Functional dissection of breast cancer risk-associated TERT promoter variants.

Sonja Helbig,Juliet D French,Karen Mccue,Thilo Dörk,Ursula Hille-Betz,Jonathan Beesley,Annick Bouendeu,Stacey L Edwards,Hilda A Pickett,Leesa Wockner,Georgia Chenevix-Trench,Roger R Reddel

doi:10.18632/oncotarget.18226

Abstract

The multi-cancer susceptibility locus at 5p15.33 includes TERT, encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT-CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.

Highlights

Telomeres consist of DNA tandem repeats and telomere binding proteins, which together form secondary nucleoprotein structures at the end of chromosomes [1, 2]
Genome-wide association studies (GWAS) have been very successful in identifying cancer susceptibility loci, but to pin-pointing the causal variants, among many correlated candidate variants, usually requires subsequent in silico annotation and functional studies [57]
Risk-associated alleles in the TERT promoter are associated with decreased overall breast cancer risk and the association is most significant with estrogen receptor (ER)-negative breast cancer [41]

Summary

Introduction

Telomeres consist of DNA tandem repeats and telomere binding proteins, which together form secondary nucleoprotein structures at the end of chromosomes [1, 2]. Due to the “end-replication problem”, the ends of www.impactjournals.com/oncotarget linear DNA molecules are not fully replicated, and this limits the number of divisions a cell can undergo before entering the state of permanent cell cycle arrest referred to as senescence [6,7,8,9,10]. Another function of telomeres is to act as a buffer against the loss of coding or regulatory DNA during cell proliferation. When cells bypass senescence and continue to proliferate, telomeres become shortened to a critical length leading to a state of crisis with chromosomal end-to-end-fusion events and genomic instability [11, 12]

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