RESUMO
In this issue of Developmental Cell, Shiraishi et al. investigate the epigenetic changes occurring during the formation of SHH medulloblastoma and show that an epigenomic shift renders Nuclear Factor I family of transcription factors oncogenic.
Assuntos
Epigênese Genética , Proteínas Hedgehog , Meduloblastoma , Fatores de Transcrição NFI , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Fatores de Transcrição NFI/metabolismo , Fatores de Transcrição NFI/genética , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , CamundongosRESUMO
Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.
Assuntos
Sistemas CRISPR-Cas , Neoplasias Cerebelares , DNA (Citosina-5-)-Metiltransferase 1 , Proteínas Hedgehog , Meduloblastoma , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Animais , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Humanos , Camundongos , Linhagem Celular Tumoral , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Técnicas de Inativação de Genes/métodosRESUMO
Medulloblastoma, the most common malignant pediatric brain tumor, is classified into four main molecular subgroups, but group 3 and group 4 tumors are difficult to subclassify and have a poor prognosis. Rapid point-of-care diagnostic and prognostic assays are needed to improve medulloblastoma risk stratification and management. N6-methyladenosine (m6A) is a common RNA modification and long non-coding RNAs (lncRNAs) play a central role in tumor progression, but their impact on gene expression and associated clinical outcomes in medulloblastoma are unknown. Here we analyzed 469 medulloblastoma tumor transcriptomes to identify lncRNAs co-expressed with m6A regulators. Using LASSO-Cox analysis, we identified a five-gene m6A-associated lncRNA signature (M6LSig) significantly associated with overall survival, which was combined in a prognostic clinical nomogram. Using expression of the 67 m6A-associated lncRNAs, a subgroup classification model was generated using the XGBoost machine learning algorithm, which had a classification accuracy > 90%, including for group 3 and 4 samples. All M6LSig genes were significantly correlated with at least one immune cell type abundance in the tumor microenvironment, and the risk score was positively correlated with CD4+ naïve T cell abundance and negatively correlated with follicular helper T cells and eosinophils. Knockdown of key m6A writer genes METTL3 and METTL14 in a group 3 medulloblastoma cell line (D425-Med) decreased cell proliferation and upregulated many M6LSig genes identified in our in silico analysis, suggesting that the signature genes are functional in medulloblastoma. This study highlights a crucial role for m6A-dependent lncRNAs in medulloblastoma prognosis and immune responses and provides the foundation for practical clinical tools that can be rapidly deployed in clinical settings.
Assuntos
Adenosina , Neoplasias Cerebelares , Meduloblastoma , RNA Longo não Codificante , Transcriptoma , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Prognóstico , Criança , Perfilação da Expressão Gênica/métodos , Masculino , Feminino , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , MetiltransferasesRESUMO
Hypothyroidism is commonly detected in patients with medulloblastoma (MB). However, whether thyroid hormone (TH) contributes to MB pathogenicity remains undetermined. Here, we find that TH plays a critical role in promoting tumor cell differentiation. Reduction in TH levels frees the TH receptor, TRα1, to bind to EZH2 and repress expression of NeuroD1, a transcription factor that drives tumor cell differentiation. Increased TH reverses EZH2-mediated repression of NeuroD1 by abrogating the binding of EZH2 and TRα1, thereby stimulating tumor cell differentiation and reducing MB growth. Importantly, TH-induced differentiation of tumor cells is not restricted by the molecular subgroup of MB, suggesting that TH can be used to broadly treat MB subgroups. These findings establish an unprecedented association between TH signaling and MB pathogenicity, providing solid evidence for TH as a promising modality for MB treatment.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Proteína Potenciadora do Homólogo 2 de Zeste , Meduloblastoma , Hormônios Tireóideos , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Meduloblastoma/genética , Humanos , Diferenciação Celular/efeitos dos fármacos , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Camundongos , Hormônios Tireóideos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/tratamento farmacológico , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores alfa dos Hormônios Tireóideos/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Effective and less toxic therapies for medulloblastoma have proved to be highly elusive. In this issue of Cancer Cell, Yang et al. show that thyroid hormone treatment leads to the activation of neurogenic differentiation factor 1 (NeuroD1) and differentiation of medulloblastoma cells through reversing EZH2-mediated transcriptional repression of NeuroD1.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular , Neoplasias Cerebelares , Proteína Potenciadora do Homólogo 2 de Zeste , Meduloblastoma , Meduloblastoma/patologia , Meduloblastoma/genética , Meduloblastoma/metabolismo , Humanos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Complexo Repressor Polycomb 2/genética , Hormônios Tireóideos/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
OTX2 is a transcription factor and known driver in medulloblastoma (MB), where it is amplified in a subset of tumours and overexpressed in most cases of group 3 and group 4 MB. Here we demonstrate a noncanonical role for OTX2 in group 3 MB alternative splicing. OTX2 associates with the large assembly of splicing regulators complex through protein-protein interactions and regulates a stem cell splicing program. OTX2 can directly or indirectly bind RNA and this may be partially independent of its DNA regulatory functions. OTX2 controls a pro-tumorigenic splicing program that is mirrored in human cerebellar rhombic lip origins. Among the OTX2-regulated differentially spliced genes, PPHLN1 is expressed in the most primitive rhombic lip stem cells, and targeting PPHLN1 splicing reduces tumour growth and enhances survival in vivo. These findings identify OTX2-mediated alternative splicing as a major determinant of cell fate decisions that drive group 3 MB progression.
