RESUMEN
BACKGROUND: PD-0332991 is an inhibitor of cyclin-dependent kinases (CDK) 4 and 6, and was evaluated to determine its anti-proliferative effects in 25 renal cell carcinoma (RCC) cell lines. MATERIALS AND METHODS: Half-maximal inhibitory concentrations (IC50) of PD-0332991 were determined with cell line proliferation assays, as were its effects on the cell cycle, apoptosis, and retinoblastoma (RB) phosphorylation. Molecular markers for response prediction, including p16, p15, cyclin D1 (CCND1), cyclin E1 (CCNE1), E2F transcription factor 1 (E2F1), RB, CDK4 and CDK6, were studied using array comparative genomic hybridization (CGH) and gene expression. RESULTS: IC50 values for PD-0332991 ranged from 25.0 nM to 700 nM, and the agent demonstrated G0/G1 cell-cycle arrest, induction of late apoptosis, and blockade of RB phosphorylation. Through genotype and expression data p16, p15 and E2F1 were identified as having significant association between loss and sensitivity to PD-0332991: p16 (p=0.021), p15 (p=0.047), and E2F1 (p=0.041). CONCLUSION: PD-0332991 has antiproliferative activity in RCC cell lines, and molecular markers predict for sensitivity to this agent.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Piperazinas/farmacología , Piridinas/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/enzimología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Renales/enzimología , Neoplasias Renales/genética , Neoplasias Renales/patología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína de Retinoblastoma/metabolismoRESUMEN
The RBM5/H37 gene is located at the most 'sought-after' tumor suppressor locus in lung cancer, 3p21.3. This region of most frequent chromosomal deletion found at the earliest stage in lung cancer development houses 19 genes, many of which may act together as a 'tumor suppressor group', representing one of the most promising opportunities for development of new diagnostics/prognostics and therapeutics for lung cancer as well as for many other types of cancers. For the past decade, we have demonstrated tumor suppressor function of RBM5 in vitro and in vivo involving cell cycle arrest and apoptosis, as well as loss of RBM5 mRNA and protein expression in primary lung tumors. Here we report our latest data suggesting that RBM5 may regulate inhibition of metastasis in lung cancer. We performed cDNA microarray to identify global gene expression changes caused by RBM5 gene knockdown. In order to identify "consensus" pathways consistently deregulated by RBM5 loss irrespective of genetic background, the experiments were repeated in three different lung cancer cell lines of varying RBM5 expression levels, a normal lung epithelial cell line, and a normal breast epithelial cell line. Both Gene Set Enrichment Analysis (GSEA) and individual gene analysis identified consistent, statistically significant gene expression changes common to all five cell pairs examined. Genes involved in the functions of cell adhesion, migration and motility, known to be important in the metastatic process, were upregulated with RBM5-knockdown. These genes include Rac1, ß-catenin, collagen, laminin and the overall gene set of the gene ontology group "proteinaceous extracellular matrix". Among these, we have focused on Rac1 and ß-catenin which play essential roles in cell movement downstream of Wnt signaling. We have confirmed increased protein expression of ß-catenin and increased protein activation of Rac1 with RBM5-knockdown. In addition, we found that RBM5 protein expression loss in primary lung tumors is correlated with increased lymph node metastasis in a small number of lung cancer patients. These data are corroborated by an independent report showing RBM5 as part of a 17-gene signature of metastasis in primary solid tumors. Taken together, the accumulated evidence suggests that RBM5 expression loss may increase the metastatic potential of tumors. Further study is warranted to evaluate the potential clinical utility of RBM5 in lung cancer diagnostics, prognostics and therapeutics.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Neoplasias Pulmonares/genética , Metástasis Linfática , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Cromosomas Humanos Par 3/genética , Proteínas de Unión al ADN/genética , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Metástasis Linfática/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Transducción de Señal/genética , Proteínas Supresoras de Tumor/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Loss of heterozygosity (LOH) at chromosome 3p21.3 is one of the most prevalent genetic disturbances occurring at the earliest stage of tumor development for a wide variety of human cancers, culminated in lung cancer. The 19 genes residing at 3p21.3 have been vigorously characterized for tumor suppressor activity and gene inactivation mechanism because of their potentially significant merits of clinical applications. Many of these 19 genes have been shown to manifest various growth inhibitory properties, however none of them are inactivated by coding mutations in their remaining allele as in the Knudson's two- hits hypothesis. Thus far the most prevailing, alternative gene inactivation mechanism known for the 3p21.3 TSGs is epigenetic silencing by promoter hypermethylation. Previously, we have focused our investigation on one of the 19 genes at 3p21.3, H37/RBM5, and demonstrated its tumor suppressor activity both in vitro and in vivo as well as its mRNA/protein expression loss from the remaining allele in a majority of the primary lung tumors examined. The current study tested our hypothesis that the H37 inactivation in primary lung tumors may, as seen in most of the other 3p21.3 TSGs, be due to hypermethylation in its promoter CpG islands. Contrary to this most plausible postulation, however, we found no evidence of epigenetic gene silencing for the H37 TSG. Here we suggest some of the possible, further- alternative means of the H37 gene expression loss in tumor, including defects in transcription and post-transcriptional/translational modifications as well as mechanisms related to haploinsufficiency.