Oestrogen‐related receptor alpha mediates chemotherapy resistance of osteosarcoma cells via regulation of ABCB1

Abstract Chemotherapy resistance is one of the major challenges for the treatment of osteosarcoma (OS). The potential roles of oestrogenic signals in the chemoresistance of OS cells were investigated. As compare to the parental cells, the doxorubicin and cisplatin (CDDP) resistant OS cells had greater levels of oestrogen‐related receptors alpha (ERRα). Targeted inhibition of ERRα by its specific siRNAs or inverse agonist XCT‐790 can restore the sensitivity of OS resistant cells to chemotherapy. This might be due to that si‐ERRα can decrease the expression of P‐glycoprotein (P‐gp, encoded by ABCB1), one important ABC membrane transporter for drug efflux. XCT‐790 can decrease the transcription and mRNA stability of ABCB1, while had no effect on protein stability of P‐gp. ERRα can bind to the transcription factor of SP3 to increase the transcription of ABCB1. Furthermore, XCT‐790 treatment decreased the expression of miR‐9, which can bind to the 3′UTR of ABCB1 and trigger its decay. Collectively, we found that ERRα can regulate the chemoresistance of OS cells via regulating the transcription and mRNA stability of ABCB1. Targeted inhibition of ERRα might be a potential approach for OS therapy.


| INTRODUCTION
Osteosarcoma (OS) is an aggressive and common type of solid bone tumours in children and adolescence. 1 Surgical resection, chemotherapy and radiotherapy have been considered as the standard treatment strategies for OS. 2 With the improvement of therapy approach, the mortality rate of OS has a huge decline in these two decades. 3 The 5-year survival rate of OS patients is about 70% within the past decades. 4 However, chemotherapy resistance, defined as tumour cells can develop resistance to a wide variety of anticancer drugs, is one of the most important formidable obstacles in the OS treatment. 2 Doxorubicin (Dox), cisplatin (CDDP), methotrexate and ifosfamide are the first-line chemotherapy drugs for OS patients. 5 Once the patient was resistance to these drugs, there are no established second-line chemotherapy drugs anymore. 1 Therefore, studies about the mechanisms responsible for chemoresistance of OS cells would be great important for developing effective therapies for OS patients.
The development of drug resistance is associated with multiple mechanisms. The most important mechanism responsible for cancer chemoresistance is the dysregulation of ABC membrane transporters. 6,7 Among all members of ABC family involved in chemoresistance, P-glycoprotein (P-gp), encoded by ABCB1 (also named MDR1), is highly expressed in the drug resistant cell lines to pump Yantao Chen and Kunshui Zhang contributed equally to this work.  8 The down-regulation of ABCB1 can sensitize OS cells to chemotherapy drugs such as Dox and CDDP. [9][10][11] Although the transcription regulation of ABCB1 is far from being completely understood, numerous studies indicated that transcription factors such as c-jun, c-fos, NF-kB (p65) and Sp3 can bind to its promoter to regulate the transcription. 12 As to the protein, P-gp is a relatively stable protein with a half-life of 14-17 hours, while modification by ubiquitin is important for degradation of P-gp. 13 Besides, ABCC1 (also named MRP1), ABCC2 (MRP2), ABCC3 and ABCG2 (BCRP) can also contribute to drug resistance of OS cells. 14,15 It has been reported that oestrogenic signals can regulate the progression of various cancers including chemotherapy resistance. [16][17][18] For example, IL-1β induced methylation of ERα is correlated with the chemoresistance in breast cancer cells. 19 E2-induced CDDP chemoresistance depends on the balance between ERα and ERβ expression and the p53 pathway. 18 Oestrogen-related receptors alpha (ERRα), which has similar structure with oestrogen receptor (ER), can bind to ERR-response elements (ERREs) and mediate the oestrogenic response in cells. 20 It has been reported that ERRα can confer methotrexate resistance of OS cells via attenuation of reactive oxygen species and p53 mediated apoptosis of OS cells. 21 Furthermore, ERRα can mediate the metabolic adaptations driving lapatinib resistance in breast cancer. 22 The reprogramming of ERRα target gene landscape can trigger the tamoxifen resistance of breast cancer cells. 23 Inhibition of ERRα by inverse agonist XCT-790 induces cell death in chemotherapeutic resistant cancer cells. 24 Our previous study revealed that ERRα participates transforming growth factor-β (TGF-β) induced epithelial-mesenchymal transition (EMT) of OS cells. 25 However, the roles of ERRα in drug resistance of OS cells were not well illustrated.
In this study, we found that the expression of ERRα was increased in Dox and CDDP resistant OS cells. Targeted inhibition of ERRα can restore the chemosensitivity of OS cells via down-regulation of ABCB1. The transcription factor SP3 and miR-9 were involved in ERRα regulated expression of ABCB1 in OS cells.

