The identification and interpretation of pathogenic non-coding mutations in pancreatic ductal adenocarcinoma.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive and lethal malignancies. Despite significant advances in medicine and surgery, pancreatic cancer still has a dismal 5-year survival rate below 8%. Much emphasis to date has been directed into identifying somatic mutations in the coding regions of the genome, identifying several known drivers including KRAS, TP53, CDNK2A, AIRD1A, and SMAD4. However, the coding regions comprise only 2% of the human genome. The other 98% comprises non-coding DNA, where somatic non-coding mutations (NCMs) are becoming increasingly associated with pathogenic roles in cancer. To date, little is known regarding somatic NCMs and their regulatory affects in PDAC. We integrate publicly available ChIP-Seq data for the enrichment of active promoter and enhancer regions, RNA-seq data for gene expression analysis and whole genome sequencing data produced by the International Cancer Genome Consortium (659 PDAC patients). Using our analytic pipeline of two independent approaches, we identify 14 regulatory regions harbouring putative functional somatic mutations or recurrent variants, arising proximal to PDAC relevant genes including an enhancer within the intron of the transcription factor FOXP1 and the promoter of the oncogenic long non-coding RNA (lncRNA), HOTAIR. Furthermore, pathway analysis identified NCMs in close proximity to genes involved in transcriptional regulation and biological processes such as cell adhesion. Using a combination of luciferase reporter and high-throughput STARR-seq assays, we assess the regulatory activity of 587 somatic NCMs with putative transcriptional factor motif gain or loss of function capabilities. Our results show 43 NCMs with significant gene reporter expression alterations. Interestingly, we identify NCMs located in a 2kb region within the intron of the cancer associated polycistronic lncRNA MIR100HG, which demonstrates an up-regulation in gene reporter activity. This site is also located proximal to the oncogenic mir-125b-1. CRISPR-Cas9 mediated deletion of this 2kb element demonstrated phenotypic alterations in PDAC cell lines, with a reduced capacity in cell migration and clonogenicity. We also observe evidence of differential expression of nearby TAD associated genes, suggesting important regulatory roles of this 2kb intronic element. This study represents a step towards unravelling the non-coding regions of the genome and to the identification of novel biomarkers and therapeutic targets in this deadly disease.