High expression of ABCG2 is associated with doxorubicin resistance of osteosarcoma

Previous studies showed overexpression of ABCG2 in a variety of tumor tissues, which could potentially indicate the probability of chemotherapy resistance. This study aimed to reveal the role of ABCG2 in the development of doxorubicin (DOX) resistance and the prognosis of OS. 68 DOX-resistant OS cell lines were induced by consecutive exposure of gradually increasing concentration of DOX to the parental cell lines. Lentivirus was used for the knockdown of ABCG2 in OS cells. Cells were treated with the gradient concentration of DOX and the viability was assessed by CCK8 assay. Total RNA was isolated from the tumor tissues or tumor cells, and the expression of ABCG2 was analyzed by qPCR. The relationship between ABCG2 expression and clinicopathological characteristics of the patients was analyzed using the Student’s t-test or the Chi-square test. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. P < 0.05 was considered statistically signicant. signicantly associated with the resistance to DOX and the overall survival of OS patients. ABCG2 may become a promising therapeutic target for OS patients.


Introduction
Osteosarcoma (OS) is the most common malignant bone tumor characterized by a high incidence of distant metastasis [1,2]. Before the advent of neo-adjuvant chemotherapy, OS patients undergoing amputation or reconstruction surgery had a low 5-year survival rates averaging approximately 15-20% [3,4]. Bene ted from the multi-agent neo-adjuvant chemotherapy, the ve-year survival rate now rise up to 70% for patients with non-metastatic OS [5]. However, it is noteworthy that patients with poor survival rate showed worse responsiveness to chemotherapy. In the past years, different approaches have been applied to investigate the mechanisms involved in chemoresistance of OS [6,7]. It was speculated that intrinsic gene expression differences may account for the low responsiveness to chemotherapy, while the precise underlying mechanisms remains poorly understood. Herein, identi cation of genes associated with chemoresistance is of great importance to personalize an effective regimen of chemotherapy.
First cloned from doxorubicin-resistant breast cancer cells, the human ABCG2 gene encodes ATP binding cassette (ABC) transporters that drive the transport of various substrates across cell membranes [8,9].
Previous studies showed that ABCG2 plays an important role in the proliferation and differentiation of stem cells [10,11]. Moreover, overexpression of ABCG2 has been observed in a variety of tumor tissues, which could potentially indicate the probability of chemotherapy resistance [12,13]. Noguchi et al [13] reported that ABCG2 was related to drug resistance in the breast cancer. Damiani et al observed that overexpression of ABCG2 could signi cantly affect the duration of complete remission, which therefore might be regarded as a prognostic factor in patients with acute myeloid leukemia [12].
To our knowledge, studies concerning the role of ABCG2 in OS chemoresistance are still limited. In this study, we aimed to investigate the role of ABCG2 in the development of doxorubicin (DOX) resistance and the prognosis of OS. Moreover, we investigated the role of genetic polymorphism of ABCG2 in the DOX resistance of OS patients.

Subjects
Under the approval by the local institutional Ethnics Committee, 68 OS patients undergoing treatment in our clinic centers between 2008 March and 2013 January were included in this study. All the patients were histologically diagnosed as OS. Baseline characteristics of the patients were collected from the medical record, including age, gender, tumor size, histologic differentiation, and Anneking Stages. Before the surgery, all the patients had received two cycles of neoadjuvant chemotherapy composed of DOX, methotrexate and cisplatin. Tumor tissues were collected from each patient during surgery. Good response to the chemotherapy was histologically de ned as more than 90% necrosis rate in the tumor tissues. All patients signed written informed consent concerning the collection of blood and tissue samples.
Cell culture and establishment of DOX resistance The human OS cell lines U2OS and HOS were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). All cell lines were cultured in Dulbecco's modi ed Eagle's medium (4.5 g/l glucose)/Ham F12 (1:1) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS).
All cells were cultured at 37 °C in a humidi ed atmosphere of 5% CO2. DOX was purchased from Sigma (Buchs, Switzerland). DOX-resistant cell lines were induced by consecutive exposure of gradually increasing concentration of DOX to the parental cell lines for 6 months as previously reported. As a starting concentration, 0.015 µM DOX was applied and the concentration was gradually increased up to 0.12 µM in a period of 3 months. One month after exposure to 0.12 µM doxorubicin, the resistance stability was de ned as not signi cantly altered IC50 of U2OS and HOS cells when incubated in DOX for a week.

