RESUMO
We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein-protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Mutação , Proteínas de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Carbazóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Cloridrato de Erlotinib , Furanos , Humanos , Proteínas de Neoplasias/genética , Fosforilação , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Estaurosporina/análogos & derivados , Estaurosporina/farmacologiaRESUMO
We describe a strategy for comprehending signaling pathways that are active in lung cancer cells and that are targeted by dasatinib using chemical proteomics to identify direct interacting proteins combined with immunoaffinity purification of tyrosine-phosphorylated peptides corresponding to activated tyrosine kinases. We identified nearly 40 different kinase targets of dasatinib. These include SRC-family kinase (SFK) members (LYN, SRC, FYN, LCK and YES), nonreceptor tyrosine kinases (FRK, BRK and ACK) and receptor tyrosine kinases (Ephrin receptors, DDR1 and EGFR). Using quantitative phosphoproteomics, we identified peptides corresponding to autophosphorylation sites of these tyrosine kinases that are inhibited in a concentration-dependent manner by dasatinib. Using drug-resistant gatekeeper mutants, we show that SFKs (particularly SRC and FYN), as well as EGFR, are relevant targets for dasatinib action. The combined mass spectrometry-based approach described here provides a system-level view of dasatinib action in cancer cells and suggests both functional targets and a rationale for combinatorial therapeutic strategies.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteômica/métodos , Pirimidinas/farmacologia , Tiazóis/farmacologia , Apoptose , Linhagem Celular Tumoral , Dasatinibe , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Peptídeos/química , Peptídeos/metabolismo , Fenótipo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Tiazóis/metabolismo , Tiazóis/uso terapêuticoRESUMO
Mutations of the epidermal growth factor receptor (EGFR) selectively activate Akt and signal transducer and activator of transcription (STAT) pathways that are important in lung cancer cell survival. Src family kinases can cooperate with receptor tyrosine kinases to signal through downstream molecules, such as phosphatidylinositol 3-kinase/PTEN/Akt and STATs. Based on the importance of EGFR signaling in lung cancer, the known cooperation between EGFR and Src proteins, and evidence of elevated Src activity in human lung cancers, we evaluated the effectiveness of a novel orally bioavailable Src inhibitor dasatinib (BMS-324825) in lung cancer cell lines with defined EGFR status. Here, we show that cell fate (death versus growth arrest) in lung cancer cells exposed to dasatinib is dependent on EGFR status. In cells with EGFR mutation that are dependent on EGFR for survival, dasatinib reduces cell viability through the induction of apoptosis while having minimal apoptotic effect on cell lines with wild-type (WT) EGFR. The induction of apoptosis in these EGFR-mutant cell lines corresponds to down-regulation of activated Akt and STAT3 survival proteins. In cell lines with WT or resistant EGFR mutation that are not sensitive to EGFR inhibition, dasatinib induces a G(1) cell cycle arrest with associated changes in cyclin D and p27 proteins, inhibits activated FAK, and prevents tumor cell invasion. Our results show that dasatinib could be effective therapy for patients with lung cancers through disruption of cell growth, survival, and tumor invasion. Our results suggest EGFR status is important in deciding cell fate in response to dasatinib.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Dasatinibe , Receptores ErbB/genética , Deleção de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Transdução de Sinais , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
Stat transcription factors are activated by cytokines and can activate pathways important in oncogenesis. Although previous studies have identified an oncogenic role of Stat3 in lung cancer cells, the role of Stat1 is unclear. Using a mutant of Stat1 with constitutive activity (Stat1C), we examined the effect of persistent Stat1 activity on lung cancer cell growth, survival and gene expression. We identified no significant effect of Stat1C alone or with interferon-gamma (IFN-gamma) on lung cancer cell growth or survival. Consistent with prior reports, Stat1C expression alone elicited minimal changes in gene expression and required costimulatory IFN-gamma for full activity. Using oligonucleotide gene arrays and quantitative real-time PCR, we identified numerous proinflammatory gene products and chemokines regulated by IFN-gamma/Stat1C signaling. These results suggest the major role of IFN-gamma and Stat1 in lung cells is to direct a proinflammatory gene expression program rather than have major effects on cell growth or survival or both.
