Epigenetic regulation affects N-myc downstream-regulated gene 1 expression indirectly in pancreatic cancer cells

Abstract

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.

Figure 1

(A) HPAF-II cells were treated with AZA or TSA at the indicated concentrations for one to three days. Both drugs time-dependently increased NDRG1 protein expression, as measured by densitometry. (B) HPAF-II cells were treated with AZA for 72 hours and/or TSA for the last 24 hours. Expression of NDRG1 was measured by Western blotting and quantified by densitometry. (C) HPAF-II cells were treated with AZA for 72 hours and/or TSA for the last 24 hours. Expression of NDRG1 mRNA was measured by real time PCR.

Figure 2

(A) Capan-2 cells were treated with AZA for 72 hours and/or TSA for the last 24 hours. (B) BxPC-3 cells were stably transfected with scrambled (Scr) or shRNA (sh4, sh6 and sh7) for NDRG1. (C) The scrambled and silenced clones were treated with TSA for 24 hours. Mock (M), TSA (T) (A–C) Expression of NDRG1 was measured by Western blotting and quantified by densitometry.

Figure 3

The NDRG1 gene contains a large CpG island framing the transcription start (+1169 to −647 bp) (insert). DNA methylation in pancreatic cancer cell lines was assessed by COBRA on a portion of the promoter CpG island +36 to −388 relative to the transcriptional start site. The hatched area shows the coverage of analysis. Pre-methylated samples served as positive control and verified the ability to detect methylation in samples with greater than 33% DNA methylation. U denotes mock digested amplicons, D denotes BstU1 digested amplicons.