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Gene Focus Concise expert focus on a gene at the heart of pharmacogenomic research
Pharmacogenomic importance of ABCG2 George Cusatis1 & Alex Sparreboom2† †
Author for correspondence 1 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA 2 Department of Pharmaceutical Sciences, St Jude Children’s Hospital, 332 North Lauderdale, DTRC, Mail Stop 313, Room I5308, Memphis, TN 38105, USA Tel.: +1 901 495 5346; Fax: +1 901 495 3125; E-mail:
[email protected]
Highlights • The ATP-binding cassette transporter ABCG2 (ABCP, BCRP and MXR) influences the absorption and disposition of a wide variety of drugs. • ABCG2 expression is associated with an individual’s susceptibility to certain drug-induced side effects and treatment efficacy. • Naturally occurring variants in ABCG2 have been identified that affect the function and expression of the encoded protein. • The frequency of many functionally variant alleles in ABCG2 is ethnically dependent. • There is increasing evidence that ABCG2 SNP analysis is a strategy that can aid in predicting systemic exposure to substrate drugs. • Additional studies are needed to evaluate the clinical utility of ABCG2 genotyping as a prospective tool to predict outcome of treatment.
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The ATP-binding cassette transporter ABCG2 (BCRP, MXR and ABCP) is highly expressed in the gastrointestinal tract and liver, and governs absorption, distribution and excretion of a wide variety of clinically important drugs. Common germline polymorphisms in the ABCG2 gene have been described that can affect expression, cellular localization and/or substrate recognition of the encoded protein. Alteration of transporter function by either of these mechanisms contributes significantly to interindividual variability in drug disposition and treatment outcome with certain, but not all, substrates for ABCG2.
Human ATP-binding cassette transporter G2 (ABCG2) The ATP-binding cassette (ABC) transporters represent the largest family of transmembrane proteins that bind ATP and use the energy to drive the transport of various molecules across cell membranes. Based on the arrangement of molecular structural components, for example, the nucleotide-binding domain and the topology of transmembrane domains, human proteins are classified into seven distinct families (ABCA to ABCG) [1]. The ABCG subfamily consists of several half transporters that are generally thought to form homo- or heterodimers to create the active transporter. The ABCG2 gene is comprised of 16 exons and 15 introns, and is located on chromosome 4q22. The gene encodes a 655 amino-acid ABC half transporter (ABCG2, also known as MXR, BCRP or ABCP) that is comprised of one nucleotide binding fold and one transmembrane region, often referred to as an NBF-TM. Like other cell membrane-localized ABC transporters, ABCG2-mediated flux is
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primarily unidirectional, and it transports substrates from the cytoplasm out of the cell. The gene product ABCG2 has been shown to be a promiscuous transporter of a large number of hydrophobic substrates, including several prescription drugs (Box 1). Various high-throughput assays for ABCG2 have been developed recently to screen large libraries of compounds [2–6], and the application of these screening systems has resulted in an explosion in the identification of novel selective inhibitors of this transporter (Box 1). Similar to the ABC transporter ABCB1 (P-glycoprotein), ABCG2 is expressed in apical membranes of multiple healthy organs, including the liver, kidney, intestine and brain [7], and is thought to play an important role in removing toxic substances from cells, in preventing excessive accumulation in certain tissues and in reducing absorption. ABCG2 expression is strongly induced in the mammary gland of various mammals during lactation [8], where it is likely involved in the secretion of certain important nutrients into milk, such as riboflavin (vitamin B2) [9].
