Tumori, 92: 236-240, 2006
UPREGULATION OF THE ONCOGENIC HELIX-LOOP-HELIX PROTEIN ID2 IN EWING SARCOMA Hye-Rim Park1, Woon Won Jung2, Hyun Sook Kim2, Eduard Santini-Araujo3, Ricardo K Kalil4, Patrizia Bacchini5, Franco Bertoni5, K Krishnan Unni6, and Yong-Koo Park7 1 Department of Pathology, College of Medicine, Hallym University, Anyang, Korea; 2My Gene Bioscience Institute, Seoul, Korea; 3Department of Pathology, University of Buenos Aires, Buenos Aires, Argentina; 4Rede SARAH de Hospitais do Aparelho, Locomotor, Bloco A-Area de Patologia, Brasilia, Distrito Federal, Brazil; 5Servizio di Anatomia Patologica, Istituto Ortopedico Rizzoli, Bologna, Italy; 6Department of Pathology, Mayo Clinic, Rochester, MN, USA; 7Department of Pathology, College of Medicine, Kyung Hee University, Seoul, Korea
Aims and background: Id helix-loop-helix proteins function as regulators of cell growth and differentiation. However, they can induce malignant transformation when overexpressed. The EWS/ETS chimeric proteins in Ewing sarcoma act as aberrant transcription factors leading to tumorigenic processes. An enhanced expression of the Id2 gene in Ewing sarcoma cells was previously shown by gene array techniques. We investigated the expression of Id2 at the protein and gene level in Ewing sarcoma. Methods: We evaluated the expression of Id2 protein using immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a total of 71 cases of Ewing sarcoma. Additionally, a Ewing sarcoma cell line was examined by real-time quantitative PCR.
Results: Id2 expression was observed in 65 cases (91.5%) of the 71 total cases examined and a high level of Id2 expression was observed in 45 of these cases (63.8%). In tumor cells, Id2 proteins displayed cytoplasmic as well as nuclear localization. The amplification of the Id2 gene was not noted in a Ewing sarcoma cell line using real-time quantitative PCR. The crossing points of Id2 in the Ewing sarcoma cell line, control fibroblast, and osteosarcoma cell line were 18.54 ± 0.16, 18.25, and 18.34, respectively. Conclusions: Our data support a role for increased Id2 protein expression in Ewing sarcoma. However, this overexpression of the Id2 protein could not be confirmed by a corresponding change at the gene level in a Ewing sarcoma cell line.
Key words: Ewing sarcoma, Id2 protein.
Introduction
Ewing sarcoma is characterized by specific balanced chromosomal translocations that result in the fusion of the N-terminus of the EWS gene product to the C-terminus of an ETS transcription factor, which includes protein family members FLI1, ERG, ETV1, E1AF or FEV1-5. Rare cases harboring FUS-ERG fusion transcripts have also been reported 6 . The EWS-FLI1 chimeric protein appears to be essential for maintaining the oncogenic property of tumor cells7. The EWS/ETS chimeric proteins work as aberrant transcription factors that interfere with legitimate cellular functions and promote oncogenesis in Ewing sarcomas. Id (inhibitor of DNA binding) proteins are helix-loophelix (HLH) proteins that lack the basic amino acid domain necessary to bind DNA. They regulate transcription factors that are involved in developmental processes such as myogenesis, neurogenesis, bone morphogenesis, lymphopoiesis, hematopoiesis, and myeloid differentiation8. Id proteins are also involved in cellular proliferation processes. Previous studies showed that Id2 was overexpressed in several types of tumors and that upregulation of Id2 was mediated by N-myc, c-myc or TCF9. The TGF-beta type II receptor, mE2-C, EAT-2, PDGF-C, p57KIP2 and c-myc genes were previously
identified as transcriptional targets for EWS/ETS proteins10-12, although the exact tumorigenic mechanism mediated by EWS/ETS remains unclear. Since the ETS proteins augment expression of various types of transcription factors, we presumed that there would be other genes that have their expression modified by these chimeric proteins. Nishimori et al. reported that the Id2 gene, which encodes a helix-loop-helix protein, is a target of transcriptional activation by EWS-FLI1 and EWS-ERG, which mediated this by directly interacting with the Id2 promoter13. We demonstrate in this report that high levels of Id2 expression occur in Ewing sarcomas, which suggests a role for Id2 in the tumorigenesis of Ewing sarcoma. Materials and methods
Patients and samples We collected 71 cases of Ewing sarcoma from the files of the Rizzoli Institute (Bologna, Italy), Sarah Network of Hospitals (Brasilia, Brazil), University of Buenos Aires (Buenos Aires, Argentina), and Kyung Hee University Hospital (Seoul, Korea). The protocol was reviewed and approved by the Institutional Review Board in the Kyung Hee University Hospital. All cases were independently reviewed by two of the authors
Correspondence to: Dr Yong-Koo Park, Department of Pathology, Kyung Hee University Hospital, #1 Hoeki-dong, Dongdaemun-ku, Seoul 130-702, Korea. Tel +82-2-958-8742; fax +82-2-957-0489; e-mail
[email protected] Received August 8, 2005; accepted February 14, 2006.
