RESUMEN
Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. SIGNIFICANCE: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Endosomas/metabolismo , Invasividad Neoplásica/patología , Podosomas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/metabolismo , Conjuntos de Datos como Asunto , Matriz Extracelular , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/patología , Ratones , Invasividad Neoplásica/prevención & control , Podosomas/patología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , Imagen de Lapso de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A general route to a series of differentially substituted bicyclo[2,2,2]octenones has been developed, making use of the in situ intramolecular Diels Alder reaction of masked ortho-benzoquinones. This approach was used to synthesize a series of thirteen key acid-containing templates from which a solution phase discovery library of 1126 diverse amides was then constructed. The rigid polycyclic nature of the templates and the prevalence of oxygenated functionality confer natural product-like qualities and three-dimensional diversity. The library was screened in HTS in vivo against a number of weed, insect and fungal model organisms leading to the discovery of a novel series of herbicidally active compounds. The development, production and biological activity of the library are described.