ABCMdb

PMID: 20345483

Kawahara H, Noguchi K, Katayama K, Mitsuhashi J, Sugimoto Y
Pharmacological interaction with sunitinib is abolished by a germ-line mutation (1291T>C) of BCRP/ABCG2 gene.
Cancer Sci. 2010 Jun;101(6):1493-500. Epub 2010 Feb 22., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Gln141Lys ABCG2 p.Phe431Leu These inhibitory effects of sunitinib were further analyzed in Q141K-, R482G-, R482S-, and F431L-variant BCRPs. Login to comment 6 ABCG2 p.Phe431Leu Intriguingly, the F431L-variant BCRP, which is expressed by a germ-line mutant allele 1291T>C, was almost insensitive to both sunitinib- and fumitremorgin C (FTC)-mediated inhibition in a cell proliferation assay. Login to comment 7 ABCG2 p.Phe431Leu Sunitinib and FTC did not inhibit 125 I-iodoarylazidoprazosin-binding to F431L-BCRP. Login to comment 20 ABCG2 p.Gln141Lys (14) We also reported variant BCRP SNP cDNAs harboring 421C>A (amino acid substitution Q141K),(15) and this SNP is physiologically important because the pharmacokinetics of diflomotecan, a new camptothecin-derivative anticancer agent, and the risk of adverse reactions, such as gefitinib-induced diarrhea, was affected in patients heterozygous for the A421 allele. Login to comment 21 ABCG2 p.Phe431Leu (16) In addition, we reported that a germ-line mutant allele 1291T>C expresses the F431L variant of BCRP with lower functional resistance to SN-38. Login to comment 22 ABCG2 p.Phe431Leu (17) This suggests that amino acid substitution F431L may affect substrate recognition of SN-38. Login to comment 37 ABCG2 p.Phe431Leu Moreover, we show for the first time that this inhibitory effect of sunitinib on BCRP is cancelled by a germ-line mutation of BCRP gene (1291T>C) that causes a single amino acid substitution of F431L. Login to comment 45 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu (17,21) To establish K562 /F431L cells, parental K562 cells were transduced with a HaBCRP retrovirus- harboring Myc-tagged human BCRP (F431L) cDNA in the Ha retrovirus vector as previously described. Login to comment 46 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu (17) After limiting dilution without any selective drugs and screening of over 700 clones, we selected two F431L-BCRP-expressing K562 cell clones K562/F431L-1 and -3. Login to comment 109 ABCG2 p.Gln141Lys ABCG2 p.Phe431Leu (13,35-37) The Q141K variant, a widespread SNP in Japanese individuals, is associated with the low protein expression of BCRP,(15) and the F431L variant, also a germ-line mutation of BCRP, shows a low level of resistance to SN-38. Login to comment 110 ABCG2 p.Arg482Thr ABCG2 p.Arg482Gly The R482T and R482G are BCRP variants identified after in vitro selection of culture cells and these variants confer DOX- and MXR-resistances. Login to comment 126 ABCG2 p.Phe431Leu marginal ability to overcome SN-38 resistance in the F431L-BCRP variant, even though the other BCRP variants were all sensitive to sunitinib with comparable efficacy. Login to comment 127 ABCG2 p.Phe431Leu To confirm these observations, we also tested the effect of sunitinib on F431L-BCRP in different cell lines. Login to comment 128 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu The F431L BCRP-expressing K562 cell clones K562/F431L-1 and -3 were established without the drug selection process. Login to comment 129 ABCG2 p.Phe431Leu FACS analysis showed that the protein expression of F431L-BCRP in K562 cells was relatively low (1/8-1 /4-fold compared with wild-type BCRP-expressing K562 cells) (Fig. 5a). Login to comment 130 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu The reason for the lower protein expression of F431L-BCRP in K562 cells is unknown, but we could not isolate K562 /F431L clones expressing high levels of F431L-BCRP protein, even after the screening of over 700 clones. Login to comment 131 ABCG2 p.Phe431Leu These additional experiments also revealed that sunitinib could not overcome SN-38 resistance conferred by F431L-BCRP (Fig. 5b). Login to comment 132 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu We also examined the inhibition of the F431L-BCRP variant by the typical BCRP inhibitor FTC and found that the F431L-BCRP variant was also resistant to FTC-mediated inhibition (Fig. 5c). Login to comment 134 ABCG2 p.Phe431Leu Effects of sunitinib on IAAP binding to F431L-BCRP. Login to comment 135 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu We next examined the effect of sunitinib on photoaffinity labeling of wild-type and F431L-BCRP with [I125 ]IAAP-binding to investigate the direct competition between the substrate IAAP and sunitinib on the F431L variant. Login to comment 136 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu Because F431L-BCRP protein expression was lower than wild-type BCRP in K562 cell lines (Fig. 6a), [I125 ]IAAP-binding to the membrane vesicles prepared from these K562/F431L cells was weaker than that from wild-type BCRP-expressing membrane vesicles. Login to comment 137 ABCG2 p.Phe431Leu Consistent with other reports, sunitinib (10 lmol /L) and FTC (10 lmol /L) inhibited [I125 ]IAAP-binding to wild-type BCRP, whereas [I125 ]IAAP-binding to the F431L variant was apparently resistant to