Assuntos
Antivirais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quimiocinas/biossíntese , Quimiocinas/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Inflamação/genética , Inflamação/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genéticaRESUMO
Wound healing and cancer are both characterized by cell proliferation, remodeling of extracellular matrix, cell invasion and migration, new blood vessel formation, and modulation of blood coagulation. The mechanisms that link wound healing and cancer are poorly understood. We report here that Stat3, a common signaling mechanism involved in oncogenesis and tissue injury, regulates a common set of genes involved in wound healing and cancer. Using oligonucleotide gene arrays and quantitative real-time PCR, we evaluated changes in global gene expression resulting from expression of Stat3 in lung epithelial cells. We report here previously uncharacterized genes induced by Stat3 implicated in signaling pathways common to both wound healing and cancer including cell invasion and migration, angiogenesis, modulation of coagulation, and repression of interferon-inducible genes. Consistent with these results, we found increased Stat3 activity associated with wound healing in chronically inflamed mouse lungs and increased Stat3 activity was identified at the leading edge of lung tumors invading adjacent nontumor stroma. These findings provide a molecular basis for understanding cancer as a deregulation of normal wound healing processes.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/farmacologia , Perfilação da Expressão Gênica , Neoplasias/genética , Neoplasias/fisiopatologia , Transativadores/genética , Transativadores/farmacologia , Cicatrização/genética , Proteínas de Fase Aguda , Animais , Movimento Celular , Transformação Celular Neoplásica , Modelos Animais de Doenças , Células Epiteliais , Inflamação , Pulmão/citologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Fator de Transcrição STAT3 , Transdução de SinaisRESUMO
PURPOSE: Signal transducers and activators of transcription 3 (Stat3), a member of the STAT family of transcription factors, regulates multiple oncogenic pathways, including pathways regulating tumor cell survival. We evaluated Stat3 activation in early stage non-small cell lung cancers (NSCLC) and how this relates to upstream epidermal growth factor receptor (EGFR) activation, tumor apoptosis, and prognosis. EXPERIMENTAL DESIGN: High-density tissue microarrays using tissues from 176 surgically resected NSCLC were evaluated for expression of phosphorylated Stat3 (pStat3) and epidermal growth factor receptor (pEGFR) along with tumor apoptosis. Using NSCLC cell lines, we evaluated how pStat3 expression relates to EGFR mutations and sensitivity of cells to gefitinib. RESULTS: We identified nuclear pStat3 expression in 54% of tumors. pStat3 expression was correlated with smaller tumors (P < 0.0001) and with limited smoking history (P = 0.02). We identified a trend toward higher pStat3 expression in adenocarcinomas compared with other tumor histology (P = 0.09). No relationship was found between pStat3 and prognosis following surgical resection. Importantly, we found a strong positive correlation between pEGFR expression and pStat3 expression (P <0.0001) and an inverse correlation between pStat3 and apoptosis (P = 0.01) consistent with less apoptosis in tumors expressing high amounts of pStat3. Cell lines with mutant EGFR have increased levels of pStat3 compared with cell lines without mutant EGFR and this correlates with their sensitivity to gefitinib. Finally, antisense-mediated knockdown of Stat3 induces apoptosis in EGFR mutant lung cancer cells. CONCLUSIONS: Early-stage NSCLC tumors have activated EGFR-Stat3 signaling with low apoptosis. Our findings suggest that pStat3 expression may be helpful in identifying patients appropriate for treatment with EGFR tyrosine kinase inhibitors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Fosforilação , Prognóstico , Quinazolinas/uso terapêutico , Taxa de Sobrevida , Análise Serial de TecidosRESUMO
Patients with lung cancers harboring anaplastic lymphoma kinase (ALK) gene fusions benefit from treatment with ALK inhibitors, but acquired resistance inevitably arises. A better understanding of proximal ALK signaling mechanisms may identify sensitizers to ALK inhibitors that disrupt the balance between prosurvival and proapoptotic effector signals. Using affinity purification coupled with mass spectrometry in an ALK fusion lung cancer cell line (H3122), we generated an ALK signaling network and investigated signaling activity using tyrosine phosphoproteomics. We identified a network of 464 proteins composed of subnetworks with differential response to ALK inhibitors. A small hairpin RNA screen targeting 407 proteins in this network revealed 64 and 9 proteins that when knocked down sensitized cells to crizotinib and alectinib, respectively. Among these, knocking down fibroblast growth factor receptor substrate 2 (FRS2) or coiled-coil and C2 domain-containing protein 1A (CC2D1A), both scaffolding proteins, sensitized multiple ALK fusion cell lines to the ALK inhibitors crizotinib and alectinib. Collectively, our data set provides a resource that enhances our understanding of signaling and drug resistance networks consequent to ALK fusions and identifies potential targets to improve the efficacy of ALK inhibitors in patients.