Assuntos
Processamento Alternativo , Neoplasias Cerebelares , Meduloblastoma , Células-Tronco Neoplásicas , Fatores de Transcrição Otx , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição Otx/genética , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Processamento Alternativo/genética , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Camundongos , Proliferação de CélulasRESUMO
Medulloblastoma (MB) is the most common malignant brain tumor in children and is stratified into three major subgroups. The Sonic hedgehog (SHH) subgroup represents â¼30% of all MB cases and has significant survival disparity depending upon TP53 status. Here, we describe a zebrafish model of SHH MB using CRISPR to create mutant ptch1, the primary genetic driver of human SHH MB. In these animals, tumors rapidly arise in the cerebellum and resemble human SHH MB by histology and comparative onco-genomics. Similar to human patients, MB tumors with loss of both ptch1 and tp53 have aggressive tumor histology and significantly worse survival outcomes. The simplicity and scalability of the ptch1-crispant MB model makes it highly amenable to CRISPR-based genome-editing screens to identify genes required for SHH MB tumor formation in vivo, and here we identify the gene encoding Grk3 kinase as one such target.
Assuntos
Modelos Animais de Doenças , Proteínas Hedgehog , Meduloblastoma , Receptor Patched-1 , Proteínas de Peixe-Zebra , Peixe-Zebra , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Humanos , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Sistemas CRISPR-Cas/genéticaRESUMO
We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.
Assuntos
Meduloblastoma , Células-Tronco Neoplásicas , Humanos , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Animais , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Camundongos , Rombencéfalo/metabolismo , Rombencéfalo/embriologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Células Endoteliais/metabolismo , Nicho de Células-Tronco , Células-Tronco/metabolismo , Técnicas de Cocultura , Estruturas Embrionárias , Metencéfalo/embriologiaRESUMO
SNCAIP duplication may promote Group 4 medulloblastoma via induction of PRDM6, a poorly characterized member of the PRDF1 and RIZ1 homology domain-containing (PRDM) family of transcription factors. Here, we investigated the function of PRDM6 in human hindbrain neuroepithelial stem cells and tested PRDM6 as a driver of Group 4 medulloblastoma. We report that human PRDM6 localizes predominantly to the nucleus, where it causes widespread repression of chromatin accessibility and complex alterations of gene expression patterns. Genome-wide mapping of PRDM6 binding reveals that PRDM6 binds to chromatin regions marked by histone H3 lysine 27 trimethylation that are located within, or proximal to, genes. Moreover, we show that PRDM6 expression in neuroepithelial stem cells promotes medulloblastoma. Surprisingly, medulloblastomas derived from PRDM6-expressing neuroepithelial stem cells match human Group 3, but not Group 4, medulloblastoma. We conclude that PRDM6 expression has oncogenic potential but is insufficient to drive Group 4 medulloblastoma from neuroepithelial stem cells. We propose that both PRDM6 and additional factors, such as specific cell-of-origin features, are required for Group 4 medulloblastoma. Given the lack of PRDM6 expression in normal tissues and its oncogenic potential shown here, we suggest that PRDM6 inhibition may have therapeutic value in PRDM6-expressing medulloblastomas.
