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BACKGROUND & AIMS: Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. METHODS: To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. RESULTS: Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. CONCLUSIONS: Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Biomarcadores Tumorais/genética , Neoplasias Colorretais/tratamento farmacológico , Carioferinas/antagonistas & inibidores , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Bases de Dados Genéticas , Células HCT116 , Células HT29 , Humanos , Indóis/administração & dosagem , Carioferinas/metabolismo , Camundongos , Morfolinas/administração & dosagem , Piperazinas/administração & dosagem , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Receptores Citoplasmáticos e Nucleares/metabolismo , Sulfonamidas/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Exportina 1RESUMO
Somatic mutations in driver genes may ultimately lead to the development of cancer. Understanding how somatic mutations accumulate in cancer genomes and the underlying factors that generate somatic mutations is therefore crucial for developing novel therapeutic strategies. To understand the interplay between spatial genome organization and specific mutational processes, we studied 3,000 tumor-normal-pair whole-genome datasets from 42 different human cancer types. Our analyses reveal that the change in somatic mutational load in cancer genomes is co-localized with topologically-associating-domain boundaries. Domain boundaries constitute a better proxy to track mutational load change than replication timing measurements. We show that different mutational processes lead to distinct somatic mutation distributions where certain processes generate mutations in active domains, and others generate mutations in inactive domains. Overall, the interplay between three-dimensional genome organization and active mutational processes has a substantial influence on the large-scale mutation-rate variations observed in human cancers.
Assuntos
Cromatina/química , Genoma Humano , Mutação , Neoplasias/genética , Linhagem Celular Tumoral , Cromossomos Humanos X/genética , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA , DNA de Neoplasias , Conjuntos de Dados como Assunto , Feminino , Humanos , Masculino , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Inativação do Cromossomo XRESUMO
Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inflamação/patologia , Telômero/patologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antibacterianos/uso terapêutico , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Caspase 1/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Criança , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Gastroenteropatias/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Interleucina-18/genética , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Mutantes , Fosforilação , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transdução de Sinais , Telomerase/genética , Telomerase/metabolismo , Proteínas de Sinalização YAPRESUMO
In the version of this article originally published, information regarding several funding sources was omitted from the Acknowledgements section. The following sentences should have been included: "This work was supported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shots Program, the UT Lung SPORE 5 P50 CA07090, and the MD Anderson Cancer Center Support Grant P30CA01667. V.P is supported by R01CA155196-01A1 from the National Cancer Institute." Also, reference 18 was incorrect. The original reference was: Kim, E. S. et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov. 1, 44-53 (2011). It should have been: Papadimitrakopoulou, V. et al. The BATTLE-2 study: a biomarker-integrated targeted therapy study in previously treated patients with advanced non-small-cell lung cancer. J Clin. Oncol. 34, 3638-3647 (2016). The errors have been corrected in the HTML and PDF versions of this article.
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Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5-55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)--a PTEN-interacting protein and negative regulator of PTEN in breast cancer--as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.
Assuntos
Genoma Humano/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Melanoma/genética , Mutação/genética , Luz Solar/efeitos adversos , Pontos de Quebra do Cromossomo/efeitos da radiação , Dano ao DNA , Análise Mutacional de DNA , Regulação Neoplásica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Mutagênese/efeitos da radiação , Mutação/efeitos da radiação , Oncogenes/genética , Raios Ultravioleta/efeitos adversosRESUMO
SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation. Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.
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Apoptose , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Dermatite/genética , Dermatite/metabolismo , Dermatite/patologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Quinase I-kappa B/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Post-translational modifications of proteins and the domains that recognize these modifications have central roles in creating a highly dynamic relay system that reads and responds to alterations in the cellular microenvironment. Here we review the common principles of post-translational modifications and their importance in signal integration underlying epidermal growth factor receptor signaling and endocytosis, DNA-damage responses and immunity.
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Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Dano ao DNA/fisiologia , Endocitose/fisiologia , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Modelos Biológicos , Oxigênio/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/imunologiaRESUMO
Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase activity and increased acetylation of alpha-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.
