ABSTRACT
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.
Subject(s)
Cell Membrane/metabolism , Cell Movement/physiology , Kangai-1 Protein/metabolism , Neoplasm Metastasis/pathology , Neoplasms/metabolism , Stress Fibers/metabolism , Tetraspanins/metabolism , Caveolin 1/metabolism , Cell Adhesion/physiology , Cell Line , Cell Line, Tumor , Humans , Mechanotransduction, Cellular/physiology , Membrane Proteins/metabolism , Neoplasms/pathology , Signal Transduction/physiology , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Docosahexaenoic Acids/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Protein Kinase C-delta/metabolism , Protein Kinase C-epsilon/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Taxoids/pharmacology , Animals , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Survival/drug effects , Docetaxel , Dose-Response Relationship, Drug , Down-Regulation , Drug Resistance, Neoplasm/drug effects , Enzyme Activation , Female , Humans , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/enzymology , Mammary Neoplasms, Experimental/pathology , Methylnitrosourea , Phosphorylation , Protein Kinase C-delta/antagonists & inhibitors , Protein Kinase C-delta/genetics , Protein Kinase C-epsilon/antagonists & inhibitors , Protein Kinase C-epsilon/genetics , Protein Kinase Inhibitors/pharmacology , RNA Interference , Rats, Sprague-Dawley , Time Factors , Transfection , Tumor Burden/drug effectsABSTRACT
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.
