RESUMO
Upregulation of the extracellular signal-regulated kinase (ERK) pathway has been shown to contribute to tumour invasion and progression. Because the two predominant ERK isoforms (ERK1 and ERK2, also known as MAPK3 and MAPK1, respectively) are highly homologous and have indistinguishable kinase activities in vitro, both enzymes were believed to be redundant and interchangeable. To challenge this view, we show that ERK2 silencing inhibits invasive migration of MDA-MB-231 cells, and re-expression of ERK2 but not ERK1 restores the normal invasive phenotype. A detailed quantitative analysis of cell movement on 3D matrices indicates that ERK2 knockdown impairs cellular motility by decreasing the migration velocity as well as increasing the time that cells spend not moving. Using gene expression arrays we found that the expression of the genes for Rab17 and liprin-ß2 was increased by knockdown of ERK2 and restored to normal levels following re-expression of ERK2, but not ERK1. Both play inhibitory roles in the invasive behaviour of three independent cancer cell lines. Importantly, knockdown of either Rab17 or liprin-ß2 restores invasiveness of ERK2-depleted cells, indicating that ERK2 drives invasion of MDA-MB-231 cells by suppressing expression of these genes.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Transporte/antagonistas & inibidores , Movimento Celular/fisiologia , Proteínas de Membrana/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Microambiente Tumoral/fisiologia , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Neoplasias da Mama/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Proteínas rab de Ligação ao GTP/biossíntese , Proteínas rab de Ligação ao GTP/genéticaRESUMO
In solid tumors, resistance to therapy inevitably develops upon treatment with cytotoxic drugs or molecularly targeted therapies. Here, we describe a system that enables pooled shRNA screening directly in mouse hepatocellular carcinomas (HCC) in vivo to identify genes likely to be involved in therapy resistance. Using a focused shRNA library targeting genes located within focal genomic amplifications of human HCC, we screened for genes whose inhibition increased the therapeutic efficacy of the multikinase inhibitor sorafenib. Both shRNA-mediated and pharmacological silencing of Mapk14 (p38α) were found to sensitize mouse HCC to sorafenib therapy and prolong survival by abrogating Mapk14-dependent activation of Mek-Erk and Atf2 signaling. Elevated Mapk14-Atf2 signaling predicted poor response to sorafenib therapy in human HCC, and sorafenib resistance of p-Mapk14-expressing HCC cells could be reverted by silencing Mapk14. Our results suggest that a combination of sorafenib and Mapk14 blockade is a promising approach to overcoming therapy resistance of human HCC.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/genética , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/genética , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Fator 2 Ativador da Transcrição/metabolismo , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais , Sorafenibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.
Assuntos
Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Movimento Celular , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/química , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de SinaisRESUMO
Here we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5ß1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5ß1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo.