RESUMO
We describe a mechanism of tumorigenesis mediated by kinase-dead BRAF in the presence of oncogenic RAS. We show that drugs that selectively inhibit BRAF drive RAS-dependent BRAF binding to CRAF, CRAF activation, and MEK-ERK signaling. This does not occur when oncogenic BRAF is inhibited, demonstrating that BRAF inhibition per se does not drive pathway activation; it only occurs when BRAF is inhibited in the presence of oncogenic RAS. Kinase-dead BRAF mimics the effects of the BRAF-selective drugs and kinase-dead Braf and oncogenic Ras cooperate to induce melanoma in mice. Our data reveal another paradigm of BRAF-mediated signaling that promotes tumor progression. They highlight the importance of understanding pathway signaling in clinical practice and of genotyping tumors prior to administering BRAF-selective drugs, to identify patients who are likely to respond and also to identify patients who may experience adverse effects.
Assuntos
Antineoplásicos/efeitos adversos , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismoRESUMO
BACKGROUND: Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors. METHODS: We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed. RESULTS: Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor. CONCLUSIONS: Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).
Assuntos
Carcinoma de Células Escamosas/genética , Genes ras , Indóis/uso terapêutico , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Neoplasias Cutâneas/genética , Sulfonamidas/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/administração & dosagem , Masculino , Camundongos , Pessoa de Meia-Idade , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Sulfonamidas/administração & dosagem , VemurafenibRESUMO
We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFß production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.
Assuntos
Imunomodulação/efeitos dos fármacos , Neuropilina-1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Linfócitos T Reguladores/imunologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
We previously showed how key pathways in cancer-related inflammation and Notch signaling are part of an autocrine malignant cell network in ovarian cancer. This network, which we named the "TNF network", has paracrine actions within the tumor microenvironment, influencing angiogenesis and the immune cell infiltrate.The aim of this study was to identify critical regulators in the signaling pathways of the TNF network in ovarian cancer cells that might be therapeutic targets. To achieve our aim, we used a systems biology approach, combining data from phospho-proteomic mass spectrometry and gene expression array analysis. Among the potential therapeutic kinase targets identified was the protein kinase Casein kinase II (CK2).Knockdown of CK2 expression in malignant cells by siRNA or treatment with the specific CK2 inhibitor CX-4945 significantly decreased Notch signaling and reduced constitutive cytokine release in ovarian cancer cell lines that expressed the TNF network as well as malignant cells isolated from high grade serous ovarian cancer ascites. The expression of the same cytokines was also inhibited after treatment with CX-4945 in a 3D organotypic model. CK2 inhibition was associated with concomitant inhibition of proliferative activity, reduced angiogenesis and experimental peritoneal ovarian tumor growth.In conclusion, we have identified kinases, particularly CK2, associated with the TNF network that may play a central role in sustaining the cytokine network and/or mediating its effects in ovarian cancer.
Assuntos
Inflamação/enzimologia , Neoplasias Ovarianas/patologia , Transdução de Sinais/imunologia , Animais , Caseína Quinase II/metabolismo , Linhagem Celular Tumoral , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Xenoenxertos , Humanos , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/imunologia , Proteômica/métodos , Biologia de Sistemas/métodos , Transcriptoma , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The cytokine IL6 has a number of tumor-promoting activities in human and experimental cancers, but its potential as an angiogenic agent has not been fully investigated. Here, we show that IL6 can directly induce vessel sprouting in the ex vivo aortic ring model, as well as endothelial cell proliferation and migration, with similar potency to VEGF. However, IL6-stimulated aortic ring vessel sprouts had defective pericyte coverage compared with VEGF-stimulated vessels. The mechanism of IL6 action on pericytes involved stimulation of the Notch ligand Jagged1 as well as angiopoietin2 (Ang2). When peritoneal xenografts of ovarian cancer were treated with an anti-IL6 antibody, pericyte coverage of vessels was restored. In addition, in human ovarian cancer biopsies, there was an association between levels of IL6 mRNA, Jagged1, and Ang2. Our findings have implications for the use of cancer therapies that target VEGF or IL6 and for understanding abnormal angiogenesis in cancers, chronic inflammatory disease, and stroke.
Assuntos
Interleucina-6/metabolismo , Interleucina-6/farmacologia , Neovascularização Patológica/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-6/genética , Proteína Jagged-1 , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular/métodos , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Técnicas de Cultura de Órgãos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Pericitos/efeitos dos fármacos , Pericitos/patologia , Ratos Wistar , Proteínas Serrate-Jagged , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas de Transporte Vesicular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Excess production of the proinflammatory IL6 has both local and systemic tumor-promoting activity in many cancers, including ovarian cancer. However, treatment of advanced ovarian cancer patients with a neutralizing IL6 antibody yielded little efficacy in a previous phase II clinical trial. Here, we report results that may explain this outcome, based on the finding that neutralizing antibodies to IL6 and STAT3 inhibition are sufficient to upregulate the EGFR pathway in high-grade serous and other ovarian cancer cells. Cell treatment with the EGFR inhibitor gefitinib abolished upregulation of the EGFR pathway. Combining neutralizing IL6 antibodies and gefitinib inhibited malignant cell growth in 2D and 3D culture. We found that ErbB-1 was localized predominantly in the nucleus of ovarian cancer cells examined, contrasting with plasma membrane localization in lung cancer cells. Treatment with anti-IL6, gefitinib, or their combination all led to partial restoration of ErbB-1 on the plasma membrane. In vivo experiments confirmed the effects of IL6 inhibition on the EGFR pathway and the enhanced activity of a combination of anti-IL6 antibodies and gefitinib on malignant cell growth. Taken together, our results offer a preclinical rationale to combine anti-IL6 and gefitinib to treat patients with advanced stage ovarian cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Receptores ErbB/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Animais , Anticorpos Neutralizantes/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Núcleo Celular , Sinergismo Farmacológico , Receptores ErbB/genética , Feminino , Gefitinibe , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Interleucina-6/fisiologia , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Transporte Proteico , Quinazolinas/administração & dosagem , Fator de Transcrição STAT3/metabolismo , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: ß-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event.
Assuntos
Anáfase , Biomarcadores Tumorais/biossíntese , Senescência Celular , Melanoma/metabolismo , Proteínas de Neoplasias/biossíntese , Nevo Pigmentado/metabolismo , Aneuploidia , Humanos , Melanoma/genética , Melanoma/patologia , Nevo Pigmentado/genética , Nevo Pigmentado/patologia , Telômero/genética , Telômero/metabolismo , Telômero/patologia , Células Tumorais CultivadasRESUMO
The small G-protein NRAS is mutated in 22% of human melanomas, whereas the related proteins KRAS and HRAS are mutated in only 2% and 1% of melanomas, respectively. We have developed a mouse model of melanoma in which Cre recombinase/LoxP technology is used to drive inducible expression of (G12V)KRAS in the melanocytic lineage. The mice develop skin hyperpigmentation, nevi, and tumors that bear many of the cardinal histopathology features and molecular characteristics of human melanoma. These tumors invade and destroy the underlying muscles and cells derived from them can grow as subcutaneous tumors and colonize the lungs of nude mice. These data establish that oncogenic KRAS can be a founder event in melanomagenesis.