RESUMO
BACKGROUND & AIMS: Gallbladder cancer (GBC) is the most common type of biliary tract cancer, but the molecular mechanisms involved in gallbladder carcinogenesis remain poorly understood. In this study, we applied integrative genomics approaches to characterise GBC and explore molecular subtypes associated with patient survival. METHODS: We profiled the mutational landscape of GBC tumours (whole-exome sequencing on 92, targeted sequencing on 98, in total 190 patients). In a subset (n = 45), we interrogated the matched transcriptomes, DNA methylomes, and somatic copy number alterations. We explored molecular subtypes identified through clustering tumours by genes whose expression was associated with survival in 47 tumours and validated subtypes on 34 publicly available GBC cases. RESULTS: Exome analysis revealed TP53 was the most mutated gene. The overall mutation rate was low (median 0.82 Mut/Mb). APOBEC-mediated mutational signatures were more common in tumours with higher mutational burden. Aflatoxin-related signatures tended to be highly clonal (present in ≥50% of cancer cells). Transcriptome-wide survival association analysis revealed a 95-gene signature that stratified all GBC patients into 3 subtypes that suggested an association with overall survival post-resection. The 2 poor-survival subtypes were associated with adverse clinicopathologic features (advanced stage, pN1, pM1), immunosuppressive micro-environments (myeloid-derived suppressor cell accumulation, extensive desmoplasia, hypoxia) and T cell dysfunction, whereas the good-survival subtype showed the opposite features. CONCLUSION: These data suggest that the tumour micro-environment and immune profiles could play an important role in gallbladder carcinogenesis and should be evaluated in future clinical studies, along with mutational profiles. LAY SUMMARY: Gallbladder cancer is highly fatal, and its causes are poorly understood. We evaluated gallbladder tumours to see if there were differences between tumours in genetic information such as DNA and RNA. We found evidence of aflatoxin exposure in these tumours, and immune cells surrounding the tumours were associated with survival.
Assuntos
Carcinogênese , Neoplasias da Vesícula Biliar , Transcriptoma , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/genética , Aflatoxinas/toxicidade , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinógenos/toxicidade , Variações do Número de Cópias de DNA , Feminino , Neoplasias da Vesícula Biliar/genética , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/mortalidade , Neoplasias da Vesícula Biliar/patologia , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Análise de Sobrevida , Sequenciamento do ExomaRESUMO
The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalized human myoblasts. Although myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3-FOXO1 and MYCN formed rapidly growing RMS tumours, whereas myoblasts expressing only PAX3-FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3-FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3-FOXO1 expression, and cell lines derived from these recurrent tumours showed transformation in the absence of doxycycline. The doxycycline-independent oncogenicity in these recurrent tumour-derived lines persisted even after PAX3-FOXO1 was inactivated with a CRISPR/Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3-FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour-derived cells without PAX3-FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3-FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3-FOXO1, recurrent tumours can develop by a PAX3-FOXO1-independent mechanism, in which rare cells are postulated to acquire secondary transforming events that were activated or selected by initial PAX3-FOXO1 expression. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
Assuntos
Carcinogênese/genética , Recidiva Local de Neoplasia/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma/genética , Translocação Genética/genética , Animais , Morte Celular , Diferenciação Celular , Linhagem Celular Tumoral , Doxiciclina/administração & dosagem , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Desenvolvimento Muscular , Mioblastos/metabolismo , Mioblastos/patologia , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma/patologiaRESUMO
Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2- to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system.
Assuntos
Ritmo Circadiano/genética , Glândula Pineal/metabolismo , RNA não Traduzido/genética , Animais , Bucladesina/farmacologia , Ritmo Circadiano/efeitos dos fármacos , Biologia Computacional , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/metabolismo , Norepinefrina/farmacologia , Glândula Pineal/efeitos dos fármacos , RNA não Traduzido/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Exocytosis of neurotransmitters and hormones occurs through the fusion of secretory vesicles with the plasma membrane. This highly regulated process involves key proteins, such as SNAREs, and specific lipids at the site of membrane fusion. Phospholipase D (PLD) has recently emerged as a promoter of membrane fusion in various exocytotic events potentially by providing fusogenic cone-shaped phosphatidic acid. We show here that PLD1 is regulated by ribosomal S6 kinase 2 (RSK2)-dependent phosphorylation. RSK2 is activated by a high K(+)-induced rise in cytosolic calcium. Expression of inactive RSK2 mutants or selective knockdown of endogenous RSK2 dramatically affects the different kinetic components of the exocytotic response in chromaffin cells. RSK2 physically interacts with and stimulates PLD activity through the phosphorylation of Thr-147 in the PLD1 amino-terminal phox homology domain. Expression of PLD1 phosphomimetic mutants fully restores secretion in cells depleted of RSK2, suggesting that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. We propose that PLD-related defects in neuronal and endocrine activities could contribute to the effect observed after the loss-of-function mutations in Rsk2 that lead to Coffin-Lowry syndrome, an X-linked form of growth and mental retardation.
