HDAC2 blockade promotes cancer stem cell phenotype in human osteosarcoma
Marcella La Noce1#, Francesca Paino2#, Luigi Mele1#, Gianpaolo Papaccio1*, Tarik Regad3, Federica Papaccio4§, Vincenzo Desiderio1§, Virginia Tirino1§*
1Dipartimento di Medicina Sperimentale, Sezione di Biotecnologie, Istologia Medica e Biologia Molecolare, Università degli Studi della Campania “L. Vanvitelli”, Napoli, via L. Armanni, 5 – 80138, Napoli, Italy2Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Milano, Via Commenda,10 – 20122, Milano, Italy3The John van Geest Cancer Research Centre, School of Science and Technology, Nottingham Trent University, Nottingham, United Kingdom4Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale “F. Magrassi”, Università degli Studi della Campania “L. Vanvitelli”, via S. Pansini-Cappella Cangiani-80131 Napoli, Italy
Background. Cancer stem cells (CSCs) play a key role in cancer initiation, progression and chemoresistance. Epigenetic alterations have been identified as prominent factors that contribute to the CSCs phenotype. Here, we investigated the effects of the HDAC inhibitor valproic acid (VPA) and the demethylating agent, 5’azacytidine (DAC) on the stem phenotype of MG63 and Saos2 osteosarcoma cell lines.
Methods. Saos2 and MG63 cells were treated with DAC and VPA, alone and in combination. Untreated and treated cells were examined for stemness phenotype by cytometry and real-time PCR. Sarcospheres and colonies formation were also evaluated. Moreover, histone modification and methylation were tested by flow cytomery and western blotting. HDAC2 depleted cells were examined for stemness phenotype and their ability to generate tumors in NOD/SCID IL2R-gamma-0 (NSG) mice .
Results. We found that DAC and VPA induce an increased expression of stem markers including CD133, OCT4, SOX2 and NANOG, and an increased ability in sarcospheres and colonies formation efficiency. Interestingly, we showed that DAC and VPA treatment decreased repressive histone markers, while increased the active ones. These histone modifications were also associated with an increase of acetylation of histones H3, a decrease of DNA global methylation, HDAC2 and DNMT3a. Furthermore, HDAC2 silenced-MG63 cells acquired a stem phenotype, and promoted in vivo tumorigenesis.
Conclusions. Collectively, our results suggest that VPA and DAC induce an expansion of osteosarcoma CSCs, and we report for the first time that HDAC2 is a key factor regulating both CSCs phenotype and in vivo cancer growth. In conclusion, we have identified HDAC2 as a potential therapeutic target in human osteosarcoma treatment.
Keywords: Cancer stem cells, osteosarcomas, methylation, HDAC2, DNMT3a