RESUMEN
Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to loss of key cellular pathways in tumorigenesis. Methyl-CpG-targeted transcriptional activation (MeTA) reactivates hypermethylation-mediated silenced genes in a different way from DNA-demethylating agents. Microarray coupled with MeTA (MeTA-array) identified seven commonly hypermethylation-mediated silenced genes in 12 pancreatic ductal adenocarcinoma (PDAC) cell lines. Among these, we focused on IRX4 (Iroquois homeobox 4) because IRX4 is located at chromosome 5p15.33 where PDAC susceptibility loci have been identified through genome-wide association study. IRX4 was greatly downregulated in all of the analyzed 12 PDAC cell lines by promoter hypermethylation. In addition, the IRX4 promoter region was found to be frequently and specifically hypermethylated in primary resected PDACs (18/28: 64%). Reexpression of IRX4 inhibited colony formation and proliferation in two PDAC cell lines, PK-1 and PK-9. In contrast, knockdown of IRX4 accelerated cell proliferation in an IRX4-expressing normal pancreatic ductal epithelial cell line, HPDE-1. Because IRX4 is a sequence-specific transcription factor, downstream molecules of IRX4 were pursued by microarray analyses utilizing tetracycline-mediated IRX4 inducible PK-1 and PK-9 cells; CRYAB, CD69, and IL32 were identified as IRX4 downstream candidate genes. Forced expression of these genes suppressed colony formation abilities for both PK-1 and PK-9. These results suggest that DNA methylation-mediated silencing of IRX4 contributes to pancreatic tumorigenesis through aberrant transcriptional regulation of several cancer-related genes.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Proliferación Celular/genética , Silenciador del Gen , Proteínas de Homeodominio/genética , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Metilación de ADN , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen/métodos , Proteínas de Homeodominio/metabolismo , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Plásmidos , Análisis por Matrices de Proteínas , Ensayo de Tumor de Célula Madre , Regulación hacia Arriba , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismo , Neoplasias PancreáticasRESUMEN
Epigenetic gene silencing by aberrant DNA methylation leads to loss of key cellular pathways in tumorigenesis. DNA methylation-mediated silenced genes in pancreatic cancer were searched for using the methyl-CpG targeted transcriptional activation (MeTA) method, and LHX6 (LIM homeobox 6), a transcription factor involved in embryogenesis and head development, was selected as a strong candidate gene. LHX6 was downregulated in most of the pancreatic cancer cell lines (83%, 10/12), mainly through promoter hypermethylation and histone deacetylation. Furthermore, LHX6 was methylated in primary pancreatic cancer specimens (57%, 16/28) in a tumor-specific manner. Re-expression of LHX6 inhibited colony formation and proliferation in LHX6 low-expressing pancreatic cancer cell lines, PK-1 and PK-9. In contrast, knockdown of LHX6 accelerated cell proliferation in LHX6 high-expressing pancreatic cancer cell lines, PCI-35 and MIA PaCa-2. In order to analyze LHX6 downstream genes, we performed microarray analyses using LHX6 inducible PK-1 and PK-9 and found that LHX6 induction upregulated several genes that had tumor suppressive functions. Among these, we focused on TFPI2 (Tissue factor pathway inhibitor-2) and found that TFPI2 was greatly downregulated in all twelve pancreatic cancer cell lines. Our present results suggest that epigenetic inactivation of LHX6 plays an important role in pancreatic tumorigenesis by promoting cell proliferation through aberrant transcriptional regulation of several cancer-related genes.
