RESUMO
Somatic KIT activating mutations drive most gastrointestinal stromal tumors (GISTs). Disease progression eventually develops with first-line imatinib, commonly due to KIT secondary mutations, and different kinase inhibitors have various levels of treatment efficacy dependent on specific acquired resistance mutations. Ripretinib is a broad-spectrum switch-control KIT/PDGFRA tyrosine kinase inhibitor for patients with advanced GIST who received prior treatment with three or more kinase inhibitors, including imatinib. Exploratory baseline circulating tumor DNA analysis from the second-line INTRIGUE trial determined that patients with advanced GIST previously treated with imatinib harboring primary KIT exon 11 mutations and secondary resistance mutations restricted to KIT exons 17/18 had greater clinical benefit with ripretinib versus sunitinib. We describe the rationale and design of INSIGHT (NCT05734105), an ongoing Phase III open-label study of ripretinib versus sunitinib in patients with advanced GIST previously treated with imatinib exclusively harboring KIT exon 11 + 17/18 mutations detected by circulating tumor DNA.Clinical Trial Registration: NCT05734105 (ClinicalTrials.gov).
Gastrointestinal stromal tumor (GIST) is rare, but it is the most common mesenchymal tumor (a type of tumor that develops from cells which give rise to soft tissues) of the gastrointestinal tract. The primary treatment for advanced GIST is medication that targets the abnormal mechanisms in cancer cells in order to block tumor growth and spread. Ripretinib is an inhibitor of a protein known as KIT, which is a member of the tyrosine kinase protein family and is involved in the growth of GIST. In a Phase III clinical trial called INTRIGUE, the effects of ripretinib and another receptor tyrosine kinase inhibitor, sunitinib, were compared in patients with advanced GIST previously treated with the drug imatinib. An exploratory analysis from the INTRIGUE trial that characterized baseline circulating tumor DNA in the blood showed a greater clinical benefit with ripretinib versus sunitinib in patients with gene mutations solely occurring in KIT exon 11 + 17 and/or 18 (exon 11 + 17/18). This article describes the rationale and design for a Phase III clinical trial called INSIGHT that will evaluate the benefit of ripretinib compared with sunitinib in patients with advanced GIST whose tumors have mutations in KIT exon 11 and KIT exon 17 and/or 18. Patients will receive ripretinib or sunitinib in 6-week cycles, and investigators will assess survival without cancer progression as the primary outcome, and overall survival, and response of the tumor to these two drugs as secondary outcomes.
RESUMO
INTRIGUE was an open-label, phase 3 study in adult patients with advanced gastrointestinal stromal tumor who had disease progression on or intolerance to imatinib and who were randomized to once-daily ripretinib 150 mg or sunitinib 50 mg. In the primary analysis, progression-free survival (PFS) with ripretinib was not superior to sunitinib. In clinical and nonclinical studies, ripretinib and sunitinib have demonstrated differential activity based on the exon location of KIT mutations. Therefore, we hypothesized that mutational analysis using circulating tumor DNA (ctDNA) might provide further insight. In this exploratory analysis (N = 362), baseline peripheral whole blood was analyzed by a 74-gene ctDNA next-generation sequencing-based assay. ctDNA was detected in 280/362 (77%) samples with KIT mutations in 213/362 patients (59%). Imatinib-resistant mutations were found in the KIT ATP-binding pocket (exons 13/14) and activation loop (exons 17/18). Mutational subgroup assessment showed 2 mutually exclusive populations with differential treatment effects. Patients with only KIT exon 11 + 13/14 mutations (ripretinib, n = 21; sunitinib, n = 20) had better PFS with sunitinib versus ripretinib (median, 15.0 versus 4.0 months). Patients with only KIT exon 11 + 17/18 mutations (ripretinib, n = 27; sunitinib, n = 25) had better PFS with ripretinib versus sunitinib (median, 14.2 versus 1.5 months). The results of this exploratory analysis suggest ctDNA sequencing may improve the prediction of the efficacy of single-drug therapies and support further evaluation of ripretinib in patients with KIT exon 11 + 17/18 mutations. ClinicalTrials.gov identifier: NCT03673501.