Assuntos
Carbazóis/farmacologia , Proteínas de Ciclo Celular , Neoplasias Pulmonares , Proteínas Associadas aos Microtúbulos , Piperidinas/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Interferência de RNA , Receptores Proteína Tirosina Quinases , Serina Endopeptidases , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quinase do Linfoma Anaplásico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Crizotinibe , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) is one mechanism of acquired resistance to inhibitors of the epidermal growth factor receptor-tyrosine kinases (EGFR-TKIs) in non-small cell lung cancer (NSCLC). The precise mechanisms of EMT-related acquired resistance to EGFR-TKIs in NSCLC remain unclear. We generated erlotinib-resistant HCC4006 cells (HCC4006ER) by chronic exposure of EGFR-mutant HCC4006 cells to increasing concentrations of erlotinib. HCC4006ER cells acquired an EMT phenotype and activation of the TGF-ß/SMAD pathway, while lacking both T790M secondary EGFR mutation and MET gene amplification. We employed gene expression microarrays in HCC4006 and HCC4006ER cells to better understand the mechanism of acquired EGFR-TKI resistance with EMT. At the mRNA level, ZEB1 (TCF8), a known regulator of EMT, was >20-fold higher in HCC4006ER cells than in HCC4006 cells, and increased ZEB1 protein level was also detected. Furthermore, numerous ZEB1 responsive genes, such as CDH1 (E-cadherin), ST14, and vimentin, were coordinately regulated along with increased ZEB1 in HCC4006ER cells. We also identified ZEB1 overexpression and an EMT phenotype in several NSCLC cells and human NSCLC samples with acquired EGFR-TKI resistance. Short-interfering RNA against ZEB1 reversed the EMT phenotype and, importantly, restored erlotinib sensitivity in HCC4006ER cells. The level of micro-RNA-200c, which can negatively regulate ZEB1, was significantly reduced in HCC4006ER cells. Our results suggest that increased ZEB1 can drive EMT-related acquired resistance to EGFR-TKIs in NSCLC. Attempts should be made to explore targeting ZEB1 to resensitize TKI-resistant tumors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Proteínas de Homeodomínio/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Antineoplásicos/farmacologia , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mutação , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Overexpression of receptor tyrosine kinases including the epidermal growth factor receptor (EGF-R) as well as nonreceptor tyrosine kinases, such as Src, have been implicated in the formation of human lung cancers. In addition, cytokines like interleukin-6 (IL-6) have been demonstrated to modulate lung cancer cell growth and elevated levels of IL-6 have been shown to be an adverse prognostic factor for patients with lung cancer. Despite a large body of evidence pointing to their potential importance, few direct studies into the role of signal transducers and activators of transcription (STAT) pathways in human lung cancer have been undertaken. Here we demonstrate that multiple nonsmall cell lung cancer cell lines demonstrate constitutive Stat3 DNA-binding activity. Stat3 DNA-binding activity is specifically upregulated by the addition of epidermal growth factor (EGF), IL-6, and hepatocyte-derived growth factor (HGF). Furthermore, the stimulation of Stat3 DNA-binding activity by EGF requires the activity of EGF-R tyrosine kinase as well as Src-kinase, while the upregulation of Stat3 activity by IL-6 or HGF requires only Src-kinase activity. Treatment of A549 lung cancer cells with PD180970 or SU6656, both pharmacological inhibitors of Src-kinase, resulted in reduced Src and Stat3 activity, cell cycle arrest in G2, and reduced viability of cells accompanied by induction of apoptosis. Treatment of Stat3-positive A549 and H358 cells with antisense Stat3 oligonucleotides results in complete loss of Stat3 DNA-binding activity and apoptosis, while Stat3-positive H1299 cells remained healthy. Finally, an adenoviral vector expressing a dominant-negative Stat3 isoform results in loss of Stat3 DNA-binding activity, apoptosis, and reduced cellular viability. These results demonstrate a role of Stat3 in transducing survival signals downstream of tyrosine kinases