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Expression and prognostic relevance of PRAME in primary osteosarcoma
Biochemical and Biophysical Research Communications 419 (2012) 801–808
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Expression and prognostic relevance of PRAME in primary osteosarcoma Pingxian Tan a, Changye Zou a, Bicheng Yong a, Ju Han b, Longjuan Zhang c, Qiao Su d, Junqiang Yin a, Jin Wang a, Gang Huang a, Tingsheng Peng b, Jingnian Shen a,⇑ a
Department of Orthopaedic Surgery, Musculoskeletal Tumor Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China Department of Pathology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China c Surgery Lab. Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China d Experimental Animal Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China b
a r t i c l e
i n f o
Article history: Received 10 February 2012 Available online 27 February 2012 Keywords: PRAME Preferentially expressed antigen of melanoma Osteosarcoma Prognosis siRNA Proliferation
a b s t r a c t The preferentially expressed antigen of melanoma (PRAME), a cancer-testis antigen with unknown function, is expressed in many human malignancies and is considered an attractive potential target for tumor immunotherapy. However, studies of its expression and function in osteosarcoma have rarely been reported. In this study, we found that PRAME is expressed in five osteosarcoma cell lines and in more than 70% of osteosarcoma patient specimens. In addition, an immunohistochemical analysis showed that high PRAME expression was associated with poor prognosis and lung metastasis. Furthermore, PRAME siRNA knockdown significantly suppressed the proliferation, colony formation, and G1 cell cycle arrest in U-2OS cells. Our results suggest that PRAME plays an important role in cell proliferation and disease progression in osteosarcoma. However, the detail mechanisms of PRAME function in osteosarcoma require further investigation. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
1. Introduction Preferentially expressed antigen in melanoma (PRAME) belongs to the cancer-testis antigen family and was first identified by Ikeda et al. in a patient with melanoma [1]. The PRAME gene encodes a HLA-A24-restricted antigenic peptide that can be recognized by autologous tumor-specific cytotoxic T lymphocyte cells and has been found to be expressed in a number of solid tumors and various leukemias [1–7]. Because PRAME is recognized by tumorspecific cytotoxic T lymphocytes and because it is not detected in normal tissues (except low expression in testicular, ovarian, endometrial and adrenal tissue) but is over-expressed in a number of human malignancies, PRAME is considered an attractive potential target for tumor immunotherapy and a diagnostic marker in many human tumors that express this antigen [8]. Though some studies have shown that PRAME expression in tumors is correlated to clinical outcome and extensive investigations into PRAME function have been reported, its specific mechanism in tumors remain elusive [9–11]. Osteosarcoma is an aggressive cancer of the bone with unknown etiology. It predominantly affects children and adolescents [12–14]. Despite the dramatic advances in osteosarcoma treatment in recent ⇑ Corresponding author. Address: Department of Orthopaedic Surgery, Musculoskeletal Tumor Center, First Affiliated Hospital of Sun Yat-Sen University, No. 58, Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. E-mail address: [email protected] (J. Shen).
years, patient survival has reached a plateau. Therefore, novel treatment modalities are urgently needed to improve survival [15–17]. The use of tumor antigens as targets for immunotherapy has emerged as a treatment strategy in recent years [18,19]. New tumor antigen markers could further improve diagnosis and predict prognosis. However, few antigenic peptides have been identified in bone or soft tissue sarcomas thus far, and the development of appropriate vaccines and therapies has been hampered by the lack of knowledge regarding the expression of tumor-specific antigens in osteosarcoma. From our previous gene microarray and proteomic analysis [20,21], we found that cancer-testis antigens family (including PRAME) expressed in most of the tested osteosarcoma tissues and a few osteosarcoma cell lines. In the present study, we further investigated the pattern of PRAME expression in osteosarcoma tissues and several cell lines, PRAME’s relevance to disease prognosis and the potential function of PRAME in osteosarcoma.
2. Materials and methods 2.1. Cell lines and cell culture The human osteosarcoma cell lines MG-63, U-2OS and Saos-2 were gifts from Dr. M. Serra (Istituti Ortopedici Rizzoli, Bologna, Italy). The human osteosarcoma cell line ZOS was established from primary tumor tissues in our institution [22]. The human osteoblast cells (h-FOB) were purchased from Born Cells Resource
0006-291X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.110
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Fig. 1. Expression levels of PRAME in osteosarcoma tissues and cell lines. (A) PRAME mRNA expression levels in osteosarcoma cell lines and human osteoblast cells. K562 cells were used as a positive control. (B) Real-time PCR revealed that 36 osteosarcoma tissue samples had increased PRAME mRNA levels compared with the muscle tissue specimens at the surgical margin. (C) PRAME protein expression levels in K562 cells, osteosarcoma cell lines and the human osteoblast cells. (D) PRAME protein expression levels in osteosarcoma tissues and muscle tissues at the resection margin.
Fig. 2. Inhibition of PRAME expression prevents osteosarcoma growth in vitro. (A) The RT-PCR and (B) Western blotting demonstrate the inhibition of PRAME expression after siRNA interference. (C) CCK8 analysis of the growth of three groups of normal, negative control and PRAME siRNA U2OS cells (⁄P < 0.01 PRAME siRNA vs. negative control and untransfected cells.). (D) Similar results were observed in ZOS, ZOS-control and ZOS-siRNA cell. (E and F) The colony formation assay revealed that PRAME knock-down reduced colony formation in U2OS and ZOS cells. U-2OS, U-2OS siRNA, ZOS and ZOS siRNA cells were seeded at a low density (500 cells/ml) and incubated in DMEM supplemented with 10% FCS for 21 days. The cell colonies were fixed and stained with crystal violet. The experiment was performed three times and reported as the mean ± SD. The sterisk indicates a significant difference between PRAME siRNA-treated and scrambled siRNA-treated U-2OS and ZOS cells.