Assuntos
Antineoplásicos , Neoplasias Gastrointestinais , Tumores do Estroma Gastrointestinal , Naftiridinas , Ureia/análogos & derivados , Adulto , Humanos , Sunitinibe/uso terapêutico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Mesilato de Imatinib/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Biomarcadores , Mutação/genética , Antineoplásicos/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologiaRESUMO
Chemokines and their receptors have been implicated in cancer biology. The CXCL12/CXCR4 axis is essential for the homing and retention of hematopoietic stem cells in bone marrow niches, and has a significant role in neonatal development. It is also implicated in multiple facets of cancer biology including metastasis, angiogenesis/neo-vasculogenesis, and immune cell trafficking at the tumor microenvironment (TME). Immunohistochemistry (IHC) is an ideal method for investigating involvement of CXCL12 in the TME. Three antibodies were evaluated here for their suitability to stain CXCL12. Both D8G6H and K15C gave apparent specific staining in both lymphoid and tumor tissue, but with converse staining patterns. D8G6H stained cells in the parafollicular zone whereas K15C showed staining of lymphoid cells in the interfollicular zone of tonsil tissue. Using a cell line with high CXCL12 expression, TOV21G, as a positive control, it was found that D8G6H gave strong staining of TOV21G cells whereas no staining was observed with K15C indicating that D8G6H specifically stains CXCL12. Significant staining of CXCL12 in the ovarian TME using tissue microarray was observed using D8G6H. These data demonstrate the importance of antibody characterization for IHC applications, and provide further evidence for the involvement of CXCL12 in ovarian cancer biology.
Assuntos
Anticorpos/análise , Quimiocina CXCL12/análise , Imuno-Histoquímica/métodos , Células A549 , Animais , Anticorpos Monoclonais/análise , Células CACO-2 , Feminino , Células HT29 , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Coelhos , Microambiente TumoralRESUMO
BACKGROUND: Induction chemotherapy is a common therapeutic option for patients with locoregionally-advanced head and neck cancer (HNC), but it remains unclear which patients will benefit. In this study, we searched for biomarkers predicting the response of patients with locoregionally-advanced HNC to induction chemotherapy by evaluating the expression pattern of DNA repair proteins. METHODS: Expression of a panel of DNA-repair proteins in formalin-fixed paraffin embedded specimens from a cohort of 37 HNC patients undergoing platinum-based induction chemotherapy prior to definitive chemoradiation were analyzed using quantitative immunohistochemistry. RESULTS: We found that XPF (an ERCC1 binding partner) and phospho-MAPKAP Kinase 2 (pMK2) are novel biomarkers for HNSCC patients undergoing platinum-based induction chemotherapy. Low XPF expression in HNSCC patients is associated with better response to induction chemoradiotherapy, while high XPF expression correlates with a worse response (pâ=â0.02). Furthermore, low pMK2 expression was found to correlate significantly with overall survival after induction plus chemoradiation therapy (pâ=â0.01), suggesting that pMK2 may relate to chemoradiation therapy. CONCLUSIONS: We identified XPF and pMK2 as novel DNA-repair biomarkers for locoregionally-advanced HNC patients undergoing platinum-based induction chemotherapy prior to definitive chemoradiation. Our study provides insights for the use of DNA repair biomarkers in personalized diagnostics strategies. Further validation in a larger cohort is indicated.
Assuntos
Biomarcadores/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Quimioterapia de Indução/normas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Células HeLa , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Quimioterapia de Indução/métodos , Medicina de Precisão/métodos , Análise de Sobrevida , Resultado do TratamentoRESUMO
Circulating tumor cells (CTCs) are shed from cancerous tumors, enter the circulatory system, and migrate to distant organs to form metastases that ultimately lead to the death of most patients with cancer. Identification and characterization of CTCs provides a means to study, monitor, and potentially interfere with the metastatic process. Isolation of CTCs from blood is challenging because CTCs are rare and possess characteristics that reflect the heterogeneity of cancers. Various methods have been developed to enrich CTCs from many millions of normal blood cells. Microfluidics offers an opportunity to create a next generation of superior CTC enrichment devices. This review focuses on various microfluidic approaches that have been applied to date to capture CTCs from the blood of patients with cancer.
