Frequent inactivation of cysteine dioxygenase type 1 contributes to survival of breast cancer cells and resistance to anthracyclines

Abstract

Purpose: Genome-wide DNA methylation analyses have identified hundreds of candidate DNA-hypermethylated genes in cancer. Comprehensive functional analyses provide an understanding of the biologic significance of this vast amount of DNA methylation data that may allow the determination of key epigenetic events associated with tumorigenesis.

Experimental design: To study mechanisms of cysteine dioxygenase type 1 (CDO1) inactivation and its functional significance in breast cancer in a comprehensive manner, we screened for DNA methylation and gene mutations in primary breast cancers and analyzed growth, survival, and reactive oxygen species (ROS) production in breast cancer cells with restored CDO1 function in the context of anthracycline treatment.

Results: DNA methylation-associated silencing of CDO1 in breast cancer is frequent (60%), cancer specific, and correlates with disease progression and outcome. CDO1 function can alternatively be silenced by repressive chromatin, and we describe protein-damaging missense mutations in 7% of tumors without DNA methylation. Restoration of CDO1 function in breast cancer cells increases levels of ROS and leads to reduced viability and growth, as well as sensitization to anthracycline treatment. Priming with 5-azacytidine of breast cancer cells with epigenetically silenced CDO1 resulted in restored expression and increased sensitivity to anthracyclines.

Conclusion: We report that silencing of CDO1 is a critical epigenetic event that contributes to the survival of oxidative-stressed breast cancer cells through increased detoxification of ROS and thus leads to the resistance to ROS-generating chemotherapeutics including anthracyclines. Our study shows the importance of CDO1 inactivation in breast cancer and its clinical potential as a biomarker and therapeutic target to overcome resistance to anthracyclines.

Figure 1

Silencing of CDO1 is associated with DNA promoter hypermethylation or repressive chromatin structure. A, appearance of CDO1 within the breast cancer hypermethylome. Cell lines were treated with either 5 μmol/L DAC for 96 hours or 300 nmol/L TSA for 18 hours. Gene expression changes (analyzed on 4 × 44 K Agilent platform) are plotted by fold change (log scale) after DAC (y-axis) or TSA (x-axis) treatment. B, quantitative mRNA expression of CDO1 in DAC- (5 μmol/L 96 hours) or TSA- (300 nmol/L 18 hours) treated cells is shown in fold change (log₂) relative to mock-treated cells. Expression of CDO1 in normal breast (NB) is shown in relation to basal expression levels of CDO1 in other cell lines. Group comparisons were carried out using Student t test. ^*, P < 0.05. C, bisulfite sequencing of the CDO1 promoter region from −192 bp to +60 bp relative to the transcription start site (TSS). White and black circles represent unmethylated and methylated CpG dinucleotides, respectively. D, ChIP at the CDO1 promoter region from −154 bp to −29 bp relative to TSS for α-H3k4me2 and α-H3k27me3. Data presented are the mean levels of enrichment relative to input obtained by real-time PCR from 2 independent experiments ± SEM.

Figure 2

DNA methylation-associated silencing of CDO1 is cancer specific, frequent, and correlates with disease progression and outcome. A, DNA methylation and protein expression status of CDO1. CDO1 promoter region from −168 bp to −45 bp relative to the TSS was assayed by MSP in a cohort of 20 normal breast tissues from non–cancer patients and 185 primary breast cancers (BC) of stages 0 (DCIS) to 4 with U and M marking unmethylated and methylated bands, respectively. Representative examples are shown for each cohort and each tumor stage. In vitro methylated DNA (IVD), DKO cells, normal lymphocytes (NL), and H₂O controls were assayed along with samples. Protein expression status of CDO1 was assayed by immunohistochemistry in normal breast and selected primary breast cancers. Note, BC013a, a DCIS sample, displays loss of CDO1 expression in hyperproliferative ductuli (HD) but expression of CDO1 in normal ductuli (ND). B, scatter plot depicting correlation between expression log₂ values (y-axis; analyzed on Agilent 244 K Custom Gene Expression G4502A-07 platform) and DNA methylation β values (x-axis; analyzed on Illumina HumanMethylation 27k platform) of CDO1 in 255 primary breast cancers from TCGA data portal. A Spearman correlation coefficient of ρ = −0.47 and P value of 1.7e-15 were calculated. A P < 0.05 was considered statistically significant. C, DNA methylation frequency (in%) of CDO1 plotted by tumor stage of 185 primary breast cancers. CDO1 methylation status and tumor stage were correlated using Pearson χ² test. ^*, P < 0.05. D, survival curve depicting prognostic value of CDO1 methylation status for outcome prediction in 185 primary breast cancers. HR for prognostic value was calculated using univariate Cox proportional hazards regression model.

