Stemness and Cell Cycle Regulators and Their Modulation by Retinoic Acid in Ewing Sarcoma.

Retinoic acid (RA) regulates stemness and differentiation in human embryonic stem cells (ESCs). Ewing sarcoma (ES) is a pediatric tumor that may arise from the abnormal development of ESCs. Here we show that RA impairs the viability of SK-ES-1 ES cells and affects the cell cycle. Cells treated with RA showed increased levels of p21 and its encoding gene, CDKN1A. RA reduced mRNA and protein levels of SRY-box transcription factor 2 (SOX2) as well as mRNA levels of beta III Tubulin (TUBB3), whereas the levels of CD99 increased. Exposure to RA reduced the capability of SK-ES-1 to form tumorspheres with high expression of SOX2 and Nestin. Gene expression of CD99 and CDKN1A was reduced in ES tumors compared to non-tumoral tissue, whereas transcript levels of SOX2 were significantly higher in tumors. For NES and TUBB3, differences between tumors and control tissue did not reach statistical significance. Low expression of CD99 and NES, and high expression of SOX2, were significantly associated with a poorer patient prognosis indicated by shorter overall survival (OS). Our results indicate that RA may display rather complex modulatory effects on multiple target genes associated with the maintenance of stem cell's features versus their differentiation, cell cycle regulation, and patient prognosis in ES.

A Case Study of a Rare Undifferentiated Spindle Cell Sarcoma of the Penis: Establishment and Characterization of Patient-Derived Models.

Rare sarcomas present significant treatment challenges compared to more prevalent soft tissue sarcomas due to limited treatment options and a poor understanding of their biology. This study investigates a unique case of penile sarcoma, providing a comprehensive morphological and molecular analysis. Through the creation of experimental patient-derived models-including patient-derived xenograft (PDX), 3D, and monolayer primary cultures-we successfully replicated crucial molecular traits observed in the patient's tumor, such as smooth muscle actin and CD99 expression, along with specific mutations in genes like TSC2 and FGFR4. These models are helpful in assessing the potential for an in-depth exploration of this tumor's biology. This comprehensive approach holds promise in identifying potential therapeutic avenues for managing this exceedingly rare soft tissue sarcoma.

Histone Methyltransferases G9a/Ehmt2 and GLP/Ehmt1 Are Associated with Cell Viability and Poorer Prognosis in Neuroblastoma and Ewing Sarcoma.

Changes in epigenetic programming have been proposed as being key events in the initiation and progression of childhood cancers. HMT euchromatic histone lysine methyltransferase 2 (G9a, EHMT2), which is encoded by the G9a (Ehmt2) gene, as well as its related protein GLP, which is encoded by the GLP/Ehmt1 gene, participate in epigenetic regulation by contributing to a transcriptionally repressed chromatin state. G9a/GLP activation has been reported in several cancer types. Herein, we evaluated the role of G9a in two solid pediatric tumors: neuroblastoma (NB) and Ewing sarcoma (ES). Our results show that G9a/Ehmt2 and GLP/Ehmt1 expression is higher in tumors with poorer prognosis, including St4 International Neuroblastoma Staging System (INSS) stage, MYCN amplified NB, and metastatic ES. Importantly, higher G9a and GLP levels were associated with shorter patient overall survival (OS) in both NB and ES. Moreover, pharmacological inhibition of G9a/GLP reduced cell viability in NB and ES cells. These findings suggest that G9a and GLP are associated with more aggressive NB and ES tumors and should be further investigated as being epigenetic targets in pediatric solid cancers.

Gynecologic and Breast Cancers: What's New in Chemoresistance and Chemosensitivity Tests?

Gynecological and breast cancers affect women's health worldwide. Although chemotherapy is one of the principal treatments for cancer, it also has limitations owing to toxicity and tumor resistance to the drugs used. Thus, individualized treatment based on personal tumor characteristics is essential for improving therapeutic outcomes and patient survival. Chemoresistance and chemosensitivity tests can be useful for predicting tumor response and guiding chemotherapy choices. This methodology has already been applied to breast, ovarian, cervical, and endometrial cancers, identifying successfully which drugs cause resistance and sensitivity responses for each individual person, influencing their progression-free survival and overall response. In addition, more recent techniques, such as organoids and patient-derived xenografts, can also recapitulate patients' tumor characteristics and contribute to chemo response evaluation. Therefore, this review compiles information on chemoresistance and chemosensitivity tests performed in gynecologic and breast cancers and their main results for women's health improvement.