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Center, Chinese Academy of Sciences (Shanghai, China), and were used as a negative control. The cell line K562 was kindly provided by Dr. Wang (Department of Hematology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China), and were used as a positive control. The cells were cultured in D-MEM or RPMI 1640 (Gibco, Invitrogen) supplemented with 10% fetal calf serum (FCS) and 1 mg/mL penicillin–streptomycin (Sigma, Saint Louis MO, USA) and incubated at 37 °C and 5% CO2 in a humidified incubator. 2.2. Patients and tissue samples The medical records of osteosarcoma patients who were seen in our department from January 1999 to December 2010 were reviewed. All the patients underwent neoadjuvant chemotherapy and definitive surgery. Osteosarcoma was confirmed histopathologically, and follow-up data were extracted from our database. For the RNA analysis, fresh tumor specimens were snap frozen in liquid nitrogen after surgical resection, and then cryopreserved at 80 °C in our tissue bank. Only tumor samples that were evaluated by pathologists and composed of >80% tumor cells were used for the study. The formalin-fixed, paraffin-embedded specimens from osteosarcoma biopsies (before any treatment was initiated) and sixteen osteofibrous dysplasia samples were collected at the tissue bank of the Department of Pathology, First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). The osteofibrous dysplasia samples were used as the negative control. Prior patient (or guardian) consent and approval from the Institutional Research Ethics Committee of Sun Yat-Sen University were obtained prior to the use of these tumor specimens and clinical materials for research purposes. 2.3. Silencing of PRAME by short interfering RNAs (siRNAs) in U2OS and ZOS cells Control and PRAME short interfering RNAs were purchased by Invitrogen. The U-2OS and ZOS cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. PRAME silencing was confirmed by RT-PCR, real-time PCR and Western-blotting. 2.4. Total RNA and protein extraction Thirty-six freshly dissected tumor specimens and muscle tissues from wide surgical margins were obtained from osteosarcoma patients treated in our institution. Total cell and tissue sample mRNA and protein were extracted using TRIzol (Invitrogen) and RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China), respectively, according to the manufacturer’s instructions and stored at 80 °C. 2.5. RT- PCR and real-time PCR A total of 500 ng of RNA was transcribed into cDNA using a PrimeScript RT reagent Kit (Invitrogen). The reverse transcription PCR and real-time PCR oligonucleotide primers for PRAME and b-actin were designed according to the literature [1]. The RT-PCR amplification protocol was as follows: denaturation at 94 °C for 10 min, 35 cycles of 45 s at 94 °C, 60 s at 64 °C and 45 s at 72 °C, and a final extension step at 72 °C for 7 min. Three percent agarose gel electrophoresis was performed to detect the amplified products. Real-time PCR reactions were performed in triplicate in a 10 lL reaction volume using standard conditions with 40 cycles of amplification. The amplification products were detected in the presence of the SYBRÒ Premix Ex TaqTM kits reagents (TaKaRa Biotechnology
CO., Dalian, China) with an ABI7900 PRISM real-time PCR instrument (Applied Biosystems, Sunnyvale, CA). The mean cycle threshold value (Ct) from triplicate samples was used to calculate gene expression. The PCR products were normalized to b-actin levels. 2.6. Western blotting Western blotting analysis was performed by using standard methods with anti-human PRAME protein (1:1000, Abcam, Cambridge, UK), anti-P27 (1:500, Beyotime Institute of Biotechnology, Shanghai, China) and anti-GAPDH antibody (1:1000, Sigma). 2.7. CCK-8 cell viability assay Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratory, Kumamoto, Japan) according to manufacturer’s instructions. U-2OS and ZOS cells were seeded in 96-well plates at a density of 3000 cells/well and grown in DMEM supplemented with 1% FBS. The cells were transfected with the siRNA and control interfering. At 24, 48, 72, 96, and 120 h after transfection, CCK-8 mixed with serum-free DMEM was added at a ratio of 1:10. After incubation for 2 h, the absorbance at 450 nm was measured. All the experiments were performed in triplicate. 2.8. Flow cytometry A cell cycle analysis of the PRAME siRNA and control siRNAtreated cells was performed by flow cytometry. At 24, 48, and 72 h after transfection, the cells were trypsinized, washed in PBS and fixed in 70% cold ethanol for 24 h. After fixation, the cells were washed, and the DNA was stained with 50 lg/mL propidium iodide (PI) solution in the presence of 250 lg/ml RNAse A (Sigma). The cellular DNA content distribution was analyzed with WinMDI 2.9 software (Becton Coulter, Brea, CA). 2.9. Immunohistochemistry Paraffin-embedded tissue samples were deparafinized in xylene and rehydrated in graded alcohols. Endogenous peroxidase was inhibited by incubating the sections in 3% hydrogen peroxide (H2O2) for 30 min. Antigens were retrieved by boiling the tissue sections in citrate buffer (pH 6) for 10 min, followed by successive rinses in phosphate-buffered saline (PBS) containing 0.5% Triton. The slides were incubated for 10 min in 0.1 M glycine (diluted in PBS) and rinsed in PBS. The slides were incubated overnight with rabbit polyclonal antibodies against the human PRAME protein (1:1000 dilution; Abcam, Cambridge, UK) at 4 °C. For the negative control, the primary antibody was replaced with normal goat serum and incubated under the same conditions before immunostaining. After washing with PBS, the slides were incubated with biotinylated goat ant-rabbit antibody for 1 h at room temperature,
Table 1 Cell cycle analysis of PRAME siRNA interference in U-2OS cell. G1 (%) 24 h Control siRNA 48 h Control siRNA 72 h Control siRNA *
p < 0.05.
47.13 ± 0.05 55.47 ± 0.35
G2 (%)
S (%)
10.4 ± 0.44 8.03 ± 1.66
42.5 ± 0.44 36.5 ± 1.31
55.2 ± 3.32* 65.5 ± 2.95*
9.83 ± 2.97 8.97 ± 2.42
34.93 ± 6.21* 24.53 ± 5.35*
64.13 ± 2.50* 73.73 ± 3.04*
10.57 ± 0.92 8.26 ± 0.83
26.4 ± 1.90* 18.03 ± 3.51*
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Fig. 3. Inhibition of PRAME expression results in U-2OS cell cycle arrest. (A) The cell cycle analysis showed that the inhibition PRAME of expression led to U-2OS cell cycle arrest in the G1 phase. (B) The western blots show the protein level changes of PRAME and p27 after PRAME siRNA transfection in U-2OS cells, p27 levels increased, while the PRAME protein levels decreased.
followed by conjugation to a horseradish peroxidase decorated dextran polymer backbone (Envision, Dako, Denmark). Staining
was performed with 3,30 -diaminobenzidine. The slides were counterstained with hematoxylin, dehydrated and mounted.
