RESUMO
Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Genoma Humano , Proteína HMGB2/metabolismo , Fator de Ligação a CCCTC/genética , Proliferação de Células , Senescência Celular , Cromatina/genética , Proteína HMGB2/genética , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3-7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.
Assuntos
Inibidores Enzimáticos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Apoptose/efeitos dos fármacos , Aurora Quinase B , Aurora Quinases , Benzotiazóis , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA/genética , Diaminas , Citometria de Fluxo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Immunoblotting , Compostos Orgânicos , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-myc/metabolismo , Quinolinas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched samples from patients with primary and metastatic SCLC to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2 (ANG-2)/Integrin ß-1-dependent pathway in tumor cells, mediated by focal adhesion kinase/Src kinase signaling. Strikingly, CRISPR-Cas9 KO of Integrin ß-1 or blocking Integrin ß-1 signaling by an anti-ANG-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched samples from patients with primary and metastatic SCLC confirmed a strong increase of Integrin ß-1 in liver metastasis in comparison with the primary tumor. We further show that ANG-2 blockade combined with PD-1-targeted by anti-PD-1 treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of ANG-2/Integrin ß-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a potentially new effective treatment strategy for patients with SCLC.
Assuntos
Angiopoietina-2 , Integrina beta1 , Neoplasias Hepáticas , Neoplasias Pulmonares , Transdução de Sinais , Carcinoma de Pequenas Células do Pulmão , Animais , Feminino , Humanos , Masculino , Camundongos , Angiopoietina-2/metabolismo , Angiopoietina-2/genética , Linhagem Celular Tumoral , Integrina beta1/metabolismo , Integrina beta1/genética , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Invasividade Neoplásica , Metástase Neoplásica , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológicoRESUMO
In cancer, genetically activated proto-oncogenes often induce "upstream" dependency on the activity of the mutant oncoprotein. Therapeutic inhibition of these activated oncoproteins can induce massive apoptosis of tumor cells, leading to sometimes dramatic tumor regressions in patients. The PI3K and MAPK signaling pathways are central regulators of oncogenic transformation and tumor maintenance. We hypothesized that upstream dependency engages either one of these pathways preferentially to induce "downstream" dependency. Therefore, we analyzed whether downstream pathway dependency segregates by genetic aberrations upstream in lung cancer cell lines. Here, we show by systematically linking drug response to genomic aberrations in non-small-cell lung cancer, as well as in cell lines of other tumor types and in a series of in vivo cancer models, that tumors with genetically activated receptor tyrosine kinases depend on PI3K signaling, whereas tumors with mutations in the RAS/RAF axis depend on MAPK signaling. However, efficacy of downstream pathway inhibition was limited by release of negative feedback loops on the reciprocal pathway. By contrast, combined blockade of both pathways was able to overcome the reciprocal pathway activation induced by inhibitor-mediated release of negative feedback loops and resulted in a significant increase in apoptosis and tumor shrinkage. Thus, by using a systematic chemo-genomics approach, we identify genetic lesions connected to PI3K and MAPK pathway activation and provide a rationale for combined inhibition of both pathways. Our findings may have implications for patient stratification in clinical trials.
Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Genótipo , Humanos , Neoplasias/enzimologia , Neoplasias/patologia , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
Purpose: Abrogation of Notch signaling, which is pivotal for lung development and pulmonary epithelial cell fate decisions was shown to be involved in the aggressiveness and the differentiation of lung carcinomas. Additionally, the transcription factors YAP and TAZ which are involved in the Hippo pathway, were recently shown to be tightly linked with Notch signaling and to regulate the cell fate in epidermal stem cells. Thus, we aim to elucidate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression in lung cancer. Methods: We investigated the effect of conditional Cre-recombinase mediated Notch1 knock-out on lung cancer cells in vivo using an autochthonous mouse model of lung adenocarcinomas driven by Kras LSL-G12V and comprehensive immunohistochemical analysis. In addition, we analyzed clinical samples and human lung cancer cell lines for TAZ expression and supported our findings by publicly available data from The Cancer Genome Atlas (TCGA). Results: In mice, we found induction of papillary adenocarcinomas and protrusions of tumor cells from the bronchiolar lining upon Notch1 deficiency. Moreover, the mutated Kras driven lung tumors with deleted Notch1 showed increased TAZ expression and focal nuclear translocation which was frequently observed in human pulmonary adenocarcinomas and squamous cell carcinomas of the lung, but not in small cell lung carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas. Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade.
