Inactivation of FBXW7/hCDC4-β expression by promoter hypermethylation is associated with favorable prognosis in primary breast cancer

Abstract

Introduction: Mutational inactivation of the FBXW7/hCDC4 tumor suppressor gene (TSG) is common in many cancer types, but infrequent in breast cancers. This study investigates the presence and impact of FBXW7/hCDC4 promoter methylation in breast cancer.

Methods: FBXW7/hCDC4-β expression and promoter methylation was assessed in 161 tumors from two independent breast cancer cohorts. Associations between methylation status and clinicopathologic characteristics were assessed by Fisher's exact test. Survival was analyzed using the Kaplan-Meier method in addition to modeling the risk by use of a multivariate proportional hazard (Cox) model adjusting for possible confounders of survival.

Results: Methylation of the promoter and loss of mRNA expression was found both in cell lines and primary tumors (43% and 51%, respectively). Using Cox modeling, a trend was found towards decreased hazard ratio (HR) for death in women with methylation of FBXW7/hCDC4-β in both cohorts (HR 0.53 (95% CI 0.23 to 1.23) and HR 0.50 (95% CI 0.23 to 1.08), respectively), despite an association between methylation and high-grade tumors (P = 0.017). Interestingly, in subgroups of patients whose tumors are p53 mutated or lymph-node positive, promoter methylation identified patients with significantly improved survival (P = 0.048 and P = 0.017, respectively).

Conclusions: We demonstrate an alternative mechanism for inactivation of the TSG FBXW7/hCDC4, namely promoter specific methylation. Importantly, in breast cancer, methylation of FBXW7/hCDC4-β is related to favorable prognosis despite its association with poorly differentiated tumors. Future work may define whether FBXW7/hCDC4 methylation is a biomarker of the response to chemotherapy and a target for epigenetic modulation therapy.

Figure 1

Differential expression of FBXW7/hCDC4-β mRNA and promoter methylation in tumor cell lines. (a) Semi-quantitative RT-PCR analysis of FBXW7/hCDC4 isoforms, left, in normal breast, immortalized mammary epithelial cells and breast cancer cell lines, right, in human cancer lines from different origins. GAPDH was amplified as an internal control. (b) Schematic map of the FBXW7/hCDC4-β promoter region around the transcription start site (TSS). CpG dinucleotides are depicted. PCR primers used for amplification of the promoter are indicated as arrows (FP1 and RP1). The translational start codon (ATG), centromere (Cen) and telomere (Tel) are marked. (c) real-time PCR showing the relationship between FBXW7/hCDC4-β expression (top) and promoter methylation (bottom); Methylation is presented as the percentage of methylated CpG sites based on bisulphate sequence analysis of genomic DNA from different cancer cell lines. The degrees of methylated cytosines or unmethylated cytosines are indicated by black or white boxes, respectively. (d) Representative gel analysis of PCR product (1.6 kb) from McrBc digested (+) genomic DNA in comparison with undigested (-) control DNA. Note the lack of amplification of McrBc digested DNA from methylated cell lines (HeLa and DLD1) in comparison to unmethylated cell lines (IME and T-ALL).

Figure 2

Inverse correlation between FBXW7/hCDC4-β mRNA expression and promoter methylation in tumor cell lines. (a) Cancer cell lines were divided into two groups based on the ratio of band intensity of McrBc digested cell line DNA divided by undigested DNA. The expression levels were lower in the methylated (M) group than in the unmethylated (UM) group (P = 0.0001). Horizontal lines, median expression value of each group. P value was calculated using the nonparametric Mann-Whitney U test. (b) 5-aza-dC treatment upregulate FBXW7/hCDC4-β mRNA levels in methylated cell lines. Mean ± SD. (c) 5-aza-dC demethylate the FBXW7/hCDC4-β promoter as evident by restoration of PCR amplification of McrBc digested DNA. (d) pGL3E luciferase reporter assay without or with FBXW7/hCDC4-β promoter region in vitro methylated using SssI enzyme (filled bars). Relative fold change in luciferase activity +/- SEM is shown.

Figure 3

Inverse correlation between FBXW7/hCDC4-β mRNA expression and promoter methylation in primary breast tumor specimens. (a) Representative analysis of FBXW7/hCDC4-β promoter methylation in normal breast (N), breast tumor specimens (T), immortalized breast epithelial cells (IME) and a cancer cell line (HeLa). Genomic DNA was incubated with (+) or without (-) McrBc enzyme and the promoter was PCR amplified (1.6 kb). The cleavage pattern indicates differential methylation in tumor samples (T1, T2; methylated and T3, T4; unmethylated) and a lack of methylation normal breast (N1, N2). (b) Relationship between FBXW7/hCDC4-β mRNA expression levels and promoter methylation in primary breast tumors. Samples were stratified by relative methylation status (UM; unmethylated group, N = 68, M; methylated group, N = 71, see materials and methods for classification of methylation). FBXW7/hCDC4-β expression levels (normalized to GAPDH mRNA) were lower in the methylated group compared to the unmethylated group (Mann-Whitney U test, P = 0.0002). Bars from each box extend to largest and smallest expression levels. Outliers are plotted as asterisks (*).

Figure 4

Kaplan-Meier analysis of overall survival in specific patient subgroups. (a) Overall survival rate for patients with methylated FBXW7/hCDC4-β promoter was significantly higher than that for patients in the unmethylated group in lymph node positive patients from cohort 1 (log rank test, P = 0.017). (b) Overall survival for patients from cohort 2 showed a similar trend as patients in cohort 1 but was not statistically significant (log rank test, P = 0.72). (c) Patients with methylated FBXW7/hCDC4-β promoter and p53 mutation have improved survival compared to patients with p53 mutation and unmethylated FBXW7/hCDC4-β promoter in cohort 2 (log rank test, P = 0.048).