Abstract B13: Epigenetic silencing alters the SLIT2/ROBO1/miR-218-1 signaling axis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early tumor dissemination from pancreatic intraepithelial neoplasia (PanIN) precursor lesions through tumor budding and perineural invasion. Therefore, it is necessary to elucidate the mechanism of transcriptional regulation of genes and signaling pathways that lead to early spread of this disease. Neuronal guidance molecules are secreted membrane-bound ligands that act as signaling cues in morphogenesis, depending on the presence of their cognate receptors. Four major classes of guidance molecules include the Ephrins and Ephrin receptors, Slits and Robo receptors, Netrins and Unc5H receptors, and Semaphorins and Plexin receptors. Mammals encode three SLIT genes (SLIT1-3). The SLIT2/miR-218-1 signaling axis has properties of a tumor suppressor pathway via the inhibition of epithelial cell growth, directional migration, ductal morphogenesis, and is epigenetically silenced in lung, colon, prostate and breast cancers. SLIT2 is also a target for polycomb repressor complex 2 protein EZH2 which enriches H3K27me3 at the SLIT2 promoter. We proposed that changes in DNA methylation patterns and chromatin dynamics lead to differences in SLIT2 and miR-218-1 expression in PDAC and may mediate ductal expansion and early spread following the conversion of PanIN precursor lesions to invasive carcinoma. The SLIT2 promoter contains a large CpG island (290 CpG sites) that extends 2,021 base pairs (bp) upstream of the transcriptional start site (TSS) through the first exon and ends 1,251 bp into the first intron (-2,021 to +1,251). In our studies, we determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 and ROBO1 in a cell autonomous manner while KRAS-independent cell lines have silenced SLIT2 expression and upregulated ROBO1 expression. We confirmed that loss of SLIT2 in KRAS-independent cell lines was due to DNA hypermethylation of its promoter using Sequenom. We cloned 1.4 kb of the SLIT2 promoter upstream of the firefly luciferase gene to evaluate activity of this region in transcriptional control. We transfected the SLIT2 reporter into pancreatic cancer cell lines. The cell lines showed relative promoter activity similar to the level of SLIT2 transcripts. Chromatin immunoprecipitation (ChIP) shows that silencing histone mark H3K27me3 is found at the TSS of the SLIT2 gene in KRAS-independent lines while activating mark H3K4me2 is found at the TSS of the SLIT2 gene in KRAS-dependent lines. Treatment with demethylating agents, but not histone deacetylase inhibitors, reactivates SLIT2 expression suggesting that DNA promoter methylation is the dominant epigenetic mark in silencing SLIT2 expression. miR-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. Most intronic miRNAs are transcribed from the host gene promoter; however, miR-218 expression is variable in pancreatic cancer cell lines and does not correlate with SLIT2 expression. Therefore, we proposed that miR-218-1 has an alternative TSS other than the SLIT2 promoter. Using ChIP for H3K4me3, which defines gene promoters, we discovered a putative alternative TSS of miR-218-1. ChIP analysis using activating mark H4ac shows that this TSS is active in all pancreatic cancer cell lines regardless of KRAS dependence suggesting that the tumor suppressive function of SLIT2 may be mediated through miR-218-1 instead. Overall, our data establishes that the SLIT2/ROBO1/miR-218-1 signaling axis is epigenetically regulated in pancreatic cancer leading to tumor expansion and progression along intrapancreatic neurons that express the SLIT2 ligand. Citation Format: Brenna Rheinheimer, Lukas Vrba, Bernard Futscher, Ronald L. Heimark. Epigenetic silencing alters the SLIT2/ROBO1/miR-218-1 signaling axis in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B13.