RESUMEN
To define the functional pathways regulating epithelial cell migration, we performed a genome-wide RNAi screen using 55,000 pooled lentiviral shRNAs targeting â¼11,000 genes, selecting for transduced cells with increased motility. A stringent validation protocol generated a set of 31 genes representing diverse pathways whose knockdown dramatically enhances cellular migration. Some of these pathways share features of epithelial-to-mesenchymal transition (EMT), and together they implicate key regulators of transcription, cellular signaling, and metabolism, as well as novel modulators of cellular trafficking, such as DLG5. In delineating downstream pathways mediating these migration phenotypes, we observed universal activation of ERKs and a profound dependence on their RSK effectors. Pharmacological inhibition of RSK dramatically suppresses epithelial cell migration induced by knockdown of all 31 genes, suggesting that convergence of diverse migratory pathways on this kinase may provide a therapeutic opportunity in disorders of cell migration, including cancer metastasis.
Asunto(s)
Movimiento Celular/genética , Estudio de Asociación del Genoma Completo , Interferencia de ARN , Proteínas Quinasas S6 Ribosómicas/metabolismo , Línea Celular Tumoral , Células Epiteliales/citología , Humanos , Proteínas de la Membrana/metabolismo , Mesodermo/citología , Reproducibilidad de los Resultados , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Epithelial-to-mesenchymal transition (EMT) plays an important role during normal embryogenesis, and it has been implicated in cancer invasion and metastasis. Here, we report that Ladybird homeobox 1 (LBX1), a developmentally regulated homeobox gene, directs expression of the known EMT inducers ZEB1, ZEB2, Snail1, and transforming growth factor beta2 (TGFB2). In mammary epithelial cells, overexpression of LBX1 leads to morphological transformation, expression of mesenchymal markers, enhanced cell migration, increased CD44(high)/CD24(low) progenitor cell population, and tumorigenic cooperation with known oncogenes. In human breast cancer, LBX1 is up-regulated in the unfavorable estrogen receptor (ER)/progesterone (PR)/HER2 triple-negative basal-like subtype. Thus, aberrant expression of LBX1 may lead to the activation of a developmentally regulated EMT pathway in human breast cancer.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Factores de Transcripción/metabolismo , Animales , Antígeno CD24/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proteínas de Homeodominio/genética , Humanos , Receptores de Hialuranos/metabolismo , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/metabolismo , Oncogenes/fisiología , Proteínas Represoras/metabolismo , Factores de Transcripción de la Familia Snail , Células Madre/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta2/metabolismo , Regulación hacia Arriba , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
UNLABELLED: Epithelial-to-mesenchymal transition (EMT) has been implicated in models of tumor cell migration, invasion, and metastasis. In a search for candidate therapeutic targets to reverse this process, nontumorigenic MCF10A breast epithelial cells were infected with an arrayed lentiviral kinome shRNA library and screened for either suppression or enhancement of a 26-gene EMT RNA signature. No individual kinase gene knockdown was sufficient to induce EMT. In contrast, grouped epithelial markers were induced by knockdown of multiple kinases, including mitogen activated protein kinase 7 (MAPK7). In breast cancer cells, suppression of MAPK7 increased E-cadherin (CDH1) expression and inhibited cell migration. In an orthotopic mouse model, MAPK7 suppression reduced the generation of circulating tumor cells and the appearance of lung metastases. Together, these observations raise the possibility that targeting kinases that maintain mesenchymal cell properties in cancer cells, such as MAPK7, may lessen tumor invasiveness. IMPLICATIONS: Suppression of MAPK7 induces epithelial markers, reduces generation of circulating tumor cells and appearance of lung metastases.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Animales , Antígenos CD , Neoplasias de la Mama/sangre , Cadherinas/biosíntesis , Cadherinas/genética , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos NOD , Proteína Quinasa 7 Activada por Mitógenos/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , TranscriptomaRESUMEN
The neural crest (NC) is a highly motile cell population that gives rise to multiple tissue lineages during vertebrate embryogenesis. Here, we identify a novel effector of the small GTPase Rap, called RADIL, and show that it is required for cell adhesion and migration. Knockdown of radil in the zebrafish model results in multiple defects in NC-derived lineages such as cartilage, pigment cells, and enteric neurons. We specifically show that these defects are primarily due to the diminished migratory capacity of NC cells. The identification of RADIL as a regulator of NC migration defines a role for the Rap pathway in this process.