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Box 1. Substrates and inhibitors of ABCG2. Substrates • 9-aminocamptothecin, abacavir, bisantrene, BNP1350, chlorin E6, CI1033, ciprofloxacin, cladribine, daunorubicin, diflomotecan (BN-80915), dipyridamole, doxorubicin, DX-8951f, edevarone (metabolites), epirubicin, erlotinib, etoposide, flavopiridol, gefitinib, gimatecan, glyburide, GW1843, heterocyclic amines, homocamptothecin, imatinib, J-107088, JNJ-7706621, leflunomide, methotrexate, mitoxantrone, NB-506, nelfinavir, nitrofurantoin, norfloxacin, ofloxacin, olmesartan, pheophorbide A, pitavastatin, posuvastatin, protoporphyrin IX, pyropheophorbide A, SN-38 (irinotecan metabolite), sulfasalazine, teniposide, tomudex, topotecan, triglutamate, UCN-01, zidovudine Inhibitors • 17-β-estradiol, 6-prenlychrysin, abacavir, acacetin, amprenavir, atazanavir, biricodar (VX-710), chrysin, CI1033, curcumin, cyclosporin A, delavirdine, diethylstilbestrol, dihydropyridines, dipyridamole, efavirenz, elacridar (GF120918), estrone, fumitremorgin C (FTC), gefitinib, genistein, ginsenosides, imatinib, Ko143 (FTC analog), lopinavir, naringenin, nelfinavir, nicardapine, nimodipine, nitrendipine, novobiocin, ortataxel, pantoprazole, quercetin, reserpine, ritonavir, rosuvastatin, saquinavir, silymarin, sirolimus (rapamycin), tacrolimus, tamoxifen, tariquidar (XR9576), techochrysin, tryprostatin A, WK-X-34
Germline variants in ABCG2 gene Interindividual variability in the pharmacokinetics of drugs is the net result of complex interactions between genetic, physiological and environmental factors. Thus, it is reasonable to assume that genetic variations in the ABCG2 gene could alter drug disposition and might have important consequences for drug-induced toxicity or efficacy. Indeed, identification of genetic factors associated with variability in the absorption and disposition of substrate drugs between different subjects is vital to predicting or eventually adapting appropriate, individualized doses [10]. Recent resequencing of the ABCG2 transporter has revealed a number of allelic variants that may dramatically affect the activity of the gene product in vivo. Some of these genetic variants may potentially modulate the ABCG2 phenotype in patients, and therefore affect their predisposition to toxicity and response to substrate drug treatment [11]. In particular, a SNP in exon 5 of the ABCG2 gene has been described, in which a 421C>A transversion results in a lysine to glutamine amino acid change at codon 141 (Q141K) [12]. Although a detailed analysis of the potential functional consequences of this
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ABCG2 variant has not yet been evaluated, several in vitro studies have indicated an altered substrate specificity and function of the mutant protein relative to the reference protein (reviewed in Gardner et al. [13]). It has also been shown that the 421A variant allele is predominantly found in Asian populations compared with Caucasian and African populations [14]. Several other SNPs have been identified in coding regions of the gene, and at least three additional nonsynonymous SNPs have been identified, occurring at positions 34 (V12M, exon 2), 616 (I206L, exon 6) and 1768 (N590Y, exon 15). Although these SNPs have not been found to confer an alteration in protein expression or function [13], some variants can affect the protein stability of ABCG2 [15]. Based on a resequencing of the ABCG2 promoter and intron 1, sequence diversity in the cis-regulatory region has been found to be a significant determinant of ABCG2 protein expression [16]. Association studies of ABCB2 pharmacogenetics Evidence suggesting that ABCG2 SNP analysis might be a useful strategy to predict systemic exposure to ABCG2 substrate drugs is becoming increasingly prevalent. Specifically, recent studies have
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demonstrated that subjects with a reduced ABCG2 activity owing to the Q141K variant are at an increased risk for gefitinib-induced diarrhea [17] (Figure 1), and altered pharmacokinetics of 9-aminocamptothecin [18], diflomotecan [19], irinotecan [20], rosuvastatin [21], sulfasalazine [22,23] and topotecan [24]. However, contradictory results have been reported for other known ABCG2 substrates, such as doxorubicin [25], imatinib [26], nelfinavir [27] and pitavastatin [28]. It should be pointed out that several studies published to date suffer from small sample sizes in relation to the allelic and genotypic frequencies of the studied variants, as well as from a host of potentially confounding factors that influence their outcome. Most important among these are environmental and physiological factors that may affect expression of the transporter, and links to other genes or variants of putative relevance for drug absorption and disposition pathways. The inclusion of data on other variants in ABCG2 [13,29], and/or the use of haplotype profiles as opposed to testing unphased SNPs to predict certain phenotypes, may have clinical ramifications for agents such as erlotinib [30] and imatinib [16], but this
remains to be clarified for most drugs. In addition, more detailed investigations into the influence of ethnicity on ABCG2 transporter function and expression in relation to substrate-specific phenotypes is urgently needed. In the field of drug resistance, the pharmacogenetic aspects of ABCG2 are still unexplored. Future perspective The ability to detect genetic alterations in ABCG2 and assess their role in the pharmacokinetics and resistance to drugs is a topic of major clinical interest, although currently no diagnostic test is available. However, the knowledge gained in the field of tumor biology encourages the adoption of strategies for treatment optimization based on the compatibility between the molecular profile of the disease and the drug to be administered. Future directions in this field will be mainly concerned with the development of genomic and proteomic techniques, and
Figure 1. Incidence of diarrhea in patients treated with the EGFR tyrosine kinase inhibitor gefitinib as a function of the ABCG2 421C>A genotype. 100
p = 0.0048 N = 95
No. of patients (%)
80
N=9
60
Grade 0 N=7
Grade >0
40
20
N = 13
0 CC
CA
ABCG2 421C>A genotype
Data were obtained from 124 patients with non-small-cell lung cancer receiving gefitinib at a dose of 250 mg once daily on the Iressa Expanded Access Program.
