RESUMO
Hydrocephalus is a common finding in newborns. In most cases, it is caused by intraventricular hemorrhage associated with prematurity, whereas in some patients the cause of hydrocephalus can be traced back to genetic changes, associated with disease syndromes such as RASopathies, lysosomal storage diseases, dystroglycanopathies, craniosynostosis but also ciliopathies. Ciliopathies are a group of diseases that can affect multiple organ systems due to dysfunction or the absence of cilia. Cilia are small organelles, extending from the cell surface. Nonmotile monocilia are ubiquitously present during cell development fulfilling chemosensory functions, whereas specialized epithelia such as the ependyma, lining the inner surface of the brain ventricles, exhibit multiciliated cells propelling fluids along the cell surface. This review highlights ciliopathies and their pathophysiology in congenital hydrocephalus. While nonmotile ciliopathies are often associated with severe prenatal hydrocephalus combined with other severe congenital brain malformations, motile ciliopathies, especially those associated with defects in multiciliogenesis can cause hydrocephalus and chronic lung disease.
Assuntos
Ciliopatias , Hidrocefalia , Malformações do Sistema Nervoso , Cílios/genética , Ciliopatias/genética , Ciliopatias/metabolismo , Humanos , Hidrocefalia/genética , Hidrocefalia/metabolismo , Recém-NascidoRESUMO
Soft-tissue sarcomas are rare, heterogeneous, and often aggressive mesenchymal cancers. Many of them are associated with poor outcome, partially because biomarkers that can identify high-risk patients are lacking. Studies on sarcomas are often limited by small sample-sizes rendering the identification of biomarkers difficult when focusing on individual cohorts. However, the increasing number of publicly available 'omics' data opens inroads to overcome this obstacle. Here, we combine transcriptome analyses, immunohistochemistry, and functional assays to show that high adenosine monophosphate deaminase 2 (AMPD2) is a robust prognostic biomarker for worse outcome in undifferentiated pleomorphic sarcoma (UPS). Gene expression and survival data for UPS from two independent studies were subjected to survival association-testing. Genes, whose high expression was significantly correlated with worse outcome in both cohorts, were considered as biomarker candidates. The best candidate, AMPD2, was validated in a tissue microarray. Analysis of DNA copy-number data and matched transcriptomes indicated that high AMPD2 expression is significantly correlated with gains at the AMPD2 locus. Gene set enrichment analyses of AMPD2 co-expressed genes in both transcriptome datasets suggested that AMPD2-high UPS are enriched in tumorigenic signatures. Consistently, knockdown of AMPD2 by RNA interference in an UPS cell line inhibited proliferation in vitro and tumorigenicity in vivo. Collectively, we provide evidence that AMPD2 may serve as a biomarker for outcome prediction in UPS. Our study exemplifies how the integration of 'omics' data, immunohistochemistry, and functional experiments can identify novel biomarkers even in a rare sarcoma, which may serve as a blueprint for biomarker identification for other rare cancers.
Assuntos
AMP Desaminase/genética , Biomarcadores Tumorais/genética , Genômica/métodos , Histiocitoma Fibroso Maligno/genética , AMP Desaminase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histiocitoma Fibroso Maligno/metabolismo , Histiocitoma Fibroso Maligno/patologia , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Prognóstico , Terapêutica com RNAi/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Adulto JovemRESUMO
Chromosomal instability (CIN) is a hallmark of cancer1. Yet, many childhood cancers, such as Ewing sarcoma (EwS), feature remarkably 'silent' genomes with minimal CIN2. Here, we show in the EwS model how uncoupling of mitosis and cytokinesis via targeting protein regulator of cytokinesis 1 (PRC1) or its activating polo-like kinase 1 (PLK1) can be employed to induce fatal genomic instability and tumor regression. We find that the EwS-specific oncogenic transcription factor EWSR1-FLI1 hijacks PRC1, which physiologically safeguards controlled cell division, through binding to a proximal enhancer-like GGAA-microsatellite, thereby promoting tumor growth and poor clinical outcome. Via integration of transcriptome-profiling and functional in vitro and in vivo experiments including CRISPR-mediated enhancer editing, we discover that high PRC1 expression creates a therapeutic vulnerability toward PLK1 inhibition that can repress even chemo-resistant EwS cells by triggering mitotic catastrophe.Collectively, our results exemplify how aberrant PRC1 activation by a dominant oncogene can confer malignancy but provide opportunities for targeted therapy, and identify PRC1 expression as an important determinant to predict the efficacy of PLK1 inhibitors being used in clinical trials.
Assuntos
Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Sarcoma de Ewing/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Criança , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Terapêutica com RNAi/métodos , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/terapia , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Quinase 1 Polo-LikeRESUMO
Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Estresse Oxidativo , Sarcoma de Ewing/patologia , Adulto , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Criança , Condrócitos/metabolismo , Metilação de DNA , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Hidrazinas/química , Células-Tronco Mesenquimais/metabolismo , Camundongos , Repetições de Microssatélites , Mitocôndrias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes , Interferência de RNA , Fatores de Transcrição SOXD/metabolismo , Sarcoma/genéticaRESUMO
A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20017-2.
RESUMO
Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.
