Prognostic relevance of Wnt-inhibitory factor-1 (WIF1) and Dickkopf-3 (DKK3) promoter methylation in human breast cancer

Abstract

Background: Secreted Wnt signaling antagonists have recently been described as frequent targets of epigenetic inactivation in human tumor entities. Since gene silencing of certain Wnt antagonists was found to be correlated with adverse patient survival in cancer, we aimed at investigating a potential prognostic impact of the two Wnt antagonizing molecules WIF1 and DKK3 in breast cancer, which are frequently silenced by promoter methylation in this disease.

Methods: WIF1 and DKK3 promoter methylation were assessed by methylation-specific PCR with bisulfite-converted DNA from 19 normal breast tissues and 150 primary breast carcinomas. Promoter methylation was interpreted in a qualitative, binary fashion. Statistical evaluations included two-sided Fisher's exact tests, univariate log-rank tests of Kaplan-Meier curves as well as multivariate Cox regression analyses.

Results: WIF1 and DKK3 promoter methylation were detected in 63.3% (95/150) and 61.3% (92/150) of breast carcinoma samples, respectively. In normal breast tissues, WIF1 methylation was present in 0% (0/19) and DKK3 methylation in 5.3% (1/19) of samples. In breast carcinomas, WIF1 methylation was significantly associated with methylation of DKK3 (p = 0.009). Methylation of either gene was not associated with clinicopathological parameters, except for DKK3 methylation being associated with patient age (p = 0.007). In univariate analysis, WIF1 methylation was not associated with clinical patient outcome. In contrast, DKK3 methylation was a prognostic factor in patient overall survival (OS) and disease-free survival (DFS). Estimated OS rates after 10 years were 54% for patients with DKK3-methylated tumors, in contrast to patients without DKK3 methylation in the tumor, who had a favorable 97% OS after 10 years (p < 0.001). Likewise, DFS at 10 years for patients harboring DKK3 methylation in the tumor was 58%, compared with 78% for patients with unmethylated DKK3 (p = 0.037). Multivariate analyses revealed that DKK3 methylation was an independent prognostic factor predicting poor OS (hazard ratio (HR): 14.4; 95% confidence interval (CI): 1.9-111.6; p = 0.011), and short DFS (HR: 2.5; 95% CI: 1.0-6.0; p = 0.047) in breast cancer.

Conclusion: Although the Wnt antagonist genes WIF1 and DKK3 show a very similar frequency of promoter methylation in human breast cancer, only DKK3 methylation proves as a novel prognostic marker potentially useful in the clinical management of this disease.

Figure 1

Methylation analysis of the human WIF1 promoter. (A) A 1.29 kb genomic sequence of the WIF1 promoter, analyzed by methprimer software [29], revealed the presence of a CpG island (blue) between relative position 604 and 1153. Position 1000 indicates the transcription start site (TSS, arrow). A region of high CpG (red vertical bars) densitiy was chosen for MSP analysis. The black bar indicates the MSP amplicon. (B) Sensitivity of the utilized MSP primers was determined by a dilution series of methylated DNA with unmethylated DNA (Epi Tect control DNA, Qiagen). At least 1% of methylated DNA (~0.1 ng) can be detected with the WIF1 MSP primers. bp, base pair marker; NTC, 'no template control'.

Figure 2

WIF1 methylation in primary breast cancer. WIF1 methylation analyses of primary breast cancer specimens. MSP was performed on bisulfite-treated DNA from breast cancer (T) and matching normal primary breast tissues (N). MSP results from three representative matched pairs and eight additional breast carcinomas (#) are shown. DNA bands in lanes labeled with U indicate MSP products amplified with primers recognizing the unmethylated promoter sequence. DNA bands in lanes labeled with M represent amplified MSP products with methylation-specific primers. Peripheral blood lymphocytes (PBL) and breast cancer cell line ZR75-1 served as positive controls for the methylation-specific reaction, respectively. Water was used as template in the 'no template control' (NTC). Note that tumor tissue usually displayed a PCR product in the U-reaction as well, due to contaminating normal tissue (stromal cells, endothelial cells) present in the tumor specimens as has also been described by Suzuki et al. [34].

Figure 3

Methylation analysis of the human DKK3 promoter. (A) A 2.0 kb genomic sequence of the DKK3 promoter, analyzed by methprimer software [29], revealed the presence of two CpG islands (blue); one between relative position 834 and 1261 and another one between position 1529 and 1917. Two alternative tissue-specific DKK3 transcripts have been described [30]. Since transcription of the shorter transcript is widely distributed in normal tissues, we chose the region of the second transcription start site (TSS, arrow) for methylation analysis. Position 1000 indicates the alternative transcription start site of the longer transcript (TSS*, arrow). A region of high CpG (red vertical bars) densitiy within the second CpG island was chosen for MSP analysis. (B) Sensitivity of the utilized MSP primers was determined by a dilution series of methylated DNA with unmethylated DNA (Epi Tect control DNA, Qiagen). At least 1% of methylated DNA (~0.1 ng) can be detected with the DKK3 MSP primers. bp, base pair marker; NTC, 'no template control'.

Figure 4

Distribution of WIF1 and DKK3 promoter methylation in primary breast carcinomas. Methylation status of either gene has been determined by MSP in the same tumors. Of n = 150 breast cancer patients, the larger fraction reveals an identical methylation status of both genes (63.3%). In the remaining smaller fraction (36.6%), methylation of only one of the two genes could be detected. In total, WIF1 methylation was significantly associated with DKK3 methylation (p = 0.009; Fisher's exact test).

Figure 5

Univariate Kaplan-Meier survival analysis of breast cancer patients in relation to WIF1 and DKK3 promoter methylation. (A) Overall survival and (B) disease-free survival are not associated with WIF1 promoter methylation in human breast cancer. Solid lines indicate methylated WIF1 promoter; dotted lines indicate unmethylated WIF1 promoter in the tumor. (C) In contrast, methylation of the DKK3 promoter in tumor tissue (solid line) is significantly associated with adverse patient overall survival, whereas patients with an unmethylated DKK3 promoter in the tumor tissue have a very favorable clinical outcome (dotted line) (p < 0.001). (D) In addition, DKK3-methylated tumors reveal a significant shorter time to recurrence (solid line), as compared to tumors harboring an unmethylated DKK3 promoter (dotted line) (p = 0.037). Vertical tick marks represent censored patients.