Epigenetic Regulation Affects N-myc Downstream-Regulated Gene 1 Expression Indirectly in Pancreatic Cancer Cells

Objectives: N-myc downstream-regulated gene 1 (NDRG1), important in tumor growth and metastasis, has recently gained interest as a potential therapeutic target. Loss of NDRG1 expression is generally associated with poor clinical outcome in pancreatic cancer (PaCa) patients. As the NDRG1 gene possesses a large promoter CpG island, we sought to determine whether its repression is epigenetically mediated in PaCa cells. Methods: Pancreatic cancer cells were treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A. Promoter methylation was assessed by genomic bisulfite sequencing and by combined bisulfite restriction analyses. Results: Treatment with 5-aza-2'-deoxycytidine and trichostatin A enhanced NDRG1 protein expression, implicating epigenetic regulation of NDRG1. However, there was no significant DNA methylation of the NDRG1 promoter CpG island, as determined by genomic bisulfite sequencing of HPAF-II cells. We further confirmed the lack of promoter methylation in 6 PaCa cell lines by combined bisulfite restriction analyses. Conclusions: These findings indicate that NDRG1 gene reactivation in PaCa cell lines by pharmacologic reversal of DNA methylation and histone deacetylation occurs via an indirect mechanism. This may occur via the altered expression of genes involved in the regulation of NDRG1 transcription or NDRG1 protein stability in PaCa cells.