PDF file, 288K, Supplemental data showing study design, quality control, and replication results.

ABCG2 encodes the breast cancer resistance protein (BCRP), an efflux membrane transporter important in the detoxification of xenobiotics. In the present study, the basal activity of the ABCG2 promoter in liver, kidney, intestine, and breast cell lines was examined using luciferase reporter assays. The promoter activities of reference and variant ABCG2 sequences were compared in human hepatocellular carcinoma cell (HepG2), human embryonic kidney cell (HEK293T), human colorectal carcinoma cell (HCT116), and human breast adenocarcinoma cell (MCF-7) lines. The ABCG2 promoter activity was strongest in the kidney and intestine cell lines. Four variants in the basal ABCG2 promoter (rs76656413, rs66664036, rs139256004, and rs59370292) decreased the promoter activity by 25%-50% in at least three of the four cell lines. The activity of these four variants was also examined in vivo using the hydrodynamic tail vein assay, and two single nucleotide polymorphisms (rs76656413 and rs59370292) significantly decreased in vivo liver promoter activity by 50%-80%. Electrophoretic mobility shift assays confirmed a reduction in nuclear protein binding to the rs59370292 variant probe, whereas the rs76656413 probe had a shift in transcription factor binding specificity. Although both rs59370292 and rs76656413 are rare variants in all populations, they could contribute to patient-level variation in ABCG2 expression in the kidney, liver, and intestine.

ABCG2 encodes the mitoxantrone resistance protein (MXR; breast cancer resistance protein), an ATP-binding cassette (ABC) efflux membrane transporter. Computational analysis of the ∼300 kb region of DNA surrounding ABCG2 (chr4:88911376-89220011, hg19) identified 30 regions with potential cis-regulatory capabilities. These putative regulatory regions were tested for their enhancer and suppressor activity in a human liver cell line using luciferase reporter assays. The in vitro enhancer and suppressor assays identified four regions that decreased gene expression and five regions that increased expression >1.6-fold. Four of five human hepatic in vitro enhancers were confirmed as in vivo liver enhancers using the mouse hydrodynamic tail vein injection assay. Two of the in vivo liver enhancers (ABCG2RE1 and ABCG2RE9) responded to 17β-estradiol or rifampin in human cell lines, and ABCG2RE9 had ChIP-seq evidence to support the binding of several transcription factors and the transcriptional coactivator p300 in human hepatocytes. This study identified genomic regions surrounding human ABCG2 that can function as regulatory elements, some with the capacity to alter gene expression upon environmental stimulus. The results from this research will drive future investigations of interindividual variation in ABCG2 expression and function that contribute to differences in drug response.

Objectives The expression and activity of the breast cancer resistance protein (ABCG2) contributes toward the pharmacokinetics of endogenous and xenobiotic substrates. The effect of genetic variation on the activity of cis-regulatory elements and nuclear response elements in the ABCG2 locus and their contribution toward ABCG2 expression have not been investigated systematically. In this study, the effect of genetic variation on the in vitro and in vivo enhancer activity of six previously identified liver enhancers in the ABCG2 locus was examined. Methods Reference and variant liver enhancers were tested for their ability to alter luciferase activity in vitro in HepG2 and HEK293T cell lines and in vivo using a hydrodynamic tail vein assay. Positive in vivo single-nucleotide polymorphisms (SNPs) were tested for association with gene expression and for altered protein binding in electrophoretic mobility shift assays. Results Multiple SNPs were found to alter enhancer activity in vitro. Four of these variants (rs9999111, rs12508471, ABCG2RE1*2, and rs149713212) decreased and one (rs2725263) increased enhancer activity in vivo. In addition, rs9999111 and rs12508471 were associated with ABCG2 expression in lymphoblastoid cell lines, lymphocytes, and T cells, and showed increased HepG2 nuclear protein binding. Conclusion This study identifies SNPs within regulatory regions of the ABCG2 locus that alter enhancer activity in vitro and in vivo. Several of these SNPs correlate with tissue-specific ABCG2 expression and alter DNA/protein binding. These SNPs could contribute toward reported tissue-specific variability in ABCG2 expression and may influence the correlation between ABCG2 expression and disease risk or the pharmacokinetics and pharmacodynamics of breast cancer resistance protein substrates.

AbstractPurpose: Sensory peripheral neuropathy is a common and sometimes debilitating toxicity associated with paclitaxel therapy. This study aims to identify genetic risk factors for the development of this toxicity.Experimental Design: A prospective pharmacogenetic analysis of patients with primary breast cancer, randomized to the paclitaxel arm of CALGB 40101, was used to identify genetic predictors of the onset and severity of sensory peripheral neuropathy. A genome-wide association study in 855 subjects of European ancestry was conducted and findings were replicated in additional European (n = 154) and African American (n = 117) subjects.Results: A single nucleotide polymorphism in FGD4 was associated with the onset of sensory peripheral neuropathy in the discovery cohort [rs10771973; HR, 1.57; 95% confidence interval (CI), 1.30–1.91; P = 2.6 × 10−6] and in a European (HR, 1.72; 95% CI, 1.06–2.80; P = 0.013) and African American (HR, 1.93; 95% CI, 1.13–3.28; P = 6.7 × 10−3) replication cohort. There is also evidence that markers in additional genes, including EPHA5 (rs7349683) and FZD3 (rs10771973), were associated with the onset or severity of paclitaxel-induced sensory peripheral neuropathy.Conclusions: A genome-wide association study has identified novel genetic markers of paclitaxel-induced sensory peripheral neuropathy, including a common polymorphism in FGD4, a congenital peripheral neuropathy gene. These findings suggest that genetic variation may contribute to variation in development of this toxicity. Validation of these findings may allow for the identification of patients at increased risk of peripheral neuropathy and inform the use of an alternative to paclitaxel and/or the clinical management of this toxicity. Clin Cancer Res; 18(18); 5099–109. ©2012 AACR.