MINI-REVIEW Molecular Pharmacology of ABCG2 and its Role ...€¦ · 2013-09-10 · 4. Structure...

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MINI-REVIEW

Molecular Pharmacology of ABCG2 and its Role in Chemoresistance

Alexandra E. Stacy, Patric J. Jansson, Des R. Richardson

Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, NSW 2006, Australia (A. E. S., P. J. J., D. R. R)

Molecular Pharmacology Fast Forward. Published on September 10, 2013 as doi:10.1124/mol.113.088609

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on September 10, 2013 as DOI: 10.1124/mol.113.088609

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Running title: ABCG2 in Chemoresistance

Authors for correspondence:

Patric J. Jansson, Phone:+61-2-9036-67120; Fax:+61-2-9351-3429; Email: [email protected];

Des R. Richardson, Phone:+61-2-9036-6548; Fax:+61-2-9036-6549; Email: [email protected]

Text pages: 47

Tables: 1

Figures: 9

References: 168

Word count:

Abstract: 218

Article: 7,328

Abbreviations:

ABC – ATP-binding cassette

ABCG2 – ABC transporter, sub-family G, isoform 2

Akt – γ-akt murine thymoma viral oncogene homolog

ALL – acute lymphocytic leukemia

AML – acute myeloid leukemia

BBB – blood brain barrier

BCRP – breast cancer resistance protein

ER – endoplasmic reticulum

HIF-1α – hypoxia inducible factor 1α

MDR – multidrug resistance

MRP1 – multidrug resistance protein 1

NFκB – nuclear factor light chain enhancer

of activated B cells

Pgp – P glycoprotein

PI3K – Phosphatidyl inositol 3 kinase

QSAR – quantitative structure activity relationships

SAR – structure activity relationships

SNP – small nucleotide polymorphisms


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Abstract The ATP-binding cassette, sub-family G, isoform 2 protein (ABCG2) is an important

member of the ABC transporter superfamily which has been suggested to be involved in

multidrug resistance (MDR) in cancer. Its diverse range of substrates includes many common

chemotherapeutics such as imatinib, doxorubicin and mitoxantrone. Physiologically, ABCG2

is highly expressed in areas such as the blood brain barrier and gastrointestinal tract, where it

is thought to play a role in protection against xenobiotic exposure. High ABCG2 expression

has also been found in a variety of solid tumors and in hematologic malignancies, and has

been correlated with poorer clinical outcomes. Furthermore, ABCG2 expression is a

characteristic feature of cancer stem cells which are able to self-renew and differentiate.

These cancer stem cells have been postulated to play an important role in MDR, where their

inherent ABCG2 expression may allow them to survive chemotherapy and re-populate the

tumor after exposure to chemotherapeutics. This observation raises the exciting possibility

that by inhibiting ABCG2, cancer stem cells and other cancers may be targeted and

eradicated, at which point conventional chemotherapeutics would be sufficient to eliminate

the remaining tumor cells. Inhibitors of ABCG2 such as tyrosine kinase inhibitors,

phosphodiesterase-5 inhibitors and the fumitremorgin-type indolyl diketopiperazine, Ko143,

could potentially be used for this purpose. However, these agents are still awaiting

comprehensive clinical assessment.


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1. Introduction- The ABC Transporters

The successful treatment of cancer can be impeded by the concurrent development of

resistance to multiple chemotherapeutics which are both structurally and mechanistically

unrelated (Robey et al., 2009). This multi-drug resistance (MDR) phenotype can be

associated with an increase in the expression of ATP-binding cassette (ABC) transporters,

which efflux substrates from the cell in an energy-dependent manner, driven by ATP

hydrolysis (Robey et al., 2009). Of the 48 ABC transporters currently recognized, there are

three which are most commonly associated with MDR: P-glycoprotein (Pgp; also known as

MDR1 or ABCB1), MDR associated protein 1 (MRP1) and breast cancer resistance protein

(BCRP) or ABC sub-family G, isoform 2 protein (ABCG2; (Robey et al., 2009).

It is well known that Pgp can transport a range of hydrophobic chemotherapeutics

such as anthracyclines, taxanes, etoposide and mitoxantrone (Gottesman et al., 2002; Juliano

and Ling, 1976), and was originally thought to be solely responsible for the MDR phenotype

(Gottesman et al., 2002; Juliano and Ling, 1976). However, it soon became clear that other

transporters could also confer resistance to chemotherapeutics. The gene encoding MRP1

(also known as ABCC1) was cloned by Cole et al in 1992, and similarly to Pgp, its over-

expression conferred resistance to a range of chemotherapeutics (Cole et al., 1992). The

discovery that Pgp and MRP1 alone could not account for the efflux of chemotherapeutic

drugs from a subset of leukemic cells subsequently lead to the identification of BCRP from

the breast cancer cell line, MCF-7/AdrVp (Doyle et al., 1998). The Human Genome

Organization Nomenclature Committee has since formally designated BCRP as ABCG2, as it

belongs to the ABC transporter superfamily and is the second member of the G sub-family.


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While their substrate specificities differ, there is considerable overlap between

ABCG2, Pgp and MRP1 in terms of their ability to transport many structural classes of agents

(Fig. 1). This raises the question of the physiological rationale for this apparent redundancy.

All three transporters appear to be involved in protecting the body from toxicity associated

with xenobiotic exposure, and the answer may lie in where the transporter is expressed in a

cell or organ. In the case of the gastrointestinal tract, Pgp and ABCG2 are apically localized

(Maliepaard et al., 2001a; Thiebaut et al., 1987), while MRP1 is found at the basolateral

surface of crypt cells (Peng et al., 1999). A potential explanation for this differential

expression is that in the gut, Pgp/ABCG2 may function to modulate the absorption of

xenobiotics, while MRP1 may protect these highly proliferating cells from xenobiotic

damage (Leslie et al., 2005).

2. Chromosomal Localization and Regulation of Expression

The human ABCG2 gene is located on band 4q22, between the markers D4S2462 and

D4S1557 (Knutsen et al., 2000). To date, no other ABC transporter has been assigned to

chromosome 4. Cytogenetic studies using the ABCG2 over-expressing cell lines, MCF-

7/AdrVp 3000 and MCF-7/MX, showed gene amplification exclusively between 4q21-4q22,

suggesting that ABCG2 homo-dimerizes to create an active transporter (Knutsen et al., 2000).

The ABCG2 gene consists of 16 exons and 15 introns, spanning 66 kb (Bailey-Dell et al.,

2001). The 5’ untranslated region is found mainly in exon 1, while the translational start site

is found in the second exon (Bailey-Dell et al., 2001). The Walker A and B motifs which are

crucial for nucleotide binding have been identified in exon 3 and 6, respectively, while the

ABC motif (ALSGGQ) is found in exon 6 (Bailey-Dell et al., 2001).