Assuntos
Cálcio/metabolismo , Células Cromafins/metabolismo , Síndrome de Coffin-Lowry/enzimologia , Exocitose , Fosfolipase D/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Animais , Células Cromafins/enzimologia , Síndrome de Coffin-Lowry/genética , Exocitose/genética , Células PC12 , Ácidos Fosfatídicos/metabolismo , Fosforilação , Ratos , Proteínas Quinases S6 Ribossômicas 90-kDa/genéticaRESUMO
E. coli GMP synthetase (GMPS) catalyzes the conversion of XMP to GMP. Ammonia, generated in the amino-terminal glutamine amidotransferase (GAT) domain, is transferred by an unknown mechanism to the ATP-pyrophosphatase (ATPP) domain, where it attacks a highly reactive adenyl-XMP intermediate, leading to GMP formation. To study the structural requirements for the activity of E. coli GMPS, we used PCR to generate a protein expression construct that contains the ATPP domain as well as the predicted dimerization domain (DD). The ATPP/DD protein is active in solution, utilizing NH (4) (+) as an NH(3) donor. Size-exclusion chromatography demonstrates a dimeric mass for the ATPP/ DD protein, providing the first evidence in solution for the structural organization of the intact GMPS. Kinetic characterization of the ATPP/DD domain protein provides evidence that the presence of the GAT domain can regulate the activity of the ATPP domain.
Assuntos
Carbono-Nitrogênio Ligases/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Pirofosfatases/química , Trifosfato de Adenosina/química , Amônia/química , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/isolamento & purificação , Clonagem Molecular , Dimerização , Cinética , Conformação Proteica , Estrutura Terciária de Proteína , Pirofosfatases/genética , Pirofosfatases/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Ribonucleotídeos/química , Soluções , XantinaRESUMO
PURPOSE: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS. EXPERIMENTAL DESIGN: We examined the biologic consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts. RESULTS: Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and nonamplified fusion-positive RMS cells via G1-phase cell-cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell-cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell-cycle arrest. Although all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and nonamplified fusion-positive RMS. CONCLUSIONS: Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell-cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Amplificação de Genes , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/farmacologia , Rabdomiossarcoma/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/genética , Cromossomos Humanos Par 12 , Modelos Animais de Doenças , Fatores de Transcrição E2F/metabolismo , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Deleção de Genes , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proto-Oncogene Mas , Interferência de RNA , Proteína do Retinoblastoma/metabolismo , Rabdomiossarcoma/tratamento farmacológico , Rabdomiossarcoma/patologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Expression of fusion oncoproteins generated by recurrent chromosomal translocations represents a major tumorigenic mechanism characteristic of multiple cancers, including one-third of all sarcomas. Oncogenic fusion genes provide novel targets for therapeutic intervention. The PAX3-FOXO1 oncoprotein in alveolar rhabdomyosarcoma (ARMS) is presented as a paradigm to examine therapeutic strategies for targeting sarcoma-associated fusion genes. AREAS COVERED: This review discusses the role of PAX3-FOXO1 in ARMS tumors. Besides evaluating various approaches to molecularly target PAX3-FOXO1 itself, this review highlights therapeutically attractive downstream genes activated by PAX3-FOXO1. EXPERT OPINION: Oncogenic fusion proteins represent desirable therapeutic targets because their expression is specific to tumor cells, but these fusions generally characterize rare malignancies. Full development and testing of potential drugs targeted to these fusions are complicated by the small numbers of patients in these disease categories. Although efforts to develop targeted therapies against fusion proteins should continue, molecular targets that are applicable to a broader tumor landscape should be pursued. A shift of the traditional paradigm to view therapeutic intervention as target-specific rather than tumor-specific will help to circumvent the challenges posed by rare tumors and maximize the possibility of developing successful new treatments for patients with these rare translocation-associated sarcomas.
Assuntos
Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma Alveolar/genética , Animais , Proteína Forkhead Box O1 , Humanos , Fator de Transcrição PAX3 , Rabdomiossarcoma Alveolar/tratamento farmacológicoRESUMO
C/EBPß is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPß whose biological functions are not well understood, inhibits cellular senescence. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPß. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPß. Covalently linked C/EBPß homodimers (ßâ¼ß) inhibited the proliferation and tumorigenicity of Ras(V12)-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas γâ¼ß heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg(-/-) MEFs. C/EBPß depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPß homodimers in Cebpg(-/-) MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg(-/-) bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPß through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Senescência Celular , Regulação da Expressão Gênica , Neoplasias/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Neoplasias/metabolismo , Multimerização ProteicaRESUMO
The nucleolus is the center of ribosome synthesis, with the nucleophosmin (NPM) and p19(ARF) proteins antagonizing one another to either promote or inhibit growth. However, basal NPM and ARF proteins form nucleolar complexes whose functions remain unknown. Nucleoli from Arf(-/)(-) cells displayed increased nucleolar area, suggesting that basal ARF might regulate key nucleolar functions. Concordantly, ribosome biogenesis and protein synthesis were dramatically elevated in the absence of Arf, causing these cells to exhibit tremendous gains in protein amounts and increases in cell volume. The transcription of ribosomal DNA (rDNA), the processing of nascent rRNA molecules, and the nuclear export of ribosomes were all increased in the absence of ARF. Similar results were obtained using targeted lentiviral RNA interference of ARF in wild-type MEFs. Postmitotic osteoclasts from Arf-null mice exhibited hyperactivity in vitro and in vivo, demonstrating a physiological function for basal ARF. Moreover, the knockdown of NPM blocked the increases in Arf(-/-) ribosome output and osteoclast activity, demonstrating that these gains require NPM. Thus, basal ARF proteins act as a monitor of steady-state ribosome biogenesis and growth independent of their ability to prevent unwarranted hyperproliferation.