Abstract 127: Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia
Authors: Rheinheimer Brenna A. , Vrba Lukas , Futscher Bernard W. , Heimark Ronald L. 
Published: May 2015

Guidance molecules from the Netrin, Slit, Ephrin, and Semaphorin gene families were originally described as cues for the directional guidance of axons in the developing nervous system. More recently, members of these families have been found to have critical roles in tumor invasion. The SLIT2/mir-218-1 signaling axis has properties of a tumor suppressor pathway via the inhibition of directional migration and is epigenetically silenced in many cancers.

We propose that mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma and inhibits tumor dissemination following the conversion of precursor lesions to invasive carcinoma. We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 mRNA while KRAS-independent lines show silenced SLIT2 expression. Using chromatin immunoprecipitation (ChIP) and Sequenom, we confirmed that loss of SLIT2 in KRAS-independent lines was due to enrichment of silencing histone mark H3K27me3 and DNA hypermethylation at the SLIT2 promoter, respectively. Activating marks H3K4me2 and H4ac are found at the SLIT2 promoter in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression in pancreatic cancer cell lines does not correlate with SLIT2 gene expression. Therefore, we examined whether mir-218-1 had an alternative promoter independent of the SLIT2 promoter. Using ChIP for H3K4me3, a marker for promoter regions, and H4ac, a marker of permissive chromatin, we discovered a putative alternative promoter of mir-218-1 in all pancreatic cancer cell lines regardless of KRAS dependence. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity. This suggests that mir-218-1 expression is important and may contain its own tumor suppressor properties independent of SLIT2 signaling.

Since pancreatic cancer is an invasive disease we examined the role of miR-218 target genes in specialized cellular structures, termed invadopodia. These structures have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases to degrade extracellular matrix. Transfection of a miR-218 antagomir enhanced invasion in a modified Boyden chamber while addition of mir-218 reduced cell invasion. Our results further demonstrate that mir-218 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that the SLIT2/mir-218-1 signaling axis is an important mediator of pancreatic cancer invasion through the extracellular matrix.

Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Alternative transcription of the SLIT2/mir-218-1 signaling axis mediates pancreatic cancer invasion through the regulation of invadopodia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 127. doi:10.1158/1538-7445.AM2014-127