RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. The aim of this study was to investigate the emerging technology of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery and spatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small-molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term-treated mice to measure drug tissue distribution. Histologic and statistical analyses were performed to correlate morphology, drug distribution, and survival. We found that erlotinib levels were significantly lower in PDAC compared with healthy tissue (P = 0.0078). Survival of long-term-treated mice did not correlate with overall levels of erlotinib or with overall histologic tumor grade but did correlate both with the percentage of atypical glands in the cancer (P = 0.021, rs = 0.59) and the level of erlotinib in those atypical glands (P = 0.019, rs = 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution of compounds in a preclinical setting and support drug imaging-based translational approaches. Mol Cancer Ther; 15(5); 1145-52. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Clorhidrato de Erlotinib/farmacocinética , Clorhidrato de Erlotinib/uso terapéutico , Modelos Biológicos , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Monitoreo de Drogas , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Clasificación del Tumor , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/mortalidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Análisis de Supervivencia , Factores de Tiempo , Distribución TisularRESUMEN
The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a regulatory factor of the cell cycle. Its transcriptional activation and protein stability are tightly controlled by several distinct mechanisms. S100A11 is a member of the S100 family of Ca²âº-binding proteins involved in several biological processes, including cell cycle progression and signal transduction. In the present study, we show that down-regulation of S100A11 results in the reduction of p21 protein in human HaCaT keratinocytes. It appears that a ubiquitin-independent proteasomal degradation process is involved in p21 degradation in S100A11 down-regulated cells. The application of a proteasome inhibitor stabilized p21 protein in these cells. Analysis of distinct signal transduction pathways revealed a disturbed phosphatidylinositol-3-kinase/Akt pathway after S100A11 knockdown. We determined that the glycogen synthase kinase-3, which is negatively regulated by phosphatidylinositol 3-kinase/Akt, was activated in cells possessing knocked-down S100A11 and appears to be involved in p21 protein destabilization. The application of a specific inhibitor of glycogen synthase kinase 3 resulted in an increase of the p21 protein level in S100A11 down-regulated HaCaT cells. Glycogen synthase kinase 3 is able to phosphorylate p21 at T57, which induces p21 proteasomal turnover. Mutation of the glycogen synthase kinase 3 site threonine 57 into alanine (T57A) stabilizes p21 in HaCaT cells lacking S100A11. Beside decreased p21 protein, down-regulation of S100A11 triggered the induction of apoptosis in HaCaT cells. These observations suggest that S100A11 is involved in the maintenance of p21 protein stability and appears to function as an inhibitor of apoptosis in human HaCaT keratinocyte cells. Thus, the data shed light on a novel pathway regulating p21 protein stability.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Queratinocitos/metabolismo , Proteínas S100/metabolismo , Regulación hacia Arriba , Apoptosis/efectos de los fármacos , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/agonistas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo/efectos de los fármacos , Silenciador del Gen , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Proteínas S100/antagonistas & inhibidores , Proteínas S100/genética , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Initiation of pancreatic ductal adenocarcinoma (PDA) is definitively linked to activating mutations in the KRAS oncogene. However, PDA mouse models show that mutant Kras expression early in development gives rise to a normal pancreas, with tumors forming only after a long latency or pancreatitis induction. Here, we show that oncogenic KRAS upregulates endogenous EGFR expression and activation, the latter being dependent on the EGFR ligand sheddase, ADAM17. Genetic ablation or pharmacological inhibition of EGFR or ADAM17 effectively eliminates KRAS-driven tumorigenesis in vivo. Without EGFR activity, active RAS levels are not sufficient to induce robust MEK/ERK activity, a requirement for epithelial transformation.
