RESUMO
The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/genética , Sequenciamento do Exoma , Mutação , Receptor ErbB-2/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/terapia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Variações do Número de Cópias de DNA , Feminino , Xenoenxertos , Humanos , Polimorfismo de Nucleotídeo Único , Neoplasias do Colo do Útero/patologiaRESUMO
All cellular proteins are derived from preexisting ones by natural selection. Because of the random nature of this process, many potentially useful protein structures never arose or were discarded during evolution. Here, we used a single round of genetic selection in mouse cells to isolate chemically simple, biologically active transmembrane proteins that do not contain any amino acid sequences from preexisting proteins. We screened a retroviral library expressing hundreds of thousands of proteins consisting of hydrophobic amino acids in random order to isolate four 29-aa proteins that induced focus formation in mouse and human fibroblasts and tumors in mice. These proteins share no amino acid sequences with known cellular or viral proteins, and the simplest of them contains only seven different amino acids. They transformed cells by forming a stable complex with the platelet-derived growth factor ß receptor transmembrane domain and causing ligand-independent receptor activation. We term this approach de novo selection and suggest that it can be used to generate structures and activities not observed in nature, create prototypes for novel research reagents and therapeutics, and provide insight into cell biology, transmembrane protein-protein interactions, and possibly virus evolution and the origin of life.
Assuntos
Proteínas de Membrana/genética , Oncogenes/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Linhagem Celular , Transformação Celular Neoplásica , Evolução Molecular , Feminino , Fibroblastos/metabolismo , Biblioteca Gênica , Humanos , Interleucina-3/metabolismo , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , RetroviridaeRESUMO
Somatic mutations in kinase genes are associated with sensitivity of solid tumors to kinase inhibitors, but patients with metastatic cancer eventually develop disease progression. In EGFR mutant lung cancer, modeling of acquired resistance (AR) with drug-sensitive cell lines has identified clinically relevant EGFR tyrosine kinase inhibitor (TKI) resistance mechanisms such as the second-site mutation, EGFR T790M, amplification of the gene encoding an alternative kinase, MET, and epithelial-mesenchymal transition (EMT). The full spectrum of DNA changes associated with AR remains unknown. We used next-generation sequencing to characterize mutational changes associated with four populations of EGFR mutant drug-sensitive and five matched drug-resistant cell lines. Comparing resistant cells with parental counterparts, 18-91 coding SNVs/indels were predicted to be acquired and 1-27 were lost; few SNVs/indels were shared across resistant lines. Comparison of two related parental lines revealed no unique coding SNVs/indels, suggesting that changes in the resistant lines were due to drug selection. Surprisingly, we observed more CNV changes across all resistant lines, and the line with EMT displayed significantly higher levels of CNV changes than the other lines with AR. These results demonstrate a framework for studying the evolution of AR and provide the first genome-wide spectrum of mutations associated with the development of cellular drug resistance in an oncogene-addicted cancer. Collectively, the data suggest that CNV changes may play a larger role than previously appreciated in the acquisition of drug resistance and highlight that resistance may be heterogeneous in the context of different tumor cell backgrounds.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Mutação INDEL , Neoplasias Pulmonares/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Polimorfismo de Nucleotídeo Único , Inibidores de Proteínas Quinases/farmacologia , Análise de Sequência de DNARESUMO
Extensive evidence implicates the urokinase plasminogen activator receptor (uPAR) in tumor growth, invasion, and metastasis. Recent studies have substantiated the importance of the interaction between uPAR and the extracellular matrix protein vitronectin (VN) for the signaling activity of the receptor in vitro, however, the possible relevance of this interaction for the activity of uPAR in tumor growth and metastasis has not been assessed. We generated a panel of HEK293 cell lines expressing mouse uPAR (muPAR(WT)), an uPAR mutant specifically deficient in VN binding (muPAR(W32A)), and a truncation variant (muPAR(ΔD1)) deficient in both VN and uPA binding. In vitro cells expressing muPAR(WT) display increased cell adhesion, spreading, migration, and proliferation associated with increased p130Cas and MAPK signaling. Disruption of VN binding or ablation of both VN and uPA binding specifically abrogates these activities of uPAR. When xenografted into SCID (severe combined immunodeficiency) mice, the expression of muPAR(WT), but not muPAR(W32A) or muPAR(ΔD1), accelerates tumor development, demonstrating that VN binding is responsible for the tumor-promoting activity of uPAR in vivo. In an orthotopic xenograft model using MDA-MB-231 cells in RAG1(-/-)/VN(-/-) mice, we document that host deficiency in VN strongly impairs tumor formation. These 2 lines of in vivo experimentation independently demonstrate an important role for VN in tumor growth even if the uPAR dependence of the effect in the MDA-MB-231 model remains to be ascertained.
