RESUMEN
Gastric cancer stem cell (GCSC) is implicated in gastric cancer relapse, metastasis and drug resistance. However, the key molecule(s) involved in GCSC survival and the targeting drugs are poorly understood. We discovered increased secreted clusterin (S-Clu) protein expression during the sphere-forming growth of GCSC via mass spectrometry. Overexpression of clusterin was detected in 69/90 (77%) of primary GC tissues and significantly associated with T stage, lymph node metastasis and TNM stage. Depletion of clusterin (Clu, the full-length intracellular clusterin) led to the declustering of GCSC tumorspheres and apoptosis of GCSC. Subsequently, we found clusterin was in complex with heat shock protein 90 beta (HSP90) and involved in regulating the cellular level of HSP90 client proteins. Furthermore, by screening a collection of drugs/inhibitors, we found that verteporfin (VP), a phototherapy drug, blocked clusterin gene expression, decreased the HSP90 client proteins and caused cell death of GCSC. VP treatment is more effective in eradicating GCSCs than in killing GC cells. Both clusterin silencing or VP treatment deterred tumor growth in human GCSC xenografts. These findings collectively suggest that GC patients can promptly benefit from clusterin-targeted therapy as well as VP treatment in combination with or subsequent to conventional chemotherapy for reducing mortality of GC.
Asunto(s)
Clusterina/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Verteporfina/farmacología , Verteporfina/uso terapéutico , Animales , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Inmunoprecipitación , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Unión Proteica/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Aurora Quinasas , Benzodiazepinas/química , Benzodiazepinas/farmacología , Furanos/química , Furanos/farmacología , Humanos , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Tiazolidinedionas/química , Tiazolidinedionas/farmacologíaRESUMEN
The epidermal growth factor receptor (EGFR) plays a critical role in the development, proliferation, and differentiation of cells of epithelial and mesenchymal origin. These EGFR-dependent cellular processes are mediated by a repertoire of intracellular signaling pathways triggered by the activation of the EGFR cytoplasmic domain, which originates from ligand binding of its extracellular domain. To understand the molecular mechanisms by which the intracellular domain of EGFR transmits mitogenic messages to the downstream signaling pathways, we used the cytoplasmic region of EGFR as bait in yeast two-hybrid screening. We found that ADP-ribosylation factor 4 (ARF4) interacts with the intracellular part of EGFR and mediates the EGF-dependent cellular activation of phospholipase D2 (PLD2) but does not mediate the activation of PLD1. In addition, ARF4-mediated PLD2 activation leads to dramatic activation of the transcription factor activator protein 1 (AP-1), and, importantly, ARF4 activity is required for EGF-induced activation of cellular AP-1. Our findings indicate that ARF4 is a critical molecule that directly regulates cellular PLD2 activity and that this ARF4-mediated PLD2 activation stimulates AP-1-dependent transcription in the EGF-induced cellular response.