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Two independent observers evaluated the specimens in a blinded fashion. Each score was discussed by all the authors to reach an overall consensus. The extent of the staining was scored as: (), indicating a negative staining of the tumor cells; (+), indicating staining of <10% of the tumor cells; (++), indicating staining of 10–50% of the cells; and (+++), indicating staining of >50% of the tumor cells. For our study, the staining index scores of (+) and (++) indicated low levels of PRAME expression, where a score of (+++) was regarded as a high level of expression. 2.10. Statistical analysis All the experimental data were analyzed by the SPSS 16.0 statistical software package (SPSS Inc., Chicago, IL). Kaplan–Meier curves were generated using the log-rank tests. Breslow and Tarone-ware tests were performed to assess the prognostic and predictive significance of PRAME expression in tumors. Descriptive statistics were used to summarize the patient characteristics and a statistical analysis of the results was performed using chi-squared tests to investigate relationships between variant PRAME expression and clinicopathological findings. Measurement data from at least two independent were expressed as means ± standard deviations (SD). P < 0.05 was considered statistically significant.
siRNA (P < 0.01) (Fig. 2C). A similar phenomenon was also observed in ZOS cells transfected with PRAME-targeting siRNAs, wild type ZOS cell and ZOS cells transfected with the scrambled non-targeting (P < 0.05) (Fig. 2D). 3.4. Inhibition of colony formation by PRAME siRNA transfection To further study the effects of PRAME knockdown on colony formation by osteosarcoma cells, we seeded PRAME knockdown cells and the negative control cells to form colonies at low densities. As shown in Fig. 2E and F, colony formation by PRAME siRNA transfected cells was significantly lower than that of control cells (P < 0.01). Therefore, the PRAME knockdown resulted in reduced colony formation of osteosarcoma U-2OS and ZOS cells. 3.5. Cell cycle As shown in Table 1 and Fig. 3A, the PRAME knockdown in U2OS cells resulted in a significant increase in the proportion of cells in the G1 phase. Western blotting analysis also showed an increase of P27 protein expression as well as the inhibition of PRAME protein expression (Fig. 3B). 3.6. PRAME over-expression is associated with poor prognosis
3. Results 3.1. Expression levels of PRAME mRNA in osteosarcoma cell lines and tissue samples We first examined the expression levels of PRAME mRNA with RT-PCR and real-time PCR in osteosarcoma cell lines and in an hFOB cell line. The results are provided in Fig.1. High expression levels of PRAME mRNA were detected in the U-2OS, HOS, ZOS, Saos-2 and K562 cell lines, low levels of expression MG-63, and no expression were observed in h-FOB (Fig. 1A). To further investigate whether the osteosacoma tissues also expressed PRAME mRNA, 36 fresh-frozen osteosarcoma specimens and their corresponding muscle tissues at the resection margin, which served as normal tissue control, were tested using real-time PCR. The median DCT (CTPRAME–CTb-actin) value in the osteosarcoma specimens was 9.36 (range, 5.57 to 16.19). For muscle tissue at the resection margin, the median DCT (CTPRAME–CT b-actin) value was 14.56 (range, 8.61– 21.52). As shown in Fig. 1C, PRAME mRNA expression was significantly higher in osteosarcoma specimen than in the muscle tissues at the resection margin (P < 0.001) (Fig. 1B). PRAME protein expression was also detected in five osteosarcoma cell lines and osteosarcoma tissues (Fig. 1C); whereas it was not found in muscle tissues at the resection margin (Fig. 1D). 3.2. PRAME-knockdown attenuates osteosarcoma cell proliferation and leads to cell cycle arrest The mRNA expression level of PRAME was significantly reduced in U-2OS cells at 24 h after siRNA transfection (Fig. 2A). Western blotting also showed that PRAME protein expression was distinctly reduced in U-2OS cells 48 h after siRNA knockdown compared with the negative control (Fig. 2B). These results indicated that the PRAME siRNA was efficient in knocking down the expression of PRAME in osteosarcoma cells at both the mRNA and protein level. 3.3. Inhibition of U-2OS and ZOS proliferation by PRAME siRNA The proliferation of PRAME knockdown U-2OS cells was remarkably reduced compared with wild type U-2OS and the negative control, which was transfected with scrambled non-targeting
3.6.1. Patient clinical characteristics The characteristics of the patients with osteosarcoma in the present study are listed in Table 2. The mean age was 17.2 years (range, 6–45 years). All the patients were followed up for a mean of 86 months (range, 6–144 months). A total of 95 specimens obtained by a preoperative biopsy from the patients who presented with osteosarcoma at diagnosis were examined for PRAME expression by immunohistochemistry. Fig. 4A–F illustrates that PRAME staining was observed both in the nucleus and in the cytoplasm of the tumor cells. PRAME staining was detected in 69 patients’ samples (72.6%). Among these, 32 patients (33.7%) showed more than 50% positive tumor cells. The Kaplan–Meier analysis indicated that the strong PRAME protein expression was positively correlated with poor prognosis (P = 0.01) (Fig. 4G). The strong PRAME expression was also a predictive marker of lung metastases (P = 0.03, Table 3 and Fig. 4H). As detailed in Table 2, the chi-squared analysis indicated no significant associations between PRAME protein expression and other clinicopathological characteristics. Table 2 Clinical characteristics of the patients, tumors, and PRAME staining. Characteristic
Negative or weak positive
Strong positive
P value
Age (years) Sex Male Female Left/right Left Right Site Femur Tibia Humerus Fibula Others Enneking stage II III Histopathology Osteoblastic Chondroblastic Fibroblastic Telagnic Others
19.59 ± 9.05 63 39(15) 24(11)
16.53 ± 6.37 32 26 6
0.0914
33 30
14 18
27 20 7 6 3
17 10 2 2 1
0.5957
59 4
26 6
0.0627
29 5 15 2 9
17 3 10 1 1
0.7306
0.0552
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Fig. 4. PRAME immunostaining correlate with disease prognosis (A–F). Strong, weak positive and negative immunochemistry staining of PRAME in osteosarcoma specimens. (A and B), staining of >50% of tumor cells at 20 and 40 magnification, respectively. (C and D), staining of <50% of the tumor cells at 20 and 40 magnification, respectively. (E and F), PRAME staining in osteofibrous dysplasia samples at 20 and 40 magnification, respectively). (G and H) The Kaplan–Meier curves illustrate a survival analysis of 95 patients with primary osteosarcoma. (G) The clinical outcome of the patients who lacked PRAME expression or showed weak PRAME expression was much better than those with strong, positive PRAME expression. (H) The patients with strong PRAME expression had a much higher risk of lung metastasis than those with weak or negative PRAME expression.
