cell death, we stained the U2OS cells with CM2-DCFHDA staining and found that MTX treatment significantly increased ROS production in U2OS cells while N-acetylcysteine (NAC), an antioxidant compound, effectively blocked its induction of ROS (Figures 2(a) and 2(b)). Furthermore, simultaneous treatment with NAC almost completely protected the cells from MTX-induced cell death (Figure 2(c)). Therefore, ROS plays a critical role in MTX-induced cell death.

ERR Confers MTX Resistance by Modulating ROS Production in Osteosarcoma Cells.
We and others demonstrated previously that ERR modulates ROS production in cancer cells [12,22,23]. We next examined whether ERR -mediated MTX resistance was through control of ROS. Results shown in Figures 3(a) and 3(b) demonstrated that overexpression of ERR could efficiently block MTX-induced ROS generation. To further demonstrate that ERR protected cells from MTXinduced cell death by downregulating ROS production, we treated U2OS parental and U2OS-ERR cells with hydrogen peroxide (H 2 O 2 ), a highly potent form of ROS. We found that ERR overexpression also strikingly protected H 2 O 2induced cell growth inhibition even at 50 mM of H 2 O 2 (Figure 3(c)). These results collectively suggested that ERR confers MTX resistance by suppression of ROS production in osteosarcoma cells.

P53-Mediated Apoptosis Pathway Is Involved in ERR Mediated MTX Resistance.
It is well established that tumor suppressor P53 and its target genes are involved in ROSinduced apoptosis in different cancer cells [24,25]. We thus examined the effect of ERR overexpression and MTX on p53 protein level and the role of ROS in this process. Using Western blotting analysis, we found that MTX dosedependently induced p53 at the protein level and that ERR overexpression blocked this induction (Figure 4(a)). Consistent with the notion that MTX induction of P53 involves ROS, the antioxidant compound NAC effectively eliminated MTX-induced p53 accumulation (Figure 4(b)). Moreover, P53 induction by H 2 O 2 could also be suppressed by ERR (Figure 4(c)). Importantly ERR overexpression also blocked MTX-induced NOXA and XAF1 expression which are p53 direct target genes for promoting cell apoptosis (Figures 4(d) and 4(e)). These results collectively indicated that ERR overexpression suppressed MTX-induced p53 activity through modulating ROS production.

Discussion
Osteosarcoma (OS) is the most common tumor of bone. Currently, the use of neoadjuvant chemotherapy combined with surgical resection is the mainstay therapy in OS [3]. The overall survival for osteosarcoma patients has been increased to over 70%. However, the 5-year survival of patients with OS metastasis still remains about 20-30%. Drug resistance is still a significant clinical problem in effective therapy of osteosarcoma. In this study, we showed that overexpressing ERR in human osteosarcoma cells promotes the survival of OS cells through inhibiting MTX-induced apoptosis. In addition, our results indicated that ERR overexpression confers osteosarcoma resistance to MTX via attenuation of reactive oxygen species production and p53-mediated apoptosis pathway in osteosarcoma U2OS cells.
The primary function of ERR is believed to be the regulation of energy metabolism. Additionally, ERR at least has been shown to play a role in the regulation of bone formation [26,27]. ERR is expressed throughout all developmental stages from early progenitors to bone forming osteoblasts [26]. It may play a functional role in osteoblast differentiation and bone formation by regulating the expression of osteocalcin (OC), bone sialoprotein (BSP), Runx2, and alkaline phosphatase (ALP) [28,29]. We and others have identified that osteopontin (OPN) and osteocalcin (OC) are ERR direct target genes in bone [30,31]. Interestingly, studies also showed that OPN and OC promote cancer progression in different cancer types [32,33]. Although it is well established that OPN and OC play important roles in bone formation, their roles in osteosarcoma remain unknown. To understand the role of ERR and its target genes in osteosarcoma progression, further investigations, including using animal xenograft models, are needed to be done to demonstrate the importance of ERR in advanced and chemotherapy resistance osteosarcoma. In current research, we demonstrated for the first time that the expression of ERR is correlated with osteosarcoma progression and that ERR overexpression can mediate MTX resistance in osteosarcoma cells. Our data collectively suggested that ERR may be a novel target for combating chemotherapy resistance and advanced osteosarcoma. protein level was assayed by western blotting. Data are expressed as mean ± SD ( = 3) and analyzed using Student's -test. Asterisks indicate significant differences: * < 0.05, * * < 0.01.