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(HRP, YKP) and the diagnoses were confirmed in all instances. Formalin-fixed and paraffin-embedded tissues were available.
Table 1 - Oligonucleotide primers Name
DNA isolation from cell lines Genomic DNA was extracted from the ES97 Ewing sarcoma cell line (American Type Culture Collection, Manassas, VA, USA), the MG63 osteosarcoma cell line, and control fibroblast from primary skin culture for molecular analysis. Cells were cultured in RPMI 1640 medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum. Each of the cultured cell lines was collected in 15 mL tubes from cell culture dishes after trypsinization. The cells were washed twice using 1.0 mL of 1X phosphate buffered saline. The tissue pellet was incubated overnight with 80 µ L lysis buffer (Roche Diagnostics GmbH, Mannheim, Germany) and 20 µL proteinase K to extract cellular contents. The supernatant obtained after a brief centrifugation was transferred to a Magna Pure LC sample cartridge and the DNA of the sample tissue was automatically extracted. Real-time PCR Oligonucleotide primers. The sequences of the primers used in this study are shown in Table 1. Primers were designed using Primer3 software and produced with a DNA synthesizer (Polygen GmbH, Mannheim, Germany). All primers were purified with an HPLC (Waters, Milford, MA, USA). The length of the amplified sequence was 100 bp for Id2 and 170 bp for the reference gene, GAPDH. DNA amplification. PCR products were amplified and quantified using SYBR green intercalation in a LightCycler system (Roche Diagnostics GmbH, Mannheim, Germany). All reactions were carried out in a total volume of 20 µL per capillary. Each reaction mixture contained 2.0 µL of 10X buffer (50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.001% gelatin), 2.0 µL of 2.5 mM MgCl2, 1.6 µL of 2.5 mM dNTP, 1.0 µL of primer (10 pmol/mL), 0.4 µL of Amplitaq (5 U/µL), 1.0
Size
Id2
5’- CGA CTG CTA CTC CAA GCT CAA G -3’ 5’- GAT GAC GTG CTG CAG GAT TTC -3’
100bp
GAPDH
5’- GTG AAG GTC GGA GTC AAC GGA -3’ 5’- ACG GTG CCA TGG AAT TTG CCA T -3’
170bp
Immunohistochemical analysis The avidin-biotin complex (ABC) method was performed on 4-µm-thick tissue sections. Sections were deparaffinized with xylene for 15 minutes. Sections were incubated (1:100 dilution) with the rabbit polyclonal antibody C-20 directed against Id2 (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). Positive (colon adenocarcinoma) and negative controls were used in each experiment. Controls using polyclonal rabbit immunoglobulin instead of primary antibody showed no evidence of staining. Semiquantitative assessment of expression was performed according to the following criteria: -, negative staining of neoplastic cells; (+), focal (50%) positivity of neoplastic cells.
Sequence
mL of SYBR Green I (Molecular Probes, Eugene, OR, USA; 10,000-fold dilution), 1.0 µ L of DNA (100 ng/µL) and 10.0 µL of sterile water. The amplification reaction conditions for Id2 were: 1 cycle at 95 °C for 0 seconds followed by 30 cycles at 60 °C for 5 seconds and 72 °C for 12 seconds. GAPDH reaction conditions were: 1 cycle at 95 °C for 0 seconds followed by 30 cycles at 60 °C for 5 seconds and 72 °C for 12 seconds. Melting curves were generated after the amplification. Each of the tissue samples was analyzed in duplicate. Data evaluation. The LightCycler apparatus measured the fluorescence of each sample in every cycle at the end of the annealing step. After proportional background adjustment, the fit point method was used to determine the cycle in which the log-linear signal was distinguishable from the background, and that cycle number was used as the crossing point value. The LightCycler software (version 3) produced the standard curve by measuring the crossing point of each standard and plotting them against the logarithmic values of concentrations. The Id2 and GAPDH gene were cloned in the pGEM-T vector (Promega Corp., Madison, WI, USA) for use in creating a standard curve. Ten-fold serial dilutions of known plasmid DNA concentrations were made. The gene copy number of Id2 was normalized by comparison with the GAPDH housekeeping gene. Results
Clinicopathological data There were 40 male and 31 female patients. The mean age at presentation was 18 years (range, 1-57 years). The most common site of disease was the pelvis (15 cases), followed by spine (13 cases), femur (11 cases), humerus (9 cases), tibia (5 cases), scapula (3 cases), rib (3 cases), and others (12 cases). Id2 expression in Ewing sarcoma tumor samples The enhanced expression of the Id2 gene was detected by immunohistochemistry. Id2 expression was observed in 65 cases (91.5%) of the 71 total cases examined and diffuse, strong expression was observed in 45 cases (63.8%; Figures 1-3). Id2 proteins displayed cytoplasmic as well as nuclear localization in the examined tumor cells. Real-time quantitative PCR using Ewing sarcoma cell line We performed real-time quantitative PCR three times using one type of Ewing sarcoma cell line, ES97. The
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Id2, an HLH protein, is generally thought to affect the balance between cell proliferation and differentiation by functioning as a dominant-negative antagonist of basic HLH transcription factors 9,14. We observed strong expression of Id2 protein in Ewing sarcoma tumor tissues. Unfortunately, we could not detect a corre-