A New Brazilian Device for Cervical Cancer Screening: Acceptability and Accuracy of Self-sampling.

OBJECTIVE: To evaluate the accuracy and patient acceptability toward self-sampling using a new device - SelfCervix® - for detecting HPV-DNA. METHODS: A total of 73 women aged 25-65 who underwent regular cervical cancer screening from March to October 2016 were included. Women performed self-sampling followed by a physician-sampling, and the samples were analyzed for HPV-DNA. After that, patients were surveyed about their acceptability of self-sampling. RESULTS: HPV-DNA detection rate of self-sampling presented high accuracy and was similar to physician-collection. Sixty-four (87.7%) patients answered the acceptability survey. Most patients (89%) considered the self-sampling comfortable, and 82.5% preferred self-sampling to physician-sampling. The reasons cited were time-saving and convenience. Fifty-one (79.7%) reported that they would recommend self-sampling. CONCLUSION: Self-sampling using the new Brazilian device SelfCervix® is not inferior in HPV-DNA detection rate compared with physician-collection, and patients are supportive of the method. Therefore, it might be an option to reach under-screened populations in Brazil.

OBJETIVO: Avaliar a acurácia e aceitabilidade da auto-coleta utilizando um novo coletor - SelfCervix® - para a detecção de DNA de HPV. MéTODOS: Foram incluídas no estudo 73 mulheres com idade entre 25­65 anos que realizaram seu rastreamento regular do câncer de colo do útero entre Março e Outubro de 2016. Estas mulheres realizaram a auto-coleta, seguida de coleta profissional e as amostras foram analisadas para a presença de DNA de HPV. Após, elas responderam um questionário sobre a experiência da auto-coleta. RESULTADOS: As taxas de detecção de DNA de HPV por auto-coleta foram altas e similares as da coleta profissional. Sessenta e quatro (87,7%) pacientes responderam o questionário de experiência. A maioria (89%) considerou a auto-coleta confortável, e 82,5% preferiram o método comparado a coleta profissional. As razões citadas foram economia de tempo e conveniência. Cinquenta e uma (79,7%) mulheres confirmaram que recomendariam a auto-coleta. CONCLUSãO: Auto-coleta utilizando o novo coletor desenvolvido no Brasil não é inferior na detecção de DNA de HPV quando comparada a coleta profissional, e apresenta uma boa aceitabilidade pelas mulheres. Desta maneira, pode ser uma opção para alcançar populações que não realizam o rastreamento padrão.

A New Brazilian Device for Cervical Cancer Screening: Acceptability and Accuracy of Self-sampling / Um Novo Dispositivo Brasileiro para Diagnóstico de Câncer Cervical: Aceitabilidade e Precisão da Autoamostragem

Abstract Objective To evaluate the accuracy and patient acceptability toward self-sampling using a new device - SelfCervix® - for detecting HPV-DNA. Methods A total of 73 women aged 25-65 who underwent regular cervical cancer screening from March to October 2016 were included. Women performed self-sampling followed by a physician-sampling, and the samples were analyzed for HPV-DNA. After that, patients were surveyed about their acceptability of self-sampling. Results HPV-DNA detection rate of self-sampling presented high accuracy and was similar to physician-collection. Sixty-four (87.7%) patients answered the acceptability survey. Most patients (89%) considered the self-sampling comfortable, and 82.5% preferred self-sampling to physician-sampling. The reasons cited were time-saving and convenience. Fifty-one (79.7%) reported that they would recommend self-sampling. Conclusion Self-sampling using the new Brazilian device SelfCervix® is not inferior in HPV-DNA detection rate compared with physician-collection, and patients are supportive of the method. Therefore, it might be an option to reach under-screened populations in Brazil.