Assuntos
Aciltransferases/metabolismo , Brônquios/patologia , Modelos Animais de Doenças , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor Notch1/fisiologia , Aciltransferases/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Brônquios/metabolismo , Carcinogênese , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Prognóstico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Células Tumorais CultivadasRESUMO
Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF)-VEGFR2 signaling has shown limited efficacy in lung cancer patients. Here, we demonstrate that inhibition of VEGFR2 in tumor cells, expressed in â¼20% of non-squamous non-small cell lung cancer (NSCLC) patients, leads to a pro-invasive phenotype. Drug-induced inhibition of tumor VEGFR2 interferes with the formation of the EphA2/VEGFR2 heterocomplex, thereby allowing RSK to interact with Serine 897 of EphA2. Inhibition of RSK decreases phosphorylation of Serine 897 EphA2. Selective genetic modeling of Serine 897 of EphA2 or inhibition of EphA2 abrogates the formation of metastases in vivo upon VEGFR2 inhibition. In summary, these findings demonstrate that VEGFR2-targeted therapy conditions VEGFR2-positive NSCLC to Serine 897 EphA2-dependent aggressive tumor growth and metastasis. These data shed light on the molecular mechanisms explaining the limited efficacy of VEGFR2-targeted anti-angiogenic treatment in lung cancer patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Receptor EphA2/metabolismo , Serina/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Invasividade Neoplásica , Metástase Neoplásica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
The ability of the low density lipoprotein receptor-related protein (LRP) to form homo-dimers was studied in mouse neuroblastoma and human neuroglioma cells as well as in primary cortical cultures from adult mouse brain. Homo-dimerization of LRP light chain (LC) was shown by several methods including co-immunoprecipitation, fluorescence lifetime imaging microscopy, and bimolecular fluorescence complementation assay. The requirement of intact NPXY motifs of LRP LC for homo-dimerization was ruled out by co-immunoprecipitation assay.
Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neurônios/metabolismo , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Animais , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Endocitose , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Processamento de Proteína Pós-Traducional , Transfecção/métodosRESUMO
FGFR1 amplification has been found in 15% of patients with breast cancer and has been postulated as a promising marker to predict response against FGFR inhibitors. However, early phase clinical trials of selective FGFR inhibitors demonstrated only limited efficacy in FGFR1-amplified breast cancer patients. We found that BGJ398, an FGFR inhibitor, effectively inhibited phosphorylation of FGFR1 and MEK/ERK signaling in FGFR1-amplified breast cancer without affecting tumor cell proliferation. However, FGFR1 knockout inhibited tumor angiogenesis in vivo. We unraveled that FGFR1 regulates the secretion of the proangiogenic vascular endothelial growth factor (VEGF) in a MAPK-dependent manner. We further found that FGF-FGFR1 signaling induces an autocrine activation of VEGF-VEGFR1 pathway that again amplifies VEGF secretion via VEGF-VEGFR1-AKT signaling. Targeting both VEGFR1 and FGFR1 resulted in synergistic anti-angiogenic treatment effects in vivo. We thus postulate synergistic treatment effects in FGFR1/VEGFR1-positive breast cancer patients by dual targeting of FGFR and VEGFR.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias da Mama/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Naftalenos/farmacologia , Neovascularização Patológica , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Quinolinas/farmacologiaRESUMO
LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas de Ligação a RNA/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Análise de SobrevidaRESUMO
The beta-amyloid (Abeta) precursor protein (APP) is cleaved sequentially by beta-site of APP-cleaving enzyme (BACE) and gamma-secretase to release the Abeta peptides that accumulate in plaques in Alzheimer's disease (AD). GGA1, a member of the Golgi-localized gamma-ear-containing ARF-binding (GGA) protein family, interacts with BACE and influences its subcellular distribution. We now report that overexpression of GGA1 in cells increased the APP C-terminal fragment resulting from beta-cleavage but surprisingly reduced Abeta. GGA1 confined APP to the Golgi, in which fluorescence resonance energy transfer analyses suggest that the proteins come into close proximity. GGA1 blunted only APP but not notch intracellular domain release. These results suggest that GGA1 prevented APP beta-cleavage products from becoming substrates for gamma-secretase. Direct binding of GGA1 to BACE was not required for these effects, but the integrity of the GAT (GGA1 and TOM) domain of GGA1 was. GGA1 may act as a specific spatial switch influencing APP trafficking and processing, so that APP-GGA1 interactions may have pathophysiological relevance in AD.