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Like other ABC transporters, the promoter region of ABCG2 (Fig. 2) is located

downstream of a putative GpC island, is TATA-less and contains several putative Sp1, AP1

and AP2 binding sites, a common feature of promoters lacking a TATA box (Bailey-Dell et

al., 2001). Additionally, several transcription factors and their cis and trans response

elements have been shown to regulate ABCG2 expression (Ee et al., 2004; Krishnamurthy et

al., 2004; Pradhan et al., 2010; Wang et al., 2008). The presence of a hypoxia response

element may contribute to MDR in some tumors, as HIF-1α is highly active within the

hypoxic interior of solid tumors and thus can increase the expression of ABCG2 via the

hypoxia response element (Krishnamurthy et al., 2004). Likewise, the hormones progesterone

and estrogen have response elements located within the ABCG2 promoter, and have been

shown to increase ABCG2 expression (Ee et al., 2004; Pradhan et al., 2010; Wang et al.,

2008). The estrogen response element is located adjacent to a NFκB response element, and

binding of the estrogen receptor and p65 (the active NFκB subunit) at these response

elements leads to a pronounced increase in ABCG2 mRNA expression (Pradhan et al., 2010).

The ability of hormones to regulate ABCG2 expression may be important during pregnancy,

as high ABCG2 expression is found in the placenta where ABCG2 protects the fetus from

exposure to harmful xenotoxins (Jonker et al., 2000).

3. Single Nucleotide Polymorphisms

Screening for single-nucleotide polymorphisms (SNPs) in ethnically diverse subjects

has identified over 80 synonymous and non-synonymous SNPs in the ABCG2 gene to date

(Sharom, 2008). The two most frequent polymorphisms identified were the G34A (resulting

in V12M) and C421A (resulting in a Q141K substitution) transitions (Fig. 3), found in 18%

and 35.5% of the studied population, respectively (Kobayashi et al., 2005).


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The functional impact of these SNPs on drug transport remains contentious as the

results are not consistent. No significant change in irinotecan pharmacokinetics was observed

in relation to the ABCG2 C421A genotype in American Caucasians, African Americans,

Africans, Han Chinese and European Caucasian patients treated with irinotecan (de Jong et

al., 2004). This was not corroborated by Morisaki et al. (2005) who found that cells with the

Q141K variant of ABCG2 had IC50 values 1.2- to 5-fold lower than cells transfected with

wild-type (Arg482) ABCG2, suggesting that the Q141K SNP affects drug transport (Morisaki

et al., 2005). Recently, studies aimed at evaluating the clinical relevance of the Q141K SNP

in patients undergoing chemotherapy have also been reported. In these studies, elevated

plasma concentrations of gefitinib (Li et al., 2007) and diflomotecan (Sparreboom et al.,

2004) and increased bioavailability of oral topotecan (Sparreboom et al., 2005) were found.

The Q141 mutation has also been associated with increased adverse effects in response to

gefitinib treatment (Cusatis et al., 2006). Additionally, the Q141K mutation has been

demonstrated to decrease ATPase activity by 1.3-fold compared to wild-type ABCG2

(Mizuarai et al., 2003). Furthermore, kinetic analysis of ATPase activity showed that the Km

value in Q141K cells was 1.4-fold higher than that of wild-type ABCG2 (Mizuarai et al.,

2003). These results suggest that this SNP may result in altered transport functions of

ABCG2 transporter which has important implications for the pharmacokinetics and drug-

resistance profiles of chemotherapeutics.

4. Structure

The 655 amino acid ABCG2 protein is in a reversed configuration to most other ABC

transporters, as the ATP-binding domain is at the N-terminal and the 6 putative trans-

membrane domains are at the C-terminal (Polgar et al., 2008) (Fig. 3). The large extracellular

loop between transmembrane domains 5 and 6 contains the only proven N-linked


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glycosylation site, Asp596 (Diop and Hrycyna, 2005). Additionally, ABCG2 is unusual

amongst ABC transporters in that it is a “half transporter” (Doyle et al., 1998). In fact, while

most ABC transporters contain two repeated halves, each containing a membrane-spanning

domain and a nucleotide-binding domain, ABCG2 contains only one membrane-spanning

domain and one nucleotide-binding domain (Allikmets et al., 1998). As it is widely accepted

that functional ABC transporters require two transmembrane domains and two nucleotide-

binding domains to form a central substrate translocation pathway, it is believed that ABCG2

must at least dimerize to become functional (Ni et al., 2010a). Similar to other related

members of the ABC family which are known heterodimers (Hillebrand et al., 2007),

ABCG2 probably dimerizes in the endoplasmic reticulum (ER) (Graf et al., 2003). Then

ABCG2 may exit through the ER to the Golgi apparatus for post-translational processing

before it is trafficked to the apical membrane of the cell (Graf et al., 2003).

4.1 Homo-Dimer or Higher Order Homo-Oligomer?

High resolution structural analysis of bacterial ABCG2 has shown that nucleotides

interact with elements of two separate nucleotide-binding domains when bound, suggesting

that ABCG2 is a homo-dimer (Reyes and Chang, 2005). Furthermore, under conditions of

reducing SDS-PAGE, ABCG2 migrates as a 70 kDa band, but in the absence of reducing

agents it migrates as a 140 kDa complex, which implies that it is a homo-dimer linked by

inter-molecular disulfide bridges (Kage et al., 2001). This is supported by studies using

chemical cross-linking agents which theoretically link the monomers and cause a shift in

molecular mass from 72 to 180 kDa (Litman et al., 2002).

While it is apparent that ABCG2 could be capable of forming homo-dimers, it has

been shown in several studies that it can also form higher oligomeric states, including


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tetramers to even a dodecamer (Xie et al., 2008; Xu et al., 2007). Additionally, electron

microscopy has shown an octameric complex organized as a tetramer of dimers in the mutant

R482G isoform of ABCG2 (McDevitt et al., 2006). This same isoform was demonstrated to

form higher order oligomers of 150 and 290 kDa (Velamakanni et al., 2008). Recently,

human recombinant ABCG2 purified from Pichia pastoris has led to structural analysis at 5

Å resolution and this has shown ABCG2 assembled as a tetramer (Rosenberg et al., 2010).

The conflicting results found between laboratories are likely due to several factors, such as

the type of detergents used and the extraction conditions. Hence, whether ABCG2 forms a

dimer or a higher order oligomer under physiological conditions remains unclear.

4.2 Sequences Involved in Dimerization

Mutational analysis has indicated that there are two regions of ABCG2 which may be

involved in dimerization (Polgar et al., 2010; Polgar et al., 2006; Wakabayashi et al., 2005).

The GXXXG sequence has been linked to dimerization in other proteins such as glycophorin

a (Langosch et al., 1996), and this motif is also located in transmembrane domain 1 of

ABCG2 (Fig. 3) (Polgar et al., 2010). Mutation of the glycine residues to leucines resulted in

impaired drug transport of several ABCG2 substrates, suggesting that this motif plays a role

in dimerization, although it also may be important in the formation of higher order complexes

(Polgar et al., 2004).

Mutational analysis of Thr402 which is located near the GXXXG motif

(TXXGXXXG) in combination with mutations of the GXXXG motif (T402L or T402R, and

G406L/G410L; Fig. 3) resulted in a reduction in protein expression and drug efflux,

alterations in glycosylation, and retention of ABCG2 in the ER (Polgar et al., 2010). While

the mutants could still be chemically cross-linked, this may be simply an indication of their


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close proximity within the ER (they must be physically close in order to dimerize) (Polgar et

al., 2010). It does not necessarily reflect their ability to form a fully-functional transporter

since functions such as drug efflux were shown to be affected by the mutations (Polgar et al.,

2010).

