Abstract 2559: Long non-coding RNAs as functional targets of germline genetic variation at ovarian cancer susceptibility loci.
Authors: Lawrenson Kate , Song Honglin , Lee Janet , Tyrer Jonathan , Ramus Susan , Pharoah Paul , Gayther Simon 
Published: August 2014

An emerging theme in genome-wide association studies (GWAS) is that the majority of common low-risk variants fall within non-protein coding DNA, with no immediately obvious functional role in disease initiation and development. As part of an international consortium (the ovarian cancer association consortium) we have identified eleven regions of the genome that harbor common single nucleotide polymorphisms (SNPs) that are associated with epithelial ovarian cancer (EOC) risk. We hypothesize that a proportion of risk SNPs identified by GWAS coincide with regulatory non-coding RNA species, such as long non-coding RNAs (lncRNAs). We used RNA sequencing (RNAseq) of five EOC cell lines and ten normal EOC precursor cell lines to identify lncRNAs that overlap EOC risk SNPs. Fine mapping of EOC susceptibility regions using 1000 Genome project data in 18,000 invasive EOC cases and 20,000 controls identified suspected functional variants at each known risk locus. We then integrated RNAseq and genotyping data to identify SNPs mapping to ncRNAs.

At the 2q31 risk locus a lncRNA gene (LOC401022) overlapped with 7 of 19 EOC risk variants, three of which were predicted to alter the sequence of the mature lncRNA transcript. RNAseq profiling showed that LOC401022 was overexpressed in EOC cell line compared to normal ovarian cancer precursor tissues; this was validated in a larger panel of 51 cancer and 67 normal cell lines (P=9.8x10-12). Analysis of copy number variation in ∼500 high-grade serous tumors profiled by The Cancer Genome Atlas (TCGA) project showed copy number gain in this region in over 40% of EOCs supporting the hypothesis that LOC401022 has oncogenic role during EOC development. Lentiviral delivery of a targeted shRNA was used to knockdown LOC401022 expression in OV90 EOC cells. LOC401022 knockdown was associated with reduced proliferation and a marked epithelial-to-mesenchymal transition compared to control cells transduced with a non-targeting shRNA. Cytokeratin expression was lost and vimentin expression upregulated following LOC401022 knockdown.

LOC401022 contains 145bp of sequence that has antisense similarity to the HOXD1 gene. However, in our EOC cell lines and in the cancer cell line encyclopedia, HOXD1 and LOC401022 expression are positively correlated (R2=0.48 and 0.62 respectively), and LOC401022 knockdown did not influence HOXD1 expression. This suggests that factors upstream of these two genes may regulate co-expression of both LOC401022 and HOXD1. We went on to perform genome wide expression profiling in OV90 cells after LOC401022 knockdown using RNAseq, to identify functional targets of this lncRNA and have identified several putative susceptibility genes which TCGA data suggest are involved EOC development. To our knowledge, this is the first instance in which a lncRNA has been implicated as a functional target of genetic variation at an ovarian cancer susceptibility locus.

Citation Format: Kate Lawrenson, Honglin Song, Janet Lee, Jonathan Tyrer, Susan Ramus, Paul Pharoah, Simon Gayther. Long non-coding RNAs as functional targets of germline genetic variation at ovarian cancer susceptibility loci. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2559. doi:10.1158/1538-7445.AM2013-2559

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.