such as Src, EGF-R, and c-Met, as well as cytokines such as IL-6, in human nonsmall cell lung cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transativadores/metabolismo , Adenoviridae/genética , Apoptose , Western Blotting , Ciclo Celular , Núcleo Celular/metabolismo , Sobrevivência Celular , DNA/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fase G2 , Genes Dominantes , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Interleucina-6/metabolismo , Modelos Biológicos , Oligonucleotídeos Antissenso/farmacologia , Ligação Proteica , Proteínas Tirosina Quinases/antagonistas & inibidores , Fator de Transcrição STAT3 , Fatores de Tempo , Transfecção , Células Tumorais CultivadasRESUMO
E2F1 induces apoptosis via both p53-dependent and p53-independent mechanisms. The direct targets in the p53-independent pathway remain enigmatic; however, the induction of this pathway does not require the transactivation domain of E2F1. Using cells that are defective in p53 activation, we show that E2F1 potently represses the expression of Mcl-1--an anti-apoptotic Bcl-2 family member whose depletion results in apoptosis. We also show that this transcriptional repression is direct and dependent upon E2F1's DNA-binding domain, but does not require the transactivation domain of E2F1. Consistent with this DNA binding requirement of E2F1 to repress Mcl-1, we show that E2F1 binds to the Mcl-1 promoter both in vitro and in vivo, and have identified the DNA element (-143/-117) within this promoter that is required for E2F1 binding and repression. Additionally, cell lines constitutively expressing Mcl-1 are resistant to E2F1-mediated apoptosis--suggesting that Mcl-1 downregulation is a necessary event in the p53-independent apoptotic process. Thus, we identify a p53 family-independent mechanism of E2F1-induced apoptosis in which E2F1 directly represses Mcl-1 expression.
Assuntos
Proteínas de Ciclo Celular , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Apoptose , Sequência de Bases , Western Blotting , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Sequência Consenso/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Genes Reporter , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Elementos de Resposta/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Non-receptor and receptor tyrosine kinases, such as Src and EGF receptor (EGFR), are major inducers of vascular endothelial growth factor (VEGF), one of the most potent mediators of angiogenesis. While tyrosine kinases signal through multiple pathways, signal transducer and activation of transcription 3 (Stat3) is a point of convergence for many of these and is constitutively activated with high frequency in a wide range of cancer cells. Here, we show that VEGF expression correlates with Stat3 activity in diverse human cancer cell lines. An activated Stat3 mutant (Stat3C) up-regulates VEGF expression and stimulates tumor angiogenesis. Stat3C-induced VEGF up-regulation is abrogated when a Stat3-binding site in the VEGF promoter is mutated. Furthermore, interrupting Stat3 signaling with dominant-negative Stat3 protein or Stat3 antisense oligonucleotide in tumor cells down-regulates VEGF expression. Consistent with an important role of Stat3 in VEGF up-regulation induced by various oncogenic tyrosine kinases, v-Src-mediated VEGF expression is inhibited when Stat3 signaling is blocked. Moreover, chromatin immunoprecipitation assays indicate that Stat3 protein binds to the VEGF promoter in vivo and mutation of a Stat3-binding site in the VEGF promoter abrogates v-Src-induced VEGF promoter activity. These studies provide evidence that the VEGF gene is regulated directly by Stat3 protein, and indicate that Stat3 represents a common molecular target for blocking angiogenesis induced by multiple signaling pathways in human cancers.