Epigenetic inactivation of the premature aging Werner syndrome gene in human cancer

Abstract

Werner syndrome (WS) is an inherited disorder characterized by premature onset of aging, genomic instability, and increased cancer incidence. The disease is caused by loss of function mutations of the WRN gene, a RecQ family member with both helicase and exonuclease activities. However, despite its putative tumor-suppressor function, little is known about the contribution of WRN to human sporadic malignancies. Here, we report that WRN function is abrogated in human cancer cells by transcriptional silencing associated with CpG island-promoter hypermethylation. We also show that, at the biochemical and cellular levels, the epigenetic inactivation of WRN leads to the loss of WRN-associated exonuclease activity and increased chromosomal instability and apoptosis induced by topoisomerase inhibitors. The described phenotype is reversed by the use of a DNA-demethylating agent or by the reintroduction of WRN into cancer cells displaying methylation-dependent silencing of WRN. Furthermore, the restoration of WRN expression induces tumor-suppressor-like features, such as reduced colony formation density and inhibition of tumor growth in nude mouse xenograft models. Screening a large collection of human primary tumors (n = 630) from different cell types revealed that WRN CpG island hypermethylation was a common event in epithelial and mesenchymal tumorigenesis. Most importantly, WRN hypermethylation in colorectal tumors was a predictor of good clinical response to the camptothecin analogue irinotecan, a topoisomerase inhibitor commonly used in the clinical setting for the treatment of this tumor type. These findings highlight the importance of WRN epigenetic inactivation in human cancer, leading to enhanced chromosomal instability and hypersensitivity to chemotherapeutic drugs.

Fig. 1.

Analysis of WRN CpG island promoter methylation status and gene function in human cancer cell lines. (A) Schematic depiction of the WRN CpG island around the transcription start site (long black arrow). CpG dinucleotides are represented as short vertical lines. Location of bisulfite genomic sequencing PCR primers and methylation-specific PCR primers are indicated as white and gray arrows, respectively. Shown are results of bisulfite genomic sequencing of 12 individual clones. Presence of a methylated or unmethylated cytosine is indicated by a black or white square, respectively. (B) Methylation-specific PCR for the WRN gene in human cancer cell lines. The presence of a PCR band under lanes M or U indicates methylated or unmethylated genes, respectively. In vitro methylated DNA (IVD) is used as positive control for methylated DNA. (C) RT-PCR analysis of WRN expression. Treatment with the demethylating agent (ADC + lanes) reactivates WRN gene expression. (D) Western blot analysis of WRN expression. The WRN hypermethylated cell lines HCT-116, MDA-MB-231, and U937 do not express the WRN protein or have minimal expression (MDA-MB-231). The treatment with the demethylating agent reactivates WRN gene expression. WS−/− cells are shown as negative control. (E) Immunofluorescence analysis of WRN expression. The methylated cell lines COLO-205 and U937 and the mutant WS−/− cells do not stain for the WRN protein, in comparison with the unmethylated MCF-7 cells. Treatment with the demethylating agent (DAC) restores protein expression. (F) Exonuclease activity assay in WRN-immunoprecipitated cell resolved on denaturing polyacrylamide gels. The 3′-recessed duplex substrate used for exonuclease studies was degraded more extensively in WRN unmethylated cells (MCF-7 and HL60) than in WRN methylated (U937 and MDA-MB-231) or mutated (WRN−/−) cells.

Fig. 2.

Tumor-suppressor-like properties of WRN reintroduction. (A) Colony-formation assay. (Upper Left) WRN expression monitored by RT-PCR in untransfected and WRN-transfected MDA-MB-231 cells monitored by RT-PCR. (Lower Left) Densitometric quantification of the colony formation density of MDA-MB-231 cells transfected with the empty vector or with WRN. Three independent experiments were carried out. (Right) Example of the colony focus assay after a 2-week selection with G418 and staining with methylene blue. (B) Effect of WRN transfection on the in vivo growth of MDA-MB-231 cells. Tumor weight and size was monitored over time. Shown are female athymic nude mice 30 days after injection of 10⁶ MDA-MB-231 cells. Note the large tumor on the left flank, corresponding to empty vector MDA-MB-231 cells, and the small tumor on the opposite flank, corresponding to WRN-MDA-MB-231 cell injection.

Fig. 3.

Hypermethylation-deficient WRN cancer cells are sensitive to inhibitors of topoisomerase I and DNA-damaging agents. (A) Induction of apoptosis measured by flow cytometry in unmethylated (MCF-7), methylated (MDA-MB-231), and mutated (WS−/−) WRN cells at increasing concentrations of camptothecin and mitomycin C. MDA-MB-231 and WS−/− cells are highly sensitive in comparison with MCF-7. Restoration of WRN expression in MDA-MB-231 cells induces resistance to apoptosis by both drugs. (B) Chromosomal breakage measured by cytogenetic analysis of metaphase chromosomes. (Upper Left) Untreated MDA-MB-231 cells have undetectable fragility. (Upper Right) 50 mg/ml mitomycin C-treated cells undergo a massive breakage (empty vector). (Lower Left) MDA-MB-231 cells transfected with the WRN gene display resistance to the genome damage. (Lower Right) Quantification of chromosomal breakage induced by mitomycin C in cells proficient (HL60 and MCF-7) or deficient in WRN function by mutation (WS−/−) or methylation (MDA-MB-231). Transfection of the WRN gene in MDA-MB-231 cells provokes resistance to the genomic damage induced by the drug. (C) siRNA assay for the WRN transcript in MCF-7 cells. (Right) Western blot of WRN-knocked-down MCF-7 cells by siRNA. (Left) Quantification of chromosomal breakage induced by mitomycin C in MCF-7 proficient cells or cells deficient in WRN function by siRNA. WRN-knocked-down MCF-7 cells are prone to chromosomal breakage upon exposure to mitomycin C.

Fig. 4.

WRN CpG island hypermethylation in primary human malignancies. (A) Analysis of WRN methylation by methylation-specific PCR. The presence of a PCR band under lane M indicates methylated genes. Normal lymphocytes (NL) and in vitro methylated DNA (IVD) are used as negative and positive control for unmethylated and methylated genes, respectively. (B) WRN protein expression in primary human tumors. (Upper) Immunohistochemistry of WRN in a normal gastric gland (Left), unmethylated at WRN, and in an unmethylated gastric tumor showing strong WRN expression (Center) and a methylated gastric tumor demonstrating loss of WRN staining (Right). (Lower) Western blot analyses of colorectal tumors showing the tight association between WRN methylation and loss of expression. (C) Kaplan–Meier analysis of WRN promoter hypermethylation in patients with colorectal cancer treated with irinotecan and its impact on survival. A significant increased overall survival is observed in patients with WRN methylation.