Cytidine Deaminase Deficiency Reveals New Therapeutic Opportunities against Cancer

Abstract

Purpose: One of the main challenges in cancer therapy is the identification of molecular mechanisms mediating resistance or sensitivity to treatment. Cytidine deaminase (CDA) was reported to be downregulated in cells derived from patients with Bloom syndrome, a genetic disease associated with a strong predisposition to a wide range of cancers. The purpose of this study was to determine whether CDA deficiency could be associated with tumors from the general population and could constitute a predictive marker of susceptibility to antitumor drugs.Experimental Design: We analyzed CDA expression in silico, in large datasets for cancer cell lines and tumors and in various cancer cell lines and primary tumor tissues using IHC, PDXs, qRT-PCR, and Western blotting. We also studied the mechanism underlying CDA silencing and searched for molecules that might target specifically CDA-deficient tumor cells using in silico analysis coupled to classical cellular experimental approaches.Results: We found that CDA expression is downregulated in about 60% of cancer cells and tissues. We demonstrate that DNA methylation is a prevalent mechanism of CDA silencing in tumors. Finally, we show that CDA-deficient tumor cells can be specifically targeted with epigenetic treatments and with the anticancer drug aminoflavone.Conclusions: CDA expression status identifies new subgroups of cancers, and CDA deficiency appears to be a novel and relevant predictive marker of susceptibility to antitumor drugs, opening up new possibilities for treating cancer. Clin Cancer Res; 23(8); 2116-26. ©2016 AACR.

Figure 1.

CDA expression levels in cancer cell lines and tissues. A, Transcriptomic datasets in log 2 values for Curie Institute breast cancer cell lines (n = 34) publicly available (see Materials and Methods section; left) and from NCI 60 cancer cell lines data miner (right; ref. 45). Mean and median values are shown as dashed and solid lines, respectively. B, Real-time qRT-PCR and Western blot analyses of CDA expression in a set of 26 cell lines for three different cancers (breast, non–small lung, and ovary) representative of the Curie Institute (left) and NCI60 (right) panels. Hsp90 and β-actin were used as loading controls for Western blotting; TBP and GAPDH were used for qRT-PCR data normalization. Western blotting and qRT-PCR data were reproduced at least twice. C, Real-time qRT-PCR quantification of human (Hs in dark) and mouse (Mm in gray) CDA transcripts relative to human and mouse TBP housekeeping gene transcripts in 66 breast-derived xenografts. D, Immunohistochemical analysis of CDA expression in six different tumor tissues (n = 50/tissue). TN, triple negative. Representative images for each tissue (top) and quantitative representations of CDA protein levels in each tissue (bottom) are shown. The results are presented as percentages of tumors expressing low (black) and high (gray) levels of CDA on the basis of the scores obtained (low, scores 0–1; high, scores 2–3). Scale bar (for all images), 50 μm. Black bars, percentage of low-CDA cancer tissues. E, Scatter dot plot with mean ∓ SD for transcriptomic data for CDA transcripts, comparing unmatched normal and tumor tissues for the liver (GSE14520), esophagus (GSE13898), cervix (GSE9750), and colon (GSE9348). The data were retrieved from the Nextbio (58) and Oncomine (59) data sources and downloaded from GEO and presented as log 2 intensities. F, CDA transcript levels relative to TBP, as quantifiedby qRT-PCR in a mini cohort of cancerous and noncancerous colon tissues. Error bars, SD. The P values calculated in unpaired two-tailed t tests are considered statistically significant at <0.05.

Figure 2.

Silencing of CDA gene expression by DNA methylation. A, Mean-centered CDA transcript intensity data for the NCI60 panel of cell lines obtained with the NCI-CellMiner analysis tool (top left), and mean CDA gene methylation levels in the data for the NCI60 panel of cell lines extracted from GEO under accession number GSE66872 (bottom left), and representation of the correlation between CDA transcript intensity and CDA promoter methylation for the cg04087271 and cg00784581 probes (Pearson correlation; right). B, qRT-PCR analysis of the induction of CDA expression relative to GAPDH in cells initially with and without CDA expression, after 96 hours of treatment with 2.5 μmol/L 5-Aza-dC. ns, not significant. Error bars represent means ∓SD for at least three independent experiments. The P values were calculated by paired t test. All P values <0.05 were considered statistically significant. C, Left, qRT-PCR analysis (top) and Western blot analysis (bottom) of the induction of CDA expression in HCC-1954 and IGROV-1 cell lines left untreated (white bars) or treated with 1 μmol/L 5-Aza-dC for 96 hours (black bars). Survival curves of the HCC-1954 (n = 3; middle) and IGROV-1 (n = 5; right) cell lines left untreated (control, blue curve) or subjected to pretreatment for 96 hours with 1 μmol/L 5-Aza-dC (5-Aza-dC, red curve) and then treated with various doses of gemcitabine for a further 72 hours. Cell viability was assessed in the MTT assay. Error bars represent means ∓SD for three or five experiments. The P values were calculated in paired t tests. P values <0.05 were considered statistically significant. D, Scatterplots showing the Pearson correlation between mRNA sequencing data for CDA expression and CDA CpG cg04087271 methylation for 10 different cancer samples from TCGA. The data are publicly available and were retrieved from the Broad Institute FireBrowse portal (27) and the CBioPortal for Cancer Genomics database (29, 30). Dashed vertical lines, mean CDA expression; dashed horizontal lines, mean methylation level. All P values <0.05 were considered statistically significant.

Figure 3.

Drug sensitivity of CDA-deficient cells. A, SCE frequency in CDA-deficient and CDA-proficient cells (left) and representation of SCE frequency in cells classified on the basis of their CDA expression status, low or high (right). ns, not significant. P values were calculated by Mann–Whitney tests for at least three independent experiments. P < 0.05 was considered statistically significant. B, Scatterplot showing a significant negative correlation between aminoflavone cytotoxicity and CDA expression (Pearson correlation) in the NCI60 panel of cell lines. Colors, origin of the cancer tissue. C, Isogenic HeLa cell lines (red, HeLa control cells; blue, CDA-depleted HeLa cells) were treated for 72 hours with the indicated concentrations of aminoflavone, and the percentage of cells surviving is shown. D, Breast (MCF-7, MDA-MB-468, and MDA-MB-231) and ovarian (SKOV-3, OVCAR-8, and IGROV-1) cancer cell lines were treated for 72 hours with the indicated concentrations of aminoflavone (AF). Blue, survival curves of cell lines with low levels of CDA expression; red, survival curves of cell lines with high levels of CDA expression. For C and D, Cell viability was assessed in MTT assays. The error bars represent means ±SD for three independent experiments. P < 0.05 was considered statistically significant.