P. Tan et al. / Biochemical and Biophysical Research Communications 419 (2012) 801–808 Table 3 The relevance of strong PRAME staining and patients prognosis. Variable Metastasis Yes No Recurrence Yes No Death Yes No
Strong positive
Negative or weak positive
P value
RR (95% CI)
OD (95% CI)
15 17
8 55
0.00
2.76 (1.65–4.61)
6.07 (2.20–16.76)
3 29 19 19 13
4 59 30 22 41
0.68
1.3 (0.53–3.22)
1.53 (0.32–7.28)
1.925 (1.08–3.43)
2.72 (1.14–6.54)
0.03
4. Discussion In this study, we found that PRAME mRNA was expressed in almost 70% of the osteosarcoma tissues and several osteosarcoma cell lines. In addition, the immunohistochemical analysis showed that the high PRAME expression in osteosarcoma tissues was associated with poor prognosis and lung metastasis. Meanwhile, we used small interfering RNA-induced knockdown of PRAME in U2OS and ZOS cell lines to investigate the potential functions of PRAME in osteosarcoma cells and found that PRAME knockdown can result in cell cycle arrest. PRAME was first found in melanoma and belongs to cancer-antigens category, it expressed in many types of solid tumors and some leukaemias but was not found in normal tissues, with the exception of placenta and male germ-line cells [7]. As PRAME can be recognized by autologous cytolytic T lymphocytes (CTL) and is not expressed in normal tissues, it may be a potential target for tumor therapy [1,7–9]. Studies that have monitored and correlated tumor PRAME expression levels with clinic outcome have been reported by many authors, but the results differed for various tumors. In hematological malignancies, the association between PRAME expression and disease prognosis was controversially [23–25]. In solid malignancies, including lung carcinoma, neuroblastoma, and breast cancer, high PRAME expression was correlated with advance stage disease and poor clinical outcome [2,3,24,26]. Haqq et al. [26] found that high PRAME expression correlated with the stage of melanoma lesions. Oberthue and Partheen [2,27] reported that high expression of PRAME was associated with the short overall survival of neuroblastoma and serous ovarian adenocarinoma patients. In medulloblastoma cases, there was no statistical association between disease prognosis and PRAME overexpression [4]. In our study, PRAME protein was found in more than in 70% of the biopsy samples from patients with osteosarcoma at diagnosis, according to immunohistochemistry analysis. Thus, the universal expression of PRAME in osteosarcoma indicated that PRAME may represent a cancer biomarker and a therapeutic target. Furthermore, the strong staining of PRAME in the immunohistochemistry analysis, which was correlated with poor prognosis and lung metastasis in osteosarcoma, was also identified in our study. PRAME may have an important functional role in tumorigenicity and pluripotency. However, the specific mechanisms of PRAME in osteosarcoma remain unclear and need further investigation. Epping [28] reported that PRAME is a dominant repressor of retinoic acid receptor signaling and that PRAME over-expression blocked RAR-mediated differentiation, including growth arrest and apoptosis in solid tumor cell line models. Oehler [29] reported that PRAME inhibits myeloid tumor cell differentiation in normal hematopoietic and leukemic pro-genitor cells. On the contrary, Steinbach et al. [30] reported that PRAME expression was not associated with the down regulation of retinoic acid signaling. Tajeddine et al. [31] reported that PRAME induces caspaseindependent cell death in leukemic cells, and PRAME can reduce
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tumorigenicity in vivo. Tanaka et al. [32] reported a significant decrease in the colony formation and growth rate in K562 cells of PRAME siRNA-treated cells, as well as G1cell cycle arrest. These reports suggest that PRAME expression has an effect on cell proliferation and differentiation. Thus, the function of PRAME in tumor cells appears to be controversial. To further investigate the potential function of PRAME, we inhibited PRAME expression in U-2OS and ZOS cells using siRNA interference. As a result of PRAME down-regulation, U-2OS and ZOS cells became arrested in G1, and we also observed a significant decrease in colony number. Considering that the PRAME knock down resulted in cell cycle arrest in U-2OS cells, we analyzed p27 protein expression and compared it with PRAME expression. The results showed that down-regulation PRAME protein expression was associated with high expression of the p27 protein, which plays an important role in cell cycle regulation. These results suggested that inhibiting the expression of PRAME suppresses tumor cell growth by regulating the cell cycle. However, the detailed mechanisms of how PRAME suppresses p27 expression have yet to be determined. In conclusion, we found that PRAME was universally expressed in osteosarcoma tissue. The inhibition of PRAME by siRNA in U-2OS cells can cause cell cycle arrest in the G1 phase and result in a decreased number of cell colonies. Furthermore, the PRAME gene may be important for maintaining the proliferation of tumor cells. Although the details of the functional mechanisms of PRAME need further investigation in vivo, insights into the function of PRAME can be expected to provide a new perspective in term of tumorigenicity, pluripotency, and novel tumor treatment. Acknowledgments We thank Dr. Zheng Yang (Department of Pathology, First Affiliated Hospital of SunYat-Sen University) for immunohistochemical score analysis. This project was supported by the National Natural Science Foundation of China (30973504, Jingnan Shen), the Sun Yat-Sen University Clinical Research 5010 Program (2007009, Jingnan Shen) and Science Technology Planning of Guangdong Province (2009B060300004, Qiao Su). References [1] H. Ikeda, B. Lethe, F. Lehmann, et al., Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor, Immunity 6 (1997) 199–208. [2] A. Oberthuer, B. Hero, R. Spitz, et al., The tumor-associated antigen PRAME is universally expressed in high-stage neuroblastoma and associated with poor outcome, Clin Cancer Res 10 (2004) 4307–4313. [3] M.T. Epping, A.A. Hart, A.M. Glas, et al., PRAME expression and clinical outcome of breast cancer, Br J Cancer 99 (2008) 398–403. [4] T.M. Vulcani-Freitas, N. Saba-Silva, A. Cappellano, et al., PRAME gene expression profile in medulloblastoma, Arq Neuropsiquiatr 69 (2011) 9–12. [5] E. Neumann, A. Engelsberg, J. Decker, et al., Heterogeneous expression of the tumor-associated antigens RAGE-1, PRAME, and glycoprotein 75 in human renal cell carcinoma: candidates for T-cell-based immunotherapies?, Cancer Res 58 (1998) 4090–4095 [6] J. Bankovic, J. Stojsic, D. Jovanovic, et al., Identification of genes associated with non-small-cell lung cancer promotion and progression, Lung Cancer 67 (2010) 151–159. [7] F. Wadelin, J. Fulton, P.A. McEwan, et al., Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia, Mol Cancer 9 (2010) 226. [8] M.T. Epping, R. Bernards, A causal role for the human tumor antigen preferentially expressed antigen of melanoma in cancer, Cancer Res 66 (2006) 10639–10642. [9] R. Proto-Siqueira, L.L. Figueiredo-Pontes, R.A. Panepucci, et al., PRAME is a membrane and cytoplasmic protein aberrantly expressed in chronic lymphocytic leukemia and mantle cell lymphoma, Leuk Res 30 (2006) 1333– 1339. [10] T. Schenk, S. Stengel, S. Goellner, et al., Hypomethylation of PRAME is responsible for its aberrant overexpression in human malignancies, Genes Chromosomes Cancer 46 (2007) 796–804. [11] S. Paydas, Is everything known in all faces of iceberg in PRAME?, Leuk Res 32 (2008) 1356–1357
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Expression and prognostic relevance of PRAME in primary osteosarcoma Pingxian Tan a, Changye Zou a, Bicheng Yong a, Ju Han b, Longjuan Zhang c, Qiao Su d, Junqiang Yin a, Jin Wang a, Gang Huang a, Tingsheng Peng b, Jingnian Shen a,⇑ a
Department of Orthopaedic Surgery, Musculoskeletal Tumor Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China Department of Pathology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China c Surgery Lab. Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China d Experimental Animal Center, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China b
a r t i c l e
i n f o
Article history: Received 10 February 2012 Available online 27 February 2012 Keywords: PRAME Preferentially expressed antigen of melanoma Osteosarcoma Prognosis siRNA Proliferation
a b s t r a c t The preferentially expressed antigen of melanoma (PRAME), a cancer-testis antigen with unknown function, is expressed in many human malignancies and is considered an attractive potential target for tumor immunotherapy. However, studies of its expression and function in osteosarcoma have rarely been reported. In this study, we found that PRAME is expressed in five osteosarcoma cell lines and in more than 70% of osteosarcoma patient specimens. In addition, an immunohistochemical analysis showed that high PRAME expression was associated with poor prognosis and lung metastasis. Furthermore, PRAME siRNA knockdown significantly suppressed the proliferation, colony formation, and G1 cell cycle arrest in U-2OS cells. Our results suggest that PRAME plays an important role in cell proliferation and disease progression in osteosarcoma. However, the detail mechanisms of PRAME function in osteosarcoma require further investigation. Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved.
