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1.
Cell ; 179(2): 543-560.e26, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31585087

RESUMO

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.


Assuntos
Carcinogênese/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Células A549 , Animais , Humanos , Espectrometria de Massas/métodos , Mutação , Fosfoproteínas/metabolismo , Fosforilação , Proteômica , Ratos , Ratos Sprague-Dawley , Peixe-Zebra
2.
Nature ; 575(7782): 355-360, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695196

RESUMO

Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal1. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal2. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.


Assuntos
Linhagem da Célula , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/genética , Complexo Mediador/deficiência , Complexo Mediador/metabolismo , Camundongos , Ligação Proteica , Transcrição Gênica
3.
Mol Syst Biol ; 17(1): e9730, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33502086

RESUMO

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies.


Assuntos
Carcinoma de Células Renais/genética , Biologia Computacional/métodos , Redes Reguladoras de Genes , Neoplasias Renais/genética , Carcinoma de Células Renais/metabolismo , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Humanos , Neoplasias Renais/metabolismo , Metabolômica , Fosfoproteínas
4.
Mol Cell ; 51(6): 707-22, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24011590

RESUMO

The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation, whereas FGF-10 promotes receptor recycling and cell migration. By combining mass-spectrometry-based quantitative proteomics with fluorescence microscopy and biochemical methods, we find that FGF-10 specifically induces the rapid phosphorylation of tyrosine (Y) 734 on FGFR2b, which leads to PI3K and SH3BP4 recruitment. This complex is crucial for FGFR2b recycling and responses, given that FGF-10 stimulation of either FGFR2b_Y734F mutant- or SH3BP4-depleted cells switches the receptor endocytic route to degradation, resulting in decreased breast cancer cell migration and the inhibition of epithelial branching in mouse lung explants. Altogether, these results identify an intriguing ligand-dependent mechanism for the control of receptor fate and cellular outputs that may explain the pathogenic role of deregulated FGFR2b, thus offering therapeutic opportunities.


Assuntos
Fator 10 de Crescimento de Fibroblastos/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Proteômica , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Movimento Celular , Ligantes , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteólise , Tirosina/metabolismo
5.
Breast Cancer Res Treat ; 135(1): 67-78, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22527100

RESUMO

Resistance to endocrine therapy is a major clinical challenge in current treatment of estrogen receptor-positive breast cancer. The molecular mechanisms underlying resistance are yet not fully clarified. In this study, we investigated whether NFκB signaling is causally involved in antiestrogen resistant cell growth and a potential target for re-sensitizing resistant cells to endocrine therapy. We used an MCF-7-derived cell model for antiestrogen resistant breast cancer to investigate dependence on NFκB signaling for antiestrogen resistant cell growth. We found that targeting NFκB preferentially inhibited resistant cell growth. Antiestrogen resistant cells expressed increased p50 and RelB, and displayed increased phosphorylation of p65 at Ser529 and Ser536. Moreover, transcriptional activity of NFκB after stimulation with tumor necrosis factor α was enhanced in antiestrogen resistant cell lines compared to the parental cell line. Inhibition of NFκB signaling sensitized tamoxifen resistant cells to the growth inhibitory effects of tamoxifen but was not sufficient to fully restore sensitivity of fulvestrant resistant cells to fulvestrant. In support of this, depletion of p65 with siRNA in tamoxifen resistant cells increased sensitivity to tamoxifen treatment. Our data provide evidence that NFκB signaling is enhanced in antiestrogen resistant breast cancer cells and plays an important role for antiestrogen resistant cell growth and for sensitivity to tamoxifen treatment in resistant cells. Our results imply that targeting NFκB might serve as a potential novel treatment strategy for breast cancer patients with resistance toward antiestrogen.


Assuntos
Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Antagonistas de Estrogênios/uso terapêutico , NF-kappa B/metabolismo , Transdução de Sinais , Antineoplásicos Hormonais/farmacologia , Linhagem Celular Tumoral , Estradiol/análogos & derivados , Estradiol/farmacologia , Estradiol/uso terapêutico , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Humanos , Células MCF-7 , Subunidade p50 de NF-kappa B/biossíntese , Fosfatidilinositol 3-Quinases , Fosforilação , Interferência de RNA , RNA Interferente Pequeno , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelB/biossíntese
6.
Cell Rep ; 40(6): 111177, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35947955

RESUMO

Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.