Additionally, the results of the mutation of Gly553 found in transmembrane domain 5

to Leu553 mimicked those found for the TXXGXXXG motif (Fig. 3) (Polgar et al., 2006).

Although the monomers of the mutant forms of ABCG2 can be cross-linked and form

disulfide bonds, the fact that they display impaired glycosylation and trafficking is consistent

with, but does not prove, the involvement of the TXXGXXXG motif and Gly553 in homo-

dimerization and formation of a fully-functional ABCG2 transporter (Polgar et al., 2006).

Mutagenesis studies targeting cysteine residues (Cys592, Cys603 and Cys608)

suggest that they are likely to be involved in the formation of intra-molecular disulfide bonds

(Fig. 3) (Wakabayashi et al., 2007). While the majority of evidence demonstrates that

Cys603, which is highly conserved amongst ABCG2 orthologues, plays a key role in the

formation of disulfide bonds, the disulfide bonds themselves do not appear to be essential for

protein function (Shigeta et al., 2010). This observation suggests that non-covalent protein-

protein interactions could also contribute to dimerization (Henriksen et al., 2005; Ni et al.,

2010b). Notably, Cys592 and Cys608 also appear to be important residues, as substituting

these residues for Gly results in reduced expression and aberrant localization to the

intracellular compartments as opposed to the plasma membrane (Wakabayashi et al., 2005).

Taken together, these results suggest that correct disulfide bond formation involves these

residues and impacts on intracellular sorting and function of the protein (Wakabayashi et al.,

2007)


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4.3 Homology Modeling

In order to further elucidate the structure of ABCG2, homology models have been

generated. Notably, models based only on computer predictions have been found to be

inconsistent with experimental topology structures (Ni et al., 2010a). Due to this problem,

three homology models of ABCG2 have been deduced which are based on experimental

topology structures, and represent different conformational states. The templates used were

MsbA (an Eschericia coli ABC transporter) for the substrate-unbound, nucleotide-free,

inward-facing apo (open) conformation (Ward et al., 2007); parts of murine Pgp to model the

substrate-bound, nucleotide-free, inward-facing holo (closed) conformation (Aller et al.,

2009); and Sav1866 (a Staphylococcus aureus ABC transporter) to model the nucleotide-

bound outward-facing conformation (Dawson and Locher, 2006). The nucleotide-binding

domains were modeled using the nucleotide-binding domains of the E. coli maltose

transporter MalK, (Chen et al., 2003a). The results for homology modeling of MsbA suggest

that in the presence of a substrate, ABCG2 displays a more closed conformation (Rosenberg

et al., 2010; Ward et al., 2007).

The models suggest that the large extracellular loop that connects transmembrane

domain 1 and transmembrane domain 2 may form contacts with the extracellular loop

connecting transmembrane domain 5 and transmembrane domain 6, and this could act to

stabilize the dimeric structure (Figs. 3 and 4) (Rosenberg et al., 2010). The large extracellular

loop which connects transmembrane domain 5 and transmembrane domain 6 might also play

a critical role in modulating substrate-binding (Özvegy-Laczka et al., 2008). This is

corroborated by evidence from Xu et al, who demonstrated that the transmembrane domain

5-loop- transmembrane domain 6 fragment plays a critical role in ABCG2 oligomerization

(Xu et al., 2007).


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Interestingly, the coupling helix 1 that is involved in the interactions between the

nucleotide-binding domain and the transmembrane domain was not predicted by the

homology models for ABCG2, although it is present in the templates and in all other ABC

efflux transporter structures (Rosenberg et al., 2010). However, there is a putative linker

region which connects the nucleotide-binding domain to the transmembrane domain (Fig. 4),

and this is much longer in ABCG2 than in other ABC transporters (Ni et al., 2010a). This

potential linker region may perform the same function as coupling helix 1, although it could

not be modeled due to a lack of appropriate templates (Rosenberg et al., 2010). A second

coupling helix is also present in the model and associates with the nucleotide-binding domain

of the opposite monomer (Rosenberg et al., 2010). This motif has been found in the

intracellular loop which connects transmembrane domain 4 and transmembrane domain 5

(Fig. 4), and may enable the flexibility of the transporter which in turn aids substrate

interactions (Rosenberg et al., 2010). On substrate-binding, the homology models predict

dramatic conformational changes. In fact, the intracellular entry of ABCG2 closes, which

alters the shape of the substrate-binding cavity to form the ‘ATP sandwich’ necessary for

ATP hydrolysis to occur (Rosenberg et al., 2010). A large V-shaped gap then forms on the

extracellular side of the transporter, and may release the substrates after ATP hydrolysis

(Rosenberg et al., 2010; Ward et al., 2007).

These homology models are in agreement with existing data. For example, Arg482 has

been found in the homology models to be located in the central cavity, with the side chain

pointing towards the drug translocation pathway (Fig. 4) (Cai et al., 2010). This residue has

been the subject of many site-directed mutagenesis studies which point to its importance in

determining substrate specificity and transport activity (Miwa et al., 2003; Özvegy-Laczka et

al., 2005; Robey et al., 2003). This observation could explain why resistance to methotrexate


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(which interacts with Arg482) is decreased by the R482G and R482T mutations (Chen et al.,

2003b), while efflux of prazosin (which binds to a separate and distinct binding site) was not

affected (Giri et al., 2009). It would therefore appear that homology models of ABCG2 do

provide relatively accurate and useful information, and may be valuable tools in the

development of ABCG2 substrates and inhibitors.

5. ABCG2 Transport

5.1 Substrates

ABCG2 transports a structurally diverse array of substrates (Fig. 5), the list of which

is constantly expanding (Polgar et al., 2008). Initially, many of the ABCG2 substrates were

reported to be chemotherapeutics such as mitoxantrone (Doyle et al., 1998), which can be

used to select for increased ABCG2 expression (Ross et al., 1999). Substrates of the wild-

type ABCG2 transporter include tyrosine kinase inhibitors such as imatinib and gefitinib

(Elkind et al., 2005), flavopiridol (Allen et al., 2002a; Elkind et al., 2005; Robey et al., 2001)

and the camptothecins: topotecan, irinotecan and its metabolite, SN-38 (Ma et al., 1998;

Maliepaard et al., 2001b; Yang et al., 1995). Some drug-selected cell lines that express

mutant forms of ABCG2, R482G and R482T (previously discussed in Section 4.3), are

considered to be gain-of-function mutants, as their altered substrate specificity increases

resistance to anthracyclines (doxorubicin, daunorubicin) and rhodamine 123 (Fig. 6) (Allen et

al., 2002a; Chen et al., 1990; Honjo et al., 2001). The residue at this position therefore

appears to play a key role in substrate recognition (Chen et al., 2003b).

