Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease

Abstract

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors.

The Role of WRN Helicase/Exonuclease in DNA Replication

“…Notably, it is not only WS patients who are more susceptible to cancer on WRN loss: epigenetic inactivation by methylation of CpG islands in the WRN gene promoter has been reported in epithelial and mesenchymal cancers with value in prognosis in colorectal cancer [9], while specific WRN SNPs have been correlated with breast cancer incidence [10], even though such genetic changes do not alter the helicase or exonuclease activities of the protein or modulate the levels expressed. WRN is therefore of interest not only to those attempting to understand the molecular basis of human ageing, but also to cancer biologistsindeed WRN knockdown is likely to promote cancer cell death and hypersensitise cells to current chemotherapeutic agents such as camptothecin that impact on DNA replication [9,11,12]. Small molecules that specifically inhibit WRN but not other RecQ helicases are therefore likely to have therapeutic potential [13].…”

EXD2 and WRN exonucleases are required for interstrand crosslink repair in Drosophila

“…Furthermore, WRN preferentially binds to DNA containing Holliday junction and/or replication fork structures [28][29][30][31], and is present at replication foci coincident with RPA and PCNA [32,33]. Cells derived from WS patients are highly sensitive to agents such as camptothecin that cause replication fork arrest or collapse, further implicating WRN in DNA replication [34][35][36]. Loss or mutation of WRN results in aberrant DNA replication [37][38][39] and hyper-recombination [40][41][42][43][44].…”

Meeting report: British Society for Research on Ageing (BSRA) annual scientific meeting 2012, Aston University, Birmingham, 3rd to 4th July 2012

“…Dr Penelope Mason (Department of Biochemistry, University of Oxford, Oxford, UK) also uses WS as a model system and has created a plasmid containing miRNA targeted against the Werner protein (WRN), addition of which results in the reversible knockdown of WRN helicase and exonuclease in cells aged in culture [27]. The presence of WRN is thought to prevent premature aging by stabilizing the genome.…”