Proteínas modificadoras da cromatina em tumores sólidos pediátricos : influência sobre o crescimento, diferenciação celular e atividade oncogênica

A atividade aberran te de genes que codificam enzimas modificadoras da cromatina, por consequência de mutação ou alterações na expressão gênica, tem sido frequentemente identificada como principal fator condutor para a perturbação de programas de desenvolvimento em cânceres pediátricos. No sentido de reverter esse estado epigenético aberrante, diversos inibidores utilizando como alvo enzimas modificadoras da cromatina têm sido desenvolvidos como novas estratégias para o tratamento do cãncer. Neste trabalho, nós investigamos se o potente inibidor de HDAC butirato de sódio (NaS) apresenta a capacidade de reprogramar as células de sarcoma de Ewing (SE) para um estado mais diferenciado, bem como afetar seu crescimento e sobrevivênda. Nós encontramos que a exposição das linhagens SK· C5 1 e RD·C5 ao NaO resulta em uma potente inibiçilo di!ll atividade global de I IDACs {1 h), bem como uma significante redução do crescimento celular de SE (72 h). NaS também afetou o ciclo celular provocando acúmulo de GO/G 1 e redução nos níveis proteicos do marcador mitótico histona 3 fosforilada em S10 (H3 phos-SlO). Os efeitos mediados por NaS envolveram supressão da prolife ração celular acompanhada pela expressão reduzida do transcrito oncogênico EWS·FL/1 e de genes associados a pfuripotência e sobrevivência, e a re·expressão do marcador de diferenciação neuronal ~lll·tubulina. Além disso, NaS promoveu um aumento na complexidade celular o que leva a alterações morfológicas características de crescimento neurítico. Por fim, nós observamos uma potente atividade de NaS para impedir o crescimento e a sobrevivência de tumoresferas de SE. Nossos resultados suportam o uso da inibição de HDAC como uma estratégia para impedir o crescimento e a sobrevivência celular e para reprogramar tumores de SE para um estado de diferenciação. Além disso, esses resultados corroboram com nossos estudos anteriores em que demonstraram que inibidores de HDAC podem modular o crescimento e a diferenciação de células de tumores pediâtricos com putativa origem de células tronco neurais. AJém deste estudo, nós caracterizamos o papel funcional da histona metiltransferase EZH2 no crescimento e diferenciação celular de neuroblasloma (NB). utilizando intervenções farmacológica e genética. Nós demonstramos que UNC- 1999, um inibidor duplo de EZH2 e EZH 1, foi mais efetivo para inibir o crescimento de células de NS (7 d). em comparação aos inibidores seletivos para EZH2 (GSK-126 e GSK-343). Os mecanismos mediados por inibidores de EZH2 envolveram uma robusta redução dos níveis globais da marca repressiva H3K27me3, e a reativação transcricional dos genes supressores tumorais CASZ1, NTRK1, RAR{J and CHD5, que controlam diferenciação em NB. Além disso, experimentos ex-vivo demonstraram que UNC-1999 possui uma robusta capacidade de reduzir a atividade oncogênica de tumores de xenoenxerto de NB e prolongar a sobrevivência murina. Foram ainda examinados os efeitos da ablação genética crônica de EZH2 (por silenciamento gênico utilizando um shRNA constitutivo para o gene EZH2) no crescimento tumoral de NB. O efeito da ablação genética crônica de EZH2, surpreendentemente, provocou um aumento na atividade oncogênica das células de NB, seguida por uma importante progressão tumoral observada em tumores de xenoenxerto de NS. Portanto, estes resultados sugerem que o crescimento de tumores de NS provavelmente não depende apenas da função de PRC2-EZH2. Todavia, a inibição catalitica dupla de EZH2 e EZH1 demonstrou-se como uma estratégia terapêutica efetiva para suprimir o crescimento das células NS e reativar programas gênicos envolvidos na diferenciação.Aberrant activity of genes encoding chromatin·modifying enzymes, as a consequence of mutation or changes in gene expression, has been frequenUy identified as a main driver of disruption of the developmental programs in pediatric cancers. In order to reverse this aberrant epigenetic state, several inhibitors targeting ch roma ti n ~mod ifying enzymes have been developed as a novel strategy for the treatment of cancer. In the first section of this study, we investigated whether the potent HDAC inhib~or sodium butyrate (NaS) has ability to reprogram Ewing sarooma {EWS) cells towards to a more differentiated state and affect their growth and survival. In this work, we found that exposure of SK-ES 1 and RD-ES cells to NaS produce a potent inhibition of global HDAC activity {1 h), as well as, a significant reduction in the growth of ES cells (72 h). NaB also affected the cell cycle inducing GO/G 1 accumulation and reducing lhe protein leveis of the mitotic marker phosphorylated histone 3 {S10) (H3 phos-S10). NaS-mediated effects involved suppression of cell proliferation accompanied by decreased expression of EWS·FL/1 oncogenic transcript and key survival and pl uri pote ncy ~associa ted genes, and lhe re·expression of the differentiation neuronal marker j3111·tubulin. Additionally, NaB induced an increase in cellular complexity, which leads to morphological changes characteristic of neuritic growth. FinaUy, we observed a potent activity of NaB to prevent growth and survival of ES tumorspheres. Our results suggest that inhibition of global HDAC acüvity may represent an effective strategy to inhibit cell growth, and to reprogram ES cells to a differentiated state. Furthermore, lhese results corroborate with our previous studies in which demonstrated that HDAC inhibitors may modulate cell growth and differentiation of childhood pediatric tumors with putative neural stem cell origin. Aside lhis study, we characterized lhe functional role of the histone methyltransferase EZH2 in neuroblastoma (NB) cell growth and differentiation, using pharmacological and genetic interventions. Here, we demonstrated that UNC- 1999, a dual EZH2 and EZH1 inhibitor, is more effective to inhibit NS cell growth (7 d) compared with EZH2- selecüve inhibitors (GSK-126 and GSK-343). The EZH2 inhibitors-mediated mechanisms involved a robust reduction in the global levei of the repressive mark H3K27me3, and the transcriptional reactivation of lhe tumor suppressor genes CASZl , NTRKl , RAR~ and CH05, that control differentiation in NB. In addition, ex vivo experiments showed that UNC·1999 has a robust ability to reduce the oncogenic activity of NB xenograft tumors and prolong murine survival. We further examined the effects of chronic EZH2 genetic ablation (by gene silencing using a constitutive shRNA targeting EZH2 gene) on NB tumor growth. The effect of chronic EZH2 genetic ablation, surprisingly, led to an increase in lhe oncogenic activity of NB cells, followed by a significant tumor progression observed in NB xenograft tumors. Therefore, these resuhs suggest that NB tumor growth probably does not rely on the PRC2-EZH2 function alone. However, dual catalytic inhibition of EZH2 and EZHl showed to be an effective therapeutic strategy for suppressing NB tumor growth and reactivating differentiation gene programs

Receptor tyrosine kinases as candidate prognostic biomarkers and therapeutic targets in meningioma

Meningioma (MGM) is the most common type of intracranial tumor in adults. The validation of novel prognostic biomarkers to better inform tumor stratification and clinical prognosis is urgently needed. Many molecular and cellular alterations have been described in MGM tumors over the past few years, providing a rational basis for the identification of biomarkers and therapeutic targets. The role of receptor tyrosine kinases (RTKs) as oncogenes, including those of the ErbB family of receptors, has been well established in several cancer types. Here, we review histological, molecular, and clinical evidence suggesting that RTKs, including the epidermal growth factor receptor (EGFR, ErbB1), as well as other members of the ErbB family, may be useful as biomarkers and therapeutic targets in MGM

EHMT2/G9a as an epigenetic target in pediatric and adult brain tumors

Epigenetic mechanisms, including post-translational modifications of DNA and histones that influence chromatin structure, regulate gene expression during normal development and are also involved in carcinogenesis and cancer progression. The histone methyltransferase G9a (euchromatic histone lysine methyltransferase 2, EHMT2), which mostly mediates mono- and dimethylation by histone H3 lysine 9 (H3K9), influences gene expression involved in embryonic development and tissue differentiation. Overexpression of G9a has been observed in several cancer types, and different classes of G9a inhibitors have been developed as potential anticancer agents. Here, we review the emerging evidence suggesting the involvement of changes in G9a activity in brain tumors, namely glioblastoma (GBM), the main type of primary malignant brain cancer in adults, and medulloblastoma (MB), the most common type of malignant brain cancer in children. We also discuss the role of G9a in neuroblastoma (NB) and the drug development of G9a inhibitors

Expression and pharmacological inhibition of TrkB and EGFR in glioblastoma

A member of the Trk family of neurotrophin receptors, tropomyosin receptor kinase B (TrkB, encoded by the NTRK2 gene) is an increasingly important target in various cancer types, including glioblastoma (GBM). EGFR is among the most frequently altered oncogenes in GBM, and EGFR inhibition has been tested as an experimental therapy. Functional interactions between EGFR and TrkB have been demonstrated. In the present study, we investigated the role of TrkB and EGFR, and their interactions, in GBM. Analyses of NTRK2 and EGFR gene expression from The Cancer Genome Atlas (TCGA) datasets showed an increase in NTRK2 expression in the proneural subtype of GBM, and a strong correlation between NTRK2 and EGFR expression in glioma CpG island methylator phenotype (G-CIMP+) samples. We showed that when TrkB and EGFR inhibitors were combined, the inhibitory effect on A172 human GBM cells was more pronounced than when either inhibitor was given alone. When U87MG GBM cells were xenografted into the flank of nude mice, tumor growth was delayed by treatment with TrkB and EGFR inhibitors, given alone or combined, only at specific time points. Intracranial GBM growth in mice was not significantly affected by drug treatments. Our findings indicate that correlations between NTRK2 and EGFR expression occur in specific GBM subgroups. Also, our results using cultured cells suggest for the first time the potential of combining TrkB and EGFR inhibition for the treatment of GBM