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with validation and subsequent clinical implementation of (nano)technologies of genotyping and phenotyping of ABCG2. Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization
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Sparreboom A, Danesi R, Ando Y, Chan J, Figg WD: Pharmacogenomics of ABC transporters and its role in cancer chemotherapy. Drug Resist. Updat. 6, 71–84 (2003). Glavinas H, Kis E, Pal A et al.: ABCG2 (breast cancer resistance protein/mitoxantrone resistanceassociated protein) ATPase assay: a useful tool to detect drug-transporter interactions. Drug Metab. Dispos. 35, 1533–1542 (2007). Henrich CJ, Robey RW, Bokesch HR et al.: New inhibitors of ABCG2 identified by high-throughput screening. Mol. Cancer Ther. 6, 3271–3278 (2007). Ivnitski-Steele I, Larson RS, Lovato DM et al.: High-throughput flow cytometry to detect selective inhibitors of ABCB1, ABCC1, and ABCG2 transporters. Assay Drug Dev. Technol. 6, 263–276 (2008). Matsson P, Englund G, Ahlin G, Bergstrom CA, Norinder U, Artursson P: A global drug inhibition pattern for the human ATP-binding cassette transporter breast cancer resistance protein (ABCG2). J. Pharmacol. Exp. Ther. 323, 19–30 (2007). Tamura A, An R, Hagiya Y et al.: Drug-induced phototoxicity evoked by inhibition of human ABC transporter ABCG2: development of in vitro high-speed screening systems. Expert. Opin. Drug Metab. Toxicol. 4, 255–272 (2008). Maliepaard M, Scheffer GL, Faneyte IF et al.: Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues. Cancer Res. 61, 3458–3464 (2001). Jonker JW, Merino G, Musters S et al.: The breast cancer resistance protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk. Nat. Med. 11, 127–129 (2005).
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10. Deeken JF, Figg WD, Bates SE, Sparreboom A: Toward individualized treatment: prediction of anticancer drug disposition and toxicity with pharmacogenetics. Anticancer Drugs 18, 111–126 (2007). 11. Lepper ER, Nooter K, Verweij J, Acharya MR, Figg WD, Sparreboom A: Mechanisms of resistance to anticancer drugs: the role of the polymorphic ABC transporters ABCB1 and ABCG2. Pharmacogenomics 6, 115–138 (2005). 12. Imai Y, Nakane M, Kage K et al.: C421A polymorphism in the human breast cancer resistance protein gene is associated with low expression of Q141K protein and low-level drug resistance. Mol. Cancer Ther. 1, 611–616 (2002). •
New findings: First demonstration of a functional inherited ABCG2 protein variant with reduced activity.
