RESUMEN
Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950-63. ©2016 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Animales , Proliferación Celular , Neoplasias Colorrectales/genética , Femenino , Humanos , Ratones , Transducción de SeñalRESUMEN
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
Epithelial-mesenchymal transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. After Snail-1 induction, epithelial markers were repressed within 6 hr, and mesenchymal genes were induced at 24 hr. Snail-1 binding to its target promoters was transient (6-48 hr) despite continued protein expression, and it was followed by both transient and long-lasting chromatin changes. Pharmacological inhibition of selected histone acetylation and demethylation pathways suppressed the induction as well as the maintenance of Snail-1-mediated EMT. Thus, EMT involves an epigenetic switch that may be prevented or reversed with the use of small-molecule inhibitors of chromatin modifiers.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Transición Epitelial-Mesenquimal , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Acetilación , Carcinogénesis/metabolismo , Epigénesis Genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Células MCF-7 , Metilación , Regiones Promotoras Genéticas , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genéticaRESUMEN
BRAF(V600E) drives tumors by dysregulating ERK signaling. In these tumors, we show that high levels of ERK-dependent negative feedback potently suppress ligand-dependent mitogenic signaling and Ras function. BRAF(V600E) activation is Ras independent and it signals as a RAF-inhibitor-sensitive monomer. RAF inhibitors potently inhibit RAF monomers and ERK signaling, causing relief of ERK-dependent feedback, reactivation of ligand-dependent signal transduction, increased Ras-GTP, and generation of RAF-inhibitor-resistant RAF dimers. This results in a rebound in ERK activity and culminates in a new steady state, wherein ERK signaling is elevated compared to its initial nadir after RAF inhibition. In this state, ERK signaling is RAF inhibitor resistant, and MEK inhibitor sensitive, and combined inhibition results in enhancement of ERK pathway inhibition and antitumor activity.
Asunto(s)
Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas ras/antagonistas & inhibidores , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Ligandos , Melanoma/metabolismo , Proteínas de la Membrana , Neurregulinas/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/fisiología , Receptores de Factores de Crecimiento/metabolismo , Sulfonamidas/farmacología , Vemurafenib , Proteínas ras/metabolismo , Proteínas ras/fisiologíaRESUMEN
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.