Cancer Letters

Cancer Letters

Volume 386, 1 February 2017, Pages 196-207
Cancer Letters

Original Article
DNA methylation profiling identifies PTRF/Cavin-1 as a novel tumor suppressor in Ewing sarcoma when co-expressed with caveolin-1

https://doi.org/10.1016/j.canlet.2016.11.020Get rights and content

Highlights

  • Methylation profiling uncovers PTRF/Cavin-1 as a tumor suppressor in ES.

  • Restoration of CAV1 and PTRF by co-expression increases caveolae formation and cell death.

  • Cell death induction is p53-dependent via MDM2 phosphorylation.

  • PTRF re-expression and caveolae restoration could serve as new therapeutic options for ES.

Abstract

Epigenetic modifications have been shown to be important in developmental tumors as Ewing sarcoma. We profiled the DNA methylation status of 15 primary tumors, 7 cell lines, 10 healthy tissues and 4 human mesenchymal stem cells lines samples using the Infinium Human Methylation 450K. Differential methylation analysis between Ewing sarcoma and reference samples revealed 1166 hypermethylated and 864 hypomethylated CpG sites (Bonferroni p < 0.05, δ-β-value with absolute difference of >0.20) corresponding to 392 and 470 genes respectively. Gene Ontology analysis of genes differentially methylated in Ewing sarcoma samples showed a significant enrichment of developmental genes. Membrane and cell signal genes were also enriched, among those, 11 were related to caveola formation. We identified differential hypermethylation of CpGs located in the body and S-Shore of the PTRF gene in Ewing sarcoma that correlated with its repressed transcriptional state. Reintroduction of PTRF/Cavin-1 in Ewing sarcoma cells revealed a role of this protein as a tumor suppressor. Restoration of caveolae in the membrane of Ewing sarcoma cells, by exogenously reintroducing PTRF, disrupts the MDM2/p53 complex, which consequently results in the activation of p53 and the induction of apoptosis.

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.

References (58)

  • Y.J. Choi et al.

    The requirement for cyclin D function in tumor maintenance

    Cancer Cell

    (2012)
  • D. Aran et al.

    DNA methylation of transcriptional enhancers and cancer predisposition

    Cell

    (2013)
  • W. Xie et al.

    Epigenomic analysis of multilineage differentiation of human embryonic stem cells

    Cell

    (2013)
  • F. Tirode et al.

    Mesenchymal stem cell features of Ewing tumors

    Cancer Cell

    (2007)
  • C.S. Aung et al.

    PTRF-cavin-1 expression decreases the migration of PC3 prostate cancer cells: role of matrix metalloprotease 9

    Eur. J. Cell Biol.

    (2011)
  • A.S. Brohl et al.

    The genomic landscape of the Ewing sarcoma family of tumors reveals recurrent STAG2 mutation

    PLoS Genet.

    (2014)
  • B.D. Crompton et al.

    The genomic landscape of pediatric Ewing sarcoma

    Cancer Discov.

    (2014)
  • F. Tirode et al.

    Genomic landscape of Ewing sarcoma defines an aggressive subtype with co-association of STAG2 and TP53 mutations

    Cancer Discov.

    (2014)
  • N. Gaspar et al.

    Ewing sarcoma: current management and future approaches through collaboration

    J. Clin. Oncol.

    (2015)
  • J.P.X. Cheng et al.

    Caveolae: one function or many?

    Trends Cell Biol.

    (2015)
  • L. Bai et al.

    Down-regulation of the cavin family proteins in breast cancer

    J. Cell Biochem.

    (2012)
  • V.J. Hernandez et al.

    Cavin-3 dictates the balance between ERK and Akt signaling

    Elife

    (2013)
  • M. Sáinz-Jaspeado et al.

    Caveolin-1 modulates the ability of Ewing's sarcoma to metastasize

    Mol. Cancer Res.

    (2010)
  • M. Sáinz-Jaspeado et al.

    EphA2-induced angiogenesis in Ewing sarcoma cells works through bFGF production and is dependent on caveolin-1

    PLoS One

    (2013)
  • O.M. Tirado et al.

    Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells

    Cancer Res.

    (2006)
  • L. Lagares-Tena et al.

    Caveolin-1 promotes Ewing sarcoma metastasis regulating MMP-9 expression through MAPK/ERK pathway

    Oncotarget

    (2016)
  • H. Heyn et al.

    DNA methylation profiling in the clinic: applications and challenges

    Nat. Rev. Genet.

    (2012)
  • C.R. Goding et al.

    Cancer: pathological nuclear reprogramming?

    Nat. Rev. Cancer

    (2014)
  • G. Raddatz et al.

    Dnmt3a protects active chromosome domains against cancer-associated hypomethylation

    PLoS Genet.

    (2012)
  • Cited by (23)

    • One oncogene, several vulnerabilities: EWS/FLI targeted therapies for Ewing sarcoma

      2021, Journal of Bone Oncology
      Citation Excerpt :

      Since widespread epigenetic reprogramming is a prerequisite to Ewing sarcoma tumorigenesis under the influence of EWS/FLI, several efforts have focused on modulating the epigenome in Ewing tumors as a therapeutic approach (Fig. 2). Ewing sarcoma cell viability has been linked to differential methylation at specific targets in tumor relative to mesenchymal stem cells [118–122]. The DNA demethylating agent 5-aza-2′-deoxycytidine causes a loss of clonogenicity in a dose-dependent manner in TC32 and TC71 Ewing sarcoma cells [123].

    • Clinical efficiency of epigenetic drugs therapy in bone malignancies

      2021, Bone
      Citation Excerpt :

      Additionally, methylation profiling comparing ES samples and normal tissues identified ES-specific inactivation of the polymerase I and transcript release factor Cavin 1 gene (CAVN1) [26]. Co-expression of EWS-FLI1 and CAVN1, an experimental model of ES, induced TP53-dependent cell death, supporting the observation that demethylating drugs may offer some benefits for the treatment of ES [27]. EWS/Fli1 oncogene also favors the development of ES by promoting the aberrant activity of histone demethylase KDM3 [28].

    View all citing articles on Scopus
    View full text