RESUMO
Atypical teratoid rhabdoid tumors (ATRT) are divided into MYC, TYR and SHH subgroups, suggesting diverse lineages of origin. Here, we investigate the imaging of human ATRT at diagnosis and the precise anatomic origin of brain tumors in the Rosa26-CreERT2::Smarcb1flox/flox model. This cross-species analysis points to an extra-cerebral origin for MYC tumors. Additionally, we clearly distinguish SHH ATRT emerging from the cerebellar anterior lobe (CAL) from those emerging from the basal ganglia (BG) and intra-ventricular (IV) regions. Molecular characteristics point to the midbrain-hindbrain boundary as the origin of CAL SHH ATRT, and to the ganglionic eminence as the origin of BG/IV SHH ATRT. Single-cell RNA sequencing on SHH ATRT supports these hypotheses. Trajectory analyses suggest that SMARCB1 loss induces a de-differentiation process mediated by repressors of the neuronal program such as REST, ID and the NOTCH pathway.
Assuntos
Neoplasias Encefálicas , Tumor Rabdoide , Teratoma , Humanos , Tumor Rabdoide/genética , Multiômica , Proteína SMARCB1/genética , Fatores de Transcrição/genética , Neoplasias Encefálicas/genética , Diagnóstico por Imagem , Teratoma/patologia , Proteínas Hedgehog/genéticaAssuntos
DNA Helicases/deficiência , DNA Helicases/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Imunidade , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Animais , Terapia Combinada/métodos , Modelos Animais de Doenças , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Camundongos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Polimorfismo de Nucleotídeo Único , Resultado do TratamentoRESUMO
Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.
Assuntos
Montagem e Desmontagem da Cromatina/imunologia , Tumor Rabdoide/genética , Tumor Rabdoide/imunologia , Linfócitos T/imunologia , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Humanos , Imuno-Histoquímica/métodos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Rhabdoid tumors (RTs) are aggressive tumors of early childhood characterized by SMARCB1 inactivation. Their poor prognosis highlights an urgent need to develop new therapies. Here, we performed a high-throughput screening of approved drugs and identified broad inhibitors of tyrosine kinase receptors (RTKs), including pazopanib, and the potassium channel inhibitor clofilium tosylate (CfT), as SMARCB1-dependent candidates. Pazopanib targets were identified as PDGFRα/ß and FGFR2, which were the most highly expressed RTKs in a set of primary tumors. Combined genetic inhibition of both these RTKs only partially recapitulated the effect of pazopanib, emphasizing the requirement for broad inhibition. CfT perturbed protein metabolism and endoplasmic reticulum stress and, in combination with pazopanib, induced apoptosis of RT cells in vitro. In vivo, reduction of tumor growth by pazopanib was enhanced in combination with CfT, matching the efficiency of conventional chemotherapy. These results strongly support testing pazopanib/CfT combination therapy in future clinical trials for RTs.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Compostos de Amônio Quaternário/farmacologia , Tumor Rabdoide/metabolismo , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Indazóis , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Proteína SMARCB1/metabolismoRESUMO
Extra-cranial rhabdoid tumors (RT) are highly aggressive malignancies of infancy, characterized by undifferentiated histological features and loss of SMARCB1 expression. The diagnosis is all the more challenging that other poorly differentiated cancers lose SMARCB1 expression, such as epithelioid sarcomas (ES), renal medullary carcinomas (RMC) or undifferentiated chordomas (UC). Moreover, late cases occurring in adults are now increasingly reported, raising the question of differential diagnoses and emphasizing nosological issues. To address this issue, we have analyzed the expression profiles of a training set of 32 SMARCB1-deficient tumors (SDT), with ascertained diagnosis of RT (n = 16, all < 5 years of age), ES (n = 8, all > 10 years of age), UC (n = 3) and RMC (n = 5). As compared with other SDT, RT are characterized by an embryonic signature, and up-regulation of key-actors of de novo DNA methylation processes. Using this signature, we then analysed the expression profiling of 37 SDT to infer the appropriate diagnosis. Thirteen adult onset tumors showed strong similarity with pediatric RT, in spite of older age; by exome sequencing, these tumors also showed genomic features indistinguishable from pediatric RT. In contrary, 8 tumors were reclassified within carcinoma, ES or UC categories, while the remaining could not be related to any of those entities. Our results demonstrate that embryonic signature is shared by all RT, whatever the age at diagnosis; they also illustrate that many adult-onset SDT of ambiguous histological diagnosis are clearly different from RT. Finally, our study paves the way for the routine use of expression-based signatures to give accurate diagnosis of SDT.
