Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Exp Med ; 217(2)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31841125

RESUMO

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.

2.
Sci Transl Med ; 11(477)2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700572

RESUMO

Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

3.
Arthritis Rheumatol ; 70(10): 1617-1625, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29697211

RESUMO

OBJECTIVE: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. METHODS: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. RESULTS: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. CONCLUSION: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.


Assuntos
Cadeias Pesadas de Imunoglobulinas/sangue , Leucócitos Mononucleares/fisiologia , Fator Reumatoide/sangue , Síndrome de Sjogren/sangue , Adulto , Linfócitos B/metabolismo , Compostos de Boro/metabolismo , Rastreamento de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica , Fator Reumatoide/imunologia , Síndrome de Sjogren/imunologia
4.
JNCI Cancer Spectr ; 2(3): pky047, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31360864

RESUMO

Background: The Myc oncogene family has been implicated in many human malignancies and is often associated with particularly aggressive disease, suggesting Myc as an attractive prognostic marker and therapeutic target. However, for epithelial ovarian cancer (EOC), there is little consensus on the incidence and clinical relevance of Myc aberrations. Here we comprehensively investigated alterations in gene copy number, expression, and activity for Myc and evaluated their clinical significance in EOC. Methods: To address inconsistencies in the literature regarding the definition of copy number variations, we developed a novel approach using quantitative polymerase chain reaction (qPCR) coupled with a statistical algorithm to estimate objective thresholds for detecting Myc gain/amplification in large cohorts of serous (n = 150) and endometrioid (n = 80) EOC. MYC, MYCN, and MYCL1 mRNA expression and Myc activity score for each case were examined by qPCR. Kaplan-Meier and Cox-regression analyses were conducted to assess clinical significance of Myc aberrations. Results: Using a large panel of cancer cell lines (n = 34), we validated the statistical algorithm for determining clear thresholds for Myc gain/amplification. MYC was the most predominantly amplified of the Myc oncogene family members, and high MYC mRNA expression levels were associated with amplification in EOC. However, there was no association between prognosis and increased copy number or gene expression of MYC/MYCN/MYCL1 or with a pan-Myc transcriptional activity score, in EOC, although MYC amplification was associated with late stage and high grade in endometrioid EOC. Conclusion: A systematic and comprehensive analysis of Myc genes, transcripts, and activity levels using qPCR revealed that although such aberrations commonly occur in EOC, overall they have limited impact on outcome, suggesting that the biological relevance of Myc oncogene family members is limited to certain subsets of this disease.

5.
Cancer Res ; 77(4): 971-981, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-27923830

RESUMO

Myc transcriptional activity is frequently deregulated in human cancers, but a Myc-driven gene signature with prognostic ability across multiple tumor types remains lacking. Here, we selected 18 Myc-regulated genes from published studies of Myc family targets in epithelial ovarian cancer (EOC) and neuroblastoma. A Myc family activity score derived from the 18 genes was correlated to MYC/MYCN/MYCL1 expression in a panel of 35 cancer cell lines. The prognostic ability of this signature was evaluated in neuroblastoma, medulloblastoma, diffuse large B-cell lymphoma (DLBCL), and EOC microarray gene expression datasets using Kaplan-Meier and multivariate Cox regression analyses and was further validated in 42 primary neuroblastomas using qPCR. Cell lines with high MYC, MYCN, and/or MYCL1 gene expression exhibited elevated expression of the signature genes. Survival analysis showed that the signature was associated with poor outcome independently of well-defined prognostic factors in neuroblastoma, breast cancer, DLBCL, and medulloblastoma. In EOC, the 18-gene Myc activity signature was capable of identifying a group of patients with poor prognosis in a "high-MYCN" molecular subtype but not in the overall cohort. The predictive ability of this signature was reproduced using qPCR analysis of an independent cohort of neuroblastomas, including a subset of tumors without MYCN amplification. These data reveal an 18-gene Myc activity signature that is highly predictive of poor prognosis in diverse Myc-associated malignancies and suggest its potential clinical application in the identification of Myc-driven tumors that might respond to Myc-targeted therapies. Cancer Res; 77(4); 971-81. ©2016 AACR.


Assuntos
Neoplasias/mortalidade , Proteínas Proto-Oncogênicas c-myc/fisiologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Humanos , Meduloblastoma/mortalidade , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias/terapia , Neoplasias Epiteliais e Glandulares/mortalidade , Neuroblastoma/mortalidade , Neoplasias Ovarianas/mortalidade , Reação em Cadeia da Polimerase , Prognóstico , Modelos de Riscos Proporcionais
6.
Oncotarget ; 7(34): 54937-54951, 2016 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-27448979

RESUMO

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Fatores de Transcrição/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Perfilação da Expressão Gênica/métodos , Humanos , Estimativa de Kaplan-Meier , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Interferência de RNA , Fatores de Transcrição/metabolismo
7.
Cancer Res ; 76(12): 3604-17, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27197171

RESUMO

The RNA-binding protein dyskerin, encoded by the DKC1 gene, functions as a core component of the telomerase holoenzyme as well as ribonuclear protein complexes involved in RNA processing and ribosome biogenesis. The diverse roles of dyskerin across many facets of RNA biology implicate its potential contribution to malignancy. In this study, we examined the expression and function of dyskerin in neuroblastoma. We show that DKC1 mRNA levels were elevated relative to normal cells across a panel of 15 neuroblastoma cell lines, where both N-Myc and c-Myc directly targeted the DKC1 promoter. Upregulation of MYCN was shown to dramatically increase DKC1 expression. In two independent neuroblastoma patient cohorts, high DKC1 expression correlated strongly with poor event-free and overall survival (P < 0.0001), independently of established prognostic factors. RNAi-mediated depletion of dyskerin inhibited neuroblastoma cell proliferation, including cells immortalized via the telomerase-independent ALT mechanism. Furthermore, dyskerin attenuation impaired anchorage-independent proliferation and tumor growth. Overexpression of the telomerase RNA component, hTR, demonstrated that this proliferative impairment was not a consequence of telomerase suppression. Instead, ribosomal stress, evidenced by depletion of small nucleolar RNAs and nuclear dispersal of ribosomal proteins, was the likely cause of the proliferative impairment in dyskerin-depleted cells. Accordingly, dyskerin suppression caused p53-dependent G1 cell-cycle arrest in p53 wild-type cells, and a p53-independent pathway impaired proliferation in cells with p53 dysfunction. Together, our findings highlight dyskerin as a new therapeutic target in neuroblastoma with crucial telomerase-independent functions and broader implications for the spectrum of malignancies driven by MYC family oncogenes. Cancer Res; 76(12); 3604-17. ©2016 AACR.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Neuroblastoma/patologia , Proteínas Nucleares/fisiologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Telomerase/fisiologia , Células Cultivadas , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Ribossomos/fisiologia , Proteína Supressora de Tumor p53/fisiologia
8.
Oncotarget ; 7(6): 6353-68, 2016 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-26840454

RESUMO

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response. To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel. We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10-5, HR=1.90, for rs7874043) associated with progression-free survival (PFS). Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B. The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1. Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer lines. PSIP1 (PC4 and SFRS1 Interacting Protein 1) is known to protect cells from stress-induced apoptosis, and high expression is associated with poor PFS in EOC patients. We therefore suggest that the minor allele of rs7874043 confers poor PFS by increasing PSIP1 expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Elementos Facilitadores Genéticos/genética , Neoplasias das Tubas Uterinas/mortalidade , Mutação em Linhagem Germinativa/genética , Neoplasias Ovarianas/mortalidade , Neoplasias Peritoneais/mortalidade , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Imunoprecipitação da Cromatina , Estudos de Coortes , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Neoplasias das Tubas Uterinas/tratamento farmacológico , Neoplasias das Tubas Uterinas/genética , Neoplasias das Tubas Uterinas/patologia , Feminino , Seguimentos , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/patologia , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas
9.
J Natl Cancer Inst ; 106(7)2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24957074

RESUMO

BACKGROUND: ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. METHODS: The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA-mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan-Meier analysis and log-rank tests. All statistical tests were two-sided. RESULTS: Associations with outcome were observed with ABC transporters of the "A" subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e-6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. CONCLUSIONS: Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid trafficking as a potentially important process in EOC.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Polimorfismo de Nucleotídeo Único , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Carcinoma Epitelial do Ovário , Movimento Celular , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Estimativa de Kaplan-Meier , Gradação de Tumores , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Epiteliais e Glandulares/patologia , Células-Tronco Neoplásicas , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
10.
Gynecol Oncol ; 131(1): 8-14, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23917080