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfocinas/metabolismo , Neovascularização Patológica , Transativadores/metabolismo , Regulação para Cima , Células 3T3 , Animais , Sequência de Bases , Linhagem Celular Transformada , Colágeno , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Combinação de Medicamentos , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Crescimento Endotelial/genética , Humanos , Laminina , Linfocinas/genética , Camundongos , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Proteína Oncogênica pp60(v-src)/metabolismo , Regiões Promotoras Genéticas/genética , Proteoglicanas , Fator de Transcrição STAT3 , Transativadores/antagonistas & inibidores , Transativadores/genética , Transfecção , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Overexpression of anti-apoptotic Bcl-2 family members and deregulation of the pathways that regulate pro-apoptotic family members have been observed in non-small cell lung cancers (NSCLC). Previous reports have identified both Bcl-2 and Bcl-x(L) proteins as survival factors in lung cancer cells since reductions in these proteins can induce apoptosis and sensitize lung cancer cells to apoptosis induced by chemotherapy agents. Myeloid cell leukemia-1 (Mcl-1), another member of the Bcl-2 family, has been found to be a critical survival factor in hematopoietic cells, yet little data exists for a role of Mcl-1 in human lung cancers. We used NSCLC cell lines to explore how Mcl-1 levels affect lung cancer cell survival and studied tumors from patients to determine expression patterns of Mcl-1. NSCLC cells express abundant Mcl-1 protein and depletion of Mcl-1 levels by antisense Mcl-1 oligonucleotides induces apoptosis in A549 and H1299 lung cancer cells. Reduction in Mcl-1 levels can sensitize lung cancer cells to apoptosis induced by cytotoxic agents as well as by ionizing radiation. Lung cancer cells overexpressing Mcl-1 are less sensitive to apoptosis induced by chemotherapeutic agents, ZD1839 (an inhibitor of EGFR tyrosine kinase) and Bcl-2 or Bcl-x(L) antisense oligonucleotides. We find that epidermal growth factor (EGF) can enhance Mcl-1 protein levels in an ERK-dependent manner. Signal transduction agents that reduce Mcl-1 levels correlated with their individual ability to induce apoptosis in lung cancer cells. Finally, NSCLC tumors taken directly from patients have elevated levels of Mcl-1 protein compared with normal adjacent lung tissue. Therefore, agents that target Mcl-1 can induce apoptosis and sensitize cells to apoptosis induced by cytotoxic agents. Mcl-1 protein is overexpressed in a subset of human NSCLC and enhanced levels of Mcl-1 may protect lung cancer cells from death induced by a variety of pro-apoptotic stimuli.
Assuntos
Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Neoplasias Pulmonares/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinazolinas/farmacologia , Tolerância a Radiação , Ativação Transcricional , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/genéticaRESUMO
INTRODUCTION: Interleukin-6 (IL-6) can activate downstream signaling pathways in lung cancer cells, such as the STAT3 pathway, and is reported to be produced by tumor cells with activating EGFR mutations. We examined IL-6/STAT3 in lung cancer tumor tissues and the effects of siltuximab, a neutralizing antibody to human IL-6, in mouse models of lung cancer. METHODS: IL-6 and STAT3 activation levels were compared with tumor histology and presence of KRAS mutations in snap-frozen, non-small-cell lung cancer tumors. The effects of siltuximab alone or in combination with erlotinib were examined in mouse xenograft models constructed using three cell line xenograft models and one primary explant mouse model. We examined the influence of cancer-associated fibroblasts (CAFs) on tumor growth and siltuximab effects. RESULTS: IL-6 levels were higher in tumors of squamous cell versus adenocarcinoma histology and were not associated with presence of KRAS mutations. Tyrosine phosphorylation status of STAT3 did not correlate with tumor IL-6 levels. Serine phosphorylation of STAT3 was correlated with KRAS mutation status. Both tumor and stromal cells contributed to total IL-6 within tumors. Siltuximab had minimal effect as a single agent in xenografts with tumor cells alone; however, in models coadministered with CAFs, siltuximab had more potent effects on tumor inhibition. We observed no effects of combined erlotinib and siltuximab. CONCLUSIONS: IL-6 is elevated in subsets of human NSCLCs, especially with squamous cell histology. Tumors supported by stromal production of IL-6 seem to be the most vulnerable to tumor growth inhibition by siltuximab.