Other drugs also act as substrates of ABCG2 and include: cimetidine (Pavek et al.,

2005), prazosin (Litman et al., 2000), statins (Hirano et al., 2005; Huang et al., 2006) and


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zidovudine (Wang et al., 2004).. Additionally, ABCG2 can also transport biological

substrates like estrone, 17β estradiol, porphyrins such as heme (Krishnamurthy et al., 2004),

protoporphyrin IX (Robey et al., 2004); and the dietary carcinogen, 2-amino-1-methyl-6-

phenylimidazo[4,5-b] pyridine (Pavek et al., 2005). It should also be noted that only sulfated

steroids have been found to be transported by ABCG2 in mammalian cells (Imai et al., 2003;

Suzuki et al., 2003), while unconjugated estradiol can be transported by ABCG2 identified in

Lactococcus lactis (Janvilisri et al., 2003).

For those studying ABCG2, the discovery of fluorescent substrates has been

extremely useful. Fluorescent substrates include rhodamine 123 (Litman et al., 2000) and

pheophorbide a (Robey et al., 2004). The ABCG2-specific substrate, pheophorbide a, is a

dietary chlorophyll breakdown product, that was discovered serendipitously in ABCG2-/-

mice, which were extremely sensitive to the compound and developed lethal photo-toxic

lesions on light-exposed skin (Jonker et al., 2002). Lysotracker®, prazosin-BODIPY and

Hoechst 33342 have also been reported to be ABCG2 substrates (Litman et al., 2000).

Although there is considerable substrate overlap between ABCG2, Pgp and MRP1

(Fig. 1), there are some characteristics of this transporter which set ABCG2 apart (Leslie et

al., 2005). ABCG2 is able to transport hydrophilic conjugated anions, especially if they have

been sulfated, in addition to hydrophobic substrates which Pgp can also transport (Kodaira et

al., 2010). This substrate overlap is thought exert a synergistic effect to limit penetration of

drugs across the blood brain barrier (BBB) and other areas (Kodaira et al., 2010), and may

affect the pharmacokinetics of substrate drugs. It is still unknown what features determine

whether a compound will be a substrate of ABCG2, as to date there has only been one group

of compounds (camptothecin analogues) where there is an example of a clear structure-


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activity relationship (SAR) (Yoshikawa et al., 2004). For instance, in the camptothecin

analogue, SN-38 (the active metabolite of irinotecan), the presence of hydroxyl or amino

groups at carbons 10 and 11 increases polarity and facilitates its recognition by ABCG2 (Fig.

7) (Yoshikawa et al., 2004).

5.2 ABCG2 Inhibitors

The list of inhibitors of ABCG2 is similarly large and diverse (Fig. 5). The first

inhibitor to be identified was fumitremorgin C, isolated from Aspergillus fumigates

(Rabindran et al., 2000). Fumitremorgin C was reported to inhibit mitoxantrone resistance in

the S1-M1-3.2 cell line, before ABCG2 had been cloned (Rabindran et al., 1998). Due to the

neurotoxicity of fumitremorgin C, several analogues were produced, of which Ko143 was

found to be the most potent and selective inhibitor of ABCG2 (Allen et al., 2002b). Unlike

fumitremorgin C and Ko143, many inhibitors of ABCG2 are not selective and inhibit other

ABC transporters as well, such as elacridar (de Bruin et al., 1999), tariquidar (Robey et al.,

2004), sildenafil (commercially marketed as Viagra™; (Shi et al., 2011) and cyclosporine A

(Gupta et al., 2006). Also of note, some inhibitors of ABCG2 are also substrates of this

molecule, suggesting that they are competitive inhibitors. For instance, the tyrosine kinase

inhibitors imatinib and gefitinib fall into this category (Houghton et al., 2004; Özvegy-

Laczka et al., 2004).

Several SAR and quantitative SAR (QSAR) studies have been carried out for ABCG2

inhibitors, allowing the prediction of some of the structural requirements for an inhibitor.

Lipophilicity has been shown to be a predictor of inhibition in both flavonoids (Zhang et al.,

2005) and fumitremorgin C analogues (van Loevezijn et al., 2001). However, this was not

shown to be the case for either tariquidar analogues (Pick et al., 2008) or propafenone


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analogues (Cramer et al., 2007). Planar structure has also been suggested to increase the

ABCG2-inhibitory properties of a drug, as evidenced by results from SARs of different

structural series of compounds. For example, the two cyclin-dependent kinase inhibitors,

purvalanol A and WHI-P180, have a planar structure and are also the two strongest ABCG2

inhibitors of this class (An et al., 2009). A planar structure including a 2,3-double bond in

ring C increases the inhibition potency for flavonoids (Zhang et al., 2005), and the same

effect is seen for rotenoid derivatives (Fig. 7) (Ahmed-Belkacem et al., 2007). Although

QSAR analysis has provided some useful information for the development of ABCG2

inhibitors, their efficacy will remain limited until a high resolution 3D structure has been

solved.

6. Localization and Physiological Function

Many studies have investigated the tissue distribution of ABCG2 expression,

providing clues as to the physiological function of the transporter (Robey et al., 2009). High

ABCG2 expression has been found in the placenta, brain, prostate, small intestine, testes,

ovaries, liver, adrenal gland, uterus and central nervous system (Doyle and Ross, 2003).

Several physiological roles have been posited for ABCG2, and in general it seems to protect

cells and organs from harmful xenobiotics (Robey et al., 2007). As a result of this function,

ABCG2 has been shown in many instances to affect the pharmacokinetics of its substrates,

making its expression relevant in a clinical setting (Fig. 8).

6.1 Placenta

High ABCG2 expression has been found on the apical membrane of

syncytiotrophoblasts of the chorionic villi, leading to the assertion that ABCG2 protects the

fetus from exposure to toxins and also assists in removing toxins from the fetus (Fig. 8)


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(Litman et al., 2002). Importantly, ABCB1a/1b-/- (Pgp knockout) mice were often used to

avoid the confounding effects of having Pgp present, as Pgp and ABCG2 have overlapping

substrate specificities (Fig. 1). The administration of oral topotecan and elacridar to

ABCB1a/1b-/- mice resulted in fetal plasma topotecan levels twice as high as the maternal

plasma levels (Jonker et al., 2000). Other groups have also similarly demonstrated that i.v.

administration of the antibiotic, nitrofurantoin, to pregnant mice leads to a 5-fold higher fetal

concentration in ABCG2-/- mice versus wild-type mice (Zhang et al., 2007). In humans, an ex

vivo study employing human placental vesicles demonstrated that fetal-to-maternal

concentration ratios of the gestational diabetes drug, glyburide, were increased 2-fold by co-

administration of the ABCG2 inhibitor, novobiocin (Gedeon et al., 2008).

There is also evidence demonstrating the transport of toxins which are ABCG2

substrates from the fetal to the maternal space (Myllynen et al., 2008; Staud et al., 2006). In

rats, the H2 histamine receptor antagonist, cimetidine, was shown to be transported against

the concentration gradient from the fetal space to the maternal space (Staud et al., 2006), and

analogous results have been obtained in perfused human placenta using the dietary

carcinogen, 14C-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (Myllynen et al., 2008).