Resumo Objetivo Avaliar a acurácia e aceitabilidade da auto-coleta utilizando um novo coletor - SelfCervix® - para a detecção de DNA de HPV. Métodos Foram incluídas no estudo 73 mulheres com idade entre 25-65 anos que realizaram seu rastreamento regular do câncer de colo do útero entre Março e Outubro de 2016. Estas mulheres realizaram a auto-coleta, seguida de coleta profissional e as amostras foram analisadas paraa presença de DNA de HPV. Após, elas responderam um questionário sobre a experiência da auto-coleta. Resultados As taxas de detecção de DNA de HPV por auto-coleta foram altas e similares as da coleta profissional. Sessenta e quatro (87,7%) pacientes responderam o questionário de experiência. A maioria (89%) considerou a auto-coleta confortável, e 82,5% preferiram o método comparado a coleta profissional. As razões citadas foram economia de tempo e conveniência. Cinquenta e uma (79,7%) mulheres confirmaram que recomendariam a auto-coleta. Conclusão Auto-coleta utilizando o novo coletor desenvolvido no Brasil não é inferior na detecção de DNA de HPV quando comparada a coleta profissional, e apresenta uma boa aceitabilidade pelas mulheres. Desta maneira, pode ser uma opção para alcançar populações que não realizam o rastreamento padrão.

Targeting the epigenome of cancer stem cells in pediatric nervous system tumors.

Medulloblastoma, neuroblastoma, and pediatric glioma account for almost 30% of all cases of pediatric cancers. Recent evidence indicates that pediatric nervous system tumors originate from stem or progenitor cells and present a subpopulation of cells with highly tumorigenic and stem cell-like features. These cancer stem cells play a role in initiation, progression, and resistance to treatment of pediatric nervous system tumors. Histone modification, DNA methylation, chromatin remodeling, and microRNA regulation display a range of regulatory activities involved in cancer origin and progression, and cellular identity, especially those associated with stem cell features, such as self-renewal and pluripotent differentiation potential. Here, we review the contribution of different epigenetic mechanisms in pediatric nervous system tumor cancer stem cells. The choice between a differentiated and undifferentiated state can be modulated by alterations in the epigenome through the regulation of stemness genes such as CD133, SOX2, and BMI1 and the activation neuronal of differentiation markers, RBFOX3, GFAP, and S100B. Additionally, we highlighted the stage of development of epigenetic drugs and the clinical benefits and efficacy of epigenetic modulators in pediatric nervous system tumors.

Cancer Stem Cells and Chemoresistance in Ewing Sarcoma.

Resistance to chemotherapy poses a major challenge for cancer treatment. Reactivating a stem cell program resembling that seen in embryonic development can lead cancer cells to acquire a stem-cell phenotype characterized by expression of stemness genes, pluripotency, high self-renewal ability, and tumor-initiating capability. These cancer stem cells (CSCs) are usually resistant to anticancer drugs and are likely involved in treatment failure in many cancer types. Ewing sarcoma (ES) is a pediatric cancer type typically resulting from a typical genetic alteration affecting bone or soft tissues. Despite advances in treatment, survival prognostic remains poor for patients with refractory or recurrent disease. Here, we review the increasing evidence indicating that ES tumors contain a CSC subpopulation expressing stem cell genes, including BM1, OCT3/4, NANOG, and SOX2, that plays a role in resistance to drug treatment, and current experimental strategies that successfully counteract chemoresistance mediated by CSCs in ES.

ZEB1 is a Subgroup-Specific Marker of Prognosis and Potential Drug Target in Medulloblastoma.

Medulloblastoma (MB) is a malignant brain tumor that afflicts mostly children and adolescents and presents four distinct molecular subgroups, known as WNT, SHH, Group 3, and Group 4. ZEB1 is a transcription factor that promotes the expression of mesenchymal markers while restraining expression of epithelial and polarity genes. Because of ZEB1 involvement in cerebellum development, here we investigated the role of ZEB1 in MB. We found increased expression of ZEB1 in MB tumor samples compared to normal cerebellar tissue. Expression was higher in the SHH subgroup when compared to all other MB molecular subgroups. High ZEB1 expression was associated with poor prognosis in Group 3 and Group 4, whereas in patients with WNT tumors poorer prognosis were related to lower ZEB1 expression. There was a moderate correlation between ZEB1 and MYC expression in Group 3 and Group 4 MB. Treatment with the immunomodulator and histone deacetylase (HDAC) inhibitor fingolimod (FTY720) reduced ZEB1 expression specifically in D283 cells, which are representative of Group 3 and Group 4 MB. These findings reveal novel subgroup-specific associations of ZEB1 expression with survival in patients with MB and suggest that ZEB1 expression can be reduced by pharmacological agents that target HDAC activity.

