Identification of Methylation-Associated Gene Expression in Neuroendocrine Pancreatic Tumor Cells

Background: CpG islands methylation is the main epigenetic modification found in human tumors leading to transcriptional silencing of certain tumor suppressor genes. Reacquisition of p16/CDKN2A tumor suppressor gene expression by 5-aza-2′-deoxycytidine results in concurrent growth inhibition of neuroendocrine pancreatic tumor cells. However, the growth suppressive effects of 5-aza-2′-deoxycytidine is unlikely to be solely attributable to the restored p16/CDKN2A function, but rather a consequence of re-expression of additional genes silenced by de novo methylation. In an effort to validate DNA methylation as an important mechanism in neuroendocrine tumorigenesis and metastatic spread, we attempted to isolate methylation-specific transcripts in neuroendocrine pancreatic tumor cells. Methods: Differentially expressed methylation-associated genes were identified by cDNA-representational difference analysis (cDNA-RDA). Differential expression was confirmed by semiquantitative RT-PCR using insert specific primers. Results: We identified 48 differently expressed gene fragments and methylation-associated expression was confirmed by semiquantitative RT-PCR. 52,3% (25 of 48) showed elevated expression levels after 5-aza-2′-deoxycytidine treatment, whereas 47.7% revealed lower expression levels. 7 fragments showed homology to genes with unknown function. Inter- estingly, 5-aza-2′-deoxycytidine treatment led to re-expression of cofillin whereas matriptase expression levels were significantly lower. Both genes have been associated with metastastic spread and tissue invasion. The other differentially expressed genes play an unknown role in the course of neuroendocrine tumorigenesis. Conclusion: DNA methylation appears to be an important molecular mechanism in the process of neuroendocrine pancreatic tumorigenesis and metastatic spread. The definition of DNA methylation patterns associated with neuroendocrine pancreatic tumors might open up the potential for a new sensitive diagnostic tool and might serve as a new antitumor target.