Assuntos
Proliferação de Células , Neoplasias/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Vitronectina/metabolismo , Vitronectina/fisiologia , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Transplante Heterólogo , Carga Tumoral/genética , Vitronectina/genéticaRESUMO
PURPOSE: The EGFR tyrosine kinase inhibitors (TKIs), erlotinib and afatinib, have transformed the treatment of advanced EGFR-mutant lung adenocarcinoma. However, almost all patients who respond develop acquired resistance on average approximately 1 year after starting therapy. Resistance is commonly due to a secondary mutation in EGFR (EGFR(T790M)). We previously found that the combination of the EGFR TKI afatinib and the EGFR antibody cetuximab could overcome EGFR(T790M)-mediated resistance in preclinical models. This combination has shown a 29% response rate in a clinical trial in patients with acquired resistance to first-generation TKIs. An outstanding question is whether this regimen is beneficial when used as first-line therapy. EXPERIMENTAL DESIGN: Using mouse models of EGFR-mutant lung cancer, we tested whether the combination of afatinib plus cetuximab delivered upfront to mice with TKI-naïve EGFR(L858R)-induced lung adenocarcinomas delayed tumor relapse and drug-resistance compared with single-agent TKIs. RESULTS: Afatinib plus cetuximab markedly delayed the time to relapse and incidence of drug-resistant tumors, which occurred in only 63.6% of the mice, in contrast to erlotinib or afatinib treatment where 100% of mice developed resistance. Mechanisms of tumor escape observed in afatinib plus cetuximab resistant tumors include the EGFR(T790M) mutation and Kras mutations. Experiments in cell lines and xenografts confirmed that the afatinib plus cetuximab combination does not suppress the emergence of EGFR(T790M). CONCLUSIONS: These results highlight the potential of afatinib plus cetuximab as an effective treatment strategy for patients with TKI-naïve EGFR-mutant lung cancer and indicate that clinical trial development in this area is warranted.
Assuntos
Adenocarcinoma/tratamento farmacológico , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Afatinib , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Quimioterapia Combinada/métodos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Mutação/efeitos dos fármacos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Oncogene-induced DNA methylation-mediated transcriptional silencing of tumor suppressors frequently occurs in cancer, but the mechanism and functional role of this silencing in oncogenesis are not fully understood. Here, we show that oncogenic epidermal growth factor receptor (EGFR) induces silencing of multiple unrelated tumor suppressors in lung adenocarcinomas and glioblastomas by inhibiting the DNA demethylase TET oncogene family member 1 (TET1) via the C/EBPα transcription factor. After oncogenic EGFR inhibition, TET1 binds to tumor suppressor promoters and induces their re-expression through active DNA demethylation. Ectopic expression of TET1 potently inhibits lung and glioblastoma tumor growth, and TET1 knockdown confers resistance to EGFR inhibitors in lung cancer cells. Lung cancer samples exhibited reduced TET1 expression or TET1 cytoplasmic localization in the majority of cases. Collectively, these results identify a conserved pathway of oncogenic EGFR-induced DNA methylation-mediated transcriptional silencing of tumor suppressors that may have therapeutic benefits for oncogenic EGFR-mediated lung cancers and glioblastomas.