Trk Inhibition Reduces Tumorsphere Formation and Changes Expression of Stemness Markers in SK-ES-1 Ewing Sarcoma Cells / Inibição de Trk Reduz a Formação de Tumoresferas e Modifica a Expressão de Marcadores Tronco Tumorais em Células de Sarcoma de Ewing SK-ES-1 / Inhibición de Trk Reduce la Formación de Esferoides Tumorales y Cambia la Expresión de Marcadores de Stemness en Células de Sarcoma de Ewing SK-ES-1

Introduction: Ewing sarcoma (ES) is a highly aggressive type of childhood cancer characterized by a chromosomal translocation resulting in fusions between the gene encoding EWS RNA Binding Protein 1 (EWSR1) and one gene of the ETS family, most frequently FLI-1, resulting in the EWS-FLI1 aberrant transcription factor. ES tumors can contain a subpopulation of cells showing cancer stem cell (CSC) features, which express stemness markers including CD133, OCT4 (Octamer-binding transcription factor 4), and NANOG, and display capacity to form tumorspheres likely enriched in CSCs. Neurotrophin (NT) receptors of the tropomyosin receptor kinase (Trk) family (TrkA, TrkB, and TrkC) may play a role in stimulating ES progression, but their possible role in CSCs remains unknown. Objective: To verify the effect of Trks inhibition on the formation of tumorspheres as well as the gene expression of stem markers. Method: The cells were dissociated and the formation of spheres was induced with supplemented culture medium and the K252a treatment was performed. After RNA extraction, mRNA expression levels of target genes Prom1 (CD133), OCT4 (POU5F1), SOX2, and Musashi-1 (MSI1) were analyzed by qPCR. Results: The pan-Trk inhibitor K252a (100 or 500 mM) hindered tumorsphere formation in human SK-ES-1 ES cell cultures. K252a also reduced mRNA expression of Prom1 (CD133-coding gene) while enhancing expression of OCT4. No changes in mRNA levels of SOX2 or Musashi-1 were observed. Conclusion: These findings provide the first evidence suggesting that Trk activity can influence stemness in ES cells

Introdução: O sarcoma de Ewing (SE) é um tipo altamente agressivo de câncer infantil caracterizado por uma translocação cromossômica que resulta em fusões entre o gene que codifica a proteína de ligação a RNA EWS 1 (EWSR1) e um gene da família ETS, mais frequentemente o FLI-1, resultando no fator de transcrição aberrante EWS-FLI1. Os tumores de SE podem conter uma subpopulação de células com características de células-tronco tumorais (CTT), que expressam marcadores de pluripotência como CD133, OCT4 e NANOG, e têm a capacidade de formar esferas tumorais provavelmente enriquecidas em CTT. Os receptores de neurotrofinas (NT) da família de receptor de quinase de tropomiosina (Trk) (TrkA, TrkB e TrkC) podem desempenhar um papel no estímulo à progressão do SE, mas seu possível papel nas CTT permanece desconhecido. Objetivo: Verificar o efeito da inibição dos Trk na formação de tumoresferas, bem como na expressão gênica de marcadores de pluripotência. Método: As células foram dissociadas, a formação de esferas com meio de cultura suplementado foi induzida e realizou-se o tratamento com K252a. Após a extração de RNA, os níveis de expressão de mRNA dos genes-alvo Prom1 (CD133), OCT4 (POU5F1), SOX2 e Musashi-1 (MSI1) foram analisados por qPCR. Resultados: O inibidor pan-Trk K252a (100 ou 500 mM) impediu a formação de esferas tumorais em culturas de células de SE humanas SK-ES-1. O K252a também reduziu a expressão de mRNA de Prom1 (o gene que codifica CD133), enquanto aumentou a expressão de OCT4. Não foram observadas mudanças nos níveis de mRNA de SOX2 ou Musashi-1. Conclusão: Essas descobertas fornecem as primeiras evidências, sugerindo que a atividade dos Trk possa influenciar a pluripotência nas células de SE

