The long range regulation of breast cancer associated genes

Abstract

PrefacePage iBreast cancer is the most common non-cutaneous cancer of women and a major cause of mortality [1]. Genetic factors are the single biggest risk factor and 75% of the risk derives from common but low penetrance single nucleotide polymorphisms (SNPs) [2]. These are found using genome-wide association studies (GWAS) and the majority localize to non-coding regions of the genome [3] The identification of CCND1 as an interacting partner of PRE1 and PRE2 was done using a candidate gene approach with 3C (chromosome conformation capture) targeted confirmation of interaction. To detect other potential targets of PRE1 and PRE2 at the 11q13 locus using an agnostic approach, the 4Cseq and 5C techniques were used. These revealed a number of local interaction regions covering six gene promoters. A novel strategy using knockdown of enhancer RNA transcribed from PRE1 then identified the IGHMBP2 and CPT1A genes as likely additional targets of PRE1. Genome editing with transcription activator-like effector nucleases (TALENS) was also used, creating isogenic cell lines to clarify the effects of the SNPs in their native genomic context. Further functional work is required, however the range of techniques employed has substantially expanded our understanding of the 11q13 breast cancer risk locus and forms a template for future investigations of GWAS risk loci.To identify noncoding RNAs expressed at the 11q13 locus that may be interacting with PRE1 or PRE2 required the use of RNA Capture-seq. RNA Capture-seq involves a targeted enrichment step that greatly increases the sequencing depth at regions of interest and can reveal lowly expressed transcripts that may have not been found by traditional RNA-seq [7]. This identified one novel long non-coding RNA expressed from the (+) strand that was named CUPID1 and a second arising from the same locus on the (-) strand named CUPID2. They were located in the nucleus, oestrogen induced and both expressed relatively highly in ERα positive breast cancer cell lines but not in ERα The 11q13 locus is amplified in around 20% of breast cancers and is thought to contain a number of driver genes including CCND1 [8]. Given that CUPID1 and CUPID2 are widely over-expressed inERα positive breast cancer cell lines, it was hypothesized that they may also have a role in driving tumour growth. Publically available RNAseq data showed CUPID2 to be highly expressed in breast and renal cancer but not in normal tissue. Stable cell lines over-expressing the lncRNAs were then generated and subsequent oncogenic assays revealed that CUPID2 increased cellular proliferation and the efficiency of colony formation in breast cancer cells. A murine xenograft model confirmed these findings in vivo by demonstrating a marked increase in tumour size for the mice injected with cells over-expressing CUPID2. There is thus evidence that CUPID2 behaves as an oncogene and may be an additional driver of the 11q13 amplicon in ERα positive breast cancer.In summary, CPT1A and IGHMBP2 were identified as potential mediat...