6.2 Blood Brain Barrier

Generally, the influx and efflux of drugs through the BBB has been thought to

depend, at least in part, on the physiochemical properties of the drug such as lipophilicity and

molecular weight (Pardridge, 2005). However, many drugs have lower BBB penetration than

would be predicted based on their physiochemical properties and this is largely due to the

expression of efflux transporters, such as the ABC proteins (Pardridge, 2005). This poor

penetration of some drugs affects the treatment of diseases such as Alzheimer’s and


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Parkinson’s disease, as well as cancers such as gliomas because concentrations high enough

to penetrate the BBB often cause systemic toxicity (Pardridge, 2005). The BBB also poses

several problems for drug development as it places significant restrictions on the size,

lipophilicity and transporter affinity of drugs (Pardridge, 2005). Of the three ABC

transporters in the BBB, ABCG2 is thought to be the most highly expressed, followed by Pgp

and then MRP1, and all are localized to the luminal side of endothelial cells lining BBB

capillaries (Fig. 8) (Eisenblätter et al., 2003).

The considerable substrate overlap between ABCG2 and Pgp (Fig. 1) makes it

difficult to determine the pharmacological and potentially the physiological roles of ABCG2

alone. This conclusion is exemplified by a study by Breedveld et al (2005) who examined the

uptake of imatinib in ABCG2-/- and ABCB1a/1b-/- (i.e., Pgp-/-) mice. In this investigation,

ABCB1a/1b-/- mice displayed a 3.6 -fold higher brain penetration of imatinib compared to

wild-type animals, and ABCG2-/- a 2.5-fold higher concentration, while inhibition with

elacridar caused a 4.2-fold increase in imatinib penetration in wild-type mice (Breedveld et

al., 2005). This suggests that while ABCG2 is highly expressed at the BBB, Pgp is perhaps

more clinically relevant. This is corroborated by another study which found that the blood-to-

plasma ratios of imatinib were increased by >10 fold in ABCB1a/1b/ABCG2-/- mice, while in

ABCB1a/1b-/- mice the increase was only approximately 2-fold, and no difference was

observed for ABCG2-/- mice (Oostendorp et al., 2009). Again, this observation suggests that

while Pgp may play a more important role in limiting the penetration of substrates at the

BBB, ABCG2 is also of importance and the combination of the two transporters may even be

synergistic (Fig. 8).


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6.3 Mammary Gland

ABCG2 has been found to be up-regulated in the lactating breast of mice, cows and

humans, where it may function to concentrate vitamins and minerals into milk for breast-

feeding young (Fig. 8) (Hogue et al., 1999). One such vitamin is riboflavin, which is

necessary for fat metabolism and many redox reactions (Van Herwaarden et al., 2007).

Unfortunately, ABCG2 may also play a role in the concentration in milk of toxins and drugs

such as the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (Jonker et

al., 2005), and the antibiotic, ciprofloxacin (Merino et al., 2006). Since offspring of ABCG2-/-

mice do not appear to suffer any abnormalities (Foraker et al., 2003), the exact function of

ABCG2 in the mammary gland remains unclear. However, considering the substrate

specificity overlap between ABCG2 and other commonly expressed transporters (e.g., P-gp

and MRP1; Fig. 1), this latter observation may be due to compensatory response of these

transporters. Such metabolic compensation has been observed for other targeted knockouts

e.g., the compensatory response observed after myoglobin deletion in the heart (Gödecke et

al., 1999).

6.4 Blood-Testis Barrier

High levels of ABCG2 expression have been detected in the apical membranes of

myoid cells surrounding the seminiferous tubules in humans (Fig. 8) (Bart et al., 2004). On

the basis of studies using ABCG2-/- mice which found that xenobiotic penetration into the

testis and epididymis was greatly increased, it has been suggested that ABCG2 may protect

developing spermatocytes (Enokizono et al., 2007). This protective effect may be particularly

relevant in respect to phytoestrogens, several of which (i.e., genisteine, daidzen and

coumestrol) are ABCG2 substrates and have been shown to impair male reproductive

function (Adeoya-Osiguwa et al., 2003).


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6.5 Gastrointestinal Tract

ABCG2 expression is found to decrease along the length of the gastrointestinal tract,

with the highest expression found in the duodenum and lowest in the rectum (Gutmann et al.,

2005). Within the gut, ABCG2 has been observed to localize to the apical membrane, which

supports the theory that in the gastrointestinal tract ABCG’s role is to limit the oral

bioavailability of its substrates (Fig. 8) (Maliepaard et al., 2001a). These substrates include

therapeutic agents, and a potential means of increasing the absorption of orally administered

drugs/substrates could involve ABCG2 inhibition. For instance, a study by Allen et al

(2002b) elegantly illustrates this principle, in which plasma topotecan levels were increased

4-6-fold in ABCB1a/1b-/- mice in the presence of orally administered Ko143 (a specific

ABCG2 inhibitor; (Allen et al., 2002b). Another potential method to increase the oral

bioavailability of drugs is to adjust their structure so that they are no longer ABCG2

substrates. Notably, it is possible that the increased bioavailability of newer camptothecin

analogues is due to the fact that they are poor ABCG2 substrates (Yoshikawa et al., 2004).

6.6 Kidneys and Liver

Hepatic and renal excretion are major mechanisms by which most drugs are cleared

from the body (Seldin, 2004; Wilkinson and Shand, 1975). As such, these processes should

involve ABCG2 if its main physiological function is to protect the body from exposure to

harmful xenobiotics. Consistent with this, ABCG2 expression is found in the canalicular

membrane of hepatocytes (Maliepaard et al., 2001a), in the bile ducts (Vander Borght et al.,

2006), the gall bladder (Aust et al., 2004), and in the apical membrane of proximal kidney

tubule epithelial cells (Huls et al., 2007) (Fig. 8). Notably, ABCG2-/- mice have also been

shown to have impaired renal or hepatic excretion of several ABCG2 substrates (Mizuno et

al., 2004).


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6.7 Subcellular Localization

ABCG2 is predominantly located in the plasma membrane of drug-selected and

ABCG2-transfected cell lines, as has been demonstrated by a number of

immunohistochemical studies and confocal microscopy drug localization assays (Maliepaard

et al., 2001a; Scheffer et al., 2000). This is somewhat unusual, as almost all other known half-

transporters are localized to intracellular membranes such as those of the mitochondrion or

the ER. Examples of such transporters include the ABC family members, ABC7 and M-

ABC1, which are localized in the mitochondria (Hogue et al., 1999), and ABCB2 and

ABCB3 that are found in the ER (Kleijmeer et al., 1992). Only one other half transporter has

been found to be at least partially localized to the plasma membrane, namely the human

homologue of the Drosophilia white gene, ABCG1 (Vaughan and Oram, 2005). The

localization of ABCG2 may in part be regulated by cell signaling pathways such as the

phosphatidyl inositol 3-kinase (PI3K)/γ-Akt murine thymoma viral oncogene homolog (Akt)

pathway. In fact, inhibition of both PI3K (Misra et al., 1998) and Akt (Chu et al., 2008) has

been shown to cause ABCG2 translocation from the plasma membrane to an intracellular

compartment. Moreover, ABCG2 may also be present in the membranes of acidic vesicles

(e.g. lysosomes), as co-localization studies with Lysotracker® demonstrated sequestration of

mitoxantrone in acidic vesicles of both S1 cells and the S1-M1-80 subline that expresses high

ABCG2 levels (Litman et al., 2000).