Knockdown of ABCG2 in OS cells
The lentivirus shRNA speci cally targeting ABCG2 (shABCG2) and the negative control scramble shRNA (shCtrl) were designed and constructed by Shanghai Genechem Company Ltd., China. Lentivirus particles were generated by co-transfecting recombined and packing vectors into 293T cells via Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). U2OS and HOS cells were then cultured in 6-well plates and transfected with shABCG2 or shCtrl. Both cells lines were cultured for 5 days. The knockdown e ciency of the target gene was further evaluated for both cell lines with qPCR analysis.

Cell proliferation assay
Cell proliferation was analyzed by the colorimetric water-soluble tetrazolium salt assay using a Cell Immunohistochemical staining Para n-embedded tissue samples were cut into 4-µm sections and de para nized for immunohistochemical staining (IHC). Slides were then incubated with primary anti ABCG2 antibody (1:400, Abcam) overnight at 4 °C, followed by incubation with secondary antibody for 30 minutes at 20 °C. All slides were stained by diaminobenzidine and then counterstained by hematoxylin. The IHC-stained tissue sections were scored by two pathologists. The signal intensity was scored as follows: score 0, no signal; score 1, weak; score 2, moderate; and score 3, marked. The staining distribution was scored as follows: score 0, no positive staining cells; score 1, 1-30% positive staining cells; score 2, 31-60% positive staining cells; score 3, 61-100% positive staining cells. The nal score calculated by multiplying the score of staining distribution with the signal intensity score. Remarkably higher staining of ABCG2 was de ned as the score more than 2.
Genotyping of functional variant DNA was extracted using the DNA extraction kit (QIAGEN Inc., Tokyo, Japan) according to the protocol of the manufacturers. SNP rs2231142 (c.421C > A) was genotyped using TaqMan SNP Genotyping Assay.
The genotyping assay was performed with ABI 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).

Statistical analysis
The results were presented as mean ± standard deviation, and analyzed with SPSS software (version 19.0; SPSS Inc, Chicago, IL, USA). The data was shown as means ± SD for continuous variables. The relationship between ABCG2 expression and clinicopathological characteristics of the patients was analyzed using the Student's t-test or the Chi-square test. The gene expression and the overall survival were compared among different genotypes of rs2231142 with One-way ANOVA test. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. P < 0.05 was considered statistically signi cant.

Demographic data
The demographic and clinical characteristics of the study subjects are shown in were 3.5 µM and 3.25 µM, respectively. By contrast, those of the untreated HOS and U2OS cells were 1.15 µM and 0.93 µM, respectively (p < 0.01). The results of qPCR analysis showed that mRNA expression level of ABCG2 was signi cantly increased in DOX-resistant cell lines (Fig. 1).

Knockdown of ABCG2 enhanced response to DOX in OS cells
As shown in Fig. 2, the mRNA level of ABCG2 was decreased by 70% and 65% respectively in ABCG2inhibited DOX-resistant HOS and DOX-resistant U2OS cells as compared with the control cells. After being incubated with DOX for 48 h, the viability of cells transfected with shABCG2 was remarkably lower than that of cells transfected with shCtrl. The IC 50 value of ShABCG2-transfected HOS cells and ShABCG2transfected U2OS were 3.25 µM and 3.05 µM, respectively. By contrast, the IC 50 value of HOS and U2OS cells in the control group were 1.05 µM and 0.97 µM, respectively (p < 0.01).
High expression level of ABCG2 was associated with a poor prognosis of OS The ABCG2 expression was analyzed in the tumor tissues and adjacent normal tissues of 68 patients. As shown in Fig. 3, ABCG2 was obviously highly expressed in the tumor tissues than in the normal tissues (P < 0.001). According to the results of IHC, 43 patients were included in the high expression group and the other 25 patients were included in the normal expression group. As shown in Table 2, we found that ABCG2 expression was signi cantly associated with tumor size (6.1 cm ± 2.7 cm vs. 3.9 cm ± 1.5 cm, p = 0.01) and the response to chemotherapy (62.4% ± 25.1% vs. 75.3% ± 18.7%, p = 0.02). As shown in Fig. 3, the disease-free survival time of patients with low ABCG2 expression was signi cantly longer than those of the high ABCG2 expression (38.9 ± 15.6 months vs. 28.4 ± 14.4 months, p < 0.01). No signi cant association was found between ABCG2 expression and other clinical features (Table 2).  (Fig. 4).