Assuntos
Cromatina , Meduloblastoma , Animais , Humanos , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Cromatina/metabolismo , Cromatina/genética , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Células Neuroepiteliais/metabolismoRESUMO
Medulloblastomas (MBs) are malignant pediatric brain tumors that are molecularly and clinically heterogenous. The application of omics technologies-mainly studying nucleic acids-has significantly improved MB classification and stratification, but treatment options are still unsatisfactory. The proteome and their N-glycans hold the potential to discover clinically relevant phenotypes and targetable pathways. We compile a harmonized proteome dataset of 167 MBs and integrate findings with DNA methylome, transcriptome and N-glycome data. We show six proteome MB subtypes, that can be assigned to two main molecular programs: transcription/translation (pSHHt, pWNT and pG3myc), and synapses/immunological processes (pSHHs, pG3 and pG4). Multiomic analysis reveals different conservation levels of proteome features across MB subtypes at the DNA methylome level. Aggressive pGroup3myc MBs and favorable pWNT MBs are most similar in cluster hierarchies concerning overall proteome patterns but show different protein abundances of the vincristine resistance-associated multiprotein complex TriC/CCT and of N-glycan turnover-associated factors. The N-glycome reflects proteome subtypes and complex-bisecting N-glycans characterize pGroup3myc tumors. Our results shed light on targetable alterations in MB and set a foundation for potential immunotherapies targeting glycan structures.
Assuntos
Meduloblastoma , Polissacarídeos , Proteoma , Meduloblastoma/metabolismo , Meduloblastoma/genética , Humanos , Polissacarídeos/metabolismo , Proteoma/metabolismo , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/genética , Metilação de DNA , Transcriptoma , Criança , Proteômica/métodos , Feminino , Regulação Neoplásica da Expressão Gênica , Masculino , Pré-Escolar , Perfilação da Expressão Gênica/métodosRESUMO
Medulloblastoma is the most common pediatric brain cancer, with about five cases per million in the pediatric population. Current treatment strategies have a 5-year survival rate of 70% or more but frequently lead to long-term neurocognitive defects, and recurrence is relatively high. Genomic sequencing of medulloblastoma patients has shown that DDX3X, which encodes an RNA helicase involved in the process of translation initiation, is among the most commonly mutated genes in medulloblastoma. The identified mutations are 42 single-point amino acid substitutions and are mostly not complete loss-of-function mutations. The pathological mechanism of DDX3X mutations in the causation of medulloblastoma is poorly understood, but several studies have examined their role in promoting cancer progression. This review first discusses the known roles of DDX3X and its yeast ortholog Ded1 in translation initiation, cellular stress responses, viral replication, innate immunity, inflammatory programmed cell death, Wnt signaling, and brain development. It then examines our current understanding of the oncogenic mechanism of the DDX3X mutations in medulloblastoma, including the effect of these DDX3X mutations on growth, biochemical functions, translation, and stress responses. Further research on DDX3X's mechanism and targets is required to therapeutically target DDX3X and/or its downstream effects in medulloblastoma progression.
Assuntos
RNA Helicases DEAD-box , Progressão da Doença , Meduloblastoma , Mutação , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Animais , Proteínas de Saccharomyces cerevisiaeRESUMO
Development of the cerebellum requires precise regulation of granule neuron progenitor (GNP) proliferation. Although it is known that primary cilia are necessary to support GNP proliferation, the exact molecular mechanism governing primary cilia dynamics within GNPs remains elusive. Here, we establish the pivotal roles for the centrosomal kinase TTBK2 (Tau tubulin kinase-2) and the E3 ubiquitin ligase HUWE1 in GNP proliferation. We show that TTBK2 is highly expressed in proliferating GNPs under Sonic Hedgehog (SHH) signaling, coinciding with active GNP proliferation and the presence of primary cilia. TTBK2 stabilizes primary cilia by inhibiting their disassembly, thereby promoting GNP proliferation in response to SHH. Mechanistically, we identify HUWE1 as a novel centrosomal E3 ligase that facilitates primary cilia disassembly by targeting TTBK2 degradation. Disassembly of primary cilia serves as a trigger for GNP differentiation, allowing their migration from the external granule layer (EGL) of the cerebellum to the internal granule layer (IGL) for subsequent maturation. Moreover, we have established a link between TTBK2 and SHH-type medulloblastoma (SHH-MB), a tumor characterized by uncontrolled GNP proliferation. TTBK2 depletion inhibits SHH-MB proliferation, indicating that TTBK2 may be a potential therapeutic target for this cancer type. In summary, our findings reveal the mechanism governing cerebellar development and highlight a potential anti-cancer strategy for SHH-MB.