Assuntos
Microfluídica , Células Neoplásicas Circulantes/patologia , Antígenos de Superfície/metabolismo , Biomarcadores Tumorais/metabolismo , Separação Celular , Humanos , Microfluídica/métodos , Neoplasias/diagnóstico , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismoRESUMO
Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P ≤ .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue.
Assuntos
Antígenos de Neoplasias/genética , Neoplasias da Mama/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Queratinas/genética , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Quimioterapia Adjuvante , Ciclofosfamida/uso terapêutico , Intervalo Livre de Doença , Feminino , Fluoruracila/uso terapêutico , Humanos , Hibridização in Situ Fluorescente , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Proteínas de Ligação a Poli-ADP-Ribose , Análise Serial de Tecidos , Resultado do TratamentoRESUMO
Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Interpretação de Imagem Assistida por Computador/normas , Imuno-Histoquímica/normas , Análise Serial de Tecidos/normas , Fixação de Tecidos/normas , Neoplasias da Mama/patologia , Calibragem , Feminino , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Ribonucleoproteína Nuclear Pequena U1/análise , Fatores de TempoRESUMO
PURPOSE: To evaluate the prognostic utility of immunohistochemical assessment of key proteins in multiple DNA repair pathways in triple-negative breast cancer (TNBC; estrogen receptor negative, progesterone receptor negative, and HER2/neu negative by immunohistochemistry). EXPERIMENTAL DESIGN: Archived clinically annotated tumor specimens from 112 women with TNBC were immunostained with antibodies against DNA repair proteins and scored using digital image analysis. The cohort was divided into training and test sets for development of a multiantibody model. Scores were combined with clinical data to assess association with outcome. RESULTS: Low XPF (P = 0.005), pMK2 (P = 0.01), MLH; P = 0.002), and FANCD2 (P = 0.001) were each associated with shorter time to recurrence (TTR) in univariate analysis. A 4-antibody model could segregate high-risk and low-risk groups on the basis of TTR in both the training (relative risk [RR] = 3.52; P = 9.05E-07) and test (RR 2.67; P = 0.019) cohorts. CONCLUSIONS: DNA repair proteins may be useful as prognostic markers in TNBC. Further study in larger, uniformly treated cohorts with additional clinical parameters is warranted.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico , Carcinoma/diagnóstico , Reparo do DNA , Adulto , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Estudos de Coortes , Reparo do DNA/fisiologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Recidiva , Estudos Retrospectivos , Fatores de TempoRESUMO
Receptor tyrosine kinases (RTKs) activate pathways mediated by serine-threonine kinases, such as the PI3K (phosphatidylinositol 3-kinase)-Akt pathway, the Ras-MAPK (mitogen-activated protein kinase)-RSK (ribosomal S6 kinase) pathway, and the mTOR (mammalian target of rapamycin)-p70 S6 pathway, that control important aspects of cell growth, proliferation, and survival. The Akt, RSK, and p70 S6 family of protein kinases transmits signals by phosphorylating substrates on an RxRxxS/T motif (R, arginine; S, serine; T, threonine; and x, any amino acid). We developed a large-scale proteomic approach to identify more than 300 substrates of this kinase family in cancer cell lines driven by the c-Met, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor alpha (PDGFRalpha) RTKs. We identified a subset of proteins with RxRxxS/T sites for which phosphorylation was decreased by RTK inhibitors (RTKIs), as well as by inhibitors of the PI3K, mTOR, and MAPK pathways, and we determined the effects of small interfering RNA directed against these substrates on cell viability. Phosphorylation of the protein chaperone SGTA (small glutamine-rich tetratricopeptide repeat-containing protein alpha) at serine-305 was essential for PDGFRalpha stabilization and cell survival in PDGFRalpha-dependent cancer cells. Our approach provides a new view of RTK and Akt-RSK-S6 kinase signaling, revealing previously unidentified Akt-RSK-S6 kinase substrates that merit further consideration as targets for combination therapy with RTKIs.