1. Introduction Preferentially expressed antigen in melanoma (PRAME) belongs to the cancer-testis antigen family and was first identified by Ikeda et al. in a patient with melanoma [1]. The PRAME gene encodes a HLA-A24-restricted antigenic peptide that can be recognized by autologous tumor-specific cytotoxic T lymphocyte cells and has been found to be expressed in a number of solid tumors and various leukemias [1–7]. Because PRAME is recognized by tumorspecific cytotoxic T lymphocytes and because it is not detected in normal tissues (except low expression in testicular, ovarian, endometrial and adrenal tissue) but is over-expressed in a number of human malignancies, PRAME is considered an attractive potential target for tumor immunotherapy and a diagnostic marker in many human tumors that express this antigen [8]. Though some studies have shown that PRAME expression in tumors is correlated to clinical outcome and extensive investigations into PRAME function have been reported, its specific mechanism in tumors remain elusive [9–11]. Osteosarcoma is an aggressive cancer of the bone with unknown etiology. It predominantly affects children and adolescents [12–14]. Despite the dramatic advances in osteosarcoma treatment in recent ⇑ Corresponding author. Address: Department of Orthopaedic Surgery, Musculoskeletal Tumor Center, First Affiliated Hospital of Sun Yat-Sen University, No. 58, Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. E-mail address: [email protected] (J. Shen).
years, patient survival has reached a plateau. Therefore, novel treatment modalities are urgently needed to improve survival [15–17]. The use of tumor antigens as targets for immunotherapy has emerged as a treatment strategy in recent years [18,19]. New tumor antigen markers could further improve diagnosis and predict prognosis. However, few antigenic peptides have been identified in bone or soft tissue sarcomas thus far, and the development of appropriate vaccines and therapies has been hampered by the lack of knowledge regarding the expression of tumor-specific antigens in osteosarcoma. From our previous gene microarray and proteomic analysis [20,21], we found that cancer-testis antigens family (including PRAME) expressed in most of the tested osteosarcoma tissues and a few osteosarcoma cell lines. In the present study, we further investigated the pattern of PRAME expression in osteosarcoma tissues and several cell lines, PRAME’s relevance to disease prognosis and the potential function of PRAME in osteosarcoma.
2. Materials and methods 2.1. Cell lines and cell culture The human osteosarcoma cell lines MG-63, U-2OS and Saos-2 were gifts from Dr. M. Serra (Istituti Ortopedici Rizzoli, Bologna, Italy). The human osteosarcoma cell line ZOS was established from primary tumor tissues in our institution [22]. The human osteoblast cells (h-FOB) were purchased from Born Cells Resource
0006-291X/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2012.02.110
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Fig. 1. Expression levels of PRAME in osteosarcoma tissues and cell lines. (A) PRAME mRNA expression levels in osteosarcoma cell lines and human osteoblast cells. K562 cells were used as a positive control. (B) Real-time PCR revealed that 36 osteosarcoma tissue samples had increased PRAME mRNA levels compared with the muscle tissue specimens at the surgical margin. (C) PRAME protein expression levels in K562 cells, osteosarcoma cell lines and the human osteoblast cells. (D) PRAME protein expression levels in osteosarcoma tissues and muscle tissues at the resection margin.
Fig. 2. Inhibition of PRAME expression prevents osteosarcoma growth in vitro. (A) The RT-PCR and (B) Western blotting demonstrate the inhibition of PRAME expression after siRNA interference. (C) CCK8 analysis of the growth of three groups of normal, negative control and PRAME siRNA U2OS cells (⁄P < 0.01 PRAME siRNA vs. negative control and untransfected cells.). (D) Similar results were observed in ZOS, ZOS-control and ZOS-siRNA cell. (E and F) The colony formation assay revealed that PRAME knock-down reduced colony formation in U2OS and ZOS cells. U-2OS, U-2OS siRNA, ZOS and ZOS siRNA cells were seeded at a low density (500 cells/ml) and incubated in DMEM supplemented with 10% FCS for 21 days. The cell colonies were fixed and stained with crystal violet. The experiment was performed three times and reported as the mean ± SD. The sterisk indicates a significant difference between PRAME siRNA-treated and scrambled siRNA-treated U-2OS and ZOS cells.
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Center, Chinese Academy of Sciences (Shanghai, China), and were used as a negative control. The cell line K562 was kindly provided by Dr. Wang (Department of Hematology, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China), and were used as a positive control. The cells were cultured in D-MEM or RPMI 1640 (Gibco, Invitrogen) supplemented with 10% fetal calf serum (FCS) and 1 mg/mL penicillin–streptomycin (Sigma, Saint Louis MO, USA) and incubated at 37 °C and 5% CO2 in a humidified incubator. 2.2. Patients and tissue samples The medical records of osteosarcoma patients who were seen in our department from January 1999 to December 2010 were reviewed. All the patients underwent neoadjuvant chemotherapy and definitive surgery. Osteosarcoma was confirmed histopathologically, and follow-up data were extracted from our database. For the RNA analysis, fresh tumor specimens were snap frozen in liquid nitrogen after surgical resection, and then cryopreserved at 80 °C in our tissue bank. Only tumor samples that were evaluated by pathologists and composed of >80% tumor cells were used for the study. The formalin-fixed, paraffin-embedded specimens from osteosarcoma biopsies (before any treatment was initiated) and sixteen osteofibrous dysplasia samples were collected at the tissue bank of the Department of Pathology, First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). The osteofibrous dysplasia samples were used as the negative control. Prior patient (or guardian) consent and approval from the Institutional Research Ethics Committee of Sun Yat-Sen University were obtained prior to the use of these tumor specimens and clinical materials for research purposes. 2.3. Silencing of PRAME by short interfering RNAs (siRNAs) in U2OS and ZOS cells Control and PRAME short interfering RNAs were purchased by Invitrogen. The U-2OS and ZOS cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. PRAME silencing was confirmed by RT-PCR, real-time PCR and Western-blotting. 2.4. Total RNA and protein extraction Thirty-six freshly dissected tumor specimens and muscle tissues from wide surgical margins were obtained from osteosarcoma patients treated in our institution. Total cell and tissue sample mRNA and protein were extracted using TRIzol (Invitrogen) and RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China), respectively, according to the manufacturer’s instructions and stored at 80 °C. 2.5. RT- PCR and real-time PCR A total of 500 ng of RNA was transcribed into cDNA using a PrimeScript RT reagent Kit (Invitrogen). The reverse transcription PCR and real-time PCR oligonucleotide primers for PRAME and b-actin were designed according to the literature [1]. The RT-PCR amplification protocol was as follows: denaturation at 94 °C for 10 min, 35 cycles of 45 s at 94 °C, 60 s at 64 °C and 45 s at 72 °C, and a final extension step at 72 °C for 7 min. Three percent agarose gel electrophoresis was performed to detect the amplified products. Real-time PCR reactions were performed in triplicate in a 10 lL reaction volume using standard conditions with 40 cycles of amplification. The amplification products were detected in the presence of the SYBRÒ Premix Ex TaqTM kits reagents (TaKaRa Biotechnology
CO., Dalian, China) with an ABI7900 PRISM real-time PCR instrument (Applied Biosystems, Sunnyvale, CA). The mean cycle threshold value (Ct) from triplicate samples was used to calculate gene expression. The PCR products were normalized to b-actin levels. 2.6. Western blotting Western blotting analysis was performed by using standard methods with anti-human PRAME protein (1:1000, Abcam, Cambridge, UK), anti-P27 (1:500, Beyotime Institute of Biotechnology, Shanghai, China) and anti-GAPDH antibody (1:1000, Sigma). 2.7. CCK-8 cell viability assay Cell proliferation was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratory, Kumamoto, Japan) according to manufacturer’s instructions. U-2OS and ZOS cells were seeded in 96-well plates at a density of 3000 cells/well and grown in DMEM supplemented with 1% FBS. The cells were transfected with the siRNA and control interfering. At 24, 48, 72, 96, and 120 h after transfection, CCK-8 mixed with serum-free DMEM was added at a ratio of 1:10. After incubation for 2 h, the absorbance at 450 nm was measured. All the experiments were performed in triplicate. 2.8. Flow cytometry A cell cycle analysis of the PRAME siRNA and control siRNAtreated cells was performed by flow cytometry. At 24, 48, and 72 h after transfection, the cells were trypsinized, washed in PBS and fixed in 70% cold ethanol for 24 h. After fixation, the cells were washed, and the DNA was stained with 50 lg/mL propidium iodide (PI) solution in the presence of 250 lg/ml RNAse A (Sigma). The cellular DNA content distribution was analyzed with WinMDI 2.9 software (Becton Coulter, Brea, CA). 2.9. Immunohistochemistry Paraffin-embedded tissue samples were deparafinized in xylene and rehydrated in graded alcohols. Endogenous peroxidase was inhibited by incubating the sections in 3% hydrogen peroxide (H2O2) for 30 min. Antigens were retrieved by boiling the tissue sections in citrate buffer (pH 6) for 10 min, followed by successive rinses in phosphate-buffered saline (PBS) containing 0.5% Triton. The slides were incubated for 10 min in 0.1 M glycine (diluted in PBS) and rinsed in PBS. The slides were incubated overnight with rabbit polyclonal antibodies against the human PRAME protein (1:1000 dilution; Abcam, Cambridge, UK) at 4 °C. For the negative control, the primary antibody was replaced with normal goat serum and incubated under the same conditions before immunostaining. After washing with PBS, the slides were incubated with biotinylated goat ant-rabbit antibody for 1 h at room temperature,
Table 1 Cell cycle analysis of PRAME siRNA interference in U-2OS cell. G1 (%) 24 h Control siRNA 48 h Control siRNA 72 h Control siRNA *
p < 0.05.
47.13 ± 0.05 55.47 ± 0.35
G2 (%)
S (%)
10.4 ± 0.44 8.03 ± 1.66
42.5 ± 0.44 36.5 ± 1.31
55.2 ± 3.32* 65.5 ± 2.95*
9.83 ± 2.97 8.97 ± 2.42
34.93 ± 6.21* 24.53 ± 5.35*
64.13 ± 2.50* 73.73 ± 3.04*
10.57 ± 0.92 8.26 ± 0.83
26.4 ± 1.90* 18.03 ± 3.51*
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Fig. 3. Inhibition of PRAME expression results in U-2OS cell cycle arrest. (A) The cell cycle analysis showed that the inhibition PRAME of expression led to U-2OS cell cycle arrest in the G1 phase. (B) The western blots show the protein level changes of PRAME and p27 after PRAME siRNA transfection in U-2OS cells, p27 levels increased, while the PRAME protein levels decreased.
followed by conjugation to a horseradish peroxidase decorated dextran polymer backbone (Envision, Dako, Denmark). Staining
was performed with 3,30 -diaminobenzidine. The slides were counterstained with hematoxylin, dehydrated and mounted.