Assuntos
Proliferação de Células , Cerebelo , Cílios , Proteínas Hedgehog , Meduloblastoma , Proteínas Serina-Treonina Quinases , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Meduloblastoma/genética , Cílios/metabolismo , Animais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Cerebelo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Camundongos , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/genética , Diferenciação Celular , Células-Tronco Neurais/metabolismoRESUMO
Medulloblastoma (MB) stands as one of the prevalent malignant brain tumors among pediatric patients. Despite its prevalence, the intricate interplay between the regulatory program driving malignancy in MB cells and their interactions with the microenvironment remains insufficiently understood. Leveraging the capabilities of single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq), the chromatin accessibility landscape is unveiled across 59,015 distinct MB cells. This expansive dataset encompasses cells belonging to discrete molecular subgroups, namely SHH, WNT, Group3, and Group4. Within these chromatin accessibility profiles, specific regulatory elements tied to individual subgroups are uncovered, shedding light on the distinct activities of transcription factors (TFs) that likely orchestrate the tumorigenesis process. Moreover, it is found that certain neurotransmitter receptors (NTRs) are subgroup-specific and can predict MB subgroup classification when combined with their associated transcription factors. Notably, targeting essential NTRs within tumors influences both the in vitro sphere-forming capability and the in vivo tumorigenic capacity of MB cells. These findings collectively provide fresh insights into comprehending the regulatory networks and cellular dynamics within MBs. Furthermore, the significance of the TF-NTR regulatory circuits is underscored as prospective biomarkers and viable therapeutic targets.
Assuntos
Neoplasias Cerebelares , Cromatina , Meduloblastoma , Análise de Célula Única , Fatores de Transcrição , Meduloblastoma/genética , Meduloblastoma/metabolismo , Humanos , Análise de Célula Única/métodos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Cromatina/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Camundongos , Animais , Linhagem Celular TumoralRESUMO
Prostaglandin F2 receptor negative regulator (PTGFRN) is a transmembrane protein associated with metastatic characteristics of certain cancer types. However, it remains poorly characterized and its direct function in cancer remains unclear. The study presented here aims to further examine whether PTGFRN expression affects a cancer cell's phenotype, as well as metastatic-like characteristics. We used stable shRNA and cDNA transfections to respectively knockdown and overexpress PTGFRN in three different cancer cell lines, two of which are representative of rare and aggressive cancers (Mesothelioma and Pediatric Medulloblastoma). We then examined the characteristics of the resulting clones and showed a decrease in proliferation, migration, colony formation, and spheroid growth capabilities in cells where PTGFRN expression had been inhibited, while cells overexpressing PTGFRN showed the opposite. In addition, we showed that PTGFRN displayed direct binding to two protein partners, Integrin ß1 and E. Cadherin, the latter of which is a novel direct binding partner to PTGFRN. Furthermore, silencing PTGFRN expression impacted the cellular process of autophagy, thereby providing another avenue by which PTGFRN potentially contributes to a cancer cell phenotype. Our findings demonstrate the potential role of PTGFRN in cancer metastasis and suggest PTGFRN as a future target for drug development in the treatment of metastatic cancers.
Assuntos
Carcinoma de Células Escamosas , Meduloblastoma , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/genética , Meduloblastoma/patologia , Linhagem Celular Tumoral , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Metástase Neoplásica , Movimento Celular , Fenótipo , Caderinas/metabolismo , Caderinas/genética , Criança , AutofagiaRESUMO
BACKGROUND: Certain paediatric nervous system malignancies have dismal prognoses. Retinoic acid (RA) is used in neuroblastoma treatment, and preclinical data indicate potential benefit in selected paediatric brain tumour entities. However, limited single-agent efficacy necessitates combination treatment approaches. METHODS: We performed drug sensitivity profiling of 76 clinically relevant drugs in combination with RA in 16 models (including patient-derived tumouroids) of the most common paediatric nervous system tumours. Drug responses were assessed by viability assays, high-content imaging, and apoptosis assays and RA relevant pathways by RNAseq from treated models and patient samples obtained through the precision oncology programme INFORM (n = 2288). Immunoprecipitation detected BCL-2 family interactions, and zebrafish embryo xenografts were used for in vivo efficacy testing. RESULTS: Group 3 medulloblastoma (MBG3) and neuroblastoma models were highly sensitive to RA treatment. RA induced differentiation and regulated apoptotic genes. RNAseq analysis revealed high expression of BCL2L1 in MBG3 and BCL2 in neuroblastomas. Co-treatments with RA and BCL-2/XL inhibitor navitoclax synergistically decreased viability at clinically achievable concentrations. The combination of RA with navitoclax disrupted the binding of BIM to BCL-XL in MBG3 and to BCL-2 in neuroblastoma, inducing apoptosis in vitro and in vivo. CONCLUSIONS: RA treatment primes MBG3 and NB cells for apoptosis, triggered by navitoclax cotreatment.