Assuntos
Leucemia Mieloide Aguda , Proteína Supressora de Tumor p53 , Apoptose , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Hidrazinas , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triazóis , Proteína Supressora de Tumor p53/metabolismo
7.
Breast Cancer Res Treat ; 121(3): 601-13, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19697122

RESUMO

The majority of breast cancers are estrogen responsive, but upon progression of disease other growth promoting pathways are activated, e.g., the ErbB receptor system. The present study focuses on resistance to the pure estrogen antagonist fulvestrant and strategies to treat resistant cells or even circumvent development of resistance. Limited effects were observed when targeting EGFR and ErbB2 with the monoclonal antibodies cetuximab, trastuzumab, and pertuzumab, whereas the pan-ErbB inhibitor CI-1033 selectively inhibited growth of fulvestrant resistant cell lines. CI-1033 inhibited Erk but not Akt signaling, which as well as Erk is important for antiestrogen resistant cell growth. Accordingly, combination therapy with CI-1033 and the Akt inhibitor SH-6 or the Protein Kinase C inhibitor RO-32-0432 was applied and found superior to single agent treatment. Further, the resistant cell lines were more sensitive to CI-1033 treatment when grown in the presence of fulvestrant, as withdrawal of fulvestrant restored signaling through the estrogen receptor alpha (ERalpha), partly overcoming the growth inhibitory effects of CI-1033. Thus, the resistant cells could switch between ERalpha and ErbB signaling for growth promotion. Although parental MCF-7 cell growth primarily depends on ERalpha signaling, a heregulin-1beta induced switch to ErbB signaling rescued MCF-7 cells from the growth inhibition exerted by fulvestrant-mediated blockade of ERalpha signaling. This interplay between ERalpha and ErbB signaling could be abrogated by combined therapy targeting both receptor systems. Thus, the present study indicates that upon development of antiestrogen resistance, antiestrogen treatment should be continued in combination with signal transduction inhibitors. Further, upfront combination of endocrine therapy with pan-ErbB inhibition may postpone or even prevent development of treatment resistance.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Linhagem Celular Tumoral , Estradiol/análogos & derivados , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Fulvestranto , Humanos , Morfolinas/farmacologia , Proteínas Oncogênicas v-erbB/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Mol Cell Oncol ; 7(1): 1692643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31993501

RESUMO

We developed a mass spectrometry-based proteomics strategy to study oncogenic phosphotyrosine signaling networks in tissues. We outlined epidermal growth factor-dependent phosphotyrosine signaling in lung tissue and discovered that cancer mutations in vicinity of phosphotyrosine sites can induce molecular switches in recruited protein complexes, which ultimately alter the signaling outcome of the network activation.

9.
Sci Signal ; 11(557)2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30459283

RESUMO

Oncogenic anaplastic lymphoma kinase (ALK) is one of the few druggable targets in neuroblastoma, and therapy resistance to ALK-targeting tyrosine kinase inhibitors (TKIs) comprises an inevitable clinical challenge. Therefore, a better understanding of the oncogenic signaling network rewiring driven by ALK is necessary to improve and guide future therapies. Here, we performed quantitative mass spectrometry-based proteomics on neuroblastoma cells treated with one of three clinically relevant ALK TKIs (crizotinib, LDK378, or lorlatinib) or an experimentally used ALK TKI (TAE684) to unravel aberrant ALK signaling pathways. Our integrated proximal proteomics (IPP) strategy included multiple signaling layers, such as the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome. We identified the signaling adaptor protein IRS2 (insulin receptor substrate 2) as a major ALK target and an ALK TKI-sensitive signaling node in neuroblastoma cells driven by oncogenic ALK. TKI treatment decreased the recruitment of IRS2 to ALK and reduced the tyrosine phosphorylation of IRS2. Furthermore, siRNA-mediated depletion of ALK or IRS2 decreased the phosphorylation of the survival-promoting kinase Akt and of a downstream target, the transcription factor FoxO3, and reduced the viability of three ALK-driven neuroblastoma cell lines. Collectively, our IPP analysis provides insight into the proximal architecture of oncogenic ALK signaling by revealing IRS2 as an adaptor protein that links ALK to neuroblastoma cell survival through the Akt-FoxO3 signaling axis.


Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Neuroblastoma/metabolismo , Proteômica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Biologia Computacional , Proteína Forkhead Box O3/metabolismo , Humanos , Espectrometria de Massas , Peptídeos/química , Fosforilação , Proteoma , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transfecção
10.
Cancer Discov ; 8(3): 354-369, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29203461

RESUMO

Triple-negative breast cancers (TNBC) are genetically characterized by aberrations in TP53 and a low rate of activating point mutations in common oncogenes, rendering it challenging in applying targeted therapies. We performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify somatic genetic alterations in mouse models of TNBCs driven by loss of Trp53 alone or in combination with Brca1 Amplifications or translocations that resulted in elevated oncoprotein expression or oncoprotein-containing fusions, respectively, as well as frameshift mutations of tumor suppressors were identified in approximately 50% of the tumors evaluated. Although the spectrum of sporadic genetic alterations was diverse, the majority had in common the ability to activate the MAPK/PI3K pathways. Importantly, we demonstrated that approved or experimental drugs efficiently induce tumor regression specifically in tumors harboring somatic aberrations of the drug target. Our study suggests that the combination of WES and RNA-seq on human TNBC will lead to the identification of actionable therapeutic targets for precision medicine-guided TNBC treatment.Significance: Using combined WES and RNA-seq analyses, we identified sporadic oncogenic events in TNBC mouse models that share the capacity to activate the MAPK and/or PI3K pathways. Our data support a treatment tailored to the genetics of individual tumors that parallels the approaches being investigated in the ongoing NCI-MATCH, My Pathway Trial, and ESMART clinical trials. Cancer Discov; 8(3); 354-69. ©2017 AACR.See related commentary by Natrajan et al., p. 272See related article by Matissek et al., p. 336This article is highlighted in the In This Issue feature, p. 253.