7. Significance of ABCG2 in Cancer

ABCG2 is over-expressed in several MDR cancer cell lines, which is indicative of its

importance in the MDR phenotype of cancer cells (Allikmets et al., 1998; Doyle et al., 1998;

Miyake et al., 1999). Given that a large number of commonly used chemotherapeutics are

ABCG2 substrates, the clinical impact of ABCG2 on MDR in cancer is being actively


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investigated (Robey et al., 2007). To date, studies which have attempted to link ABCG2

expression to clinical outcomes have been inconclusive and often contradictory (Doyle and

Ross, 2003). However, the possibility remains that modulation of ABCG2 may increase the

oral bioavailability of chemotherapeutics or overcome MDR.

7.1 Expression in Hematologic Malignancies

ABCG2 has been found to be frequently expressed on malignant hematopoietic and

lymphoid cells, and has been most thoroughly investigated in acute myelogenous leukemia

(AML; Table 1) (Doyle and Ross, 2003). In AML, ABCG2 has been found to show potential

as a prognostic indicator despite the fact that common first line therapies for AML are not

ABCG2 substrates (Natarajan et al., 2012). To date, all AML patients examined have

possessed the wild-type ABCG2 sequence (Plasschaert et al., 2003; Suvannasankha et al.,

2004), and these patients should therefore not be resistant to anthracyclines as they are not

substrates of wild-type ABCG2 (Fig. 6) (Honjo et al., 2001). However, it has been found that

there is a correlation between ABCG2 expression and failure to achieve complete remission

in AML patients after induction therapy with the cytotoxic agent, cytarabine (Steinbach et al.,

2002). This is likely due to Pgp expression, as high levels of Pgp have been found in 45% of

AML cases when the patient is aged over 55 (Leith et al., 1997), although this was not

directly investigated. High ABCG2 levels in AML have also been correlated with a

relapsed/refractory disease state, and lower complete response rate and shorter survival

(Table 1) (Benderra et al., 2004; Benderra et al., 2005).

These latter results are inconsistent with those found by other investigators who found

a correlation with survival in AML patients, but not the complete response rate (Uggla et al.,

2005). Interestingly, ABCG2 expression has been observed in small sub-populations of AML


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cells (Abbott et al., 2002; Suvannasankha et al., 2004), and seems to be most highly

expressed on CD34+/CD38- “cancer stem cells” (see Section 7.3) which have an immature

phenotype (de Figueiredo‐Pontes et al., 2008; Raaijmakers et al., 2005). It should be noted

that the detection of ABCG2 in AML cells has been afflicted by methodological issues.

These problems have potentially contributed to the discordance observed between studies

correlating ABCG2 expression to prognostic outcomes (Table 1).

The role of ABCG2 in acute lymphoblastic leukemia (ALL) drug resistance has also

been investigated (Table 1). As in AML, high ABCG2 expression has been correlated with

resistance to the cytotoxic agent, cytarabine, a non-ABCG2 substrate (Stam et al., 2003).

Many common drugs used to treat ALL are not ABCG2 substrates, but this latter observation

suggests that in both AML and ALL, ABCG2 expression may be a marker of resistance, as

opposed to a mechanism that is directly involved in this process (Natarajan et al., 2012).

Results linking ABCG2 expression to prognostic outcomes in ALL have been inconsistent,

particularly between adults and children. For example, ABCG2 expression in adults has been

correlated with poorer disease-free survival (Suvannasankha et al., 2004), while in children,

ABCG2 expression has shown not to be related to relapse-free survival time (Table 1)

(Sauerbrey et al., 2002). In contrast, low expression of ABCG2 is correlated to a higher

incidence of death due to drug toxicity in children (Cortez et al., 2009). This observation may

simply be due to the fact that children have better clinical outcomes than adults in ALL.

7.2 Expression in Solid Tumors

The expression of ABCG2 in solid tumors was initially not detected by

immunohistochemical studies (Scheffer et al., 2000), although with the use of a different

monoclonal antibody (BXP21 as opposed to BXP34), ABCG2 was subsequently identified


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by the same group of researchers in over 40% of tumors examined (Diestra et al., 2002).

These tumors represented more than 21 different types of untreated cancer, such as colon,

esophageal, endometrial, breast, pancreatic, and lung cancers (Diestra et al., 2002).

Like hematological malignancies, ABCG2 expression in some types of solid tumors

has been correlated with poorer outcomes (Mo and Zhang, 2012). In small cell lung cancer,

high ABCG2 expression has been associated with poor clinical response and progression-free

survival to platinum drug (carboplatin and cisplatin) -containing regimens (Kim et al., 2009),

and lower overall survival in patients with non-small cell lung cancer (Ota et al., 2009).

Unfortunately, the results of studies examining this latter tumor-type and ABCG2 expression

lack consistency (Burger et al., 2003; Faneyte et al., 2002; Kanzaki et al., 2001), as do those

for breast cancer (Burger et al., 2003; Kanzaki et al., 2001) and so it is impossible to draw

firm conclusions on whether there is a link between ABCG2 and clinical outcomes in solid

tumors (Burger et al., 2003; Faneyte et al., 2002; Kanzaki et al., 2001). The majority of recent

studies have moved away from attempting to assess this link, and have instead focused on

ABCG2 and its role in the cancer stem cell phenotype (see below).

7.3 Cancer Stem Cells and MDR

While the idea of cancer stem cells is still controversial, the theory has gained

increasing traction in recent years (Visvader and Lindeman, 2008). Cancer stem cells are a

subset of tumor cells so named in order to reflect their shared properties with normal stem

cells as they retain the ability to self-renew and differentiate, although to a limited extent

(Visvader and Lindeman, 2008). The first evidence of cancer stem cells was found in

immune-deficient mice, where it was ascertained that a small subset (0.1-1%) of AML cells

transplanted into these mice could induce leukemia (Lapidot et al., 1994). Cancer stem cells


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have also been separated from solid tumors, and usually express specific markers which have

been useful in their isolation (Hirschmann-Jax et al., 2004). These markers include CD24

(Al-Hajj et al., 2003), CD44 (Al-Hajj et al., 2003) and CD133 (Singh et al., 2004). Notably,

ABCG2 is also a marker of cancer stem cells, as the efflux of the ABCG2 substrate, Hoechst

33342, is exploited to isolate them (Zhou et al., 2001). Cancer stem cells are often referred to

as a ‘side population’ because they display low Hoechst fluorescence upon flow cytometry

and appear as negatively stained cells to one side of the main population (Zhou et al., 2001)..

Although Hoechst 33342 is also a Pgp substrate, it has been established that the side

population phenotype is due to ABCG2 expression since the side population is still present in

ABCB1a/1b-/- (Pgp knockout) cells (Zhou et al., 2001).

