Cysteine dioxygenase 1 is a tumor suppressor gene silenced by promoter methylation in multiple human cancers

Abstract

The human cysteine dioxygenase 1 (CDO1) gene is a non-heme structured, iron-containing metalloenzyme involved in the conversion of cysteine to cysteine sulfinate, and plays a key role in taurine biosynthesis. In our search for novel methylated gene promoters, we have analyzed differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine. Among the genes identified, the CDO1 promoter was found to be differentially methylated in primary CRC tissues with high frequency compared to normal colon tissues. In addition, a statistically significant difference in the frequency of CDO1 promoter methylation was observed between primary normal and tumor tissues derived from breast, esophagus, lung, bladder and stomach. Downregulation of CDO1 mRNA and protein levels were observed in cancer cell lines and tumors derived from these tissue types. Expression of CDO1 was tightly controlled by promoter methylation, suggesting that promoter methylation and silencing of CDO1 may be a common event in human carcinogenesis. Moreover, forced expression of full-length CDO1 in human cancer cells markedly decreased the tumor cell growth in an in vitro cell culture and/or an in vivo mouse model, whereas knockdown of CDO1 increased cell growth in culture. Our data implicate CDO1 as a novel tumor suppressor gene and a potentially valuable molecular marker for human cancer.

Figure 1. Methylation of the CDO1 promoter in CRC.

A, Expression of CDO1 in CRC cell lines was examined by RT-PCR analysis. No basal expression of CDO1 was seen in all CRC cell lines, and all these cell lines harbored CDO1 methylation (M). Silenced CDO1 was reactivated after treatment with the demethylating agent, 5-Aza-dC, indicating that CDO1 methylation correlates tightly with loss of gene expression in CRC cell lines. β-actin was used as a loading control. m, cells treated with vehicle only; a, cells treated with 5-Aza-dC (5 µM for three days). L, 1 Kb Plus DNA ladder. B, Methylation status of individual CpGs of the island in 5 CRC cell lines and 21 pairs of primary CRC (PT) and their corresponding normal colon tissues (PN) is shown. A total of 38 CGs were numbered from the first to last CG in the sequences as indicated. Black circle, methylation; white circle, unmethylation; grey circle, co-existence of methylated and unmethylated alleles; dashed circle, undetermined. C, Analysis of CDO1 promoter activity by luciferase reporter assay in CDO1-negative (HCT116) and -positive (HEK293) cells. The promoter constructs (pGL2-CDO1-#1 and -#2) were pre-treated with or without SssI methylase for 8 hrs before transfection. High activity of the CDO1 promoter was detected in HEK293 where CDO1 was expressed. Data are expressed as fold increase over pGL2-basic activity. Experiments were done in triplicate, and values indicate means ± SD. Mean values are presented. D. Scatter plot of CDO1 methylation levels in tissues and cell lines (CL) (left). TaqMan methylation values (TaqMeth V) is described in Materials and Method. TaqMan-MSP was performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results. PT, primary CRC; PN, matched normal colon tissues from colon cancer patients; NN, normal colon epithelium from non-cancer patients. Line indicates the optimal cut-off value for CDO1 calculated from ROC analysis. Sample numbers showing TaqMeth V over the cut-off are indicated. The overall TaqMeth V detected in PT was significantly higher than that in PN (right). TaqMan value of two NN (22%, 2/9) was above the cut-off value (24.56 and 17.86 each), suggesting that a low level of CDO1 methylation can be caused by other unknown mechanisms. E, ROC curve analysis of TaqMeth V of CDO1 in CRC. The Area under the ROC (AUROC) conveys the accuracy in distinguishing matched normal colon (PN) from CRC (PT) in terms of its sensitivity and specificity (P<0.001). Solid line, CDO1; dashed line, no discrimination. F, Methylation levels of normal (PN) and tumor tissues (PT) in individual patients.