Introducción: El sarcoma de Ewing (SE) es un tipo de cáncer infantil altamente agresivo caracterizado por una translocación cromosómica que resulta en fusiones entre el gen que codifica la proteína de unión a RNA EWS 1 (EWSR1) y un gen de la familia ETS, más frecuentemente FLI-1, lo que resulta en el factor de transcripción aberrante EWS-FLI1. Los tumores del SE pueden contener una subpoblación de células que presentan características de células madre cancerosas (CMC), las cuales expresan marcadores de pluripotencia como CD133, OCT4 y NANOG, y muestran la capacidad de formar esferas tumorales probablemente enriquecidas en CMC. Los receptores de neurotrofinas (NT) de la familia del receptor de quinasa de tropomiosina (Trk) (TrkA, TrkB y TrkC) podrían desempeñar un papel en el estímulo de la progresión del SE, pero su posible papel en las CMC aún es desconocido. Objetivo: Verificar el efecto de la inhibición de los Trk en la formación de esferoides tumorales, así como en la expresión génica de marcadores de pluripotencia. Método: Las células fueron disociadas e inducidas a formar esferas con un medio de cultivo suplementado y se realizó el tratamiento con K252a. Después de la extracción de ARN, los niveles de expresión de ARNm de los genes objetivo Prom1 (CD133), OCT4 (POU5F1), SOX2 y Musashi-1 (MSI1) se analizaron mediante qPCR. Resultados: El inhibidor pan-Trk K252a (100 o 500 mM) evitó la formación de esferas tumorales en cultivos de células de SE humanas SK-ES-1. El K252a también redujo la expresión de ARNm de Prom1 (el gen que codifica CD133), mientras que aumentaba la expresión de OCT4. No se observaron cambios en los niveles de ARNm de SOX2 o Musashi-1. Conclusión: Estos hallazgos proporcionan las primeras evidencias que sugieren que la actividad de Trk puede influir en la pluripotencia en las células del SE

Toll-like Receptors 2, 3, and 4 in Childhood Acute Lymphocytic Leukemia / Receptores do Tipo Toll 2, 3 e 4 em Leucemia Linfocítica Aguda Infanti / Receptores Tipo Toll 2, 3 y 4 en la Leucemia Linfocítica Aguda Infantil

Introduction: Acute lymphocytic leukemia (ALL) is the most common cancer type in children and accounts for 80% of pediatric leukemias. Novel targets are necessary to improve survival rates for refractory and relapsed disease. There is accumulating evidence that Toll-like Receptor (TLR) signaling may be associated with outcomes in cancer however little has been described in leukemias. Objective: Analyze the expression and contribution of TLRs to the development of childhood ALL. Method: To evaluate the effect of specific TLR2, TLR3, and TLR4 agonists on the viability and proliferation of childhood ALL cell lines and to analyzed the mRNA expression of these types of TLR in bone marrow blast cells at diagnosis (D0) and induction (D35) in pediatric ALL patients. Results: Treatment with TLR agonists reduced the cell viability of Jurkat and Sup-B15 cell lines. Cell cycle distribution in Jurkat was altered, reducing polyploid cells and increasing sub-G1 phase. Conclusion: It was observed that the cell viability of the cell lines responded with different sensitivities to the agonists. The polyploidy associated with tumor malignancy was reduced, in addition to the increase in the sub-G1 phase indicating an increase in apoptosis. There were differences in TLR expression at D35 between groups at risk of the disease. Patients with high expression of TLR2 and low expression of TLR4 on D35 demonstrated a worse prognosis

