Original ArticleDNA methylation profiling identifies PTRF/Cavin-1 as a novel tumor suppressor in Ewing sarcoma when co-expressed with caveolin-1
Introduction
In contrast to the vast majority of tumor types, Ewing sarcoma (ES), a pediatric cancer characterized by the presence of the fusion protein EWS/FLI1, has a very low mutation burden [1], [2], [3]. This suggests a role for EWS/FLI1 as the key promoter in the development of ES [4]. Besides acting as a direct modulator of transcription, EWS/FLI1 appears to exert its oncogenic functions through epigenetic modifications on the transcriptome [5], [6].
Caveolae are small flask-shaped invaginations approximately 60–80 nm in diameter on the plasma membrane, which are involved in signal transduction, cholesterol transport, mechano-sensing and clathrin-independent endocytosis [7]. Although presence of these structures in cancer cells is mostly related to tumor suppression [8], [9], the expression of their two main components, caveolin-1 (CAV1) and Polymerase I and transcription released factor (PTRF, also known as Cavin-1), is associated with tumor suppression as well as oncogenesis [10]. Our group has been working extensively in revealing the mechanisms linking CAV1 over-expression, presumably outside caveolae, in ES with tumor progression by promoting migration, invasion, angiogenesis and resistance to chemotherapy [11], [12], [13], [14].
DNA methylation profiles have been shown to be a very useful tool for clinical predictions in terms of therapy response and/or prognosis for multiple tumor types [15], [16]. Two patterns of DNA methylation changes have been observed: (i) global hypomethylation associated with increased chromosomal instability, reactivation of transposable elements and loss of imprinting [17], [18] and (ii) hypermethylation of CpG islands located in promoter regions of tumor suppressor genes, conventionally associated with transcriptional silencing [19]. Therefore, the identification of specific DNA methylation markers would be helpful for understanding the pathogenetic mechanism as well as for developing new therapeutic strategies for ES.
In the current study, we analyze the methylome of several ES tumors and cell lines in comparison to a significant number of healthy tissues and cells as reference. Our results point to PTRF, known to interact with caveolin-1 to form caveolae, as a novel tumor suppressor in ES.
Section snippets
Materials and methods
Supplementary Materials and Methods, with more detailed explanations, are available in the online version of this article.
Methylation profile of ES
In order to generate a global view of the DNA methylation landscape of ES, we investigated 15 primary ES tumors, 7 ES cell lines, 10 healthy tissues and 4 human mesenchymal stem cells (hMSC) lines samples using the Infinium Human Methylation 450K (HM450K) BeadChip. After quality control analysis and data filtering, the methylation status of a total of 467,273 CpGs and 3091 non-CpGs was quantified in all ES derived samples. We used publicly available data for the reference (detailed in
Discussion
DNA methylation signatures point toward disease mechanisms, useful biomarkers and therapeutic targets. Methylation profiles can be useful to unveil common patterns among cancer entities. One major constraint that has to be taken into account is that cell culture in vitro induces methylation changes per se, meaning that cell lines and tissue samples generally cluster separately [24], [35]. In spite of this phenomenon, we identified a characteristic methylation signature for ES. Like Patel et al.
Funding
OMT: Instituto de Salud Carlos III and EU's Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa/A way to achieve Europe” (CES12/021; PI11/00038; PI15/00035). EdA, JM & OMT: Fundación científica de la AECC (GCB13131578DE Á). SR-V: Marie Curie COFUND-Beatriu De Pinòs Researcher (BP-B 00109). SR-V & SG-M: Fundación Alba Pérez lucha contra el cáncer infantil. DH-M: Fundación Científica de la AECC. RB: FPI fellow (BES-2012-055368). JA: Instituto de Salud Carlos III (PI12/00816)
Author contributions
OMT conceived and supervised the study. JHM designed and performed the main experiments and coordinated the data analysis. FC, SR-V, DH-M, OA-R, MS-J, SG-M, LL-T, RB, LH-P, RL-A and SM contributed to the design and conduct of experiments. MAP, ME and XGM contributed to the data analysis. FC, SM and DM performed bioinformatics data analyses. AS, DA and XS performed histopathological analyses of the samples. JR, SG, JM, JA and EA provided tumor samples. JHM, SR-V, PHG and OMT wrote the
Data and materials availability
Data bases from the methylome analysis are publicly accessible at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89041.
Acknowledgments
The authors thank the donors, Seville's HUVR-IBiS Biobank (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT13/0010/0056) and Hospital Infantil Universitario Niño Jesús at Madrid, Spain, for the human specimens used in this study.
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