Cancer stem cells offer an alternative to the conventional model of acquired MDR, in

which MDR occurs by cells gaining mutations which confer drug resistance, giving them an

advantage over other cells in the tumor population by allowing them to survive chemotherapy

(Fig. 9a) (Dean et al., 2005). The cancer stem cell model of MDR differs in that the resistant

cancer stem cells are inherently present in the tumor cell population, and following

chemotherapy these cells survive (possibly due to the presence of ABCG2 and/or Pgp), and

ultimately re-populate the tumor with both cancer stem cells and more differentiated cells

(Fig. 9b) (An and Ongkeko, 2009). In an additional model of cancer stem cell MDR, some

cancer stem cells acquire new mutations which further increase their resistance resulting in a

drug-resistant phenotype (Fig. 9c) (Dean et al., 2005). This model has been shown to occur in

imatinib-resistant CML through acquisition of BCR-ABL mutations (O'Hare et al., 2007). The

final model, which can be found in renal cell cancer, is one of intrinsic resistance where both

the cancer stem cells and variably differentiated cells are drug-resistant, and therapies

therefore have no effect on their survival (Fig. 9d) (Fojo et al., 1987). Through these varied


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routes, ABCG2 expressed on cancer stem cells may contribute to the development and

propagation of MDR cancer.

7.4 Pharmacological Targeting of ABCG2 in Cancer

The expression of ABCG2 by cancer stem cells raises the possibility of selectively

targeting cancer stem cells in chemoresistant tumors. As of yet, this remains an elusive ideal.

ABCG2 is expressed on normal hematopoietic stem cells, as well as in the BBB, so any drug

would need to be carefully titrated to ensure that toxicity is minimized. Serious adverse

events related to altered pharmacokinetics of co-administered cytotoxics are one of the many

reasons why clinical trials of Pgp inhibitors have been disbanded (Nobili et al., 2006). The

first ABCG2 inhibitor, fumitremorgin C, is not used in clinical settings due to its severe

neurotoxic effects, and this has led to the development of a series of structural analogues, of

which Ko143 is the most potent and does not cause neurotoxicity in mice (Allen et al.,

2002b). These inhibitors of ABCG2 have not been tested in clinical trials to date, but remain

a promising possibility if cell-type specificity were to be achieved.

As ABCG2 inhibitors, tyrosine kinase inhibitors are an interesting option for

pharmacological targeting of ABCG2. Clearly, these agents have already been in clinical

trials, and thus, it may be theoretically easier to gain approval for their use, making them a

more tenable possibility than untested inhibitors such as Ko143. In cell-based assays, tyrosine

kinase inhibitors have been shown to be substrates of ABCG2 (possibly binding at the

prazosin-binding site(Özvegy-Laczka et al., 2004) and at high concentrations can inhibit

ABCG2 transport (Shi et al., 2009; Yang et al., 2005). Erlotinib, gefitinib and imatinib have

all been shown to increase the accumulation, and therefore, cytotoxicity of ABCG2 substrates


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such as mitoxantrone and methotrexate in vitro (Misra et al., 1998; Özvegy-Laczka et al.,

2004; Yang et al., 2005).

In addition to inhibiting ABCG2, tyrosine kinase inhibitors may also down-regulate the

expression of ABCG2 via the PI3K-Akt pathway (Nakanishi et al., 2006). These results

suggest that combination therapies involving tyrosine kinase inhibitors and conventional

chemotherapeutics may prevent MDR and enhance the efficacy of both treatments. This

intervention has yet to be trialed, although co-administration of imatinib and the

photodynamic therapy, 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide a, has been tested in

vivo (Liu et al., 2007). Many photosensitizers are ABCG2 substrates, meaning that their

intracellular accumulation is decreased in tumors that express the transporter (Robey et al.,

2005). The combination of imatinib and 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide a,

significantly enhanced the efficacy of the photosensitizer, halving the rate of tumor growth

(Liu et al., 2007). These promising results warrant further investigation in clinical trials.

The potential of phosphodiesterase-5 inhibitors as potential chemosensitizers has also

been investigated (Black et al., 2008; Das et al., 2010). In fact, sildenafil has been shown to

inhibit ABCG2 and Pgp at clinically achievable concentrations, and reverse the efflux of

mitoxantrone, paclitaxel and methotrexate (Shi et al., 2011). There are currently no published

clinical trial results examining the effect of phosphodiesterase-5 inhibitors on ABC

transporters. However, studies in animal models have demonstrated that the use of sildenafil

in combination with doxorubicin results in improved transport of doxorubicin across the BBB

(Black et al., 2008) and improved efficacy of doxorubicin in prostate cancer (Das et al.,

2010). It is possible that the increased activity of doxorubicin is due at least in part to the


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inhibition of ABCG2 and Pgp by sildenafil, and this suggests that phosphodiesterase-5

inhibitors may prove to be useful chemoadjuvants in the future (Tiwari and Chen, 2013).

8. Conclusion

While a plethora of research has been conducted examining ABCG2 biochemistry and

pharmacology with respect to substrates, inhibitors, protein structure, function and gene

regulation, there are still vast gaps in our knowledge. For example, it is still not known what

form higher order oligomers of ABCG2 take, as a high resolution crystal structure has yet to

be solved. Hence, this shortcoming limits the ability to predict structural requirements for

developing new substrates and inhibitors. Such information would be invaluable in exploiting

ABCG2 as a therapeutic target, although this is another area which has not been thoroughly

investigated. Probably the major reason for this is the failure of Pgp inhibitors in clinical

trials, principally due to methodological errors such as not selecting patients based on tumor

expression of Pgp (Robey et al., 2009). This failure has lead to an unwillingness to invest in

targeted treatments for MDR in cancer, and to date, the importance of ABCG2 is cancer

biology and treatment remains unclear.

Recent research has suggested that combination therapies involving tyrosine kinase

inhibitors and phosphodiesterase-5 inhibitors with conventional chemotherapeutics have

potential to treat MDR tumors, and this is currently the best prospect for exploiting ABCG2

in the clinic, providing lessons are learnt from past errors. Until this and further translational

research has been completed, it will remain difficult to establish the impact of ABCG2 on

chemotherapy outcomes.


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Acknowledgments The authors would like to thank Dr Darius Lane, Dr Vera Richardson and Ms Nicole

Seebacher of the Molecular Pharmacology and Pathology Program (University of Sydney)

for their critical appraisal of the manuscript before submission.

Authorship Contributions Wrote or contributed to the writing of the manuscript: Stacy, Jansson, Richardson


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is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7(9): 1028-1034.


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Footnotes

This work was supported by the National Health and Medical Research Council of Australia

(NHMRC) [Project Grant #1021607 and #1021601, Senior Principal Research Fellowship

#571123]. A.E.S. sincerely appreciates an Australian Postgraduate Award from the Sydney

Medical School, University of Sydney.

Patric J. Jansson and Des R. Richardson contributed equally as co-corresponding senior authors.

Reprint requests to be sent to Prof. Des R. Richardson (Molecular Pharmacology and

Pathology Program, Department of Pathology, Blackburn Building D06, University of

Sydney, NSW 2006, Australia). Email: [email protected].

The authors declare no conflict of interest.


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Figure Legends

Figure 1 There is considerable substrate overlap between the ABC transporters, Pgp, MRP1

and ABCG2. *Indicates substrates transported by ABCG2 mutant R482G.