sunitinib- and FTC-mediated inhibition (Fig. 6b,c). Login to comment 138 ABCG2 p.Phe431Leu These data clearly showed that the F431L variant has decreased affinity for physical interactions with sunitinib and FTC. Login to comment 173 ABCG2 p.Arg482Gly ABCG2 p.Arg482Ser ABCG2 p.Gln141Lys ABCG2 p.Phe431Leu Reversal ratios are shown for wild-type (gray circles), and Q141K (open squares), F431L (filled circles), R482S (open diamonds), and R482G (open triangles) BCRP variant-expressing cells. Login to comment 175 ABCG2 p.Phe431Leu Moreover, the germ-line BCRP variant F431L showed decreased affinity for sunitinib and therefore would be irrelevant to the pharmacological and physical interaction between BCRP and sunitinib. Login to comment 180 ABCG2 p.Phe431Leu Second, we investigated the effect of sunitinib on mutants of BCRP, and found that the F431L variant conferred resistance to sunitinib-mediated suppression. Login to comment 188 ABCG2 p.Phe431Leu Sunitinib failed to overcome F431L-breast cancer resistance protein (BCRP)-mediated drug resistance in K562 cells. Login to comment 189 ABCG2 p.Phe431Leu Protein expression of BCRP in K562 / F431L cells was analyzed by flow cytometry (a). Login to comment 190 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu The F431L-BCRP-expressing K562 cell lines K562 / F431L-1 and -3 were cultured for 5 days with SN-38 and sunitinib (b) or fumitremorgin C (FTC) (c). Login to comment 193 ABCG2 p.Phe431Leu Effect of sunitinib on photoaffinity labeling of wild-type and F431L-breast cancer resistance protein (BCRP) with [125 I]IAAP. Login to comment 194 ABCG2 p.Phe431Leu Protein expression of BCRP was analyzed by western blotting (a) in F431L-BCRP-transduced K562 cells. Login to comment 195 ABCG2 p.Phe431Leu Membrane vesicles (90 lg / mL) from K562 / BCRP and K562 / F431L-3 cells were pre-incubated for 5 min with sunitinib or fumitremorgin C (FTC) (10 lmol / L each) and then 10 nmol / L of [125 I]IAAP was added for a further 10 min. Login to comment 202 ABCG2 p.Phe431Leu The germ-line mutant F431L-BCRP was previously shown to have low ability to confer drug-resistance to SN-38. Login to comment 203 ABCG2 p.Phe431Leu (34) Our present study also showed that the F431L mutation in BCRP compromised the pharmacological and physical interactions with sunitinib and FTC. Login to comment 206 ABCG2 p.Phe431Leu (26) Thus F431L substitution may reduce substrate /inhibitor-recognition efficacy or may be an important amino acid residue involved in the functional transporter activity of BCRP. Login to comment 208 ABCG2 p.Phe431Ser ABCG2 p.Phe431Ser Li et al. reported that another mutant, F431S-BCRP, is still functional because pheophorbide A transport by F431S-BCRP was completely suppressed by FTC. Login to comment 210 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu Our observations also indicate that the F431L-BCRP protein forms a dimer (data not shown), but our data appeared to be inconsistent with their conclusion because the F431L-BCRP variant showed reduced sensitivity to FTC. Login to comment 211 ABCG2 p.Phe431Leu ABCG2 p.Phe431Leu Unfortunately, we failed to confirm the inhibitory effects of sunitinib and FTC on F431L-BCRP-mediated drug transport using the in vitro vesicle transport assay because the membrane vesicles prepared from K562 /F431L cells did not show good transport activity in vitro (data not shown). Login to comment 212 ABCG2 p.Phe431Leu However, we also monitored anti-BCRP antibody 5D3 reactivity to F431L-BCRP in the presence or absence of FTC because the direct interaction between FTC and BCRP is thought to stimulate the binding efficacy of the anti-BCRP antibody 5D3 by inducing a conformational change in BCRP. Login to comment 213 ABCG2 p.Phe431Leu (46) In this 5D3 reactivity test, the fluorescence intensity associated with 5D3 antibody binding was changed by FTC treatment in K562 /BCRP cells, but not in K562/F431L cells (Fig. S3, Supporting information). Login to comment 214 ABCG2 p.Phe431Leu This experiment suggested that F431L-BCRP is resistant to the FTC-induced conformational change required for 5D3 antibody-binding to BCRP. Login to comment 215 ABCG2 p.Phe431Leu Overall, we suspect that the F431L variant shows compromised physical interaction with FTC and sunitinib, or altered conformational dynamics that are required for substrate recognition and transport cycling by BCRP. Login to comment 217 ABCG2 p.Phe431Leu (31) Although F431L-BCRP had lower transporter activity than wild-type BCRP, this mutant BCRP still conferred significant drug-resistance in both PA317 and K562 cells, so that we should pay attention to functional relevance between drug-drug interaction and this mutant BCRP. Login to comment 218 ABCG2 p.Phe431Leu Importantly, our findings demonstrate that germ-line mutations of the BCRP /ABCG2 gene 1291T>C (F431L), affect its pharmacological interaction with sunitinib. Login to comment 221 ABCG2 p.Phe431Leu In future personalized medicine, functional analysis of germ-line mutation affecting efficacy of drug-drug interactions such as F431L-BCRP with sunitinib would contribute to design for the evidence-based optimized chemotherapy regimen in each patient. Login to comment