RESUMEN
Cancer cells are dependent on protein quality-control mechanisms, including protein chaperones, the ubiquitin-proteasome system, and autophagy. The p62 receptor is a classical, ubiquitously expressed receptor, involved in many signal transduction pathways. Upregulation and/or reduced degradation of p62 have been implicated in tumor formation and resistance to therapy. PTX80 is a first-in-class novel inhibitor of protein degradation, developed by Pi Therapeutics for the treatment of cancer. PTX80 binds to p62, inducing a decrease in soluble p62 and formation of insoluble p62 aggregates, and failure of polyubiquitinated proteins to colocalize with p62. PTX80 induces proteotoxic stress and activation of unfolded protein response, which, in turn, leads to apoptosis. Targeting p62, which is a major protein degradation hub, may serve as a novel and beneficial strategy for the treatment of cancer.
Asunto(s)
Neoplasias , Complejo de la Endopetidasa Proteasomal , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Humanos , Neoplasias/tratamiento farmacológico , Proteína Sequestosoma-1/metabolismo , UbiquitinaRESUMEN
The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.
Asunto(s)
Aparato de Golgi/metabolismo , Ionóforos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteostasis/fisiología , Animales , Apoptosis/efectos de los fármacos , Autoantígenos/metabolismo , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Aparato de Golgi/efectos de los fármacos , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Ionóforos/farmacología , Proteínas de la Membrana/metabolismo , Ratones , Monensina/farmacología , Monensina/uso terapéutico , Mieloma Múltiple/patología , Proteolisis/efectos de los fármacos , Proteostasis/efectos de los fármacos , Ubiquitinación/efectos de los fármacos , Proteína que Contiene Valosina/metabolismoRESUMEN
The Na+-K+-ATPase is specifically inhibited by cardiac glycosides, some of which may also function as endogenous mammalian hormones. Previous studies using Xenopus oocytes, yeast cells, or purified isoforms demonstrated that affinities of various cardiac glycosides for three isoforms of the Na+-K+-ATPase (α1-α3ß1) may differ, a finding with potential clinical implication. The present study investigates isoform selectivity and effects of cardiac glycosides on cultured mammalian cells under more physiological conditions. H1299 cells (non-small cell lung carcinoma) were engineered to express only one α-isoform (α1, α2, or α3) by combining stable transfection of isoforms and silencing endogenous α1. Cardiac glycoside binding was measured by displacement of bound 3H-ouabain. The experiments confirm moderate α1/α3:α2 selectivity of ouabain, moderate α2:α1 selectivity of digoxin, and enhanced α2:α1 selectivity of synthetic derivatives (Katz A, Tal DM, Heller D, Haviv H, Rabah B, Barkana Y, Marcovich AL, Karlish SJD. J Biol Chem 289: 21153-21162, 2014). Relative α2:α1 selectivity of digoxin vs. ouabain was also manifested by enhanced internalization of α2 in response to digoxin. Cellular proliferation assays of H1299 cells confirmed the patterns of α2:α1 selectivity for ouabain, digoxin, and a synthetic derivative and reveal a crucial role of surface pump density on sensitivity to cardiac glycosides. Because cardiac glycosides are being considered as drugs for treatment of cancer, effects of ouabain on proliferation of 12 cancer and noncancer cell lines, with variable plasma membrane expression of α1, have been tested. These demonstrated that sensitivity to ouabain indeed depends linearly on the plasma membrane surface density of Na+-K+-ATPase irrespective of status, malignant or nonmalignant.
Asunto(s)
Antineoplásicos/farmacología , Glicósidos Cardíacos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias/enzimología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Antineoplásicos/metabolismo , Unión Competitiva , Glicósidos Cardíacos/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Digoxina/metabolismo , Digoxina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Humanos , Isoenzimas , Neoplasias/genética , Neoplasias/patología , Ouabaína/metabolismo , Ouabaína/farmacología , Unión Proteica , Interferencia de ARN , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , TransfecciónRESUMEN
Internalization of the Na(+)/K(+)-ATPase (the Na(+) pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na(+)/K(+)-ATPase molecule or more generally by the disruption of cation homeostasis (Na(+), K(+), Ca(2+)) due to the partial inhibition of active Na(+) and K(+) transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K(+)-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na(+)/K(+)-ATPase complex.