rs2231142 (421 C>A, Q141K) Is More Functionally Influential than rs2231137 (34 G>A, V12M) on Anticancer Drug Resistance Mediated by the ABCG2 Haplotype In Vitro

Abstract

The ATP-binding cassette transporter ABCG2 plays a critical role in drug pharmacokinetics and multidrug resistance in cancer therapy. Two common nonsynonymous polymorphisms, rs2231137 (V12M) and rs2231142 (Q141K), are associated with altered ABCG2 function, drug response, and disease susceptibility. However, the functional impact of their haplotype remains poorly understood. In this study, we established Flp-In™-293 cell lines stably expressing ABCG2 (12M/141K) and systematically compared their expression and drug resistance profiles with those of cells expressing ABCG2 (12V/141Q) (WT), ABCG2 (12M/141Q), and ABCG2 (12V/141K). The mRNA of ABCG2 (12M/141K) was expressed at levels comparable to those of the other variants in cells. Cells expressing ABCG2 (12M/141K) exhibited significantly higher resistance to mitoxantrone (10.7-fold) and SN-38 (5.99-fold) than the mock cells. While ABCG2 (12M/141Q) conferred the highest resistance among the tested variants, the ABCG2 (12M/141K) haplotype showed a trend toward higher mitoxantrone resistance than the ABCG2 (12V/141Q) (WT) (p = 0.066), suggesting a haplotype-specific effect. These findings provide novel insights into haplotype-based modulation of ABCG2 function and its contribution to multidrug resistance, with potential implications for optimizing personalized chemotherapy strategies.

Keywords: ABCG2; ATP-binding cassette (ABC) transporter; BCRP; MXR.

Figure 1

Schematic illustration of human ABCG2 and the locations of SNPs rs2231137 (34 C>A, V12M) and rs2231142 (421 C>A, Q141K). Arrows, locations of SNPs on the ABCG2 protein; ABC, ATP-binding cassette (nucleotide-binding domain).

Figure 2

(A) Schematic illustration of the pcDNA5/FRT/ABCG2 expression vector. Partial cDNA sequences of ABCG2 at nucleotide positions 25–45 (V12M) and 412–432 (Q141K) are shown. The variant nucleotides at positions 34 (G>A, V12M) and 421 (C>A, Q141K) are underlined in bold text. ABCG2, ATP-binding cassette subfamily G member 2; BGH, bovine growth hormone; CMV, cytomegalovirus; pA, polyadenylation signal; pUC ori, pUC vector origin of replication; SV40, simian virus 40. (B) Electropherograms confirming nucleotide substitutions (C>A) at positions 34 and 421 in the ABCG2 cDNA. Arrows indicate the positions of the substituted nucleotides.

Figure 3

mRNA expression levels of ABCG2 in Flp-In™-293 cells expressing ABCG2 haplotypes (12V/141Q, 12M/141Q, 12V/141K, and 12M/141K). Expression levels are shown as ratios to GAPDH mRNA and normalized to the ABCG2/GAPDH ratio in WT cells. Data are presented as the mean ± S.D. (n = 5). Relative ABCG2 expression levels were calculated by normalizing to GAPDH and are shown relative to the 12V/141Q group (WT), which was set as 1.0. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test (* p < 0.01 compared to the mock group).

Figure 4

Expression status (A) and levels (B) of ABCG2 in Flp-In™-293 cells expressing ABCG2 haplotypes (12V/141Q, 12M/141Q, 12V/141K, and 12M/141K). (A) Expression status of ABCG2 haplotypes (12V/141Q, 12M/141Q, 12V/141K, and 12M/141K). (B) Expression levels of ABCG2 and GAPDH determined by Western blot, as described in the Materials and Methods section. The experiments were independently performed on more than two occasions. Data are presented as the mean ± S.D. (n = 3). Statistical analysis was conducted using one-way ANOVA followed by Tukey’s HSD test. Bars with different lowercase letters (a, b, c) indicate statistically significant differences (p < 0.05). Relative ABCG2 expression levels were calculated by normalizing to GAPDH and are shown relative to the 12V/141Q group (WT), which was set as 1.0.

Figure 5

Anticancer drug resistance profiles of Flp-In™-293 cells expressing the ABCG2 haplotypes (12V/141Q, 12M/141Q, 12V/141K, and 12M/141K). (A) Representative dose–response curves from five independent experiments are shown. Data are presented as the mean ± S.D. (n = 4). (B) EC₅₀ values calculated from five independent experiments. Data are presented as the mean ± S.D. (n = 5). Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s honest significant difference test. Different letters indicate statistically significant differences between the groups (p < 0.05).