Breast cancer resistant protein (BCRP) functions as a drug efflux transporter that mediates drug resistance. Topoisomerase I inhibitors including SN-38 (an active metabolite of irinotecan) are substrates effluxed by BCRP. The underlying mechanisms for overexpression of BCRP during acquisition of drug resistance remain unclear. The aims of this study are to examine a correlation of altered methylation status in the promoter region of BCRP with BCRP expression and drug resistance in lung and colorectal cancers, and to establish a methylation-specific PCR (MSP) method for BCRP. Treatment of non-BCRP-expressing PC-6 cells with DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, induced BCRP re-expression at the mRNA and protein levels. Bisulfite sequencing analysis and subsequent comparisons between bisulfite-modified and unmodified DNA sequences at CpG sites in the promoter region of BCRP (spanning from nt -1381 to +261) revealed that both alleles at all 89 CpG sites were completely methylated in PC-6, while those at 79 CpG sites (at nt -720 to +261) in BCRP-expressing cells of PC-6/SN2-5H were unmethylated. These results indicate a correlation between demethylation of the promoter region of BCRP and BCRP re-expression, and suggest a mechanism for SN-38 drug resistance. Analysis using MSP we developed also revealed an inverse correlation between methylation status of the promoter region of BCRP and BCRP mRNA expression in several lung and colorectal cancers. Examination of methylation status of the promoter region of BCRP using MSP before chemotherapy may provide a new biomarker for predicting drug resistance and responsibility.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]