Introdução: A leucemia linfoblástica aguda (LLA) é o tipo de câncer mais comum em crianças e representa 80% das leucemias pediátricas. Novos alvos são necessários para melhorar as taxas de sobrevivência para doença refratária e recidivante. Há evidências acumuladas de que a sinalização de receptores Toll-Like (TLR) pode estar associada a resultados em câncer, embora pouco tenha sido descrito em leucemias. Objetivo: Analisar a expressão e a contribuição dos TLR para o desenvolvimento da LLA infantil. Método: Avaliar o efeito de agonistas específicos de TLR2, TLR3 e TLR4 na viabilidade e proliferação de linhagens celulares de LLA infantil e analisar a expressão do RNAm desses tipos de TLR em células blásticas da medula óssea no diagnóstico (D0) e na indução (D35) em pacientes LLA pediátricos. Resultados: O tratamento com agonistas de TLR reduziu a viabilidade celular das linhagens celulares Jurkat e Sup-B15. A distribuição do ciclo celular em Jurkat foi alterada, reduzindo as células poliploides e aumentando a fase sub-G1. Houve aumento na expressão dos receptores entre D0 e D35 em amostras de pacientes. Conclusão: Observou-se que a viabilidade celular das linhagens celulares respondeu com diferentes sensibilidades aos agonistas. A poliploidia associada à malignidade tumoral foi reduzida, além de o aumento da fase sub-G1 indicar aumento da apoptose. Houve diferenças na expressão de TLR em D35 entre os grupos de risco da doença. Pacientes com alta expressão de TLR2 e baixa expressão de TLR4 no D35 demonstraram pior prognóstico.

Introducción: La leucemia linfocítica aguda (LLA) es el tipo de cáncer más común en los niños y representa el 80 % de las leucemias pediátricas. Se necesitan nuevos objetivos para mejorar las tasas de supervivencia de la enfermedad refractaria y recidivante. Cada vez hay más pruebas de que la señalización del receptor Toll-Like (TLR) puede estar asociada con resultados en el cáncer, aunque se ha descrito poco en las leucemias. Objetivo: Analizar la expresión y la contribución de los TLR al desarrollo de la LLA infantil. Método: Evaluar el efecto de agonistas específicos de TLR2, TLR3 y TLR4 en la viabilidad y proliferación de líneas celulares de LLA infantil y analizar la expresión de ARNm de estos tipos de TLR en células blásticas de médula ósea en el momento del diagnóstico (D0) y la inducción (D35) en pacientes pediátricos con LLA. Resultados: El tratamiento con agonistas de TLR redujo la viabilidad celular de las líneas celulares Jurkat y sup-B15. Se alteró la distribución del ciclo celular en Jurkat, reduciendo las células poliploides y aumentando la fase sub-G1. Hubo un aumento en la expresión de los receptores entre D0 y D35 en muestras de pacientes. Conclusión: Se observó que la viabilidad celular de las líneas celulares respondía con distintas sensibilidades a los agonistas. Se redujo la poliploidía asociada con la malignidad del tumor, además de un aumento de la fase sub-G1 que indica un aumento de la apoptosis. Hubo diferencias en la expresión de TLR en D35 entre los grupos de riesgo de enfermedad. Los pacientes con alta expresión de TLR2 y baja expresión de TLR4 en D35 mostraron peor pronóstico

G9a/EHMT2 is a Potential Prognostic Biomarker and Molecular Target in SHH Medulloblastoma.

Changes in epigenetic programming are associated with cancer development during childhood. Components of the epigenetic machinery involved in normal embryonic development and hijacked by pediatric cancers include enzymes mediating post-translational modifications of DNA and histones that regulate chromatin structure, such as histone methyltransferases (HMTs). Overexpression of the HMT G9a (euchromatic histone lysine methyltransferase 2, EHMT2) has been described in several cancer types. Medulloblastoma (MB), the main type of malignant brain tumor afflicting children, is currently classified into four molecular subgroups. Here, we show that expression level of the G9a/Ehmt2 gene is higher in MB tumors belonging to the SHH, Group 3, and Group 4 subgroups, compared to Wnt tumors. Remarkably, high G9a expression was significantly associated with shorter overall survival in MB patients. We also present evidence that G9a inhibition dose-dependently reduces MB cell viability. Our findings suggest that higher transcription of G9a may be a predictor of poor prognosis in patients with SHH MB, and that inhibiting G9a activity can display antitumor effects in MB.