Assuntos
Neoplasias Mamárias Experimentais/genética , Mutação , Neoplasias de Mama Triplo Negativas/genética , Animais , Proteína BRCA1 , Feminino , Fusão Gênica , Humanos , Sistema de Sinalização das MAP Quinases/genética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos Mutantes , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Análise de Sequência de RNA , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Sequenciamento do Exoma
11.
Nat Commun ; 9(1): 4904, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30464169

RESUMO

Therapeutic options for the treatment of glioblastoma remain inadequate despite concerted research efforts in drug development. Therapeutic failure can result from poor permeability of the blood-brain barrier, heterogeneous drug distribution, and development of resistance. Elucidation of relationships among such parameters could enable the development of predictive models of drug response in patients and inform drug development. Complementary analyses were applied to a glioblastoma patient-derived xenograft model in order to quantitatively map distribution and resulting cellular response to the EGFR inhibitor erlotinib. Mass spectrometry images of erlotinib were registered to histology and magnetic resonance images in order to correlate drug distribution with tumor characteristics. Phosphoproteomics and immunohistochemistry were used to assess protein signaling in response to drug, and integrated with transcriptional response using mRNA sequencing. This comprehensive dataset provides simultaneous insight into pharmacokinetics and pharmacodynamics and indicates that erlotinib delivery to intracranial tumors is insufficient to inhibit EGFR tyrosine kinase signaling.


Assuntos
Antineoplásicos/farmacocinética , Cloridrato de Erlotinib/farmacocinética , Glioblastoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/administração & dosagem , Feminino , Imageamento por Ressonância Magnética , Camundongos Nus , Transplante de Neoplasias , Proteínas Tirosina Quinases/metabolismo , Análise de Sequência de RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
Mol Cancer Ther ; 16(11): 2572-2585, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28830985

RESUMO

Approximately 10% of non-small cell lung cancer (NSCLC) patients in the United States and 40% of NSCLC patients in Asia have activating epidermal growth factor receptor (EGFR) mutations and are eligible to receive targeted anti-EGFR therapy. Despite an extension of life expectancy associated with this treatment, resistance to EGFR tyrosine kinase inhibitors and anti-EGFR antibodies is almost inevitable. To identify additional signaling routes that can be cotargeted to overcome resistance, we quantified tumor-specific molecular changes that govern resistant cancer cell growth and survival. Mass spectrometry-based quantitative proteomics was used to profile in vivo signaling changes in 41 therapy-resistant tumors from four xenograft NSCLC models. We identified unique and tumor-specific tyrosine phosphorylation rewiring in tumors resistant to treatment with the irreversible third-generation EGFR-inhibitor, osimertinib, or the novel dual-targeting EGFR/Met antibody, JNJ-61186372. Tumor-specific increases in tyrosine-phosphorylated peptides from EGFR family members, Shc1 and Gab1 or Src family kinase (SFK) substrates were observed, underscoring a differential ability of tumors to uniquely escape EGFR inhibition. Although most resistant tumors within each treatment group displayed a marked inhibition of EGFR as well as SFK signaling, the combination of EGFR inhibition (osimertinib) and SFK inhibition (saracatinib or dasatinib) led to further decrease in cell growth in vitro This result suggests that residual SFK signaling mediates therapeutic resistance and that elimination of this signal through combination therapy may delay onset of resistance. Overall, analysis of individual resistant tumors captured unique in vivo signaling rewiring that would have been masked by analysis of in vitro cell population averages. Mol Cancer Ther; 16(11); 2572-85. ©2017 AACR.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/genética , Piperazinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-met/genética , Acrilamidas , Proteínas Adaptadoras de Transdução de Sinal/genética , Compostos de Anilina , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/efeitos adversos , Benzodioxóis/administração & dosagem , Benzodioxóis/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Humanos , Camundongos , Mutação , Inibidores de Proteínas Quinases/imunologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Quinazolinas/administração & dosagem , Quinazolinas/efeitos adversos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/genética
13.
Mol Cancer Ther ; 16(1): 88-101, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28062706

RESUMO

Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma-specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1 We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1-dependent manner. Mol Cancer Ther; 16(1); 88-101. ©2016 AACR.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Terapia de Alvo Molecular , Animais , Antígenos CD , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Humanos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína EWS de Ligação a RNA/antagonistas & inibidores , Receptor IGF Tipo 1 , Receptor de Insulina/antagonistas & inibidores , Receptores de Somatomedina/antagonistas & inibidores , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Sci Signal ; 8(374): ra40, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25921289

RESUMO

SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines. Our findings suggest that Cbl-b limits NGF-TrkA signaling to control the length of neurites.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Diferenciação Celular , Sistema de Sinalização das MAP Quinases , Fator de Crescimento Neural/metabolismo , Neuroblastoma/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptor trkA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Humanos , Fator de Crescimento Neural/genética , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Proto-Oncogênicas c-cbl/genética , Receptor trkA/genética
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