Assuntos
Receptores ErbB/genética , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma de Pulmão , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/enzimologia , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Sistema de Sinalização das MAP Quinases , Oxigenases de Função Mista/metabolismo , Mutação , Oncogenes , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
Metastatic EGFR-mutant lung cancers are sensitive to the first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) gefitinib, erlotinib, and afatinib, but resistance develops. Acquired resistance to gefitinib or erlotinib occurs most commonly (>50%) via the emergence of a second-site EGFR mutation, T790M. Two strategies to overcome T790M-mediated resistance are dual inhibition of EGFR with afatinib plus the anti-EGFR antibody cetuximab (A+C), or mutant-specific EGFR inhibition with AZD9291. A+C and AZD9291 are now also being tested as first-line therapies, but whether these therapies will extend progression-free survival or induce more aggressive forms of resistance in this setting remains unknown. We modeled resistance to multiple generations of anti-EGFR therapies preclinically to understand the effects of sequential treatment with anti-EGFR agents on drug resistance and determine the optimal order of treatment. Using a panel of erlotinib/afatinib-resistant cells, including a novel patient-derived cell line (VP-2), we found that AZD9291 was more potent than A+C at inhibiting cell growth and EGFR signaling in this setting. Four of four xenograft-derived A+C-resistant cell lines displayed in vitro and in vivo sensitivity to AZD9291, but four of four AZD9291-resistant cell lines demonstrated cross-resistance to A+C. Addition of cetuximab to AZD9291 did not confer additive benefit in any preclinical disease setting. This work, emphasizing a mechanistic understanding of the effects of therapies on tumor evolution, provides a framework for future clinical trials testing different treatment sequences. This paradigm is applicable to other tumor types in which multiple generations of inhibitors are now available.
Assuntos
Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular , Mutação/genética , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Afatinib , Idoso , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Camundongos Nus , Quinazolinas/farmacologia , Quinazolinas/uso terapêuticoRESUMO
Resistance to targeted therapies has emerged as a major hurdle for the successful use of drugs in the clinic. Therefore, understanding the underlying molecular mechanisms of drug resistance is crucial for the identification of strategies to prevent and overcome it. Given the defined nature of the oncogenic lesions present in genetically engineered mouse models (GEMMs) and the relative ease of sample collection and analysis, they are ideal systems in which to recapitulate the response and subsequent emergence of resistance to targeted therapies. When agents that are very effective at eradicating tumors are used in GEMMs, obtaining drug-resistant tumors can be a challenge. One approach to generating such tumors is the use of a suboptimal intermittent dosing strategy to treat the animals, which allows for periods of tumor growth and progression in the absence of drug. This intermittent dosing strategy has been used successfully to study resistance to the tyrosine kinase erlotinib in lung cancer models and is described here. Although this protocol is specific for this experimental system, the concepts and general design can be adapted for use with GEMMs of other cancers.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Animais , Relação Dose-Resposta a Droga , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Patients with EGFR-mutant lung adenocarcinomas (LUADs) who initially respond to first-generation tyrosine kinase inhibitors (TKIs) develop resistance to these drugs. A combination of the irreversible TKI afatinib and the EGFR antibody cetuximab can be used to overcome resistance to first-generation TKIs; however, resistance to this drug combination eventually emerges. We identified activation of the mTORC1 signaling pathway as a mechanism of resistance to dual inhibition of EGFR in mouse models. The addition of rapamycin reversed resistance in vivo. Analysis of afatinib-plus-cetuximab-resistant biopsy specimens revealed the presence of genomic alterations in genes that modulate mTORC1 signaling, including NF2 and TSC1. These findings pinpoint enhanced mTORC1 activation as a mechanism of resistance to afatinib plus cetuximab and identify genomic mechanisms that lead to activation of this pathway, revealing a potential therapeutic strategy for treating patients with resistance to these drugs.