RESUMO
New disease specific biomarkers, especially for cancer, are urgently needed to improve individual diagnosis, prognosis, and treatment selection, that is, for personalized medicine. Genetic mutations that affect protein function drive cancer. Therefore, the detection of such mutations represents a source of cancer specific biomarkers. Here we confirm the implementation of the mutant protein specific immuno-SRM (where SRM is selective reaction monitoring) mass spectrometry method of RAS proteins reported by Wang et al. [Proc. Natl. Acad. Sci. USA 2011, 108, 2444-2449], which exploits an antibody to simultaneously capture the different forms of the target protein and the resolving power and sensitivity of LC-MS/MS and improve the technique by using a more sensitive mass spectrometer. The mutant form G12D was quantified by SRM on a QTRAP 5500 mass spectrometer and the MIDAS workflow was used to confirm the sequence of the targeted peptides. This assay has been applied to quantify wild type and mutant RAS proteins in patient tumors, xenografted human tissue, and benign human epidermal tumors at high sensitivity. The limit of detection for the target proteins was as low as 12 amol (0.25 pg). It requires low starting amounts of tissue (ca.15 mg) that could be obtained from a needle aspiration biopsy. The described strategy could find application in the clinical arena and be applied to the study of expression of protein variants in disease.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/química , Espectrometria de Massas/métodos , Proteínas Mutantes/análise , Proteínas de Neoplasias/análise , Neoplasias Pancreáticas/química , Animais , Calibragem , Neoplasias Colorretais/genética , Feminino , Humanos , Imunoprecipitação , Modelos Lineares , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/genética , Peptídeos/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise Serial de Tecidos , Proteínas ras/análise , Proteínas ras/genéticaRESUMO
PURPOSE: Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%, supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor. EXPERIMENTAL DESIGN: Transcriptional profiling and gene set enrichment analysis (baseline and posttreatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, (18)F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts. RESULTS: In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G(1), and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups. CONCLUSION: These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzimidazóis/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Animais , Apoptose/efeitos dos fármacos , Benzimidazóis/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Perfilação da Expressão Gênica , Inibidores de Histona Desacetilases/administração & dosagem , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ligantes , Camundongos , Camundongos Nus , Ressonância Magnética Nuclear Biomolecular , Tomografia por Emissão de Pósitrons , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais/efeitos dos fármacos , Vorinostat , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismoRESUMO
Mutant K-ras activity leads to the activation of the RAS/RAF/MEK/ERK pathway in approximately 44% of colorectal cancer (CRC) tumors. Accordingly, several inhibitors of the MEK pathway are under clinical evaluation in several malignancies including CRC. The aim of this study was to develop and characterize predictive biomarkers of response to the MEK1/2 inhibitor AZD6244 in CRC in order to maximize the clinical utility of this agent. Twenty-seven human CRC cell lines were exposed to AZD6244 and classified according to the IC(50) value as sensitive (≤ 0.1 µmol/L) or resistant (>1 µmol/L). All cell lines were subjected to immunoblotting for effector proteins, K-ras/BRAF mutation status, and baseline gene array analysis. Further testing was done in cell line xenografts and K-ras mutant CRC human explants models to develop a predictive genomic classifier for AZD6244. The most sensitive and resistant cell lines were subjected to differential gene array and pathway analyses. Members of the Wnt signaling pathway were highly overexpressed in cell lines resistant to AZD6244 and seem to be functionally involved in mediating resistance by shRNA knockdown studies. Baseline gene array data from CRC cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which predicted with 71% accuracy which of a test set of patient-derived K-ras mutant CRC explants would respond to AZD6244, providing the basis for a patient-selective clinical trial. These results also indicate that resistance to AZD6244 may be mediated, in part, by the upregulation of the Wnt pathway, suggesting potential rational combination partners for AZD6244 in CRC.
Assuntos
Benzimidazóis/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Algoritmos , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Camundongos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas Proto-Oncogênicas p21(ras) , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The effect of vascular endothelial growth factor (VEGF) ligands and cediranib on tumor cell proliferation, migration, and invasion was determined. It has recently been suggested that autocrine signaling through the VEGF receptor (VEGFR) pathway may play a role in tumor cell survival, invasion, and migration. The purpose of the present study was to determine the expression of VEGFRs and VEGFR ligands in a panel of gastrointestinal carcinoma cells. Additionally, we evaluated the effects of VEGF autocrine signaling on tumor cell proliferation, migration, and invasion utilizing cediranib (AZD2171), a pan-VEGFR inhibitor. Five colorectal, three pancreatic, and two hepatocellular carcinoma cell lines were screened for VEGFR and VEGF expression by several methods. Expression of VEGFR-1 and VEGFR-3 was cell line-dependent, whereas VEGFR-2 was not detected. Secretion of VEGF-A was detected in the supernatants of all cell lines whereas VEGF-C secretion was detected in the Panc-1, MiaPaca2, and Hep1 cells only. Tumor cells showed increased migratory activity, but not proliferation, when stimulated with VEGFs. The pan-VEGFR inhibitor cediranib (100 nmol/L) inhibited tumor cell migration and invasion, with no effects on proliferation. Cediranib decreased VEGFR-1 and VEGFR-3 phosphorylation as well as activation of downstream effectors. VEGFR-1 and VEGFR-3 expression was detected in all the gastrointestinal carcinoma cells evaluated. Although activation of the VEGF pathway did not affect cell proliferation, our data indicate that this pathway seems to play a role in tumor cell migration and invasion in these cell lines. Therefore, inhibition of VEGFR by cediranib may represent a clinically relevant treatment option for gastrointestinal tumors.