Abstract A79: Post-transcriptional regulatory networks of pancreatic tumor invasion
Authors: Rheinheimer Brenna A. , Vrba Lukas , Futscher Bernard W. , Heimark Ronald L. 
Published: October 2015

Guidance molecules from the Slit gene family 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 play critical roles in epithelial development, angiogenesis and tumorigenesis. SLIT2 has properties of a potential tumor suppressor, is mutated at a low frequency in pancreatic tumors and is epigenetically silenced in many cancers. mir-218-1 is an intronic microRNA found within intron 15 of the SLIT2 gene. We propose that cell intrinsic mir-218-1 expression and function is independent of SLIT2 signaling in pancreatic ductal adenocarcinoma. This dynamic expression of mir-218-1 can inhibit tumor dissemination following the conversion of precursor lesions to invasive carcinoma.

In normal pancreatic tissue, SLIT2 is highly expressed in the ductal compartment and expressed at a reduced level in the majority of primary pancreatic ductal adenocarcinomas (PDACs). We determined that KRAS-dependent pancreatic cancer cell lines express SLIT2 while KRAS-independent cell lines show silenced SLIT2 expression. Sequenom and chromatin immunoprecipitation (ChIP) confirmed that loss of SLIT2 in KRAS-independent lines was due to DNA hypermethylation and enrichment of silencing histone marks at the SLIT2 promoter and transcriptional start site with an inverse correlation between SLIT2 and EZH2 expression. Activating histone marks are found at both the SLIT2 promoter and transcriptional start site in KRAS-dependent lines suggesting that SLIT2 is lost during pancreatic cancer progression due to epigenetic silencing.

Many intronic microRNAs are transcribed from the host gene promoter; however, mir-218-1 expression is variable in pancreatic cancer cell lines and 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, we discovered an alternative promoter of mir-218-1. ChIP for H4ac and Sequenom showed that the chromatin structure within this putative alternative promoter is acetylated, unmethylated, and, thus, permissive for transcription. The putative alternative mir-218-1 promoter was also found to have basal transcription via enhanced luciferase reporter activity and enrichment of RNA polymerase II at the H3K4me3/H4ac peak. In silico analysis of the mir-218-1 alternative promoter region indicated that DNA binding sites for transcriptional repressors MYC/MAX and ZEB1 are present. In silico analysis also showed the presence of DNA binding sites for transcriptional activators CDX1, LEF1/TCF1 and NF-κB. The transcription factor NF-κB and oncogene KRAS frequently function cooperatively to promote tumor progression; however, maintenance of ductal architecture is also important in pancreatic cancer invasion and metastasis. Site-directed mutagenesis of the NF-κB binding site decreased luciferase reporter activity suggesting that NF-κB is an activator of mir-218-1 expression at this alternative promoter. This suggests that mir-218-1 expression is uncoupled from SLIT2 expression and may contain its own tumor suppressor properties independent of SLIT2 signaling.

Matrix metalloproteinases (MMPs) are associated with tumor initiation and progression by targeting extracellular matrix (ECM) components. Invadopodia are structures that have been found to facilitate the invasive phenotype of many cancer types and are dynamic actin-rich cellular projections that localize proteases such as MMP2, MMP9, and MMP14 to degrade the ECM. In addition to ROBO1, we identified additional novel mir-218-1 target genes that control PDAC cell invasion. Our results further demonstrate that mir-218-1 functions in the suppression of invadopodia maturation and metalloproteinase matrix degradation. Overall, our data establishes that mir-218-1 is a key independent mediator of pancreatic cancer invasion through the extracellular matrix.

Citation Format: Brenna A. Rheinheimer, Lukas Vrba, Bernard W. Futscher, Ronald L. Heimark. Post-transcriptional regulatory networks of pancreatic tumor invasion. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A79.