Assuntos
Fatores de Ribosilação do ADP/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Química Encefálica , Linhagem Celular , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Rim/citologia , Rim/embriologia , Camundongos , Neuroblastoma/patologia , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Relação Estrutura-Atividade , TransfecçãoRESUMO
sorLA is a recently identified neuronal receptor for amyloid precursor protein (APP) that is known to interact with APP and affect its intracellular transport and processing. Decreased levels of sorLA in the brain of Alzheimer's disease (AD) patients and elevated levels of amyloid-beta peptide (Abeta) in sorLA-deficient mice point to the importance of the receptor in this neurodegenerative disorder. We analyzed APP cleavage in an APP-shedding assay and found that both sorLA and, surprisingly, a sorLA tail construct inhibited APP cleavage in a beta-site APP-cleaving enzyme (BACE)-dependent manner. In line with this finding, sorLA and the sorLA tail significantly reduced secreted Abeta levels when BACE was overexpressed, suggesting that sorLA influences beta-cleavage. To understand the effect of sorLA on APP cleavage by BACE, we analyzed whether sorLA interacts with APP and/or BACE. Because both full-length sorLA and sorLA C-terminal tail constructs were functionally relevant for APP processing, we analyzed sorLA-APP for a potential cytoplasmatic interaction domain. sorLA and C99 coimmunoprecipitated, pointing toward the existence of a new cytoplasmatic interaction site between sorLA and APP. Moreover, sorLA and BACE also coimmunoprecipitate. Thus, sorLA interacts both with BACE and APP and might therefore directly affect BACE-APP complex formation. To test whether sorLA impacts BACE-APP interactions, we used a fluorescence resonance energy transfer assay to evaluate BACE-APP interactions in cells. We discovered that sorLA significantly reduced BACE-APP interactions in Golgi. We postulate that sorLA acts as a trafficking receptor that prevents BACE-APP interactions and hence BACE cleavage of APP.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mapeamento de Interação de Proteínas , Receptores de LDL/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/química , Animais , Ácido Aspártico Endopeptidases , Biotinilação , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Citosol , Endocitose , Endopeptidases/química , Complexo de Golgi/metabolismo , Humanos , Rim , Proteínas Relacionadas a Receptor de LDL , Proteínas de Membrana Transportadoras/química , Complexos Multiproteicos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Transporte Proteico , Receptores de LDL/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Genomic translocation events frequently underlie cancer development through generation of gene fusions with oncogenic properties. Identification of such fusion transcripts by transcriptome sequencing might help to discover new potential therapeutic targets. We developed TRUP (Tumor-specimen suited RNA-seq Unified Pipeline) (https://github.com/ruping/TRUP), a computational approach that combines split-read and read-pair analysis with de novo assembly for the identification of chimeric transcripts in cancer specimens. We apply TRUP to RNA-seq data of different tumor types, and find it to be more sensitive than alternative tools in detecting chimeric transcripts, such as secondary rearrangements in EML4-ALK-positive lung tumors, or recurrent inactivating rearrangements affecting RASSF8.