Figure 3

Restoration of CDO1 function reduces growth and viability of cancer cells and their capacity to detoxify ROS. A, tumor cell clonogenicity was assessed on plastic and in soft-agar. Cells were transiently transfected with pcDNA3.1 (empty vector), pcDNA3.1-CDO1-WT (wild-type CDO1), or pcDNA3.1-CDO1-MU (mutant CDO1), and replated 24 hours posttransfection for selection with Geneticin/G418. After 18 days of selection, colonies were stained with Giemsa and counted. Data presented are the mean of 2 independent experiments ± SEM. Group comparisons were carried out using Student t test and trend test. ^*, P < 0.05. Reexpression of CDO1 was confirmed 48 and 96 hours posttransfection by Western blot analysis using α-V5 antibody, targeting the V-5-His tag of recombinant CDO1 protein, and α-GAPDH as a control. ROS production and cell viability were assayed in tetracycline-inducible CDO1-stable MDA-MB-231 cells before and after treatment with 0.5 μg/mL doxycycline (Dox; B), and 293 cells before and after the treatment with 0.5 mmol/L BSO (for depletion of glutathione; C) by fluorescence of the CM-H2DCFDA probe. Obtained values were normalized to untreated or treated empty vector controls and plotted as % relative to untreated MDA-MB-231-CDO1-WT or untreated 293 cells. Data presented are the mean of 3 independent experiments ± SEM. Group comparisons were carried out using Student t test. ^*, P < 0.05; n.s., not significant. Reexpression of CDO1 in MDA-MB-231 cells 48 hours after doxycycline treatment or downregulation of CDO1 expression in 293 cells 24, 48, and 72 hours after glutathione depletion with BSO was assessed by Western blot analysis using α-CDO1 antibody and α-β-actin as a control.

Figure 4

CDO1-induced reduction in ROS detoxification sensitizes breast cancer cells to anthracycline treatment. A, cell viability of doxycycline-induced/not doxycycline-induced CDO1-stable MDA-MB-231 cells before and 48 hours after treatment with different doses of anthracycline (doxorubicin) was measured by MTS assay. Data presented are the mean of 2 independent experiments ± SEM. Group comparisons were carried out using Student t test. ^*, P < 0.05. B, ROS production, using CM-H2DCFDA probe and cell viability of same cells before and 48 hours after treatment with 0.078 μmol/L doxorubicin was measured. Data presented are the mean of 2 independent experiments ± SEM. Obtained values for A and B were normalized to anthracycline-untreated or -treated doxycycline-induced/not doxycycline-induced empty vector control cells and plotted as % relative to anthracycline-untreated and not doxycycline-induced MDA-MB-231-CDO1-WT cells. Group comparisons were carried out using Student t test. ^*, P < 0.05; n.s., not significant.

Figure 5

Reactivation of epigenetically silenced CDO1 through priming with 5-azacytidine contributes to the sensitization of breast cancer cells to anthracycline treatment. A, cell viability of MDA-MB-231, MCF7, and T47-D cells primed with 5-azacytidine at doses ranging from 1 μmol/L to 5 μmol/L for 72 hours and subsequently treated with doxorubicin at doses ranging from 0.078 μmol/L to 20 μmol/L for 48 hours. Obtained values are plotted as % relative to doxorubicin-untreated cells. Group comparisons were carried out using Student t test. ^*, P < 0.05 for 1 μmol/L 5-azacytidine in MCF7 and T47-D cells or 2 μmol/L in MDA-MB-231 cells. ⁺, P < 0.05 for 2 μmol/L 5-azacytidine in MCF7 and T47-D cells or 5 μmol/L in MDA-MB-231 cells. B, quantitative reexpression of CDO1 in 5-azacytidine (1 μmol/L, 2 μmol/L, or 5 μmol/L for 72 hours)-treated cells prior doxorubicin treatment is shown in fold change (log₂) relative to mock-treated cells. Group comparisons were carried out using Student t test. ^*, P < 0.05. C, cell viability of doxycycline-induced CDO1-stable MDA-MB-231 cells (doxycyline was supplemented every 24 hours throughout the entire experiment) primed with 5-azacytidine at a dose of 2 μmol/L for 72 hours and subsequently treated with doxorubicin at a dose of 1.25 μmol/L for 48 hours. Obtained values are plotted as % relative to doxorubicin-untreated cells. As a control, cell viability was measured in CDO1-stable MDA-MB-231 cells that were not treated with 5-azacytidine. D, restoration of CDO1 protein expression in MDA-MB-231 cells 72 hours posttreatment with doxycycline and with or without 5-azacytidine was confirmed by Western blot using α-CDO1 antibody and α-β-actin as a control.