Aberrant Methylation and Reduced Expression of RASSF1A in Ewing Sarcoma

Pathology Department, Tel Aviv Sourasky Medical Center, Tel Aviv University, Tel Aviv, Israel
Pediatric Blood & Cancer (Impact Factor: 2.39). 12/2009; 53(6):1023 - 1028. DOI: 10.1002/pbc.22115


Ewing sarcoma (ES) is the second most common solid bone and soft tissue malignancy in children and young adults with low cure rates indicating the need to identify further prognostic markers. The importance of methylation in the inactivation of key tumor suppressor genes including RASSF1A has begun to be appreciated in context of cancer development, prognosis and therapy. However there is lack of similar broad based studies in ES. The objective of this study was to analyze RASSF1A methylation and assess its clinical significance in ES.ProcedureThe methylation of RASSF1A was determined 31 ES tumor samples and 4 ES cell lines. ES cell lines were also treated with demethylating agent 5-aza-2′-deoxycytidine to ascertain its effect on methylation. RASSF1A expression was studied in 12 ES tumors. The association between RASSF1A methylation, clinical parameters and outcome was also analyzed.ResultsMethylation of RASSF1A was observed in 21/31 (68%) tumors and in 3/4 ES cell lines. A significant correlation of methylation to reduced expression of RASSF1A was observed in 12 ES tumors analyzed (P = 0.0013) and in all cell lines. ES patients with methylated RASSF1A had worse prognosis compared to the unmethylated group (P = 0.049). Treatment with 5-aza-2′-deoxycytidine resulted in the re-expression of the unmethylated form of RASSF1A in two ES cell lines.ConclusionRASSF1A is frequently methylated in ES. Pediatr Blood Cancer 2009;53:1023–1028. © 2009 Wiley-Liss, Inc.

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    • "These results were similar to a report by Harada et al. in 2002, which did not demonstrate RASSF1A methylation in 8 EWS tumors [33]. However, our findings contradict a more recent report by Avigad et al. [34]. One possible explanation for this discrepancy is that the MSP primers used to investigate methylation status used by Avigad et al. are located considerably farther downstream from the transcription start site (+220 to +300) compared to our MSP primer set and the bead chip assay (−76 to +96 and +116, resp.). "
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    ABSTRACT: The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs) using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA)-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1,) and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.
    Full-text · Article · Sep 2012 · Sarcoma
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    • "Failure to detect CDKN2A promoter methylation in this study can be reconciled with the results of the larger studies described above based simply on insufficient sample size. With respect to RASSF1A, these results are more difficult to reconcile with the high frequency of RASSF1A methylation in a previously published report [80]; Avigad et al. identified 21 of 31 (68%) patient samples and 1 of 4 (25%) cell lines with hemizygous promoter methylation and 2 of 4 (50%) cell lines with homozygous promoter methylation. This larger study also correlated reduced RASSF1A expression with promoter methylation in 12 tumors checked. "
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    ABSTRACT: Ewing's sarcoma is the second most common bone malignancy affecting children and young adults. The prognosis is especially poor in metastatic or relapsed disease. The cell of origin remains elusive, but the EWS-FLI1 fusion oncoprotein is present in the majority of cases. The understanding of the molecular basis of Ewing's sarcoma continues to progress slowly. EWS-FLI1 affects gene expression, but other factors must also be at work such as mutations, gene copy number alterations, and promoter methylation. This paper explores in depth two molecular aspects of Ewing's sarcoma: copy number alterations (CNAs) and methylation. While CNAs consistently have been reported in Ewing's sarcoma, their clinical significance has been variable, most likely due to small sample size and tumor heterogeneity. Methylation is thought to be important in oncogenesis and balanced karyotype cancers such as Ewing's, yet it has received only minimal attention in prior studies. Future CNA and methylation studies will help to understand the molecular basis of this disease.
    Full-text · Article · Mar 2011 · Sarcoma
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    ABSTRACT: Ewing sarcoma family tumors (ESFT) are a group of aggressive solid bone and soft tissue malignancies of children and young adults characterized by specific chromosomal translocations that give rise to EWS-ETS aberrant transcription factors. Identification of EWS-ETS target genes and their role in tumor signaling networks together with the unravelling of the cell of origin will facilitate the translation into new treatment modalities for these neoplasms.
    Full-text · Article · May 2010 · Cancer biology & therapy
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