RESUMO

OBJECTIVE: ABCB1 encodes the multi-drug efflux pump P-glycoprotein (P-gp) and has been implicated in multi-drug resistance. We comprehensively evaluated this gene and flanking regions for an association with clinical outcome in epithelial ovarian cancer (EOC). METHODS: The best candidates from fine-mapping analysis of 21 ABCB1 SNPs tagging C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642) were analysed in 4616 European invasive EOC patients from thirteen Ovarian Cancer Association Consortium (OCAC) studies and The Cancer Genome Atlas (TCGA). Additionally we analysed 1,562 imputed SNPs around ABCB1 in patients receiving cytoreductive surgery and either 'standard' first-line paclitaxel-carboplatin chemotherapy (n=1158) or any first-line chemotherapy regimen (n=2867). We also evaluated ABCB1 expression in primary tumours from 143 EOC patients. RESULT: Fine-mapping revealed that rs1128503, rs2032582, and rs1045642 were the best candidates in optimally debulked patients. However, we observed no significant association between any SNP and either progression-free survival or overall survival in analysis of data from 14 studies. There was a marginal association between rs1128503 and overall survival in patients with nil residual disease (HR 0.88, 95% CI 0.77-1.01; p=0.07). In contrast, ABCB1 expression in the primary tumour may confer worse prognosis in patients with sub-optimally debulked tumours. CONCLUSION: Our study represents the largest analysis of ABCB1 SNPs and EOC progression and survival to date, but has not identified additional signals, or validated reported associations with progression-free survival for rs1128503, rs2032582, and rs1045642. However, we cannot rule out the possibility of a subtle effect of rs1128503, or other SNPs linked to it, on overall survival.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Carboplatina/administração & dosagem , Carcinoma Epitelial do Ovário , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Epiteliais e Glandulares/cirurgia , Neoplasias Ovarianas/cirurgia , Paclitaxel/administração & dosagem , Farmacogenética , Polimorfismo de Nucleotídeo Único , Modelos de Riscos Proporcionais
11.
J Natl Cancer Inst ; 103(16): 1236-51, 2011 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-21799180

RESUMO

BACKGROUND: Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux. METHODS: A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN(1/-), 205 hMYCN(+/1) mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan-Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided. RESULTS: Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the "poor prognosis" expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001). CONCLUSION: ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.


Assuntos
Antineoplásicos/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Adolescente , Animais , Western Blotting , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Criança , Pré-Escolar , Modelos Animais de Doenças , Intervalo Livre de Doença , Regulação para Baixo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares/metabolismo , Razão de Chances , Proteínas Oncogênicas/metabolismo , Reação em Cadeia da Polimerase , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , RNA Interferente Pequeno/metabolismo , Recidiva , Fatores de Tempo , Transfecção , Regulação para Cima , Adulto Jovem
12.
J Biol Chem ; 285(16): 11800-9, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20167605

RESUMO

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.


Assuntos
Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Estabilidade Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transativadores/genética , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
13.
Breast Cancer Res ; 10(2): R28, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18373870

RESUMO

INTRODUCTION: Estrogens play a pivotal role in the initiation and progression of breast cancer. The genes that mediate these processes are not fully defined, but potentially include the known mammary oncogene MYC. Characterization of estrogen-target genes may help to elucidate further the mechanisms of estrogen-induced mitogenesis and endocrine resistance. METHODS: We used a transcript profiling approach to identify targets of estrogen and c-Myc in breast cancer cells. One previously uncharacterized gene, namely HBV pre-S2 trans-regulated protein 3 (HSPC111), was acutely upregulated after estrogen treatment or inducible expression of c-Myc, and was selected for further functional analysis using over-expression and knock-down strategies. HSPC111 expression was also analyzed in relation to MYC expression and outcome in primary breast carcinomas and published gene expression datasets. RESULTS: Pretreatment of cells with c-Myc small interfering RNA abrogated estrogen induction of HSPC111, identifying HSPC111 as a potential c-Myc target gene. This was confirmed by the demonstration of two functional E-box motifs upstream of the transcription start site. HSPC111 mRNA and protein were over-expressed in breast cancer cell lines and primary breast carcinomas, and this was positively correlated with MYC mRNA levels. HSPC111 is present in a large, RNA-dependent nucleolar complex, suggesting a possible role in ribosomal biosynthesis. Neither over-expression or small interfering RNA knock-down of HSPC111 affected cell proliferation rates or sensitivity to estrogen/antiestrogen treatment. However, high expression of HSPC111 mRNA was associated with adverse patient outcome in published gene expression datasets. CONCLUSION: These data identify HSPC111 as an estrogen and c-Myc target gene that is over-expressed in breast cancer and is associated with an adverse patient outcome.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estrogênios/genética , Antígenos de Superfície da Hepatite B/metabolismo , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Adulto , Idoso , Neoplasias da Mama/mortalidade , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc , Antígenos de Superfície da Hepatite B/genética , Humanos , Immunoblotting , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fase S , Análise de Sobrevida , Regulação para Cima
14.
Cell Cycle ; 6(24): 3070-7, 2007 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-18073532