Assuntos
Sequenciamento do Exoma/métodos , Perfilação da Expressão Gênica/métodos , Tumor Rabdoide/diagnóstico , Tumor Rabdoide/genética , Proteína SMARCB1/deficiência , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Metilação de DNA , Diagnóstico Diferencial , Redes Reguladoras de Genes , Humanos , Lactente , Masculino , Adulto JovemRESUMO
Rhabdoid tumours (RTs) are highly aggressive tumours of infancy, frequently localized in the central nervous system (CNS) where they are termed atypical teratoid/rhabdoid tumours (AT/RTs) and characterized by bi-allelic inactivation of the SMARCB1 tumour suppressor gene. In this study, by temporal control of tamoxifen injection in Smarcb1(flox/flox);Rosa26-Cre(ERT2) mice, we explore the phenotypes associated with Smarcb1 inactivation at different developmental stages. Injection before E6, at birth or at 2 months of age recapitulates previously described phenotypes including embryonic lethality, hepatic toxicity or development of T-cell lymphomas, respectively. Injection between E6 and E10 leads to high penetrance tumours, mainly intra-cranial, with short delays (median: 3 months). These tumours demonstrate anatomical, morphological and gene expression profiles consistent with those of human AT/RTs. Moreover, intra- and inter-species comparisons of tumours reveal that human and mouse RTs can be split into different entities that may underline the variety of RT cells of origin.
Assuntos
Neoplasias Encefálicas/genética , Proteínas Cromossômicas não Histona/genética , Tumor Rabdoide/genética , Animais , Neoplasias Encefálicas/induzido quimicamente , Neoplasias Encefálicas/metabolismo , Pré-Escolar , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Camundongos , Tumor Rabdoide/induzido quimicamente , Tumor Rabdoide/metabolismo , Proteína SMARCB1 , Tamoxifeno/toxicidade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Rapidly proliferating cells promote glycolysis in aerobic conditions, to increase growth rate. Expression of specific glycolytic enzymes, namely pyruvate kinase M2 and hexokinase 2, concurs to this metabolic adaptation, as their kinetics and intracellular localization favour biosynthetic processes required for cell proliferation. Intracellular factors regulating their selective expression remain largely unknown. Here we show that the peroxisome proliferator-activated receptor gamma transcription factor and nuclear hormone receptor contributes to selective pyruvate kinase M2 and hexokinase 2 gene expression in PTEN-null fatty liver. Peroxisome proliferator-activated receptor gamma expression, liver steatosis, shift to aerobic glycolysis and tumorigenesis are under the control of the Akt2 kinase in PTEN-null mouse livers. Peroxisome proliferator-activated receptor gamma binds to hexokinase 2 and pyruvate kinase M promoters to activate transcription. In vivo rescue of peroxisome proliferator-activated receptor gamma activity causes liver steatosis, hypertrophy and hyperplasia. Our data suggest that therapies with the insulin-sensitizing agents and peroxisome proliferator-activated receptor gamma agonists, thiazolidinediones, may have opposite outcomes depending on the nutritional or genetic origins of liver steatosis.
