RESUMEN
The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.
Asunto(s)
Membrana Celular/metabolismo , Movimiento Celular/fisiología , Proteína Kangai-1/metabolismo , Metástasis de la Neoplasia/patología , Neoplasias/metabolismo , Fibras de Estrés/metabolismo , Tetraspaninas/metabolismo , Caveolina 1/metabolismo , Adhesión Celular/fisiología , Línea Celular , Línea Celular Tumoral , Humanos , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo , Neoplasias/patología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismoRESUMEN
Taxanes can induce drug resistance by increasing signaling pathways such as PI3K/Akt and ERK, which promote survival and cell growth in human cancer cells. We have previously shown that long chain n-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA, 22:6n-3) decrease resistance of experimental mammary tumors to anticancer drugs. Our objective was to determine whether DHA could increase tumor sensitivity to docetaxel by down-regulating these survival pathways. In docetaxel-treated MDA-MB-231 cells, phosphorylated-ERK1/2 levels were increased by 60% in membrane and nuclear compartments, compared to untreated cells. Our data showed that ERK1/2 activation depended on PKC activation since: i) enzastaurin (a pan-PKC inhibitor) blocked docetaxel-induced ERK1/2 phosphorylation ii) docetaxel increased PKC activity by 30% and phosphatidic acid level by 1.6-fold iii) inhibition of PKCε and PKCδ by siRNA resulted in reduced phosphorylated ERK1/2 levels. In DHA-supplemented cells, docetaxel was unable to increase PKCε and δ levels in membrane and nuclear fractions, resulting in diminished ERK1/2 phosphorylation and increased docetaxel efficacy. Reduced membrane level of PKCε and PKCδ was associated with significant incorporation of DHA in all phospholipids, including phosphatidylcholine which is a major source of phosphatidic acid. Additionally, examination of the Akt pathway showed that DHA could repress docetaxel-induced Ser473Akt phosphorylation. In rat mammary tumors, dietary DHA supplementation during docetaxel chemotherapy repressed ERK and Akt survival pathways and in turn strongly improved taxane efficacy. The P-ERK level was negatively correlated with tumor regression. These findings are of potential clinical importance in treating chemotherapy-refractory cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Ácidos Docosahexaenoicos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Taxoides/farmacología , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Docetaxel , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Resistencia a Antineoplásicos/efectos de los fármacos , Activación Enzimática , Femenino , Humanos , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Metilnitrosourea , Fosforilación , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/genética , Proteína Quinasa C-epsilon/antagonistas & inhibidores , Proteína Quinasa C-epsilon/genética , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Ratas Sprague-Dawley , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacosRESUMEN
Knowledge of Aurora A kinase functions is limited to premetaphase events, particularly centrosome maturation, G2/M transition, and mitotic spindle assembly. The involvement of Aurora A in events after metaphase has only been suggested because appropriate experiments are technically difficult. We report here the design of the first human Aurora A kinase (as-AurA) engineered by chemical genetics techniques. This kinase is fully functional biochemically and in cells, and is rapidly and specifically inhibited by the ATP analogue 1-Naphthyl-PP1 (1-Na-PP1). By treating cells exclusively expressing the as-AurA with 1-Na-PP1, we discovered that Aurora A is required for central spindle assembly in anaphase through phosphorylation of Ser 19 of P150Glued. This paper thus describes a new Aurora A function that takes place after the metaphase-to-anaphase transition and a new powerful tool to search for and study new Aurora A functions.
