RESUMEN
Oncolytic viruses are generally designed to be cancer selective on the basis of a single genetic mutation. JX-594 is a thymidine kinase (TK) gene-inactivated oncolytic vaccinia virus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and lac-Z transgenes that is designed to destroy cancer cells through replication-dependent cell lysis and stimulation of antitumoral immunity. JX-594 has demonstrated a favorable safety profile and reproducible tumor necrosis in a variety of solid cancer types in clinical trials. However, the mechanism(s) responsible for its cancer-selectivity have not yet been well described. We analyzed the replication of JX-594 in three model systems: primary normal and cancer cells, surgical explants, and murine tumor models. JX-594 replication, transgene expression, and cytopathic effects were highly cancer-selective, and broad spectrum activity was demonstrated. JX-594 cancer-selectivity was multi-mechanistic; replication was activated by epidermal growth factor receptor (EGFR)/Ras pathway signaling, cellular TK levels, and cancer cell resistance to type-I interferons (IFNs). These findings confirm a large therapeutic index for JX-594 that is driven by common genetic abnormalities in human solid tumors. This appears to be the first description of multiple selectivity mechanisms, both inherent and engineered, for an oncolytic virus. These findings have implications for oncolytic viruses in general, and suggest that their cancer targeting is a complex and multifactorial process.
Asunto(s)
Neoplasias/metabolismo , Virus Oncolíticos/fisiología , Poxviridae/fisiología , Transducción de Señal/fisiología , Replicación Viral/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Leucocitos Mononucleares , Ratones , Ratones Desnudos , Neoplasias/genética , Viroterapia Oncolítica , Virus Oncolíticos/genética , Poxviridae/genética , Transducción de Señal/genética , Replicación Viral/genéticaRESUMEN
Cell migration involves a multitude of signals that converge on cytoskeletal reorganization, essential for development, immune responses, and tissue repair. Here, we show that the microtubule-associated Ste20 kinase SLK, required for cell migration, interacts with the LIM domain binding transcriptional cofactor proteins Ldb1/CLIM2 and Ldb2/CLIM1/NLI. We demonstrate that Ldb1 and 2 bind directly to the SLK carboxy-terminal AT1-46 homology domain in vitro and in vivo. We find that Ldb1 and -2 colocalize with SLK in migrating cells and that both knockdown and overexpression of either factor results in increased motility. Supporting this, knockdown of Ldb1 increases focal adhesion turnover and enhances migration in fibroblasts. We propose that Ldb1/2 function to maintain SLK in an inactive state before its activation. These findings highlight a novel function for Ldb1 and -2 and expand their role to include the control of cell migration.
Asunto(s)
Movimiento Celular , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Sitios de Unión , Western Blotting , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Inmunoprecipitación , Proteínas con Dominio LIM , Ratones , Microscopía Confocal , Mutación , Células 3T3 NIH , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Cell migration involves a multitude of signals that converge on cytoskeletal reorganization, essential for development, immune responses and tissue repair. Using knockdown and dominant negative approaches, we show that the microtubule-associated Ste20-like kinase SLK is required for focal adhesion turnover and cell migration downstream of the FAK/c-src complex. Our results show that SLK co-localizes with paxillin, Rac1 and the microtubules at the leading edge of migrating cells and is activated by scratch wounding. SLK activation is dependent on FAK/c-src/MAPK signaling, whereas SLK recruitment to the leading edge is src-dependent but FAK independent. Our results show that SLK represents a novel focal adhesion disassembly signal.