Figure 2. Methylation of the CDO1 promoter in multiple types of human cancer. A

, Quantitative methylation levels of CDO1 were determined in primary tissues derived from breast, esophagus, lung, bladder, and stomach. TaqMan methylation values (TaqMeth V) is described in Materials and Method. PT, primary tumor; PN, matched normal tissues; NN, normal tissues from non-cancer patients; CL, cell lines. Lines indicate the optimal cut-off value for each tissue. Arrow, MCF-12A, a non-tumorigenic cell line, harbored a low level of CDO1 methylation (TaqMeth V, 6.2). All assays were performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results. B, ROC analysis (PT vs. PN) of CDO1 in multiple human cancers. Solid line, CDO1; dashed line, no discrimination.

Figure 3. CDO1 mRNA levels in different types of cancer. A

, Expression of CDO1 in cell lines was examined by RT-PCR or qRT-PCR analyses. m, mock treatment. a, 5-Aza-dC treatment (5 µM for three days). β-actin was used as a loading control. Methylation status of CDO1 promoter in each cell line was examined and indicated as M for methylation, U for unmethylation, and M/U for co-existence of methylated and unmethylated alleles. CDO1 was completely methylated in all cancer cell lines since only cytosine peaks were observed in CpGs sequenced (100% methylation) while it was not methylated in HEK293 since only thymidine peaks were observed (0% methylation). CDO1 was partially methylated in MCF-12A since both methylated and unmethylated alleles were observed in 10 CpGs of the CDO1 promoters examined. When a cytosine peak were compared with a thymidine peak in each CpG of the 10 CpGs, cytosine peaks were dominant (methylated), but since these “methylated CpGs” were found in less than 50% of total CpGs (10/34), it was considered as “methylation-negative” according to the criteria described in Materials and Methods. B. qRT-PCR was performed in cDNAs derived from patients with colon, breast, esophagus, bladder and stomach cancer (T) and patients without cancer (NN) (upper). Relative expression (Fold) was calculated by comparing the ratios of mRNA expression of CDO1 to an internal control gene, β-actin. The CDO1 expression level was determined in 10 lung cancer patients and 10 patients without cancer (lower). 2?-()*100, the expression of CDO1 relative to β-actin calculated based on the threshold cycle (C_t) as 2^−ΔCt (ΔCt = C_t,CDO1 - C_t,β-actin). Experiments were done in duplicate, and values indicate means ± SD. *, P<0.05 in T-test. C, The CDO1 expression level was examined in five pairs (A ∼ E) of matched cDNA prepared from patients with colon and lung cancer. PT, primary tumor; PN, matched normal tissues.

Figure 4. Investigation of CDO1 expression with Cancer Profiling Arrays.

Cancer profiling arrays II was performed to compare CDO1 expression between tumor (T) and matched normal control (N) tissues of multiple tissue types. The array was hybridized with the CDO1 cDNA probe labeled with ³²P-α-deoxycytidine triphosphate according to the manufacturer’s protocol. Tissue type and the number of cases with down-regulation of CDO1 vs. total cases are indicated. Arrow, downregulation of CDO1 in tumor compared to normal tissue. Ubiquitin cDNA (Ubi) was used as a control. n/d, not determined.

Figure 5. Immunohistochemical analysis of CDO1 in colon and esophagus cancer tissue array. A

, Strong expression of CDO1 in non-malignant colon tissues. The expression of CDO1 protein was positive in 6 out of 6 patients. NN1, a patient without cancer, and two more cases are shown in Figure S2A. B, A group of samples were derived from a single patient consist of colon adenocarcinomas (AD), matched cancer adjacent normal appearing tissue (NAT) and matched cancer adjacent tissues (Adjacent). CDO1 expression was investigated in a total of 36 groups of samples. Patients were numbered arbitrarily (Pt1 ∼ Pt3). CRC Pt1, a patient with CRC. Two more cases are shown in Figure S2B. C, CDO1 expression in ESCC. PT, ESCC; PN, matched normal appearing tissues. ESCC Pt1, a patient with ESCC. Three more cases are shown in Figure S2C. D, CDO1 expression in colon adenocarcinoma (AD) with different tumor grades. Mu-AD, mucinous adenocarcinomas. E, CDO1 expression in ESCC with different tumor grades. EAD, esophageal adenocarcinomas; Me-ESCC, metastatic ESCC.