sponding increase in Id2 gene copy number by realtime quantitative PCR amplification in the examined Ewing sarcoma cell line. It is important to comment that this study focused on the gene product expression of Id2 and the Id2 gene copy number was examined in only one Ewing sarcoma cell line, ES97. Northern blot analysis using Ewing sarcoma tumor samples would have provided more comparable results. Nishimori et al.13 showed that Id2 is induced at a significantly higher level in cells expressing EWS/ETS fusions than in cells expressing their corresponding normal ETS transcription factor. It was also shown that induction of Id2 is dependent on the expression of EWSETS proteins in their model cells. These authors suggested that EWS-ETS fusion proteins transactivate the Id2 gene in the Ewing family of tumor (EFT) cells in a direct manner and may play an important role in the oncogenesis of EFT cells. Fukuma et al.15 demonstrated direct transactivation of Id2 by EWS/ETS chimeric proteins through direct interaction with the Id2 promoter by EWS/FLI-1 in vivo. The substantial increase in Id2 expression induced by the EWS/ETS chimeric proteins seemed to be functionally mediated through ETS recognition sequences and the E-box. The chimeric proteins directly activated the Id2 gene. Moreover, the EWS/ETS proteins induce c-myc expression, which further augmented expression of Id2. Consequently, the high levels of Id2 interfered with the growth-suppressive activity of the RB protein family and cellular differentiation regulated by bHLH transcription factors. The high levels of Id2 protein expression observed in our results may explain the high malignancy and poor prognosis of this tumor. The oncogenic process of Ewing sarcoma may be attributable to Id2-mediated inhibition of RB, which has tumor suppressor activity in a number of cancers15. Immunohistochemical analysis of primary neuroblastoma from 47 patients showed that expression of Id2 is strongly predictive of poor outcome, irrespective of other clinical and biological vari-
Figure 2 - Positive control for immunohistochemistry.
Figure 3 - Negative control for immunohistochemistry.
Figure 1 - Overexpression of Id2 in Ewing sarcoma tumor sample. Diffuse and strong expression was noted in both cytoplasm and nucleus of round primitive tumor cells.
DNA amplification curve for Id2 is shown in Figure 4. The crossing point of Id2 (corrected by the GAPDH data) was 18.54 ± 0.16 in the Ewing sarcoma cell line. The crossing points of Id2 in the control fibroblast and osteosarcoma cell line, MG63, were 18.25 and 18.34, respectively. The amplification of the Id2 gene was not observed in this particular Ewing sarcoma cell line. Discussion
Fluorescence (F1)
UPREGULATION OF ID2 IN EWING SARCOMA 0.13 0.12 0.11 0.10 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0.00 -0.01
0 2
4 5 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle number
Figure 4 - Id2-specific DNA amplification curves in Ewing sarcoma cell line, control fibroblast and osteosarcoma cell line by real-time quantitative PCR. The RelQuant program (version 1.00) was used to calculate the amount of Id2 DNA amplification. The amplification of the Id2 gene was not noted in this Ewing sarcoma cell line, ES97.
ables9. The high level of Id2 expression may explain certain characteristics of Ewing sarcoma, such as primitive morphological features and the expression of neural molecules15. Wang et al.16 reported that Id2 mRNA and protein expression was highly variable and independent of the MYCN amplification status and mRNA expression in 10 human-derived neuroblastoma cell lines. In addition, Id2 mRNA expression was not associated with the MY-
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CN gene amplification status or MYCN expression in 131 separate diagnostic primary neuroblastoma samples analyzed by real-time quantitative RT-PCR. These data suggest that transcriptional regulation of Id2 by the MYCN oncoprotein is unlikely to be a seminal molecular event resulting in a highly malignant neuroblastoma phenotype. Vandesompele et al.17 also found no correlation between MYCN and Id2 expression in neuroblastoma cell lines or tumor specimens. Importantly, they could not confirm the reported prognostic power of Id2 expression in neuroblastoma18. The target genes of EWS/ETS oncoproteins have been extensively investigated to try and understand the tumorigenic mechanisms involved in Ewing sarcoma. EWS/FLI1 is a strong transactivator of the c-myc promoter19. Several genes such as EAT-220, stromelysin21, and fringe10 have been reported to be upregulated, while TGF-beta type II receptor22, p2123, and p57KIP224 are downregulated. Based on the protein structure of EWSFLI1, its transforming activity is mainly thought to depend on its DNA binding ability13. Therefore, isolation of downstream targets of EWS-ETS proteins could lead to the identification of genes involved in the initiation and progression of the Ewing family of tumors. In conclusion, we demonstrated overexpression of the Id2 protein by immunohistochemistry in 91.5% of the Ewing sarcomas examined and discussed a role for Id2 in tumorigenesis of Ewing sarcomas.
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