Assuntos
Pontos de Quebra do Cromossomo , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Fusão Oncogênica , Transcriptoma , Translocação Genética , Sequência de Bases , Linhagem Celular Tumoral , Análise por Conglomerados , Biologia Computacional/métodos , Inativação Gênica , Genômica , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
UNLABELLED: We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-ß3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. SIGNIFICANCE: CD74NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease.
Assuntos
Adenocarcinoma Mucinoso/genética , Adenocarcinoma/genética , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Histocompatibilidade Classe II/genética , Neoplasias Pulmonares/genética , Neuregulina-1/genética , Proteínas de Fusão Oncogênica/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Células NIH 3T3 , Proteínas de Fusão Oncogênica/metabolismo , Análise de Sequência de DNA , Transdução de Sinais/genéticaRESUMO
The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Retroalimentação Fisiológica/efeitos dos fármacos , Neoplasias Pulmonares/irrigação sanguínea , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/farmacologia , Benzamidas , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Difenilamina/análogos & derivados , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Piperidinas , Quinazolinas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
PURPOSE: ALK rearrangement-positive lung cancers can be effectively treated with ALK inhibitors. However, the magnitude and duration of response is heterogeneous. In addition, acquired resistance limits the efficacy of ALK inhibitors, with most upfront resistance mechanisms being unknown. EXPERIMENTAL DESIGN: By making use of the Ba/F3 cell line model, we analyzed the cytotoxic efficacy of ALK kinase inhibitors as a function of different EML4-ALK fusion variants v1, v2, v3a, and v3b as well as of three artificially designed EML4-ALK deletion constructs and the ALK fusion genes KIF5b-ALK and NPM1-ALK. In addition, the intracellular localization, the sensitivity to HSP90 inhibition and the protein stability of ALK fusion proteins were studied. RESULTS: Different ALK fusion genes and EML4-ALK variants exhibited differential sensitivity to the structurally diverse ALK kinase inhibitors crizotinib and TAE684. In addition, differential sensitivity correlated with differences in protein stability in EML4-ALK-expressing cells. Furthermore, the sensitivity to HSP90 inhibition also varied depending on the ALK fusion partner but differed from ALK inhibitor sensitivity patterns. Finally, combining inhibitors of ALK and HSP90 resulted in synergistic cytotoxicity. CONCLUSIONS: Our results might explain some of the heterogeneous responses of ALK-positive tumors to ALK kinase inhibition observed in the clinic. Thus, targeted therapy of ALK-positive lung cancer should take into account the precise ALK genotype. Furthermore, combining ALK and HSP90 inhibitors might enhance tumor shrinkage in EML4-ALK-driven tumors.
Assuntos
Neoplasias Pulmonares , Proteínas de Fusão Oncogênica , Proteínas Tirosina Quinases , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Adenocarcinoma , Adenocarcinoma de Pulmão , Quinase do Linfoma Anaplásico , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Crizotinibe , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Células NIH 3T3 , Nucleofosmina , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Estabilidade Proteica/efeitos dos fármacos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacosRESUMO
Small-cell lung cancer (SCLC) is an aggressive lung tumor subtype with poor prognosis. We sequenced 29 SCLC exomes, 2 genomes and 15 transcriptomes and found an extremely high mutation rate of 7.4±1 protein-changing mutations per million base pairs. Therefore, we conducted integrated analyses of the various data sets to identify pathogenetically relevant mutated genes. In all cases, we found evidence for inactivation of TP53 and RB1 and identified recurrent mutations in the CREBBP, EP300 and MLL genes that encode histone modifiers. Furthermore, we observed mutations in PTEN, SLIT2 and EPHA7, as well as focal amplifications of the FGFR1 tyrosine kinase gene. Finally, we detected many of the alterations found in humans in SCLC tumors from Tp53 and Rb1 double knockout mice. Our study implicates histone modification as a major feature of SCLC, reveals potentially therapeutically tractable genomic alterations and provides a generalizable framework for the identification of biologically relevant genes in the context of high mutational background.
