Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/pharmacodynamics

Abstract

Breast cancer resistance protein (BCRP) is a half-molecule ATP-binding cassette transporter that forms a functional homodimer and pumps out various anticancer agents, such as 7-ethyl-10-hydroxycamptothecin, topotecan, mitoxantrone and flavopiridol, from cells. Estrogens, such as estrone and 17beta-estradiol, have been found to restore drug sensitivity levels in BCRP-transduced cells by increasing the cellular accumulation of such agents. Furthermore, synthetic estrogens, tamoxifen derivatives and phytoestrogens/flavonoids have now been identified that can effectively circumvent BCRP-mediated drug resistance. Transcellular transport experiments have shown that BCRP transports sulfated estrogens and various sulfated steroidal compounds, but not free estrogens. The kinase inhibitor gefitinib inhibited the transporter function of BCRP and reversed BCRP-mediated drug resistance both in vitro and in vivo. BCRP-transduced human epidermoid carcinoma A431 (A431/BCRP) and BCRP-transduced human non-small cell lung cancer PC-9 (PC-9/BCRP) cells showed gefitinib resistance. Physiological concentrations of estrogens (10-100 pM) reduced BCRP protein expression without affecting its mRNA levels. Two functional polymorphisms of the BCRP gene have been identified. The C376T (Q126Stop) polymorphism has a dramatic phenotype as active BCRP protein cannot be expressed from a C376T allele. The C421A (Q141K) polymorphism is also significant as Q141K-BCRP-transfected cells show markedly low protein expression levels and low-level drug resistance. Hence, individuals with C376T or C421A polymorphisms may express low levels of BCRP or none at all, resulting in hypersensitivity of normal cells to BCRP-substrate anticancer agents. In summary, both modulators of BCRP and functional single nucleotide polymorphisms within the BCRP gene affect the transporter function of the protein and thus can modulate drug sensitivity and substrate pharmacokinetics and pharmacodynamics in affected cells and individuals.

Figure 1

Schematic structure of the breast cancer resistance protein, representing the positions of amino acid changes in mutants, single nucleotide polymorphisms and a splicing variant.

Figure 2

Resistance to gefitinib of breast cancer resistance protein (BCRP)‐transduced human epidermoid carcinoma A431 (A431/BCRP) and BCRP‐transduced human non‐small cell lung cancer PC‐9 (PC‐9/BCRP) cells. Cells were cultured for 5 days with increasing concentrations of gefitinib. Cell numbers were counted with a Coulter counter. Data are represented as mean ± SD from triplicate determinations.

Figure 3

Putative mechanism of the suppression of breast cancer resistance protein (BCRP) expression by estrogens. Estrogens, such as estradiol at physiological concentrations (10–100 pM), markedly decrease BCRP expression in estrogen‐responsive and estrogen receptor (ER)α‐positive cells. Estrogen does not affect the BCRP mRNA expression in ERα‐positive cells. The BCRP downregulation seems to be associated with alterations in protein maturation, but not protein turnover.

Figure 4

Effect of C376T (Q126Stop) and C421A (Q141K) single nucleotide polymorphisms in the breast cancer resistance protein (BCRP) gene on protein expression. (a) Upper panel: western blotting of BCRP in PA317 cells and BCRP transfectants. PA317 cells transfected with wild‐type, G34A, C421A and Δ944‐949 BCRP cDNAs were termed PA/WT, PA/V12M, PA/Q141K and PA/Δ315‐6, respectively. Western blot analysis processed under non‐reducing conditions. The BCRP dimer was detected as a band at approximately 140 kDa (ref. 42). Lower panel: northern blot analysis of PA317 cells and BCRP transfectants. The blot was hybridized with an internal BCRP cDNA probe (ref. 42). (b) Putative BCRP expression levels in the C376T and/or C421A allele carriers. Left, putative BCRP expression levels relative to that of homozygous wild‐type allele carriers. Right, putative frequencies of each genotype with respect to nucleotides 376 and 421 of the BCRP gene.