Assuntos
Adenocarcinoma/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/química , Adenocarcinoma/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Cloridrato de Erlotinib , Feminino , Fibroblastos , Humanos , Interleucina-6/análise , Interleucina-6/imunologia , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Quinazolinas/administração & dosagem , Fator de Transcrição STAT3/metabolismo , Serina/metabolismo , Tirosina/metabolismo , Proteínas ras/genéticaRESUMO
DDR2 mutations occur in approximately 4% of lung squamous cell cancer (SCC) where the tyrosine kinase inhibitor dasatinib has emerged as a new therapeutic option. We found that ERK and AKT phosphorylation was weakly inhibited by dasatinib in DDR2-mutant lung SCC cells, suggesting that dasatinib inhibits survival signals distinct from other oncogenic receptor tyrosine kinases (RTK) and/or compensatory signals exist that dampen dasatinib activity. To gain better insight into dasatinib's action in these cells, we assessed altered global tyrosine phosphorylation (pY) after dasatinib exposure using a mass spectrometry-based quantitative phosphoproteomics approach. Overlaying protein-protein interaction relationships upon this dasatinib-regulated pY network revealed decreased phosphorylation of Src family kinases and their targets. Conversely, dasatinib enhanced tyrosine phosphorylation in a panel of RTK and their signaling adaptor complexes, including EGFR, MET/GAB1, and IGF1R/IRS2, implicating a RTK-driven adaptive response associated with dasatinib. To address the significance of this observation, these results were further integrated with results from a small-molecule chemical library screen. We found that dasatinib combined with MET and insulin-like growth factor receptor (IGF1R) inhibitors had a synergistic effect, and ligand stimulation of EGFR and MET rescued DDR2-mutant lung SCC cells from dasatinib-induced loss of cell viability. Importantly, we observed high levels of tyrosine-phosphorylated EGFR and MET in a panel of human lung SCC tissues harboring DDR2 mutations. Our results highlight potential RTK-driven adaptive-resistant mechanisms upon DDR2 targeting, and they suggest new, rationale cotargeting strategies for DDR2-mutant lung SCC.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Neoplasias de Células Escamosas/tratamento farmacológico , Pirimidinas/administração & dosagem , Receptores Proteína Tirosina Quinases/genética , Receptores Mitogênicos/genética , Tiazóis/administração & dosagem , Imunidade Adaptativa/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dasatinibe , Receptores com Domínio Discoidina , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/imunologia , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Mutação , Neoplasias de Células Escamosas/imunologia , Neoplasias de Células Escamosas/patologia , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Mitogênicos/metabolismo , Receptores de Somatomedina/antagonistas & inibidores , Transdução de Sinais/genéticaRESUMO
A growing number of gene mutations, which are recognized as cancer drivers, can be successfully targeted with drugs. The redundant and dynamic nature of oncogenic signaling networks and complex interactions between cancer cells and the microenvironment, however, can cause drug resistance. While these challenges can be addressed by developing drug combinations or polypharmacology drugs, this benefits greatly from a detailed understanding of the proteome-wide target profiles. Using mass spectrometry-based chemical proteomics, we report the comprehensive characterization of the drug-protein interaction networks for the multikinase inhibitors dasatinib and sunitinib in primary lung cancer tissue specimens derived from patients. We observed in excess of 100 protein kinase targets plus various protein complexes involving, for instance, AMPK, TBK1 (sunitinib), and ILK (dasatinib). Importantly, comparison with lung cancer cell lines and mouse xenografts thereof showed that most targets were shared between cell lines and tissues. Several targets, however, were only present in tumor tissues. In xenografts, most of these proteins were of mouse origin suggesting that they originate from the tumor microenvironment. Furthermore, intersection with subsequent global phosphoproteomic analysis identified several activated signaling pathways. These included MAPK, immune, and integrin signaling, which were affected by these drugs in both cancer cells and the microenvironment. Thus, the combination of chemical and phosphoproteomics can generate a systems view of proteins, complexes, and signaling pathways that are simultaneously engaged by multitargeted drugs in cancer cells and the tumor microenvironment. This may allow for the design of novel anticancer therapies that concurrently target multiple tumor compartments.
Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , Modelos Moleculares , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multiple studies from independent groups find evidence for signal transducer and activator of transcription 3 (Stat3) activation in nearly 50% of lung cancers, suggesting a functional role for this target in subsets of lung cancer. On the basis of the existing evidence, we hypothesized that bioavailable curcuminoid complex may modulate lung carcinogenesis, primarily by inhibiting Stat3 activation. With the safety of this being botanically well established, the objective of these studies was to test our hypothesis in vitro and in vivo in an effort to inform the design of a phase II chemoprevention trial in former smokers. We treated non-tumor-derived, normal (but immortalized) human bronchial epithelial cells (AALE) (Lundberg et al., 2002; Pillai et al., 2011) and lung adenocarcinoma-derived cells (H441) with bioactive curcumin C3 complex. Asynchronous cells in each case were treated with curcumin for 24 h, followed by immunoblotting for Stat3 and activated Stat3-P, prior signal of which was used for normalization. We also completed a preclinical trial in which 12 mice were randomly divided into three groups and subjected to 3 days or 9 days of curcumin intraperitoneal injections, followed by analysis of lung tissues for Stat3-P changes and growth suppressive effects of the curcumin. The growth suppressive effects were measured using Cyclin D1 and the replicative helicase subunit, Mcm2, as surrogates for the proliferative capacity of the tissues. In-vitro studies with curcuminoid complex demonstrated that the activity of Stat3 in both normal bronchoepithelial cells and lung cancer-derived cells is sensitive to curcumin exposure. In a dose-dependent manner, curcumin treatment resulted in significant suppression of Stat3 phosphorylation and reduction in the proliferative capacity of both cell types. In the preclinical trial with rodent models, curcumin reduced Stat3-P and the proliferative markers CycD1 and Mcm2 in mice lung tissues in vivo. These culture and preclinical studies indicate that the activity of the Stat3 pathway can be suppressed by curcumin treatment, concomitant with a reduction in cell proliferation, supporting our hypothesis that inhibition of the Stat3 pathway represents at least one important mechanism by which curcumin elicits its effects on the bronchoepithelium. These data provide a rationale for the use of curcumin as a promising chemopreventive agent in high-risk populations such as former smokers.
Assuntos
Curcumina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias Pulmonares/prevenção & controle , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Linhagem Celular , Feminino , Humanos , Camundongos , Camundongos Nus , Distribuição AleatóriaRESUMO
Members of the signal transducer and activator of transcription (STAT) family of transcription factors are potential targets for the treatment and prevention of cancers including non-small-cell lung cancer. STAT proteins can be phosphorylated and activated by diverse upstream kinases including cytokine receptors and tyrosine kinases. We examined STAT protein activation in lung cancer cell lines including those with activating mutations in the EGFR and examined upstream kinases responsible for STAT3 phosphorylation and activation using small molecules, antibodies, and RNA interference. We found more pronounced STAT3 activation in cells with activating EGFR mutations, yet inhibition of EGFR activity had no effect on STAT3 activation. Inhibition of JAK1 with small molecules or RNA interference resulted in loss of STAT3 tyrosine phosphorylation and inhibition of cell growth. An interleukin-6 neutralizing antibody, siltuximab (CNTO 328) could inhibit STAT3 tyrosine phosphorylation in a cell-dependent manner. Siltuximab could completely inhibit STAT3 tyrosine phosphorylation in H1650 cells, and this resulted in inhibition of lung cancer cell growth in vivo. Combined EGFR inhibition with erlotinib and siltuximab resulted in dual inhibition of both tyrosine and serine STAT3 phosphorylation, more pronounced inhibition of STAT3 transcriptional activity, and translated into combined effects on lung cancer growth in a mouse model. Our results suggest that JAK1 is responsible for STAT3 activation in lung cancer cells and that indirect attacks on JAK1-STAT3 using an IL-6 neutralizing antibody with or without EGFR inhibition can inhibit lung cancer growth in lung cancer subsets.