RESUMO

The cellular response to DNA damage is critical for maintenance of genomic integrity and inhibition of tumorigenesis. Mutations or aberrant expression of the E3 ubiquitin ligase EDD have been observed in a number of carcinomas and we recently reported that EDD modulates activity of the DNA damage checkpoint kinase, CHK2. Here, we demonstrate that EDD is necessary for G(1)/S and intra S phase DNA damage checkpoint activation and for the maintenance of G(2)/M arrest after double strand DNA breaks. Defective checkpoint activation in EDD-depleted cells led to radio-resistant DNA synthesis, premature entry into mitosis, accumulation of polyploid cells, and cell death via mitotic catastrophe. In addition to decreased CHK2 activation in EDD-depleted cells, the expression of several key cell cycle mediators including Cdc25A/C and E2F1 was altered, suggesting that these checkpoint defects may be both CHK2-dependent and -independent. These data support a role for EDD in the maintenance of genomic stability, emphasising the potential importance of dysregulated EDD expression and/or function in the evolution of cancer.


Assuntos
Dano ao DNA/fisiologia , Fase G2/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Fase S/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Ciclo Celular/fisiologia , Quinase do Ponto de Checagem 2 , Instabilidade Genômica/fisiologia , Células HeLa , Humanos , Fosforilação
15.
Cancer Res ; 67(18): 8942-51, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17875737

RESUMO

Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)-negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH(2)-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest.


Assuntos
Neoplasias da Mama/patologia , Moduladores de Receptor Estrogênico/farmacologia , Progestinas/farmacologia , Receptores de Progesterona/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Interações de Medicamentos , Estradiol/análogos & derivados , Estradiol/farmacologia , Fulvestranto , Regulação Neoplásica da Expressão Gênica , Humanos , Pregnenodionas/farmacologia , Proteínas Proto-Oncogênicas c-myc , Receptores de Progesterona/biossíntese , Receptores de Progesterona/genética , Transcrição Genética
16.
J Biol Chem ; 281(52): 39990-40000, 2006 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-17074762

RESUMO

EDD, the human orthologue of Drosophila melanogaster "hyperplastic discs," is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.


Assuntos
Dano ao DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Ativação Enzimática/genética , Células HeLa , Humanos , Ligação Proteica/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/metabolismo
17.
Oncogene ; 22(32): 5070-81, 2003 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-12902990

RESUMO

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.


Assuntos
Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Peptídeo Sintases/genética , Ubiquitina-Proteína Ligases , Aberrações Cromossômicas , Cromossomos Humanos Par 8 , Feminino , Humanos , Repetições de Microssatélites , Neoplasias/genética , Peptídeo Sintases/biossíntese
18.
J Biol Chem ; 277(29): 26468-78, 2002 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-12011095

RESUMO

The ubiquitin-protein ligase EDD encodes an orthologue of the hyperplastic discs tumor suppressor gene, which has a critical role in Drosophila development. Frequent allelic imbalance at the EDD chromosomal locus in human cancers suggests a role in tumorigenesis. In addition to a HECT (homologous to E6-AP carboxyl terminus) domain, the EDD protein contains a UBR1 zinc finger motif and ubiquitin-associated domain, each of which indicates involvement in ubiquitinylation pathways. This study shows that EDD interacts with importin alpha 5 through consensus basic nuclear localization signals and is localized in cell nuclei. EDD also binds progesterone receptor (PR) and potentiates progestin-mediated gene transactivation. This activity is comparable with that of the coactivator SRC-1, but, in contrast, the interaction between EDD and PR does not appear to involve an LXXLL receptor-binding motif. EDD also binds calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein, a potential target of ubiquitin-mediated proteolysis, and an altered association is found between EDD and calcium- and integrin-binding protein/DNA-dependent protein kinase-interacting protein in response to DNA damage. The data presented here demonstrate a role for EDD in PR signaling but also suggest a link to cancer through DNA damage response pathways.


Assuntos
Proteínas de Ligação ao Cálcio , Dano ao DNA , Peptídeo Sintases/fisiologia , Receptores de Progesterona/metabolismo , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Linhagem Celular , Núcleo Celular/fisiologia , Humanos , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido , alfa Carioferinas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA