Expressão de BMI1 e ativação ERK colaboram na manutenção do fenótipo indiferenciado em células de meduloblastoma

Meduloblastoma (MB), um tumor cerebral prevalentemente infantil, compreende um grupo de tumores com diferentes subtipos moleculares e prognósticos. Esses tumores apresentam alterações em vias de sinalização importantes para o desenvolvimento do cerebelo. Recentemente, estudos genômicos mostraram a importância de fatores epigenéticos para a formação e manutenção de MB. Esses fatores, que regulam processos associados a pluripotência, contribuem para a indução de um fenótipo mais indiferenciado e mais agressivo. Essas células indiferenciadas, as células tronco tumorais (CSC), são identificadas por marcadores como CD133, pela capacidade de formar esferas não aderentes em culturas livres de soro fetal bovino e pela tumorigenicidade. CSC participam nos processos iniciais do tumor, na progressão, na metástase e na recidiva tumoral. Muitos desses processos podem ser explicados pelo fenótipo indiferenciado, ou seja, pela modulação de mecanismos associados a células tronco normais. A ativação de ERK, por exemplo, importante durante o desenvolvimento do cerebelo, está aumentada em biópsias de MB comparada ao tecido normal do cérebro e regula processos como crescimento e migração nesse tumor. O objetivo dessa tese foi avaliar o impacto da modulação epigenética no fenótipo indiferenciado e, consequentemente, na manutenção de células de MB humano. No capítulo I, destaca-se que a inibição de histonas deacetilases (HDACs) por butirato de sódio (NaB) e consequente aumento na acetilação de histonas é capaz de diminuir marcadores de pluripotência de células de MB-SHH e MB-não-WNT/SHH. A ativação da via de ERK contribui para esse efeito e a inibição dessa via intensifica o efeito antiproliferativo de NaB, provavelmente por também modulador a capacidade “tromco”. No capítulo II, ressalta-se a importância da ativação de ERK no MB-Grupo 4. Tumores desse subgrupo com a assinatura gênica específica BMIhigh/CHD7low apresentam um fenótipo de maior malignidade (alta proliferação e indiferenciados) que está relacionado a ativação aumentada da sinalização de ERK. Juntos, os dados dessa tese demonstram que o fator epigenético BMI1 e a ativação da via de sinalização de ERK estão envolvidos na manutenção de um fenótipo indiferenciado em células tumorais de MB, fortalecendo as evidências de que eles devem ser considerados potenciais alvos terapêuticos nesse tumor.Medulloblastoma (MB), a prevalent type of childhood brain cancer, comprises a tumor group with different molecular subtypes and outcomes. These tumors present modifications in essential pathways in cerebellar development. Recently, integrative genomics studies showed the importance of epigenetic factors to the formation and maintenance of MB. These factors support the malignant and undifferentiated cellular phenotype by regulation of pluripotency-associated process. These cells, named cancer stem cells (CSC), are identified by expression of the marker CD133, by their ability to form non-adherent spheres in fetal bovine serum free medium and by their tumorigenicity. CSCs participate in tumor initiation process, progression, metastasis and relapse. Most of these processes are explained by stemness phenotype derived from modulation of mechanisms associated to normal stem cell. ERK signaling, for example, wich is important for cerebellar development, is upregulated in MB biopsies compare to normal brain. In MB, ERK activation also modulates processes such as growth and migration. The aim of this thesis was to evaluate the impact of epigenetic modulation on the stemness phenotype and, consequently, on the maintenance of human MB cells. In chapter I, it is noteworthy that the inhibition of histone deacetylases (HDACs) by sodium butyrate (NaB) and the consequent increase in histone acetylation is able to decrease stem cell markers in MBSHH and MB-non-WNT / SHH cells. ERK pathway activation contributes to this effect and the inhibition of this pathway enhances the antiproliferative effect of NaB, probably by modulating stemness. In chapter II, we higlighted the importance of ERK activation in MB-Group 4. Tumors in this subgroup harbouring the specific genetic signature BMIhigh/CHD7low have a higher malignancy (high proliferation and undifferentiated status) phenotype that is related to increased activation of ERK signaling. Together, data from this thesis demonstrate that the epigenetic factor BMI1 and the activation of ERK signaling are involved in maintaining an undifferentiated phenotype in MB tumor cells, supporting the view they should be considered as potential therapeutic targets in this tumor type

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

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field

Reverse engineering of Ewing Sarcoma regulatory network uncovers PAX7 and RUNX3 as master regulators associated with good prognosis

Ewing Sarcoma (ES) is a rare malignant tumor occurring most frequently in adolescents and young adults. The ES hallmark is a chromosomal translocation between the chromosomes 11 and 22 that results in an aberrant transcription factor (TF) through the fusion of genes from the FET and ETS families, commonly EWSR1 and FLI1. The regulatory mechanisms behind the ES transcriptional alterations remain poorly understood. Here, we reconstruct the ES regulatory network using public available transcriptional data. Seven TFs were identified as potential MRs and clustered into two groups: one composed by PAX7 and RUNX3, and another composed by ARNT2, CREB3L1, GLI3, MEF2C, and PBX3. The MRs within each cluster act as reciprocal agonists regarding the regulation of shared genes, regulon activity, and implications in clinical outcome, while the clusters counteract each other. The regulons of all the seven MRs were differentially methylated. PAX7 and RUNX3 regulon activity were associated with good prognosis while ARNT2, CREB3L1, GLI3, and PBX3 were associated with bad prognosis. PAX7 and RUNX3 appear as highly expressed in ES biopsies and ES cell lines. This work contributes to the understanding of the ES regulome, identifying candidate MRs, analyzing their methilome and pointing to potential prognostic factors