Assuntos
Apoptose , Sinergismo Farmacológico , Meduloblastoma , Neuroblastoma , Tretinoína , Peixe-Zebra , Humanos , Animais , Tretinoína/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Meduloblastoma/genética , Apoptose/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Neuroblastoma/metabolismo , Neuroblastoma/genética , Linhagem Celular Tumoral , Compostos de Anilina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Sulfonamidas/farmacologia , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Camundongos , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína Proto-Oncogênica N-MycRESUMO
BACKGROUND: Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current treatments have increased overall survival but can lead to devastating side effects and late complications in survivors, emphasizing the need for new, improved targeted therapies that specifically eliminate tumor cells while sparing the normally developing brain. METHODS: Here, we used a sonic hedgehog (SHH)-MB model based on a patient-derived neuroepithelial stem cell system for an unbiased high-throughput screen with a library of 172 compounds with known targets. Compounds were evaluated in both healthy neural stem cells (NSCs) and tumor cells derived from the same patient. Based on the difference of cell viability and drug sensitivity score between normal cells and tumor cells, hit compounds were selected and further validated in vitro and in vivo. RESULTS: We identified PF4708671 (S6K1 inhibitor) as a potential agent that selectively targets SHH-driven MB tumor cells while sparing NSCs and differentiated neurons. Subsequent validation studies confirmed that PF4708671 inhibited the growth of SHH-MB tumor cells both in vitro and in vivo, and that knockdown of S6K1 resulted in reduced tumor formation. CONCLUSIONS: Overall, our results suggest that inhibition of S6K1 specifically affects tumor growth, whereas it has less effect on non-tumor cells. Our data also show that the NES cell platform can be used to identify potentially effective new therapies and targets for SHH-MB.
Assuntos
Neoplasias Cerebelares , Ensaios de Triagem em Larga Escala , Meduloblastoma , Células-Tronco Neurais , Animais , Humanos , Camundongos , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Normal cells coordinate proliferation and differentiation by precise tuning of gene expression based on the dynamic shifts of the epigenome throughout the developmental timeline. Although non-mutational epigenetic reprogramming is an emerging hallmark of cancer, the epigenomic shifts that occur during the transition from normal to malignant cells remain elusive. Here, we capture the epigenomic changes that occur during tumorigenesis in a prototypic embryonal brain tumor, medulloblastoma. By comparing the epigenomes of the different stages of transforming cells in mice, we identify nuclear factor I family of transcription factors, known to be cell fate determinants in development, as oncogenic regulators in the epigenomes of precancerous and cancerous cells. Furthermore, genetic and pharmacological inhibition of NFIB validated a crucial role of this transcription factor by disrupting the cancer epigenome in medulloblastoma. Thus, this study exemplifies how epigenomic changes contribute to tumorigenesis via non-mutational mechanisms involving developmental transcription factors.