Assuntos
Peptídeos/química , Proteínas Proto-Oncogênicas c-akt/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/química , Proteínas Quinases S6 Ribossômicas 70-kDa/química , Serina-Treonina Quinases TOR/química , Motivos de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Chaperonas Moleculares , Peptídeos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Especificidade por Substrato , Serina-Treonina Quinases TOR/metabolismoRESUMO
The noncanonical IKK family member IKKepsilon is essential for regulating antiviral signaling pathways and is a recently discovered breast cancer oncoprotein. Although several IKKepsilon targets have been described, direct IKKepsilon substrates necessary for regulating cell transformation have not been identified. Here, we performed a screen for putative IKKepsilon substrates using an unbiased proteomic and bioinformatic approach. Using a positional scanning peptide library assay, we determined the optimal phosphorylation motif for IKKepsilon and used bioinformatic approaches to predict IKKepsilon substrates. Of these potential substrates, serine 418 of the tumor suppressor CYLD was identified as a likely site of IKKepsilon phosphorylation. We confirmed that CYLD is directly phosphorylated by IKKepsilon and that IKKepsilon phosphorylates serine 418 in vivo. Phosphorylation of CYLD at serine 418 decreases its deubiquitinase activity and is necessary for IKKepsilon-driven transformation. Together, these observations define IKKepsilon and CYLD as an oncogene-tumor suppressor network that participates in tumorigenesis.
Assuntos
Neoplasias da Mama , Transformação Celular Neoplásica , Quinase I-kappa B/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Enzima Desubiquitinante CYLD , Feminino , Genes Reporter , Humanos , Quinase I-kappa B/genética , Dados de Sequência Molecular , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Alinhamento de Sequência , Serina/metabolismo , Especificidade por Substrato , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.
Assuntos
Proteínas 14-3-3/metabolismo , Apoptose/fisiologia , Membrana Celular/metabolismo , Homeostase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas 14-3-3/genética , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/efeitos da radiação , Proteínas Supressoras de Tumor/fisiologia , Raios Ultravioleta , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Motivos de Aminoácidos/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/fisiologia , Dano ao DNA/efeitos da radiação , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Imunoprecipitação , Fosfopeptídeos/imunologia , Fosfopeptídeos/isolamento & purificação , Fosfopeptídeos/fisiologia , Fosforilação/efeitos da radiação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Especificidade por Substrato/genética , Especificidade por Substrato/efeitos da radiação , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
To identify novel molecular mechanisms by which ceramide regulates cell differentiation, we examined its effect on adipogenesis of 3T3-L1 preadipocytes. Hormonal stimulation of 3T3-L1 preadipocytes induced formation of triacylglycerol-laden adipocytes over 7 days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins alpha, beta and delta (C/EBP-alpha, -beta and -delta) and peroxisome-proliferator-activated receptor gamma (PPARgamma). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPalpha and PPARgamma, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPbeta, which is required for expression of C/EBPalpha and PPARgamma, but significantly blocked its transcriptional activity from a promoter construct after 24 h. The ceramide-induced decrease in the transcriptional activity of C/EBPbeta correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24 h. However, ceramide did not change the nuclear level of C/EBPbeta after a period of 4 or 16 h, suggesting that it was not affecting nuclear import. CRM1 (more recently named 'exportin-1') is a nuclear membrane protein that regulates protein export from the nucleus by binding to a specific nuclear export sequence. Leptomycin B is an inhibitor of CRM1/exportin-1, and reversed the ceramide-induced decrease in nuclear C/EBPbeta at 24 h. Taken together, these data support the hypothesis that ceramide may inhibit adipogenesis, at least in part, by enhancing dephosphorylation and premature nuclear export of C/EBPbeta at a time when its maximal transcriptional activity is required to drive adipogenesis.