| Drug sensitivity assay
The chemotherapy sensitivity of OS cells was analyzed by use of the Cell Counting Kit-8 (CCK-8) (Beyotime, Haimen, China) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 4 × 10 3 well into 96-well plates. After treatment and incubated with different anticancer drugs at varying concentrations, 10 μL CCK-8 was added to each well and cultured for an additional 2 hours. The absorbance at 450 nm was measured by using a microplate reader (Model-550; Bio-Rad Laboratories, Hercules, CA, USA).
Each experiment was performed in triplicate.

| Western blot analysis
After treatment, cells were homogenized and lysed by use of cold RIPA buffer. Next, 30 μg of total protein was separated with a 4%-

| Cell transfection
The negative control, siRNAs for ERRα and SP3, and miR-9 inhibitor and mimic were purchased from Guangzhou Ribo BioCoLTD After transfection for 6 hours, the medium was replaced by full medium containing 10% FBS.

| Dox efflux assay
Effects of ERRα on the Dox efflux were tested by use of flow cytometry according to the previous study. 27 Cells were treated with 5 μmol/L Dox for 1 hour in darkness at 37°C. After treatment, cells were washed and collected for analysis by use of a flow cytometer (BD Biosciences, San Jose, CA, USA) using an argon laser of 15 mW at 488 nm.

| Promoter activity assay
The promoter of ABCB1 (−1000 to −1 bp) was cloned to the luciferase promoter to generate pTL-MDR1. Cells were transfected with pTL-MDR1 and pBABE-puro using Lipofectamine 2000 reagent (Invitrogen) and further treated with or without XCT-790 for the indicated time periods, the luciferase activity was normalized to total proteins determined by use of BCA assay.

| Immunoprecipitation assay
The binding between ERRα and SP3 was measured by immunoprecipitation assay in OS cells. Cell lysis was incubated with IgG or ERRα antibody over night at 4°C. After further incubated with protein A agarose beads for 4 hours, the immunoprecipitate was washed for three times and subjected to western blot analysis. The antigen-antibody complexes were visualized by chemiluminescence.

| ERRα was up-regulated in chemoresistant OS cells
Our previous study showed that ERRα participates TGF-β induced EMT of OS cells. 25

| ERRα was involved in the chemoresistance of OS cells
In order to investigate whether ERRα was involved in the chemoresis-

| ERRα regulated the expression of P-gp in OS chemoresistant cells
We further checked the effects of ERRα on the expression of various ABC membrane transporters including ABCB1, ABCC1, ABCC2,

| ERRα bound to SP3 to increase the transcription of ABCB1
It has been suggested that SP3 can regulate the transcription of ABCB1 in various human tissues. 28 We then investigated SP3 was involved in ERRα regulated transcription of ABCB1. The results showed that ERRα can directly bind with SP3 in MG-63 cells, further, the binding between ERRα and SP3 was increased in MG-63/ Dox cells ( Figure 6A). Similarly, the binding between ERRα and SP3 was increased in HOS/Dox cells as compared with that in HOS cells ( Figure 6B). We then knocked down the expression of SP3 in MG-63/Dox cells by use of its specific siRNA ( Figure 6C). Our data showed that in cells transfected with si-SP3, the XCT-790 suppressed transcription of ABCB1 was abolished ( Figure 6D).

F I G U R E 5
Oestrogen-related receptors alpha (ERRα) regulated the transcription and mRNA stability of ABCB1. MG-63/Dox cells were treated with or without 1 μmol/L XCT-790 for 24 h, the mRNA (A) and protein (B) levels of P-gp was measured, respectively; (C) MG-63/Dox cells were treated with 10 μg/mL CHX together with or without 1 μmol/L XCT-790 for the indicated time periods, the expression of P-gp was measured by western blot analysis; (D) MG-63/Dox cells were treated with 5 μg/mL Act-D together with or without 1 μmol/L XCT-790 for the indicated time periods, the expression of ABCB1 was measured; MG-63/Dox cells were treated with si-ERRα (E) or XCT-790 (1 μmol/L) (F) for 24 h, the relative promoter activity of ABCB1 was analyzed by a dual-luciferase assay kit. Data are presented as means ± SD of three independent experiments. **P < 0.01 compared with control Furthermore, Western blot analysis confirmed that si-SP3 can attenuate XCT-790 suppressed expression of P-gp ( Figure 6E). These results suggested that ERRα can bind to SP3 to increase the transcription of ABCB1.