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Two independent observers evaluated the specimens in a blinded fashion. Each score was discussed by all the authors to reach an overall consensus. The extent of the staining was scored as: (), indicating a negative staining of the tumor cells; (+), indicating staining of <10% of the tumor cells; (++), indicating staining of 10–50% of the cells; and (+++), indicating staining of >50% of the tumor cells. For our study, the staining index scores of (+) and (++) indicated low levels of PRAME expression, where a score of (+++) was regarded as a high level of expression. 2.10. Statistical analysis All the experimental data were analyzed by the SPSS 16.0 statistical software package (SPSS Inc., Chicago, IL). Kaplan–Meier curves were generated using the log-rank tests. Breslow and Tarone-ware tests were performed to assess the prognostic and predictive significance of PRAME expression in tumors. Descriptive statistics were used to summarize the patient characteristics and a statistical analysis of the results was performed using chi-squared tests to investigate relationships between variant PRAME expression and clinicopathological findings. Measurement data from at least two independent were expressed as means ± standard deviations (SD). P < 0.05 was considered statistically significant.
siRNA (P < 0.01) (Fig. 2C). A similar phenomenon was also observed in ZOS cells transfected with PRAME-targeting siRNAs, wild type ZOS cell and ZOS cells transfected with the scrambled non-targeting (P < 0.05) (Fig. 2D). 3.4. Inhibition of colony formation by PRAME siRNA transfection To further study the effects of PRAME knockdown on colony formation by osteosarcoma cells, we seeded PRAME knockdown cells and the negative control cells to form colonies at low densities. As shown in Fig. 2E and F, colony formation by PRAME siRNA transfected cells was significantly lower than that of control cells (P < 0.01). Therefore, the PRAME knockdown resulted in reduced colony formation of osteosarcoma U-2OS and ZOS cells. 3.5. Cell cycle As shown in Table 1 and Fig. 3A, the PRAME knockdown in U2OS cells resulted in a significant increase in the proportion of cells in the G1 phase. Western blotting analysis also showed an increase of P27 protein expression as well as the inhibition of PRAME protein expression (Fig. 3B). 3.6. PRAME over-expression is associated with poor prognosis
3. Results 3.1. Expression levels of PRAME mRNA in osteosarcoma cell lines and tissue samples We first examined the expression levels of PRAME mRNA with RT-PCR and real-time PCR in osteosarcoma cell lines and in an hFOB cell line. The results are provided in Fig.1. High expression levels of PRAME mRNA were detected in the U-2OS, HOS, ZOS, Saos-2 and K562 cell lines, low levels of expression MG-63, and no expression were observed in h-FOB (Fig. 1A). To further investigate whether the osteosacoma tissues also expressed PRAME mRNA, 36 fresh-frozen osteosarcoma specimens and their corresponding muscle tissues at the resection margin, which served as normal tissue control, were tested using real-time PCR. The median DCT (CTPRAME–CTb-actin) value in the osteosarcoma specimens was 9.36 (range, 5.57 to 16.19). For muscle tissue at the resection margin, the median DCT (CTPRAME–CT b-actin) value was 14.56 (range, 8.61– 21.52). As shown in Fig. 1C, PRAME mRNA expression was significantly higher in osteosarcoma specimen than in the muscle tissues at the resection margin (P < 0.001) (Fig. 1B). PRAME protein expression was also detected in five osteosarcoma cell lines and osteosarcoma tissues (Fig. 1C); whereas it was not found in muscle tissues at the resection margin (Fig. 1D). 3.2. PRAME-knockdown attenuates osteosarcoma cell proliferation and leads to cell cycle arrest The mRNA expression level of PRAME was significantly reduced in U-2OS cells at 24 h after siRNA transfection (Fig. 2A). Western blotting also showed that PRAME protein expression was distinctly reduced in U-2OS cells 48 h after siRNA knockdown compared with the negative control (Fig. 2B). These results indicated that the PRAME siRNA was efficient in knocking down the expression of PRAME in osteosarcoma cells at both the mRNA and protein level. 3.3. Inhibition of U-2OS and ZOS proliferation by PRAME siRNA The proliferation of PRAME knockdown U-2OS cells was remarkably reduced compared with wild type U-2OS and the negative control, which was transfected with scrambled non-targeting
3.6.1. Patient clinical characteristics The characteristics of the patients with osteosarcoma in the present study are listed in Table 2. The mean age was 17.2 years (range, 6–45 years). All the patients were followed up for a mean of 86 months (range, 6–144 months). A total of 95 specimens obtained by a preoperative biopsy from the patients who presented with osteosarcoma at diagnosis were examined for PRAME expression by immunohistochemistry. Fig. 4A–F illustrates that PRAME staining was observed both in the nucleus and in the cytoplasm of the tumor cells. PRAME staining was detected in 69 patients’ samples (72.6%). Among these, 32 patients (33.7%) showed more than 50% positive tumor cells. The Kaplan–Meier analysis indicated that the strong PRAME protein expression was positively correlated with poor prognosis (P = 0.01) (Fig. 4G). The strong PRAME expression was also a predictive marker of lung metastases (P = 0.03, Table 3 and Fig. 4H). As detailed in Table 2, the chi-squared analysis indicated no significant associations between PRAME protein expression and other clinicopathological characteristics. Table 2 Clinical characteristics of the patients, tumors, and PRAME staining. Characteristic
Negative or weak positive
Strong positive
P value
Age (years) Sex Male Female Left/right Left Right Site Femur Tibia Humerus Fibula Others Enneking stage II III Histopathology Osteoblastic Chondroblastic Fibroblastic Telagnic Others
19.59 ± 9.05 63 39(15) 24(11)
16.53 ± 6.37 32 26 6
0.0914
33 30
14 18
27 20 7 6 3
17 10 2 2 1
0.5957
59 4
26 6
0.0627
29 5 15 2 9
17 3 10 1 1
0.7306
0.0552
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Fig. 4. PRAME immunostaining correlate with disease prognosis (A–F). Strong, weak positive and negative immunochemistry staining of PRAME in osteosarcoma specimens. (A and B), staining of >50% of tumor cells at 20 and 40 magnification, respectively. (C and D), staining of <50% of the tumor cells at 20 and 40 magnification, respectively. (E and F), PRAME staining in osteofibrous dysplasia samples at 20 and 40 magnification, respectively). (G and H) The Kaplan–Meier curves illustrate a survival analysis of 95 patients with primary osteosarcoma. (G) The clinical outcome of the patients who lacked PRAME expression or showed weak PRAME expression was much better than those with strong, positive PRAME expression. (H) The patients with strong PRAME expression had a much higher risk of lung metastasis than those with weak or negative PRAME expression.