Figure 6. Tumor suppressive role of CDO1. A

. The MTT assay was performed in HCT116 clones stably expressing CDO1 or control clones. Cell growth was expressed as absorbance (Abs) at 570 nm wavelength. Two independent experiments were done in triplicate, and values are expressed as means ± SD. *, P<0.05 in T-test. B, Colony focus assays were performed in the HCT116 clones after incubation in the presence of G418 for 10 days. Colonies were stained with 0.4% crystal violet solution (MeOH/10% Acetic acid, 3∶1). After air-drying, colonies were photographed under a microscope (right). Values are expressed as means ± SD and are derived from experiments done in triplicate. C, Inhibitory ability of CDO1 in anchorage-independent cell growth was determined by the soft agar assay. Colonies at size >0.5 mm were counted (left). Colonies were photographed under phase-contrast microscope (middle) or under UV after staining with ethidium bromide in 1 × PBS/0.1% Triton X-100 solution overnight (right). Scale bar, 500 µm. D, The MTT, colony focus (E) and soft agar assays (F) were performed in DLD-1 clones stably expressing CDO1 or control clones. F, Colonies were photographed under a phase-contrast microscope (right top) or under UV after staining with ethidium bromide in 1 X PBS/0.1% Triton X-100 solution overnight (right bottom). G, DLD-1-pCDO1-#2 and DLD-1-p3.1 cells were injected on the right flank of 6-week-old nude mice (n = 5 each), and the time course of tumor growth was measured once a week for 4 weeks with caliper (left). Each point represents the mean ± S.D. of tumor volumes of mice in each group. At day 28 after injection, pictures were taken before mice were sacrificed (right). *, P<0.05 in T-test.

Figure 7. Increased cell growth by CDO1 knockdown.

A, siRNAs targeting CDO1 mRNA (siR-1 ∼ -4) and a non-targeting control siRNA (siR-Cont) were transfected into CDO1-expressing HEK293 cells, and CDO1 gene knockdown was examined by RT-PCR analysis (left) and cell growth was determined by the MTT assay (right). β-actin was used as a loading control. Two independent experiments were done in triplicate, and values are expressed as means ± SD. *, P<0.05 in T-test. Both siR-1 and 2 reduced the CDO1 mRNA, but only siR-2 displayed increased HEK293 cell growth (left). B, The morphology of HEK293 cells were examined under a phase-contrast microscope after siRNA-transfected cells were grown on Matrigel beds for 3 days. MG alone, a picture of Matrigel without cells. Scale bar, 500 µm. C, HepG2 cells express CDO1, but do not harbor the gene methylation (data not shown). CDO1 siRNA (−1 and −2) or control siRNA were transfected into HepG2 cells, and RT-PCR and MTT assays were performed. N, cells without transfection. Increased cell growth was observed in cells transfected only with siR-2. D, Colony focus assays were performed in HepG2 cells after transfection with siR-2 and controls. Colonies were grown for 13 days and stained with crystal violet solution. After air-drying, colonies were counted (left) and photographed (right). Two independent experiments were done in triplicate, and values are expressed as means ± SD. *, P<0.05 in T-test. E, The in vitro cell invasion assay was performed in the HepG2 cells after transfection with siR-2 and controls. Cells were incubated for 16 hrs, and after fixation and staining, invading cells were counted at 100 X magnification (left). Cell growth for 16 hrs determined by MTT assay was not significant (right).