Assuntos
Anticorpos Neutralizantes/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Interleucina-6/imunologia , Janus Quinase 1/metabolismo , Neoplasias Pulmonares/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Interleucina-6/antagonistas & inibidores , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/genética , Camundongos , Camundongos Nus , Mutação , Fosforilação , Interferência de RNA , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Targeted agents such as tyrosine kinase inhibitors have been extensively studied in preclinical systems and in advanced-stage patients. Little is known about levels of kinase inhibitors found in tumors as opposed to plasma. Similarly, effects of inhibitors on tumor signaling pathways in patient-based materials are unclear. To explore these questions, we conducted a trial of a brief course of preoperative gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, in early-stage non-small cell lung cancer. METHODS: Patient with early-stage non-small cell lung cancer received 4 weeks of gefitinib 250 mg daily before surgical resection. Pre- and posttreatment computerized tomography scans and positron emission tomography scans were used to assess clinical response. Gefitinib and surgical toxicity were evaluated. Tumor tissue was evaluated for gefitinib levels and was compared with plasma gefitinib levels. Activated signaling molecules including EGFR, STAT3, ERK, and AKT were examined in surgically resected tumor tissue. RESULTS: Twenty-three patients participated in the study, and all had surgical resection of tumors. No toxicities unrelated to known effects of gefitinib or surgery were encountered. Twenty-two patients had stable disease, and one had progression in tumor size. There was no correlation with positron emission tomography response and computerized tomography response. Tumor levels of gefitinib were approximately 40-fold higher than plasma levels, indicating potential tumor concentration of gefitinib. Tyrosine phosphorylated STAT3 was abundant in the surgically resected tumor tissue, indicating potential role in primary resistance in vivo. CONCLUSIONS: This study confirms previous preclinical observations that tumor tissues concentrate gefitinib. Persistent STAT3 may be leading to primary resistance to EGFR inhibitors in vivo.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Idoso , Antineoplásicos/farmacocinética , Western Blotting , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Estadiamento de Neoplasias , Fosforilação/efeitos dos fármacos , Projetos Piloto , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacocinética , Fator de Transcrição STAT3/metabolismo , Taxa de Sobrevida , Distribuição Tecidual , Tirosina/metabolismoRESUMO
PURPOSE: Src family kinase (SFK) proteins are frequently activated in cancer and can coordinate tumor cell growth, survival, invasion, and angiogenesis. Given the importance of SFK signaling in cancer, known cooperation between SFK and epidermal growth factor receptor (EGFR) signaling, and efficacy of EGFR inhibitors, we performed a phase I trial combining dasatinib, an SFK and multikinase inhibitor, with erlotinib, an EGFR inhibitor, in patients with advanced non-small-cell lung cancer. PATIENTS AND METHODS: Patients received erlotinib for 1 week before addition of dasatinib; pharmacokinetics were performed after weeks 1 and 2. Four cohorts were examined, including twice-daily and daily dasatinib dosing. Responses were assessed after 8 weeks. Plasma levels of angiogenic markers (vascular endothelial growth factor [VEGF], interleukin-8, and basic fibroblast growth factor [bFGF]) were determined before and during treatment. RESULTS: Thirty-four patients were enrolled. The average duration of treatment was 73 days. The main adverse events include GI (diarrhea, anorexia, and nausea), skin rash, cytopenias, pleural effusions, and fatigue. No effect of escalating doses of dasatinib was observed on erlotinib pharmacokinetics. Two partial responses and one bone response were observed, and the disease control rate was 63%. Reductions in plasma VEGF and bFGF were observed, and reductions in VEGF correlated with disease control. CONCLUSION: The combination of erlotinib and dasatinib is tolerable, with adverse effects consistent with the two agents. Disease control and inhibition of plasma angiogenesis markers were observed. Personalized strategies for deployment of SFK should receive further attention.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Farmacológicos/sangue , Dasatinibe , Esquema de Medicação , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Feminino , Fator 2 de Crescimento de Fibroblastos/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/efeitos adversos , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Quinazolinas/administração & dosagem , Quinazolinas/efeitos adversos , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , Tiazóis/administração & dosagem , Tiazóis/efeitos adversos , Tiazóis/farmacocinética , Tiazóis/farmacologia , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
Src family kinases (SFKs) have a critical role in cell adhesion, invasion, proliferation, survival, and angiogenesis during tumor development. SFKs comprise nine family members that share similar structure and function. Overexpression or high activation of SFKs occurs frequently in tumor tissues and they are central mediators in multiple signaling pathways that are important in oncogenesis. SFKs can interact with tyrosine kinase receptors, such as EGFR and the VEGF receptor. SFKs can affect cell proliferation via the Ras/ERK/MAPK pathway and can regulate gene expression via transcription factors such as STAT molecules. SFKs can also affect cell adhesion and migration via interaction with integrins, actins, GTPase-activating proteins, scaffold proteins, such as p130(CAS) and paxillin, and kinases such as focal adhesion kinases. Furthermore, SFKs can regulate angiogenesis via gene expression of angiogenic growth factors, such as fibroblast growth factor, VEGF, and interleukin 8. On the basis of these important findings, small-molecule SFK inhibitors have been developed and are undergoing early phase clinical testing. In preclinical studies these agents can suppress tumor growth and metastases. The agents seem to be safe in humans and could add to the therapeutic arsenal against subsets of cancers.