Werner Syndrome Helicase Is Required for the Survival of Cancer Cells with Microsatellite Instability

Abstract

Werner syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss-of-function mutations in WRN led to premature aging and predisposition to cancer. We evaluated synthetic lethal (SL) interactions between WRN and another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN was SL with a DNA mismatch repair protein MutL homolog 1, loss of which is associated with high microsatellite instability (MSI-H). MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles, and decreased viability in response to WRN knockdown, in contrast to microsatellite stable (MSS) lines, which tolerated depletion of WRN. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors.

Keywords: Biological Sciences; Cancer; Molecular Biology.

Graphical abstract

Figure 1

MSI cell lines are sensitive to WRN knockdown. (A) Terminal cell counts in a 10-day proliferation assay after transfection of A549 cells with control, WRN (left) or BLM (right), and three independent MLH1 siRNAs. Excess over Bliss for WRN and MLH siRNA 1 (MLH1.1) = 0.48, WRN and MLH siRNA 2 (MLH1.2) = 0.47, and WRN and MLH siRNA 3 (MLH1.3) = 0.38. One-way ANOVA ***p ≤ 0.0001, **p ≤ 0.001, *p ≤ 0.01. (B) Graphs showing terminal cell counts in 7- to 10-day proliferation assays following transfection of five MSI cell lines (HCT116, LoVo, RKO, SW48, LS174T) and three MSS cell lines (SW620, SW948, and T84). Cells were transfected with control, BLM, WRN, or Kif11 (essential gene and positive control for decreased proliferation). Data are representative of three biological experiments. Error bars represent SEM.

Figure 2

WRN Helicase Domain Rescues the WRN Knockdown Loss of Proliferation Phenotype in MSI Cells (A) Schematic representation of endogenous WRN wild-type (WT) and siRNA-resistant exogenous transcripts (WT, E84A [exonuclease-dead], K577R [helicase-dead], and E84A/K577R [enzymatically dead]). The endogenous transcript contains a 5′ UTR that can be selectively targeted by an siRNA (WRN 5′UTR siRNA). An internally targeting siRNA (WRN siRNA) leads to a decrease in both endogenous and exogenous WRN transcripts. (B) WRN and tubulin immunoblots of HCT116 rescue cells transfected with the indicated siRNAs. Numbers on blots indicate where molecular weight (kD) bands of protein ladder would be. (C) Growth curves of MSI (HCT116, RKO, LoVo) cells transfected with the indicated siRNAs. Relative caspase activity (raw caspase activity/cell number) over time in HCT116 rescue cell lines transfected with the indicated siRNAs (second row). Error bars represent SEM. Data are representative of three biological experiments.

Figure 3

WRN Knockdown Increases γH2AX and p21 Levels and Alters the Cell Cycle of MSI Cells (A) Representative immunofluorescence images of HCT116 cells transfected for 72 h with WRN (top row) or control (bottom row) siRNAs and incubated with Hoechst stain (DNA), p21, and γH2AX antibodies. Individual channels are shown in gray scale and overlay of all three channels in pseudo color. Scale bars, 50 μm. (B) Graphs showing quantification of γH2AX and p21 staining from multiple images collected from two MSI cell lines (HCT116 and RKO) and one MSS cell line (SW620). Error represents STDEV. (C) Flow cytometry data measuring cell cycles states of HCT116 cells 48 and 72 h after transfection with control or WRN siRNA. Data are representative of two biological experiments.

Figure 4

Model of WRN Function in MSS versus MSI cells During replication, non-canonical DNA structures (hairpin) such as G4 quadruplexes in MSS cells are resolved by WRN and/or other helicases (left). DNA mismatches (open triangle) are repaired by MMR proteins. In MSI cells (right), there is an increase in non-canonical secondary structures creating a requirement for WRN for their resolution. When WRN expression is decreased, the replication machinery runs into the unresolved structures eventually leading to an increase in double-stranded breaks. Cells either arrest in G2 or proceed into mitosis with DNA damage. The latter cells fail to complete cytokinesis leading to G1 arrest or apoptosis.