Assuntos
Genoma Humano , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Substituição de Aminoácidos , Animais , Proteína de Ligação a CREB/genética , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Proteína p300 Associada a E1A/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Histona-Lisina N-Metiltransferase , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Knockout , Modelos Moleculares , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , PTEN Fosfo-Hidrolase/genética , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional/genética , Proteína do Retinoblastoma/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
PURPOSE: EML4-ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4-ALK-positive tumors. EXPERIMENTAL DESIGN: By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684. RESULTS: Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4-ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066. CONCLUSIONS: Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4-ALK fusions and ALK mutations.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Fusão Oncogênica/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Serina Endopeptidases/genética , Quinase do Linfoma Anaplásico , Linhagem Celular Tumoral , Crizotinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Polimorfismo de Nucleotídeo Único , Receptores Proteína Tirosina Quinases/antagonistas & inibidoresRESUMO
UNLABELLED: While genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations which drive squamous cell lung cancer. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of squamous cell lung cancers and cell lines. Squamous lung cancer cell lines harboring DDR2 mutations were selectively killed by knock-down of DDR2 by RNAi or by treatment with the multi-targeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation which was blocked by dasatinib. A squamous cell lung cancer patient with a response to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. As dasatinib is already approved for use, these findings could be rapidly translated into clinical trials. SIGNIFICANCE: DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Dasatinibe , Receptores com Domínio Discoidina , Cloridrato de Erlotinib , Humanos , Neoplasias Pulmonares/enzimologia , Camundongos , Camundongos Nus , Mutação , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Tiazóis/farmacologia , Tiazóis/uso terapêutico , Quinases da Família src/genéticaRESUMO
Despite the successful introduction of potent anti-cancer therapeutics, most of these drugs lead to only modest tumor-shrinkage or transient responses, followed by re-growth of tumors. Combining different compounds has resulted in enhanced tumor control and prolonged survival. However, methods querying the efficacy of such combinations have been hampered by limited scalability, analytical resolution, statistical feasibility, or a combination thereof. We have developed a theoretical framework modeling cellular viability as a stochastic lifetime process to determine synergistic compound combinations from high-throughput cellular screens. We apply our method to data derived from chemical perturbations of 65 cancer cell lines with two inhibitors. Our analysis revealed synergy for the combination of both compounds in subsets of cell lines. By contrast, in cell lines in which inhibition of one of both targets was sufficient to induce cell death, no synergy was detected, compatible with the topology of the oncogenically activated signaling network. In summary, we provide a tool for the measurement of synergy strength for combination perturbation experiments that might help define pathway topologies and direct clinical trials.
Assuntos
Antineoplásicos/farmacologia , Modelos Teóricos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Processos EstocásticosRESUMO
Reversible epidermal growth factor receptor (EGFR) inhibitors are the first class of small molecules to improve progression-free survival of patients with EGFR-mutated lung cancers. Second-generation EGFR inhibitors introduced to overcome acquired resistance by the T790M resistance mutation of EGFR have thus far shown limited clinical activity in patients with T790M-mutant tumors. In this study, we systematically analyzed the determinants of the activity and selectivity of the second-generation EGFR inhibitors. A focused library of irreversible as well as structurally corresponding reversible EGFR-inhibitors was synthesized for chemogenomic profiling involving over 79 genetically defined NSCLC and 19 EGFR-dependent cell lines. Overall, our results show that the growth-inhibitory potency of all irreversible inhibitors against the EGFR(T790M) resistance mutation was limited by reduced target inhibition, linked to decreased binding velocity to the mutant kinase. Combined treatment of T790M-mutant tumor cells with BIBW-2992 and the phosphoinositide-3-kinase/mammalian target of rapamycin inhibitor PI-103 led to synergistic induction of apoptosis. Our findings offer a mechanistic explanation for the limited efficacy of irreversible EGFR inhibitors in EGFR(T790M) gatekeeper-mutant tumors, and they prompt combination treatment strategies involving inhibitors that target signaling downstream of the EGFR.