Discussion
Chemotherapy regimen composed of DOX, methotrexate and cisplatin is most commonly applied for patients with OS [5,14]. However, resistance to chemotherapeutic drugs remains the primary obstacle to achieve favorable outcome of human cancer. Various factors leading to chemoresistance have been reported in previous studies, such as alterations in the drug target, abnormal induction of cell death pathways and overexpression of ABC membrane transporter family members [15][16][17]. Nevertheless, there is still a lack of methods to predict chemoresistance in OS effectively. Obviously, identifying new contributing factors of chemoresistance in OS is of great importance to improve the therapeutic e cacy and predict the drug response.
High ABCG2 expression has been identi ed in a variety of solid tumors and has been correlated with poorer clinical outcomes [8][9][10]. For the rst time, we investigated the role of ABCG2 in the development of chemoresistance in OS. In the current study, DOX-resistant OS cell lines were successfully established through a gradually increasing dose of DOX. Comparably, through Genechip analysis, Walters et al [18] identi ed increased expression of ABCG2 in the OS cell lines treated by doxorubicin. Compared with their parental DOX-sensitive cells, DOX-resistant cells were observed to have remarkably elevated expression level of ABCG2. Furthermore, knockdown of ABCG2 level in DOX-resistant cell lines by lentivisurs could effectively recover the therapeutic e cacy of DOX treatment. Of note, we found that ABCG2 expression level was signi cantly increased in patients with decreased disease free survival and poor response to chemotherapy. It was in accordance with previous study reporting that elevated levels of ABCG2 have a vital role in OS prognosis [19]. On the basis of these data, we proposed that ABCG2 may become a promising therapeutic target for OS patients. These characteristics of ABCG2 enable it a promising molecular target to guide the chemotherapy treatment of OS.
The genetic variants of ABCG2 were reported to be involved in cancer risk and chemotherapeutic response [20,21]. To illustrate the mechanism underlying the regulation of ABCG2 expression in OS tissues, a functional polymorphism rs2231142 of ABCG2 were genotyped in our cohort of patients. We con rmed that genotype CC of rs2231142 was indicative of remarkably higher expression of ABCG2. For OS patients receiving a therapeutic program that includes DOX, those with genotype CC could had poor outcome, which was in line with our nding that high expression of ABCG2 was associated with lower survival rate. Comparably, Morisaki et al [22] found that cells transfected with the allele A of rs2231142 showed remarkably higher resistance to chemotherapy drugs than those transfected with wild-type, suggesting that rs2231142 may affected drug transport. Additionally, the rs2231142 has also been reported to be associated with increased adverse effects in response to chemotherapy treatment. Mizuarai et al [23] found that rs2231142 mutant may decrease ATPase activity by 1.3-fold as compared with the wild-type ABCG2. Taken together, these results suggested that rs2231142 may result in altered transport functions of ABCG2 transporter, which has important implications for the pharmacokinetics and drug-resistance pro les of chemotherapeutics. To conclude, we have provided the rst data concerning the impact of ABCG2 polymorphism in the prognosis of patients with OS. Early detection of this variant could be helpful in driving the choice of other alternative drugs to be used in chemotherapy.
Two limitations of this study should be addressed here. Due to the inherent drawback of retrospective study, the sample size of the OS patients was relatively small. In the future study, more OS patients need to be recruited for a stronger statistical power. Second, the mechanism underlying the in uence of rs2231142 on expression level of ABCG2 remains undetermined, which is worthy of further investigation in the future study.

Conclusions
ABCG2 expression level was signi cantly associated with the response to chemotherapy and the overall survival of OS patients. Allele C of rs2231142 was indicative of higher ABCG2 expression and associated with poor prognosis of OS. ABCG2 may become a promising therapeutic target for OS patients. Presence of allele C of rs2231142 could suggest the choice of other alternative drugs to be used in chemotherapy.

Abbreviations
Osteosarcoma: OS; ATP binding cassette: ABC; Doxorubicin: DOX; Cell Counting Kit-8: CCK8 Declarations Con ict of interest statement No bene ts in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

Figure 1
Upregulated expression of ABCG2 in DOX-resistant OS cells a. Successful establishment of DOX-resistant OS cell lines was veri ed by CCK8 assay. Cell viability of DOX-treated HOS cells and U2OS cells was remarkably higher than those not treated by DOX; b. Through qPCR, we con rmed that mRNA expression level of ABCG2 was signi cantly increased in DOX-resistant cell lines as compared with normal cell lines. Relationship between ABCG2 expression and the prognosis of OS patients. a. Analysis of gene expression in OS tissues showed remarkably higher expression of ABCG2 as compared with adjacent normal tissues (p <0.01); b. The disease-free survival time of patients with high expression level of ABCG2 had obviously decreased survival time than the patients with low expression (p <0.01).