Ewing sarcoma (ES) is the second most common bone tumor in childhood. ES harbors a characteristic gene translocation that gives rise to a fusion protein, most commonly EWS/FLI1 (EF). Caveolin-1 (CAV1) is a direct target of EF, it is overexpressed in ES and has an oncogenic role. CAV1 and the Polymerase I and transcript release factor (PTRF) interact at the plasma membrane and are essential for caveolae formation. The methylome analysis of ES samples and cell lines revealed a hypermethylation in the N-shore islands of the PTRF promoter compared to normal cells. We hypothesize that, as ES cells have very few caveolae and do not express PTRF, the oncogenic role of CAV1 in ES would be driven by the impossibility of being recruited at caveolae.
Epigenetic silencing of PTRF was confirmed by bisulfite genomic sequencing on ES cells and human samples. We verified that ES cells do not express PTRF by western blot. Demethylating agent 5-aza (decitabine) treatment of A673 cells resulted in re-expression of PTRF and enhancement of caveolae formation as shown by electron microscopy. Immunofluorescent imaging in TC252 cells stably transfected with PTRF showed co-localization with CAV1 at the plasma membrane. Immunoprecipitation assays also confirmed their interaction. PTRF transfection in TC252 cells promoted an increase in cell death up to 50% in a caspase-3 dependent way. Furthermore, PTRF-expressing xenografts had a lower capacity to form tumors compared to controls. CAV1-PTRF pro-apoptotic interaction was verified also in EW7 cells.
These results pointed that CAV1-PTRF interaction and caveolae formation promote cell death in ES cells. The presence of caveolae has been related with stress and p53 activation, due to the MDM2 binding to CAV1, that causes the release and activation of p53. To elucidate the role of p53 in the PTRF-mediated cell death, PTRF-transfected TC252 cells were treated with a p53 siRNA and cell death was significantly reduced. Our results suggest that PTRF acts as a tumor suppressor in ES. PTRF re-expression enhances caveolae formation in ES cells, thus modulating CAV1 localization that results in p53-mediated cell death.
Citation Format: Juan Huertas-Martínez, Frank Court, Santiago Rello-Varona, David Herrero Martin, Olga Almacellas, Miguel Sáinz-Jaspeado, Silvia Garcia-Monclús, Laura Lagares-Tena, Raqeul Buj, Lourdes Hontecillas-Prieto, Silvia Mateo-Lozano, Ana Sastre, Daniel Azorín, Jaune Mora, Josep Roma, Sebastian Moran, Soledad Gallego, Miguel Angel Peinado, Xavier Garcia del Muro, Javier Alonso, Enrique de Alava, Dave Monk, Manel Esterller, Oscar M Tirado. Epigenetic profiling uncovers the suppressive role of caveolae in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A18.