Assuntos
Proteínas de Transporte/biossíntese , Fígado Gorduroso/metabolismo , Regulação Enzimológica da Expressão Gênica , Hexoquinase/biossíntese , Proteínas de Membrana/biossíntese , PPAR gama/metabolismo , Hormônios Tireóideos/biossíntese , Animais , Proliferação de Células , Glicólise , Humanos , Imuno-Histoquímica/métodos , Insulina/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tiazolidinedionas/farmacologia , Proteínas de Ligação a Hormônio da TireoideRESUMO
Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Regeneração Hepática/fisiologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sirolimo/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Genótipo , Hepatectomia , Hepatócitos/citologia , Hepatócitos/fisiologia , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/citologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos , Proteínas/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Serina-Treonina Quinases TORRESUMO
It is now widely recognized that translation factors are involved in cancer development and that components of the translation machinery that are deregulated in cancer cells may become targets for cancer therapy. The eukaryotic Release Factor 3 (eRF3) is a GTPase that associates with eRF1 in a complex that mediates translation termination. eRF3a/GSPT1 first exon contains a (GGC)n expansion coding for proteins with different N-terminal extremities. Herein we show that the longer allele (12-GGC) is present in 5.1% (7/137) of the breast cancer patients analysed and is absent in the control population (0/135), corresponding to an increased risk for cancer development, as revealed by Odds Ratio analysis. mRNA quantification suggests that patients with the 12-GGC allele overexpress eRF3a/GSPT1 in tumor tissues relative to the normal adjacent tissues. However, using an in vivo assay for translation termination in HEK293 cells, we do not detect any difference in the activity of the eRF3a proteins encoded by the various eRF3a/GSPT1 alleles. Although the connection between the presence of eRF3a/GSPT1 12-GGC allele and tumorigenesis is still unknown, our data suggest that the presence of the 12-GGC allele provides a potential novel risk marker for various types of cancer.
Assuntos
Neoplasias da Mama/genética , Fatores de Terminação de Peptídeos/genética , Polimorfismo Genético , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Razão de Chances , Fatores de Terminação de Peptídeos/metabolismo , Prognóstico , Interferência de RNA , Medição de Risco , Fatores de Risco , TransfecçãoRESUMO
In eukaryotes, eRF1 and eRF3 are associated in a complex that mediates translation termination. The regulation of the formation of this complex in vivo is far from being understood. In mammalian cells, depletion of eRF3a causes a reduction of eRF1 level by decreasing its stability. Here, we investigate the status of eRF3a when not associated with eRF1. We show that eRF3a forms altered in their eRF1-binding site have a decreased stability, which increases upon cell treatment with the proteasome inhibitor MG132. We also show that eRF3a forms altered in eRF1 binding as well as wild-type eRF3a are polyubiquitinated. These results indicate that eRF3a is degraded by the proteasome when not associated with eRF1 and suggest that proteasomal degradation of eRF3a controls translation termination complex formation by adjusting the eRF3a level to that of eRF1.
Assuntos
Terminação Traducional da Cadeia Peptídica , Fatores de Terminação de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Leupeptinas/farmacologia , Poliadenilação , Inibidores de ProteassomaRESUMO
Girolline is a 2-aminoimidazole derivative with cytotoxic activity. It affects the survival of exponentially growing leukaemic cultured cells and has a significant antitumour activity on grafted murine tumours in vivo. In vitro studies showed that girolline affected protein synthesis by interfering with the translation termination process. Here, we investigate the effect of girolline on translation termination in human cultured cells. We show that girolline neither induces an increase in translational readthrough of stop codons nor affects the polysome profile in treated cells. This suggests that girolline does not act on translation in vivo. Then, we examine the effect of girolline on cell-cycle progression and we show that girolline induces an arrest of the cell cycle at the G2 stage.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Imidazóis/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Linhagem Celular , Códon de Terminação , Humanos , Cinética , Óperon Lac/efeitos dos fármacos , Polirribossomos/efeitos dos fármacosRESUMO
Eukaryotic release factor 3 (eRF3) is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. Studies have related eRF3 with cell cycle regulation, cytoskeleton organization, and tumorigenesis. In mammals, two genes encode two distinct forms of eRF3, eRF3a and eRF3b, which differ in their N-terminal domains. eRF3a is the major factor acting in translation termination, and its expression level controls termination complex formation. Here, we investigate the role of eRF3a in cell cycle progression using short interfering RNAs and flow cytometry. We show that eRF3a depletion induces a G1 arrest and that eRF3a GTP-binding activity, but not the eRF3a N-terminal domain, is required to restore G1-to-S-phase progression. We also show that eRF3a depletion decreases the global translation rate and reduces the polysome charge of mRNA. Finally, we show that two substrates of the mammalian TOR (mTOR) kinase, 4E-BP1 and protein kinase S6K1, are hypophosphorylated in eRF3a-depleted cells. These results strongly suggest that the G1 arrest and the decrease in translation induced by eRF3a depletion are due to the inhibition of mTOR activity and hence that eRF3a belongs to the regulatory pathway of mTOR activity.