P. Tan et al. / Biochemical and Biophysical Research Communications 419 (2012) 801–808 Table 3 The relevance of strong PRAME staining and patients prognosis. Variable Metastasis Yes No Recurrence Yes No Death Yes No
Strong positive
Negative or weak positive
P value
RR (95% CI)
OD (95% CI)
15 17
8 55
0.00
2.76 (1.65–4.61)
6.07 (2.20–16.76)
3 29 19 19 13
4 59 30 22 41
0.68
1.3 (0.53–3.22)
1.53 (0.32–7.28)
1.925 (1.08–3.43)
2.72 (1.14–6.54)
0.03
4. Discussion In this study, we found that PRAME mRNA was expressed in almost 70% of the osteosarcoma tissues and several osteosarcoma cell lines. In addition, the immunohistochemical analysis showed that the high PRAME expression in osteosarcoma tissues was associated with poor prognosis and lung metastasis. Meanwhile, we used small interfering RNA-induced knockdown of PRAME in U2OS and ZOS cell lines to investigate the potential functions of PRAME in osteosarcoma cells and found that PRAME knockdown can result in cell cycle arrest. PRAME was first found in melanoma and belongs to cancer-antigens category, it expressed in many types of solid tumors and some leukaemias but was not found in normal tissues, with the exception of placenta and male germ-line cells [7]. As PRAME can be recognized by autologous cytolytic T lymphocytes (CTL) and is not expressed in normal tissues, it may be a potential target for tumor therapy [1,7–9]. Studies that have monitored and correlated tumor PRAME expression levels with clinic outcome have been reported by many authors, but the results differed for various tumors. In hematological malignancies, the association between PRAME expression and disease prognosis was controversially [23–25]. In solid malignancies, including lung carcinoma, neuroblastoma, and breast cancer, high PRAME expression was correlated with advance stage disease and poor clinical outcome [2,3,24,26]. Haqq et al. [26] found that high PRAME expression correlated with the stage of melanoma lesions. Oberthue and Partheen [2,27] reported that high expression of PRAME was associated with the short overall survival of neuroblastoma and serous ovarian adenocarinoma patients. In medulloblastoma cases, there was no statistical association between disease prognosis and PRAME overexpression [4]. In our study, PRAME protein was found in more than in 70% of the biopsy samples from patients with osteosarcoma at diagnosis, according to immunohistochemistry analysis. Thus, the universal expression of PRAME in osteosarcoma indicated that PRAME may represent a cancer biomarker and a therapeutic target. Furthermore, the strong staining of PRAME in the immunohistochemistry analysis, which was correlated with poor prognosis and lung metastasis in osteosarcoma, was also identified in our study. PRAME may have an important functional role in tumorigenicity and pluripotency. However, the specific mechanisms of PRAME in osteosarcoma remain unclear and need further investigation. Epping [28] reported that PRAME is a dominant repressor of retinoic acid receptor signaling and that PRAME over-expression blocked RAR-mediated differentiation, including growth arrest and apoptosis in solid tumor cell line models. Oehler [29] reported that PRAME inhibits myeloid tumor cell differentiation in normal hematopoietic and leukemic pro-genitor cells. On the contrary, Steinbach et al. [30] reported that PRAME expression was not associated with the down regulation of retinoic acid signaling. Tajeddine et al. [31] reported that PRAME induces caspaseindependent cell death in leukemic cells, and PRAME can reduce
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tumorigenicity in vivo. Tanaka et al. [32] reported a significant decrease in the colony formation and growth rate in K562 cells of PRAME siRNA-treated cells, as well as G1cell cycle arrest. These reports suggest that PRAME expression has an effect on cell proliferation and differentiation. Thus, the function of PRAME in tumor cells appears to be controversial. To further investigate the potential function of PRAME, we inhibited PRAME expression in U-2OS and ZOS cells using siRNA interference. As a result of PRAME down-regulation, U-2OS and ZOS cells became arrested in G1, and we also observed a significant decrease in colony number. Considering that the PRAME knock down resulted in cell cycle arrest in U-2OS cells, we analyzed p27 protein expression and compared it with PRAME expression. The results showed that down-regulation PRAME protein expression was associated with high expression of the p27 protein, which plays an important role in cell cycle regulation. These results suggested that inhibiting the expression of PRAME suppresses tumor cell growth by regulating the cell cycle. However, the detailed mechanisms of how PRAME suppresses p27 expression have yet to be determined. In conclusion, we found that PRAME was universally expressed in osteosarcoma tissue. The inhibition of PRAME by siRNA in U-2OS cells can cause cell cycle arrest in the G1 phase and result in a decreased number of cell colonies. Furthermore, the PRAME gene may be important for maintaining the proliferation of tumor cells. Although the details of the functional mechanisms of PRAME need further investigation in vivo, insights into the function of PRAME can be expected to provide a new perspective in term of tumorigenicity, pluripotency, and novel tumor treatment. Acknowledgments We thank Dr. Zheng Yang (Department of Pathology, First Affiliated Hospital of SunYat-Sen University) for immunohistochemical score analysis. This project was supported by the National Natural Science Foundation of China (30973504, Jingnan Shen), the Sun Yat-Sen University Clinical Research 5010 Program (2007009, Jingnan Shen) and Science Technology Planning of Guangdong Province (2009B060300004, Qiao Su). References [1] H. Ikeda, B. Lethe, F. Lehmann, et al., Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor, Immunity 6 (1997) 199–208. [2] A. Oberthuer, B. Hero, R. Spitz, et al., The tumor-associated antigen PRAME is universally expressed in high-stage neuroblastoma and associated with poor outcome, Clin Cancer Res 10 (2004) 4307–4313. [3] M.T. Epping, A.A. Hart, A.M. Glas, et al., PRAME expression and clinical outcome of breast cancer, Br J Cancer 99 (2008) 398–403. [4] T.M. Vulcani-Freitas, N. Saba-Silva, A. Cappellano, et al., PRAME gene expression profile in medulloblastoma, Arq Neuropsiquiatr 69 (2011) 9–12. [5] E. Neumann, A. Engelsberg, J. Decker, et al., Heterogeneous expression of the tumor-associated antigens RAGE-1, PRAME, and glycoprotein 75 in human renal cell carcinoma: candidates for T-cell-based immunotherapies?, Cancer Res 58 (1998) 4090–4095 [6] J. Bankovic, J. Stojsic, D. Jovanovic, et al., Identification of genes associated with non-small-cell lung cancer promotion and progression, Lung Cancer 67 (2010) 151–159. [7] F. Wadelin, J. Fulton, P.A. McEwan, et al., Leucine-rich repeat protein PRAME: expression, potential functions and clinical implications for leukaemia, Mol Cancer 9 (2010) 226. [8] M.T. Epping, R. Bernards, A causal role for the human tumor antigen preferentially expressed antigen of melanoma in cancer, Cancer Res 66 (2006) 10639–10642. [9] R. Proto-Siqueira, L.L. Figueiredo-Pontes, R.A. Panepucci, et al., PRAME is a membrane and cytoplasmic protein aberrantly expressed in chronic lymphocytic leukemia and mantle cell lymphoma, Leuk Res 30 (2006) 1333– 1339. [10] T. Schenk, S. Stengel, S. Goellner, et al., Hypomethylation of PRAME is responsible for its aberrant overexpression in human malignancies, Genes Chromosomes Cancer 46 (2007) 796–804. [11] S. Paydas, Is everything known in all faces of iceberg in PRAME?, Leuk Res 32 (2008) 1356–1357
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