GGAA-Microsatellites of NR0B1 Promoter Region in Ewing's Sarcoma Patients and Healthy Individuals of a Southern Brazilian Population / Microssatélites GGAA na Região Promotora de NR0B1 em Pacientes com Sarcoma de Ewing e Indivíduos Saudáveis de uma População no Sul do Brasil / Microsatélites GGAA en la Región Promotora de NR0B1 en Pacientes con Sarcoma de Ewing y Individuos Sanos de una Población del Sur de Brasil

Introduction: The very aggressive soft tissue and bone pediatric tumor Ewing's sarcoma (ES) is caused in most cases by the chromosomal translocation t(11;22)(q24;q12), which encodes an aberrant chimeric transcription factor (EWS-FLI1) that regulates target genes, including the critical oncogene NR0B1 (Xp21.2),via GGAA-microsatellites. Objective: Analyze the GGAA-microsatellites of NR0B1promoter region of ES patients and healthy subjects in the population investigated. Method: Ten male ES patients and 71 adult healthy males from Rio Grande do Sul state, Brazil, were included in this study. DNA from peripheral blood samples was extracted, amplified by PCR, sequenced by the Sanger method and analyzed by capillary electrophoresis. Total number of GGAA-motifs, length of microsatellite in base pairs, number of segments separated by "A" insertions, and the greatest number of consecutive GGAA-motifs were analyzed as well. Statistical analyses were performed in the SPSS statistical software and p-value <0.05 was considered significant. Results: A total of 21 different alleles was identified in the 81 subjects, with 24.2 allele [(GGAA)7A(GGAA)7A(GGAA)10] being the most frequent, but when comparing the data between the two groups, no significant difference was found. Conclusion: The sample investigated had a wide variation of microsatellite structure, including the presence of rare alleles, allowing the opportunity to describe this population as an essential step to identify genetic implications in ES tumorigenesis

Introdução: O sarcoma de Ewing (ES) é um tumor pediátrico de ossos e partes moles muito agressivo, causado, na maioria das vezes, pela translocação cromossômica t(11;22)(q24;q12), codificando um fator de transcrição quimérico aberrante (EWS-FLI1) que regula genes-alvo, incluindo o oncogene NR0B1 (Xp21.2), via microssatélites GGAA. Objetivo: Analisar os microssatélites GGAA da região promotora de NR0B1 em pacientes com ES e indivíduos saudáveis da população em investigação. Método: Foram incluídos dez pacientes do sexo masculino com diagnóstico de ES e 71 indivíduos adultos hígidos do sexo masculino do Estado do Rio Grande do Sul, Brasil. O DNA foi extraído de sangue periférico e amplificado por PCR, sequenciado pelo método de Sanger e analisado por eletroforese capilar. Foram analisados o número total de repetições GGAA, comprimento total do microssatélite em pares de bases, número de segmentos separados por inserções "A" e maior número de repetições GGAA consecutivas. As análises estatísticas foram realizadas no software estatístico SPSS e o valor de p<0,05 foi considerado significativo. Resultados: Um total de 21 alelos diferentes foi identificado nos 81 indivíduos, com o alelo 24,2 [(GGAA)7A(GGAA)7A(GGAA)10], sendo o mais frequente; mas, ao comparar os dados entre os dois grupos, nenhuma diferença significativa foi encontrada. Conclusão: A amostra estudada é altamente variável em termos de estrutura de microssatélites, incluindo a presença de alelos raros, dando a oportunidade de descrever essa população, o que é uma etapa fundamental na identificação de implicações genéticas na tumorigênese do ES

Introducción: El sarcoma de Ewing (ES) es un tumor pediátrico de huesos y tejidos blandos muy agresivo, que se presenta con mayor frecuencia por translocación cromosómica t(11;22)(q24;q12), que codifica un factor de transcripción quimérico aberrante (EWS-FLI1) que regula los genes diana, incluido el oncogén NR0B1 (Xp21.2), a través de microsatélites GGAA. Objetivo: Analizar los microsatélites GGAA de la región promotora de NR0B1en pacientes con ES y personas sanas de la población investigada. Método: Este estudio incluyó a diez pacientes varones con diagnóstico de ES y 71 varones adultos del estado de Rio Grande do Sul, Brasil. El ADN se extrajo de sangre periférica y se amplificó por PCR, secuenciado por el método de Sanger y analizado por electroforesis capilar. El número total de repeticiones GGAA, longitud total de microsatélites en pares de bases, número de segmentos separados por inserciones "A" y el mayor número de repeticiones GGAA consecutivas fueran analizados. Los análisis estadísticos se realizaron con el software estadístico SPSS y se consideró significativo un valor de p<0,05. Resultados: Se identificaron un total de 21 alelos diferentes en los 81, siendo el alelo 24,2 [(GGAA)7A(GGAA)7A(GGAA)10] el más frecuente, pero al comparar los datos entre los dos grupos, no hubo diferencia estadísticamente significativa. Conclusión: La muestra estudiada es muy variable en cuanto a estructura de microsatélites, incluyendo la presencia de alelos raros, lo que nos permite la oportunidad de describir la población estudiada, lo cual es un paso fundamental en la identificación de implicaciones genéticas en la tumorigénesis de ES

