Methylation patterns of genes coding for drug-metabolizing enzymes in tamoxifen-resistant breast cancer tissues

The biological mechanisms underlying resistance to tamoxifen are of considerable clinical significance. However, little is known about the correlation between tamoxifen resistance and methylation of genes related to drug-metabolizing enzymes. To address this issue, we examined the methylation pattern and expression of the selected genes coding for drug-metabolizing enzymes, including COMT, CYP1A1, CYP2D6, NAT1, and SULT1A1 in tamoxifen-resistant and control breast cancers. Bisulfite genomic sequencing and methylation-specific PCR were carried out to evaluate the methylation patterns of the five genes from control (n = 74) and tamoxifen-resistant tissues (n = 37) chosen by an age-matched sampling method. Also, end-point reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR were performed to determine RNA expression of the genes. Bisulfite genomic sequencing revealed methylation of the NAT1 gene in 25 of the control cancers (33.8%) and 23 of the resistant tumors (62.2%). Of the five genes, only NAT1 showed a significant lower methylation rate in the control group than in the resistant group (p = 0.004). No significant difference of the methylation rate was found in the other four genes including COMT, CYP1A1, CYP2D6, and SULT1A1 (p > 0.05). Furthermore, the expression rate of NAT1 mRNA was lower in the tumors from the resistant group than in control tumors (28.6% vs. 65.2%, p = 0.031). Real-time RT-PCR analysis demonstrated that the NAT1 gene was more down-regulated in resistant tissues than in control group (p = 0.023). Moreover, malignant cells from the resistant cases demonstrated a higher percentage of positive staining for Ki67 (p = 0.001) and cyclin D1 (p = 0.043) than those from the control group. Taken together, the higher methylation rate of the NAT1 gene is related to tamoxifen resistance, and this fact supports the hypothesis that hypermethylation of the NAT1 gene might affect the initiation of tamoxifen resistance.