| miR-9 was involved in ERRα regulated mRNA stability of ABCB1
Since ERRα can regulate the mRNA stability of ABCB1, we then investigated whether miRNAs were involved in this process. The expression of miR-9, 26 miR-200c, 29 miR-206, 30 and miR-495 31 was investigated in cells treated XCT-790 due to they can directly bind to 3′UTR of ABCB1 to regulate its stability. Our data showed that XCT-790 can increase the expression of miR-9, while not others, in both MG-63/Dox ( Figure 7A) and HOS/Dox ( Figure 7B ERRα has been reported to regulate the proliferation, migration and epithelial to mesenchymal transition of cancer cells. 32,33 Recently, studies also suggested that ERRα mediates metabolic adaptations driving drug resistance in breast cancer. 23,34 Our study revealed that the expression of ERRα was up-regulated in OS resistant cells as compared to their parental cells. This might be  inverse agonist or siRNAs can restore the chemotherapy sensitivity. This was consistent with recent study that ERRα can confer methotrexate resistance of OS cells. 21 It has been reported that ERRα is a transcriptional activator during bond development 36 and can regulate the expression of osteopontin in OS cells. 37 Our previous study also suggested that ERRα is involved in TGF-β induced EMT of OS cells. 25 All these data indicated that targeted inhibition of ERRα can suppress the malignancy of OS and might be a potential therapy approach for OS patients. Our data showed that ERRα can regulate the expression of Pgp via suppression its transcription and regulating its mRNA stability. Over expression of P-gp is one of the major causes of drug resistance for various cancer cells including OS. 38 Inhibition the expression of ABCB1 (P-gp) can overcome the drug resistance of OS cells. 39 We found that ERRα can bind with SP3 to regulate the transcription of ABCB1 in OS resistant cells. It has been reported that SP3 may recruit TFIID to the ABCB1 promoter by binding to the second activation domain of Sp3. 28 In our study, XCT-790 suppressed transcription of ABCB1 was abolished in cells transfected with si-SP3. In addition, miR-491-3p, which can down-regulate the expression of ABCB1 and its transcription factor Sp3 by directly targeting their 3′-UTR, attenuated multidrug resistance of hepatocellular carcinoma. 40 Furthermore, we found that XCT-790 can increase the expression of miR-9 and then trigger the decay of ABCB1 mRNA. Various miRNAs such as miR-9, 26 miR-200c, 29 miR-206, 30 and miR-495 31 can regulate the expression of ABCB1 in cancer cells. It has been confirmed by luciferase activity analysis that miR-9 can direct bind to the 3′UTR of ABCB1 and then trigger its decay in cancer cells. 26 As a transcription factor, ERRα might occupy the conserved ERRE in the promoter of miRNAs and then regulate their expression. 41 The mechanisms that XCT-790 induced up-regulation of miR-9 should be further investigated in the future.
In summary, our study suggested that ERRα mediated the chemoresistance of OS cells via regulating the transcription and mRNA stability of ABCB1. Although further in vivo and clinical studies are needed to confirm the clinical significances, our data, together with published data, suggested that targeted inhibition of ERRα can suppress the malignancy of OS and might be a potential therapy target of OS.
F I G U R E 7 miR-9 was involved in oestrogen-related receptors alpha (ERRα) regulated mRNA stability of ABCB1. MG-63/Dox (A) or HOS/ Dox (B) were treated with or without 1 μmol/L XCT-790 for 24 h, the expression of miRNAs was measured; (C) MG-63/Dox cells were transfected with RNA scramble control or miR-9 mimics for 12 h and then further treated with Act-D for the indicated times, the mRNA of ABCB1 was measured; MG-63/Dox cells were transfected with si-NC or miR-9 inhibitor for 12 h and then further treated with or without 1 μmol/L XCT-790 for 24 h, the mRNA (D) or protein (E) expression of P-gp was measured and quantitatively analyzed. (F) The schematic of ERRα regulated ABCB1 expression via binding with SP3 and suppressing miR-9 expression in OS cells. Data are presented as means ± SD of three independent experiments. **P < 0.01 compared with control

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