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.

Oncogenic functions of ZEB1 in pediatric solid cancers: interplays with microRNAs and long noncoding RNAs.

The transcription factor Zinc finger E-box binding 1 (ZEB1) displays a range of regulatory activities in cell function and embryonic development, including driving epithelial-mesenchymal transition. Several aspects of ZEB1 function can be regulated by its functional interactions with noncoding RNA types, namely microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). Increasing evidence indicates that ZEB1 importantly influences cancer initiation, tumor progression, metastasis, and resistance to treatment. Cancer is the main disease-related cause of death in children and adolescents. Although the role of ZEB1 in pediatric cancer is still poorly understood, emerging findings have shown that it is expressed and regulates childhood solid tumors including osteosarcoma, retinoblastoma, neuroblastoma, and central nervous system tumors. Here, we review the evidence supporting a role for ZEB1, and its interplays with miRNAs and lncRNAs, in pediatric cancers.

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.

The effect of hospital care volume on overall survival of children with cancer in Southern Brazil.

PURPOSE: The purpose of this study was to analyze the effect of hospital care volume on the overall survival of children with cancer in Southern Brazil. PATIENTS AND METHODS: We performed a retrospective cohort study of 1378 cancer patients aged 0-19 years, diagnosed with cancer between August 1, 2009 and December 31, 2015 in Rio Grande do Sul, who received hospital treatment in institutions affiliated with the Universal Health Care System (Sistema Único de Saúde [SUS]). RESULTS: Most children and adolescents were male (56.9%) and White (75.8%). The most common types of cancer in our cohort were acute leukemia (40.7%), followed by lymphoma (15.9%) and central nervous system tumors (8.8%). Ninety-five percent of the patients were treated in specialized pediatric oncology centers. The cumulative probability of survival at 5 years for all patients was 73.8% (95% confidence interval [CI] 71.4-76.0%). Survival was significantly higher for patients younger than 4 years of age (P = .012) compared to all other age groups. Patients treated in institutions with a pediatric oncology patient volume of less than 15 patients/year were 41% more likely to die than patients treated in institutions with a volume of 60 patients/year or more (P = .029). CONCLUSION: Cancer is the leading cause of death by natural causes in all age groups in Brazil, but, even so, childhood tumors are rare. This complexity makes childhood cancer care a challenge. In this study, we reiterate that pediatric cancer patients demonstrate better overall survival when treated in high-volume hospitals.

Neurotrophin Signaling in Medulloblastoma.

Neurotrophins are a family of secreted proteins that act by binding to tropomyosin receptor kinase (Trk) or p75NTR receptors to regulate nervous system development and plasticity. Increasing evidence indicates that neurotrophins and their receptors in cancer cells play a role in tumor growth and resistance to treatment. In this review, we summarize evidence indicating that neurotrophin signaling influences medulloblastoma (MB), the most common type of malignant brain cancer afflicting children. We discuss the potential of neurotrophin receptors as new therapeutic targets for the treatment of MB. Overall, activation of TrkA and TrkC types of receptors seem to promote cell death, whereas TrkB might stimulate MB growth, and TrkB inhibition displays antitumor effects. Importantly, we show analyses of the gene expression profile of neurotrophins and their receptors in MB primary tumors, which indicate, among other findings, that higher levels of NTRK1 or NTRK2 are associated with reduced overall survival (OS) of patients with SHH MB tumors.