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma de Pulmão , Afatinib , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Linhagem Celular Tumoral , Cetuximab , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Nus , Camundongos Transgênicos , Mutação , Quinazolinas/administração & dosagem , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: Approximately half of EGFR-mutant non-small cell lung cancer (NSCLC) patients treated with small-molecule EGFR kinase inhibitors develop drug resistance associated with the EGF receptor (EGFR) T790M "gatekeeper" substitution, prompting efforts to develop covalent EGFR inhibitors, which can effectively suppress EGFR T790M in preclinical models. However, these inhibitors have yet to prove clinically efficacious, and their toxicity in skin, reflecting activity against wild-type EGFR, may limit dosing required to effectively suppress EGFR T790M in vivo. While profiling sensitivity to various kinase inhibitors across a large cancer cell line panel, we identified indolocarbazole compounds, including a clinically well-tolerated FLT3 inhibitor, as potent and reversible inhibitors of EGFR T790M that spare wild-type EGFR. These findings show the use of broad cancer cell profiling of kinase inhibitor efficacy to identify unanticipated novel applications, and they identify indolocarbazole compounds as potentially effective EGFR inhibitors in the context of T790M-mediated drug resistance in NSCLC. SIGNIFICANCE: EGFR-mutant lung cancer patients who respond to currently used EGFR kinase inhibitors invariably develop drug resistance, which is associated with the EGFR T790M resistance mutation in about half these cases. We unexpectedly identified a class of reversible potent inhibitors of EGFR T790M that do not inhibit wild-type EGFR, revealing a promising therapeutic strategy to overcome T790M-associated drug-resistant lung cancers.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/farmacologia , Substituição de Aminoácidos , Animais , Área Sob a Curva , Sequência de Bases , Carbazóis/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Cloridrato de Erlotinib , Humanos , Immunoblotting , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Interferência de RNA , Estaurosporina/análogos & derivados , Estaurosporina/farmacocinética , Estaurosporina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
EGF receptor (EGFR)-mutant lung cancers eventually become resistant to treatment with EGFR tyrosine kinase inhibitors (TKI). The combination of EGFR-TKI afatinib and anti-EGFR antibody cetuximab can overcome acquired resistance in mouse models and human patients. Because afatinib is also a potent HER2 inhibitor, we investigated the role of HER2 in EGFR-mutant tumor cells. We show in vitro and in vivo that afatinib plus cetuximab significantly inhibits HER2 phosphorylation. HER2 overexpression or knockdown confers resistance or sensitivity, respectively, in all studied cell line models. FISH analysis revealed that HER2 was amplified in 12% of tumors with acquired resistance versus only 1% of untreated lung adenocarcinomas. Notably, HER2 amplification and EGFR(T790M) were mutually exclusive. Collectively, these results reveal a previously unrecognized mechanism of resistance to EGFR-TKIs and provide a rationale to assess the status and possibly target HER2 in EGFR-mutant tumors with acquired resistance to EGFR-TKIs.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Receptor ErbB-2/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Afatinib , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cetuximab , Classe I de Fosfatidilinositol 3-Quinases , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Interferência de RNA , RNA Interferente Pequeno , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genéticaRESUMO
Intercellular junctions mediate adhesion and communication between adjoining cells. Although formed by different molecules, tight junctions (TJs) and adherens junctions (AJs) are functionally and structurally linked, but the signalling pathways behind this interaction are unknown. Here we describe a cell-specific mechanism of crosstalk between these two types of structure. We show that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4-beta-catenin transcriptional repressor complex. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of beta-catenin to the nucleus. These results offer a molecular basis for the link between AJs and TJs and explain why VE-cadherin inhibition may cause a marked increase in permeability.