RESUMO
KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.
Assuntos
Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , GTP Fosfo-Hidrolases/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Mutação , Neoplasias/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Feminino , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Ratos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with pulmonary tuberculosis develop pleural effusions with a high protein content. Pleural mesothelial adherens junctions promote mesothelial cell-cell adhesion and contribute to pleural integrity. In the present study we have investigated the effect of mycobacterium (BCG) on mesothelial cell adherens junction proteins and pleural permeability. BCG enhanced pleural mesothelial cell (PMC) release of vascular endothelial growth factor (VEGF), and decreased electrical resistance across the PMC monolayer. Neutralizing antibodies to VEGF significantly restored the drop in PMC electrical resistance caused by BCG. BCG infection down regulated beta-catenin (adherens junction protein) expression and caused increased permeability across confluent mesothelial monolayer. Our results suggest that in TB pleurisy, mycobacteria cause VEGF release from mesothelial cells and leads to protein exudation by altering mesothelial adherens junction proteins.
Assuntos
Proteínas do Citoesqueleto/biossíntese , Mycobacterium bovis , Transativadores/biossíntese , Tuberculose Pleural/metabolismo , Western Blotting , Caderinas/biossíntese , Regulação para Baixo , Impedância Elétrica , Fatores de Crescimento Endotelial/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitélio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/metabolismo , Permeabilidade , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , beta CateninaRESUMO
Migration of polymorphonuclear neutrophils (PMNL) from the vascular compartment into the pleural space occurs rapidly during the development of parapneumonic effusions. This study investigated the polarized secretion of interleukin (IL)-8 in activated pleural mesothelial cells (PMC) and the migration of PMNL across resting, activated PMC monolayers. Results show that PMC produce IL-8 in a polar manner. When PMC were stimulated with Staphylococcus aureus or IL-1beta at the basal or at the apical surface, significantly (P< .05) more IL-8 was released toward the apical surface. This polarized production of IL-8 was confirmed by in situ hybridization. PMNL migration was higher from the basilar to apical than from the apical to basilar surface of PMC. Neutralizing antibodies against IL-8 and intercellular adhesion molecule (ICAM)-1 significantly (P< .001) blocked PMNL migration across activated monolayers. Thus, during pleural inflammation, PMC regulate the influx of PMNL into the pleural space by polar production of IL-8 and expression of ICAM-1.