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Receptores ErbB/metabolismo , Histona Desacetilases/metabolismo , Complexos Multiproteicos/metabolismo , Transdução de Sinais/fisiologia , Acetilação , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Desacetilase 6 de Histona , Humanos , Imunoprecipitação , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , RNA/genética , Transfecção , Tubulina (Proteína)/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: Endothelial progenitor cells (EPCs) comprise a heterogeneous population of cells, which improve therapeutic neovascularization after ischemia. The neovascularization-promoting potential of progenitor cells depends on survival and retention of the infused cells to the tissue. Caspases mediate apoptosis but are also involved in other critical biological processes. Therefore, we aimed to address the role of caspases in proangiogenic cells. METHODS AND RESULTS: The caspase-8 inhibitor zIETD abrogated the ex vivo formation of EPCs, inhibited EPC adhesion and migration, and reduced their capacity to improve neovascularization in vivo. Consistently, cells isolated from caspase-8-deficient mice exhibited a reduced capacity for enhancing neovascularization when transplanted into mice after hindlimb ischemia. Because inhibition of Caspase-8 reduced the adhesion and homing functions of EPCs, we further determined the surface expression of integrins and receptors involved in cell recruitment to ischemic tissues. Pharmacological inhibition of caspase-8 and genetic depletion of caspase-8 reduced the expression of the fibronectin receptor subunits alpha5 and beta1 and the SDF-1 receptor CXCR4. Moreover, we identified the E3 ubiquitin ligase Cbl-b, which negatively regulates integrin and receptor-mediated signaling, as a potential Caspase-8 substrate. CONCLUSIONS: In summary, our data demonstrate a novel apoptosis-unrelated role of caspase-8 in proangiogenic cells.
Assuntos
Caspase 8/metabolismo , Células Endoteliais/enzimologia , Isquemia/enzimologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Células-Tronco/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Síndrome de Alstrom , Animais , Caspase 8/genética , Inibidores de Caspase , Adesão Celular , Movimento Celular , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/transplante , Membro Posterior , Humanos , Integrina alfaV/metabolismo , Integrina beta1/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Camundongos , Camundongos Knockout , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , RNA Mensageiro/metabolismo , Receptores CXCR4/metabolismo , Receptores de Fibronectina/metabolismo , Transplante de Células-Tronco , Células-Tronco/efeitos dos fármacosRESUMO
The CIN85/CMS (human homologs of mouse SH3KBP1/CD2AP) family of endocytic adaptor proteins has the ability to engage multiple effectors and couple cargo trafficking with the cytoskeleton. CIN85 and CMS (Cas ligand with multiple Src homology 3 (SH3) domains) facilitate the formation of large multiprotein complexes required for an efficient internalization of cell surface receptors. It has recently been shown that c-Cbl/Cbl-b could mediate the formation of a ternary complex between one c-Cbl/Cbl-b molecule and two SH3 domains of CIN85, important for the ability of Cbl to promote epidermal growth factor receptor down-regulation. To further investigate whether multimerization is conserved within the family of adaptor proteins, we have solved the crystal structures of the CMS N-terminal SH3 domain-forming complexes with Cbl-b- and CD2-derived peptides. Together with biochemical evidence, the structures support the notion that, despite clear differences in the interaction surface, both Cbl-b and CD2 can mediate multimerization of N-terminal CMS SH3 domains. Detailed analyses on the interacting surfaces also provide the basis for a differential Cbl-b molecular recognition of CMS and CIN85.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Peptídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Domínios de Homologia de srcRESUMO
The ubiquitin ligases c-Cbl and Cbl-b play a crucial role in receptor downregulation by mediating multiple monoubiquitination of receptors and promoting their sorting for lysosomal degradation. Their function is modulated through interactions with regulatory proteins including CIN85 and PIX, which recognize a proline-arginine motif in Cbl and thus promote or inhibit receptor endocytosis. We report the structures of SH3 domains of CIN85 and beta-PIX in complex with a proline-arginine peptide from Cbl-b. Both structures reveal a heterotrimeric complex containing two SH3 domains held together by a single peptide. Trimerization also occurs in solution and is facilitated by the pseudo-symmetrical peptide sequence. Moreover, ternary complexes of CIN85 and Cbl are formed in vivo and are important for the ability of Cbl to promote epidermal growth factor receptor (EGFR) downregulation. These results provide molecular explanations for a novel mechanism by which Cbl controls receptor downregulation.