Assuntos
Epigenoma , Meduloblastoma , Fatores de Transcrição NFI , Meduloblastoma/genética , Meduloblastoma/patologia , Meduloblastoma/metabolismo , Animais , Fatores de Transcrição NFI/metabolismo , Fatores de Transcrição NFI/genética , Camundongos , Humanos , Regulação Neoplásica da Expressão Gênica , Progressão da Doença , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Epigênese Genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proliferação de Células/genética , Diferenciação Celular/genéticaRESUMO
OBJECTIVE: To investigate the characteristics of the CD8+ T cells infiltration from the 4 subtypes in medulloblastoma (MB), to analyze the relationship between CD8+ T cells infiltration and prognosis, to study the function of C-X-C motif chemokine ligand 11 (CXCL11) and its receptor in CD8+ T cells infiltration into tumors and to explore the potential mechanism, and to provide the necessary clinicopathological basis for exploring the immunotherapy of MB. METHODS: In the study, 48 clinical MB samples (12 cases in each of 4 subtypes) were selected from the multiple medical center from 2012 to 2019. The transcriptomics analysis for the tumor of 48 clinical samples was conducted on the NanoString PanCancer IO360TM Panel (NanoString Technologies). Immunohistochemistry (IHC) staining of formalin-fixed, paraffin-embedded sections from MB was carried out using CD8 primary antibody to analyze diffe-rential quantities of CD8+ T cells in the MB four subtypes. Through bioinformatics analysis, the relationship between CD8+T cells infiltration and prognosis of the patients and the expression differences of various chemokines in the different subtypes of MB were investigated. The expression of CXCR3 receptor on the surface of CD8+T cells in MB was verified by double immunofluorescence staining, and the underlying molecular mechanism of CD8+T cells infiltration into the tumor was explored. RESULTS: The characteristic index of CD8+T cells in the WNT subtype of MB was relatively high, suggesting that the number of CD8+T cells in the WNT subtype was significantly higher than that in the other three subtypes, which was confirmed by CD8 immunohistochemical staining and Gene Expression Omnibus (GEO) database analysis by using R2 online data analysis platform. And the increase of CD8+T cells infiltration was positively correlated with the patient survival. The expression level of CXCL11 in the WNT subtype MB was significantly higher than that of the other three subtypes. Immunofluorescence staining showed the presence of CXCL11 receptor, CXCR3, on the surface of CD8+T cells, suggesting that the CD8+T cells might be attracted to the MB microenvironment by CXCL11 through CXCR3. CONCLUSION: The CD8+T cells infiltrate more in the WNT subtype MB than other subtypes. The mechanism may be related to the activation of CXCL11-CXCR3 chemokine system, and the patients with more infiltration of CD8+T cells in tumor have better prognosis. This finding may provide the necessary clinicopathological basis for the regulatory mechanism of CD8+T cells infiltration in MB, and give a new potential therapeutic target for the future immunotherapy of MB.
Assuntos
Linfócitos T CD8-Positivos , Quimiocina CXCL11 , Meduloblastoma , Receptores CXCR3 , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Meduloblastoma/imunologia , Meduloblastoma/patologia , Meduloblastoma/classificação , Meduloblastoma/genética , Meduloblastoma/metabolismo , Receptores CXCR3/metabolismo , Receptores CXCR3/genética , Quimiocina CXCL11/metabolismo , Quimiocina CXCL11/genética , Prognóstico , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias Cerebelares/imunologia , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/metabolismo , Masculino , FemininoRESUMO
The RE1 silencing transcription factor (REST) is a driver of sonic hedgehog (SHH) medulloblastoma genesis. Our previous studies showed that REST enhances cell proliferation, metastasis and vascular growth and blocks neuronal differentiation to drive progression of SHH medulloblastoma tumors. Here, we demonstrate that REST promotes autophagy, a pathway that is found to be significantly enriched in human medulloblastoma tumors relative to normal cerebella. In SHH medulloblastoma tumor xenografts, REST elevation is strongly correlated with increased expression of the hypoxia-inducible factor 1-alpha (HIF1α)-a positive regulator of autophagy, and with reduced expression of the von Hippel-Lindau (VHL) tumor suppressor protein - a component of an E3 ligase complex that ubiquitinates HIF1α. Human SHH-medulloblastoma tumors with higher REST expression exhibit nuclear localization of HIF1α, in contrast to its cytoplasmic localization in low-REST tumors. In vitro, REST knockdown promotes an increase in VHL levels and a decrease in cytoplasmic HIF1α protein levels, and autophagy flux. In contrast, REST elevation causes a decline in VHL levels, as well as its interaction with HIF1α, resulting in a reduction in HIF1α ubiquitination and an increase in autophagy flux. These data suggest that REST elevation promotes autophagy in SHH medulloblastoma cells by modulating HIF1α ubiquitination and stability in a VHL-dependent manner. Thus, our study is one of the first to connect VHL to REST-dependent control of autophagy in a subset of medulloblastomas.