Putative DNA/RNA helicase Schlafen-11 (SLFN11) sensitizes cancer cells to DNA-damaging agents

Abstract

DNA-damaging agents (DDAs) constitute the backbone of treatment for most human tumors. Here we used the National Cancer Institute Antitumor Cell Line Panel (the NCI-60) to identify predictors of cancer cell response to topoisomerase I (Top1) inhibitors, a widely used class of DDAs. We assessed the NCI-60 transcriptome using Affymetrix Human Exon 1.0 ST microarrays and correlated the in vitro activity of four Top1 inhibitors with gene expression in the 60 cell lines. A single gene, Schlafen-11 (SLFN11), showed an extremely significant positive correlation with the response not only to Top1 inhibitors, but also to Top2 inhibitors, alkylating agents, and DNA synthesis inhibitors. Using cells with endogenously high and low SLFN11 expression and siRNA-mediated silencing, we show that SLFN11 is causative in determining cell death and cell cycle arrest in response to DDAs in cancer cells from different tissues of origin. We next analyzed SLFN11 expression in ovarian and colorectal cancers and normal corresponding tissues from The Cancer Genome Atlas database and observed that SLFN11 has a wide expression range. We also observed that high SLFN11 expression independently predicts overall survival in a group of ovarian cancer patients treated with cisplatin-containing regimens. We conclude that SLFN11 expression is causally associated with the activity of DDAs in cancer cells, has a broad expression range in colon and ovarian adenocarcinomas, and may behave as a biomarker for prediction of response to DDAs in the clinical setting.

Fig. 1.

SLFN11 expression is highly correlated with the in vitro antiproliferative activity of Top1 inhibitors. (A) Scatterplot showing the correlation between SLFN11 expression (y axis, Log₂ intensity) and CPT antiproliferative activity (x axis, negative Log₁₀ growth inhibitory molar concentration 50%, GI50) in the NCI-60. (B) Mean centered bar charts representing SLFN11 expression in the NCI-60 and the antiproliferative activities of CPT, topotecan, irinotecan, and the indenoisoquinoline Top1 inhibitor in clinical trial NSC724998. Color codes correspond to tissues of origin.

Fig. 2.

Silencing SLFN11 significantly reduces sensitivity to different classes of DNA-damaging agents in cells expressing high endogenous SLFN11 levels. (A) Cytotoxicity curves of the prostate cancer cell line DU-145 (high SLFN11 expresser) transfected with nontargeting (ctrl) or SLFN11-targeting siRNAs and treated for 72 h with CPT, etoposide, cisplatin, taxol, or staurosporine (STS). Mean values ± SD are shown (one representative experiment performed in triplicate). (Lower Right) Western blot showing SLFN11 knockdown 3 and 6 d after transfection with SLFN11-targeting siRNAs. (B) (Left) Representative image of a clonogenic assay (100 nM CPT for 1 d). (Right) Number of colonies formed after 24 h treatment with CPT followed by a 15-d release (average of three independent experiments). (C) Cytotoxicity curves of the breast cancer cell line MDA-MB-231 (low SLFN11 expresser) transfected with nontargeting (ctrl) or SLFN11-targeting siRNAs and treated for 72 h with CPT, etoposide, and cisplatin. Mean values ± SD are shown (one representative experiment performed in triplicate). Whiskers in all charts represent SDs (when not visible they were within the symbol size). (D) FACS analysis showing sustained cell cycle progression and lack of apoptosis in SLFN11 knockdown DU-145 cells treated with 100 nM CPT for 16 h.

Fig. 3.

SLFN11 expression varies over a wide range in human colon and ovarian carcinoma compared with healthy tissues and may predict overall survival in ovarian cancer patients. (A) SLFN11 expression in healthy colon mucosa (n = 19) and colon adenocarcinoma (n = 37, Left; y axis: Log₂ 75th percentile normalized data obtained using Agilent 244k expression microarrays), and ovarian healthy tissue (n = 8) and ovarian cystoadenocarcinoma (n = 38; Right; y axis: Log₂ GC-robust multiarray normalized data from Affymetrix Human Exon 1.0 ST v2 expression microarrays). Medians are represented as horizontal lines, and whiskers show the interquartile expression ranges. (B) Kaplan–Meyer curves of 110 patients affected by ovarian cancer and treated with a first-line cisplatin-containing regimen. Patients are stratified as having higher or lower than average SLFN11 expression levels in that cohort (y axis: percentage survival; x axis: overall survival in months from diagnosis).