RESUMO
Ewing sarcoma (EwS) is an aggressive cancer characterized by chromosomal translocations generating fusions of the EWSR1 gene with ETS transcription factors (in 85% FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity correlates with the number of consecutive GGAA-repeats. Herein we investigate the role of the secretory neuropeptide CALCB (calcitonin-related polypeptide ß) in EwS, which signals via the CGRP (calcitonin gene-related peptide) receptor complex, containing RAMP1 (receptor activity modifying protein 1) as crucial part for receptor specificity. Analysis of 2678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed that CALCB is specifically and highly overexpressed in EwS. Time-course knockdown experiments showed that CALCB expression is tightly linked to that of EWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that of CALCB indicated that it is co-expressed with other EWSR1-FLI1 target genes and associated with signatures involved in stemness and proliferation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for FLI1 and histone marks from EwS cell lines demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close to CALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass spectrometric analyses of EwS cell culture supernatants demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediated CALCB knockdown had no effect on proliferation and clonogenic growth of EwS cells in vitro, its long-term knockdown decreased EwS growth in vitro and in vivo. Similarly, knockdown of RAMP1 reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the RAMP1-comprising CGRP receptor reduced growth of EwS. Collectively, our findings suggest that CALCB is a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1 axis may offer a new therapeutic strategy for inhibition of EwS growth.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Proteína 1 Modificadora da Atividade de Receptores/metabolismo , Sarcoma de Ewing/patologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Peptídeo Relacionado com Gene de Calcitonina/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Repetições de Microssatélites/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Proteína 1 Modificadora da Atividade de Receptores/antagonistas & inibidores , Proteína 1 Modificadora da Atividade de Receptores/genética , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transplante HeterólogoRESUMO
Pediatric malignancies including Ewing sarcoma (EwS) feature a paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome. Here, we demonstrate in EwS how cooperation of dominant oncogenes and regulatory germline variants determine tumor growth, patient survival and drug response. Binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2 mediating these phenotypes. Whole-genome and RNA sequencing reveals that variability at this locus is inherited via the germline and is associated with variable inter-tumoral MYBL2 expression. High MYBL2 levels sensitize EwS cells for inhibition of its upstream activating kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a putative biomarker for anti-CDK2-therapy. Collectively, we establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and highlight the importance of integrating the regulatory genome in precision medicine.
Assuntos
Mutação em Linhagem Germinativa/genética , Neoplasias/genética , Neoplasias/terapia , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Repetições de Microssatélites/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fenótipo , Polimorfismo Genético , Transativadores , Resultado do Tratamento , Regulação para Cima/genéticaRESUMO
Cytokinesis is the final event of the cell cycle dividing one cell into two daughter cells. The protein regulator of cytokinesis (PRC)1 is essential for cytokinesis and normal cell cleavage. Deregulation of PRC1 causes cytokinesis defects that promote chromosomal instability (CIN) and thus tumor heterogeneity and cancer evolution. Consistently, abnormal PRC1 expression correlates with poor patient outcome in various malignancies, which may be caused by PRC1-mediated CIN and aneuploidy. Here, we review the physiological functions of PRC1 in cell cycle regulation and its contribution to tumorigenesis and intratumoral heterogeneity. We discuss targeting PRC1 within the complementary approaches of either normalizing CIN in aneuploid cancers or creating chromosomal chaos in genomically stable cancers to induce apoptosis.
Assuntos
Proteínas de Ciclo Celular/genética , Evolução Clonal/genética , Citocinese/genética , Neoplasias/genética , Aneuploidia , Apoptose/genética , Carcinogênese/genética , Instabilidade Cromossômica/genética , Humanos , Mitose/genética , Neoplasias/patologiaRESUMO
Ewing sarcoma is an undifferentiated small-round-cell sarcoma. Although molecular detection of pathognomonic EWSR1-ETS fusions such as EWSR1-FLI1 enables definitive diagnosis, substantial confusion can arise if molecular diagnostics are unavailable. Diagnosis based on the conventional immunohistochemical marker CD99 is unreliable due to its abundant expression in morphological mimics. To identify novel diagnostic immunohistochemical markers for Ewing sarcoma, we performed comparative expression analyses in 768 tumors representing 21 entities including Ewing-like sarcomas, which confirmed that CIC-DUX4-, BCOR-CCNB3-, EWSR1-NFATc2-, and EWSR1-ETS-translocated sarcomas are distinct entities, and revealed that ATP1A1, BCL11B, and GLG1 constitute specific markers for Ewing sarcoma. Their high expression was validated by immunohistochemistry and proved to depend on EWSR1-FLI1-binding to highly active proximal super-enhancers. Automated cut-off-finding and combination-testing in a tissue-microarray comprising 174 samples demonstrated that detection of high BCL11B and/or GLG1 expression is sufficient to reach 96% specificity for Ewing sarcoma. While 88% of tested Ewing-like sarcomas displayed strong CD99-immunoreactivity, none displayed combined strong BCL11B- and GLG1-immunoreactivity. Collectively, we show that ATP1A1, BCL11B, and GLG1 are EWSR1-FLI1 targets, of which BCL11B and GLG1 offer a fast, simple, and cost-efficient way to diagnose Ewing sarcoma by immunohistochemistry. These markers may significantly reduce the number of misdiagnosed patients, and thus improve patient care.
RESUMO
Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Immunogenic peptides encoded by cancer-specific genes (CSGs) may enable targeted immunotherapy, even of oligo-mutated cancers, which lack neo-antigens generated by protein-coding missense mutations. Here, we describe an algorithm and user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues) that automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from transcriptome data. We applied RAVEN to a dataset assembled from 2,678 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on oligo-mutated pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs, and identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated in cancer cell lines and in a comprehensive tissue-microarray. Subsequently, RAVEN identified likely immunogenic CSG-encoded peptides by predicting their affinity to MHCs and excluded sequence identity to abundantly expressed proteins by interrogating the UniProt protein-database. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed binding-kinetics like a very immunogenic influenza control peptide. Collectively, we provide an exquisitely curated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer types, and a freely available software (https://github.com/JSGerke/RAVENsoftware) to easily apply our algorithm to any gene expression dataset. We anticipate that our peptide libraries and software constitute a rich resource to advance anti-cancer immunotherapy.