13. Gardner ER, Figg WD, Sparreboom A: Pharmacogenomics of the human ATP-binding cassette transporter ABCG2. Curr. Pharmacogenom. 4, 331–344 (2006). 14. de Jong FA, Marsh S, Mathijssen RH et al.: ABCG2 pharmacogenetics: ethnic differences in allele frequency and assessment of influence on irinotecan disposition. Clin. Cancer Res. 10, 5889–5894 (2004). 15. Nakagawa H, Tamura A, Wakabayashi K et al.: Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2. Biochem. J. 411, 623–631 (2008). 16. Poonkuzhali B, Lamba J, Strom S et al.: Association of breast cancer resistance protein/ABCG2 phenotypes and novel promoter and intron 1 single nucleotide
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or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. polymorphisms. Drug Metab. Dispos. 36, 780–795 (2008). •
New findings: Identification of promoter and intron 1 variants in ABCG2 associated with altered function.
17. Cusatis G, Gregorc V, Li J et al.: Pharmacogenetics of ABCG2 and adverse reactions to gefitinib. J. Natl. Cancer Inst. 98, 1739–1742 (2006). •
New findings: First description of a link between genetic variation in ABCG2 and drug-induced toxicity profiles.
18. Zamboni WC, Ramanathan RK, McLeod HL et al.: Disposition of 9-nitrocamptothecin and its 9-aminocamptothecin metabolite in relation to ABC transporter genotypes. Invest. New Drugs 24, 393–401 (2006). 19. Sparreboom A, Gelderblom H, Marsh S et al.: Diflomotecan pharmacokinetics in relation to ABCG2 421C>A genotype. Clin. Pharmacol. Ther. 76, 38–44 (2004). 20. Zhou Q, Sparreboom A, Tan EH et al.: Pharmacogenetic profiling across the irinotecan pathway in Asian patients with cancer. Br. J. Clin. Pharmacol. 59, 415–424 (2005). 21. Zhang W, Yu BN, He YJ et al.: Role of BCRP 421C>A polymorphism on rosuvastatin pharmacokinetics in healthy Chinese males. Clin. Chim. Acta 373, 99–103 (2006). 22. Urquhart BL, Ware JA, Tirona RG et al.: Breast cancer resistance protein (ABCG2) and drug disposition: intestinal expression, polymorphisms and sulfasalazine as an in vivo probe. Pharmacogenet. Genomics 18, 439–448 (2008). •
New findings: Demonstration of the first validated agent to determine individual ABCG2 activity in vivo.
23. Yamasaki Y, Ieiri I, Kusuhara H et al.: Pharmacogenetic characterization of sulfasalazine disposition based on NAT2
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and ABCG2 (BCRP) gene polymorphisms in humans. Clin. Pharmacol. Ther. 84(1), 95-103 (2008). •
Important confirmation: Confirms an association of variant ABCG2 and sulfasalazine disposition.
24. Sparreboom A, Loos WJ, Burger H et al.: Effect of ABCG2 genotype on the oral bioavailability of topotecan. Cancer Biol. Ther. 4, 650–658 (2005). 25. Lal S, Wong ZW, Sandanaraj E et al.: Influence of ABCB1 and ABCG2 polymorphisms on doxorubicin disposition in Asian breast cancer patients. Cancer Sci. 99, 816–823 (2008).
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26. Gardner ER, Burger H, van Schaik RH et al.: Association of enzyme and transporter genotypes with the pharmacokinetics of imatinib. Clin. Pharmacol. Ther. 80, 192–201 (2006). 27. Colombo S, Soranzo N, Rotger M et al.: Influence of ABCB1, ABCC1, ABCC2, and ABCG2 haplotypes on the cellular exposure of nelfinavir in vivo. Pharmacogenet. Genomics 15, 599–608 (2005). 28. Ieiri I, Suwannakul S, Maeda K et al.: SLCO1B1 (OATP1B1, an uptake transporter) and ABCG2 (BCRP, an efflux transporter) variant alleles and pharmacokinetics of pitavastatin in healthy volunteers. Clin. Pharmacol. Ther. 82, 541–547 (2007).
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29. Yoshioka S, Katayama K, Okawa C et al.: The identification of two germ-line mutations in the human breast cancer resistance protein gene that result in the expression of a low/non-functional protein. Pharm. Res. 24, 1108–1117 (2007). 30. Rudin CM, Liu W, Desai A et al.: Pharmacogenomic and pharmacokinetic determinants of erlotinib toxicity. J. Clin. Oncol. 26, 1119–1127 (2008).
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