RESUMEN
In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13). Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/genética , MicroARNs/biosíntesis , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mesodermo/metabolismo , Mesodermo/patología , MicroARNs/genética , Terapia Molecular Dirigida , Proteínas de Neoplasias/biosíntesis , Neuraminidasa/biosíntesis , Neuraminidasa/genética , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , GemcitabinaRESUMEN
BACKGROUND: Among the solid tumors, human pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis. Gemcitabine is the standard first line of therapy for pancreatic cancer but has limited efficacy due to inherent or rapid development of resistance and combining EGFR inhibitors with this regimen results in only a modest clinical benefit. The goal of this study was to identify molecular targets that are activated during gemcitabine therapy alone or in combination with an EGFR inhibitor. METHODS: PDAC cell lines were used to determine molecular changes and rates of growth after treatment with gemcitabine or an EGFR inhibitor, AG1478, by Western blot analysis and MTT assays respectively. Flow cytometric analysis was performed to study the cell cycle progression and rate of apoptosis after gemcitabine treatment. ShRNA was used to knockdown STAT3. An in vivo orthotopic animal model was used to evaluate STAT3 as a target. Immunohistochemical analysis was performed to analyze Ki67 and STAT3 expression in tumors. RESULTS: Treatment with gemcitabine increased the levels of EGFRTyr1068 and ERK phosphorylation in the PDAC cell lines tested. The constitutive STAT3Tyr705 phosphorylation observed in PDAC cell lines was not altered by treatment with gemcitabine. Treatment of cells with gemcitabine or AG1478 resulted in differential rate of growth inhibition. AG1478 efficiently blocked the phosphorylation of EGFRTyr1068 and inhibited the phosphorylation of down-stream effectors AKT and ERKs, while STAT3Tyr705 phosphorylation remained unchanged. Combining these two agents neither induced synergistic growth suppression nor inhibited STAT3Tyr705 phosphorylation, thus prompting further studies to assess whether targeting STAT3 improves the response to gemcitabine or AG1478. Indeed, knockdown of STAT3 increased sensitivity to gemcitabine by inducing pro-apoptotic signals and by increasing G1 cell cycle arrest. However, knockdown of STAT3 did not enhance the growth inhibitory potential of AG1478. In vivo orthotopic animal model results show that knockdown of STAT3 caused a significant reduction in tumor burden and delayed tumor progression with increased response to gemcitabine associated with a decrease in the Ki-67 positive cells. CONCLUSIONS: This study suggests that STAT3 should be considered an important molecular target for therapy of PDAC for enhancing the response to gemcitabine.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Desoxicitidina/farmacología , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Fosforilación , Procesamiento Proteico-Postraduccional , Quinazolinas/farmacología , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/genética , Tirfostinos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The newly described iron transporter, ferroportin (MTP1, IREG1), is expressed in a variety of tissues including the duodenum and cells of the mononuclear phagocyte system (MPS). In the MPS, ferroportin is hypothesized to be a major exporter of iron scavenged from senescent erythrocytes. Changes in iron metabolism, including the sequestration of iron in the MPS, are characteristic of both acute and chronic inflammation and these conditions induce changes in ferroportin expression. In a mouse model of acute inflammation, LPS administration is associated with reduced MPS ferroportin protein and mRNA expression. In addition, the ferroportin 5' UTR also has an iron-responsive element that binds to the iron-response proteins, but whether there is a role for this IRE in inflammation induced regulation of ferroportin has been unclear. A luciferase reporter gene under the control of the mouse ferroportin promoter and 5' UTR was used to determine if this 5' UTR conferred IRE-dependent regulation on this reporter gene. Stimulation of reporter gene transfected RAW 264.7 cells (a mouse macrophage cell line) with LPS resulted in IRE-dependent inhibition of luciferase production. Inhibitors of nitric oxide synthase abrogated the IRE-dependent effect of LPS. In addition, direct treatment of RAW 264.7 and with NO donor S-nitroso-N-acetylpenicillamine resulted in IRE-dependent down-regulation of luciferase expression. The effect of NO was consistent with IRP1/IRE mediated translation block. There are most likely both inflammation-mediated transcriptional and post-transcriptional (IRE-dependent) mechanisms for inhibiting ferroportin expression in MPS cells.