Figure 2 The ABCG2 promoter has multiple cis regulatory elements, SP1, AP1 and AP2

sites, the position of which is depicted in relation to the transcription start site of the first

ABCG2 exon. Abbreviations: Hypoxia response element (HRE), estrogen response element

(ERE), aryl hydrocarbon response element (AhRE), NFκB response element (NFκBRE).

Figure 3 Schematic illustration of the membrane topology of ABCG2 and highlights of

residues or mutations which affect the functioning of the transporter. Val12M and Q141K are

SNPs thought to affect the pharmacokinetics of ABCG2 substrates. Notably, T402, G406 and

G410 are near or part of the GXXXG motif and are thought to play a role in dimerization, as

do Gly553 Cys592, 603 and 608. The R482G isoform of ABCG2 appears in drug-selected

cell lines and displays altered substrate specificity to the wild-type transporter. Asp596 is the

sole N-linked glycosylation site.

Figure 4 Homology modeling of ABCG2 predicts that substrate-binding induces significant

conformational change which brings the nucleotide-binding domains closer together and

closes the substrate-binding cavity. This structural alteration possibly allows these domains to

interact and results in the transport of substrates through the central translocation pathway.

The transmembrane domains are numbered 1-6, and possible areas of significance to the

transporter’s function are highlighted. This figure was adapted from (Rosenberg et al., 2010).

Figure 5 The structural diversity of selected ABCG2 substrates and inhibitors.


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Figure 6 Drug-selected cell lines can express the mutant form of ABCG2-R428G. This

isoform has altered substrate specificities as compared to wild-type ABCG2, possibly

because some substrates interact with the amino acid at position 428. This figure was adapted

from (Sarkadi et al., 2004).

Figure 7 Substitution of polar groups at carbons in positions 10 and 11 (circled in red) of

camptothecins analogs has been found to increase the substrate recognition by ABCG2, as

demonstrated by the increase in IC50 for SN-38 compared to SN-443 in the ABCG2-

expressing cell line PC-6/SN2-5H2. A planar structure due to double bonds (circled in red)

has been found to increase the potency of both flavonoids (between carbons 2 and 3) and

rotenoids (between carbons 6 and 12). This is illustrated by the lower EC60 for increasing

mitoxantrone accumulation in MCF-7 MX100 ACBG2-expressing cells for flavone

compared to flavanone; and the increase in maximal mitoxantrone accumulation for

boeravinone G compared to boeravinone C in HEK-293/ABCG2 cells.

Figure 8 ABCG2 is highly expressed in the placenta, blood brain barrier (BBB),

gastrointestinal tract, liver, kidney, testis and the lactating breast. Generally, ABCG2 it is

found on the apical surface of cells where it is thought to actively transport substrates out of

the tissues so that they may be excreted. Hence, this prevents exposure to harmful

xenobiotics. This figure was adapted from (Vlaming et al., 2009).

Figure 9 Different models of tumor MDR. (a) In the conventional model of MDR, sporadic

mutations arise which confer drug resistant clones (yellow). After chemotherapy these cells

survive and re-populate the tumor. (b) The cancer stem cell model indicates that tumors


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contain small populations of cancer stem cells (red) and their descendants (blue) which are

committed to a particular lineage. The cancer stem cells expressing ABC transporters are able

to survive chemotherapy and proliferate to form a heterogeneous tumor population of cancer

stem cells and differentiated descendant cells. (c) The acquired resistance cancer stem cell

model is similar to b. However, following chemotherapy, where the variably differentiated

cells die, mutations arise in the surviving cancer stem cells (orange) which further enhances

their drug resistance and that of their differentiated descendants (purple). (d) In the intrinsic

resistance model, both the cancer stem cells (orange) and the variably differentiated cells

(purple) are inherently drug resistant, so chemotherapy has no effect on their survival.


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Table 1 Summary of Clinical Studies of ABCG2 Expression in Human Leukemia

Study Sample Results

Acute myelogenous leukemia

Sargent et al 20 blast samples 27% of samples were ABCG2 positive.

van der Kolt et al 20 paired (pretreatment and

refractory/relapse) samples

Low ABCG2 expression, no increase in relapse samples.

Abbott et al 40 de novo samples 78% of samples had higher ABCG2 expression than

normal blood and bone marrow, 7% had levels within the

range of drug resistant cell lines.

van Den Huevel-Eibrink et

al

20 paired (de novo and

refractory/relapsed) samples

Relapsed samples had higher ABCG2 expression, ABCG2

expression associated with Pgp expression.

van der Pol et al 45 paired samples NoABCG2 detected.

Galimberti et al 51 AML samples ABCG2 expression detected in 56% of samples,

intermediate levels in 48.2%. ABCG2 expression

associated with Pgp expression.

Suvannasankha et al 31 pretreatment blast samples No correlation between function, protein or gene

expression.

Steinbach et al 59 untreated AML, 9 relapse

samples

Median ABCG more than 10 times higher in patients who

did not achieve remission, compared to responders.

ABCG2 expression higher at relapse than diagnosis. High

ABCG2 linked to worse prognosis.

Benderra et al 149 de novo samples ABCG2 expression and ABCG2 expression associated

with Pgp function were correlated with poorer treatment

outcomes: achievement of complete remission, 4-year

disease-free survival and 4-year overall survival.

Uggla et al 40 AML samples Patients with higher ABCG2 expression who respond to

therapy had shorter overall survival. No difference in

expression between responders and non-responders.

Acute lymphocytic leukemia

Sauerbrey et al 47 de novo, 20 relapsed samples No correlation between expression and response, ABCG2

expression lower in T-cell lineages.

Plasschaert et al 46 de novo samples BXP-34 positivity in B-cell lineage 2.4-fold higher than T-

cell lineage.

Stam et al 13 non-infant and 13 infant

samples

ABCG2 expression 2.4-fold less in infant samples.

Expression correlated with Ara-C resistance.

Suvannasankha et al 30 pretreatment samples 43% of cases BXP-21 positive. BXP-21 staining predictive

of shorter disease-free survival.


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MRP1

ABCG2

Pgp

LysotrackerPheophorbide a

Riboflavin

CyclophosphamideHydroxyurea

Cisplatin

PaclitaxelColchicineMitomycin

Hoechst 33342Topotecan Rifampicin

MethotrexateFolic acid

Estrone-3-sulfate

VincristineVinblastineRitonavir


Estrone-3-sulfate

Hoescht 3342TopotecanRifamipicin


Estrone-3-sulfateDoxorubicin*Mitoxantrone

Etoposide*

Figure 1


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Figure 2

-28 NFκBRE-116 HRE

-188 ERE/PRE

-198/-189 AhRE -49 Sp1

-160 Sp1

-127 Sp1

-222 Sp1 -50 AP2-360 Sp1

-400 CpG island -250


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Figure 3

N-terminus

C-terminus

Nucleotidebinding domain

Q141K

Gly553

Val12M

Cys603T402 G406 G410

Asp596

Cys608

Cys592GXXXG motif

R482G

1 2 3 4 5 6


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Figure 4

Arg482

Coupling helix

1

2 64

56 4

12

2

21

13 3

445 5

66

3LipidBilayer

Nucleotide binding domains

3

Extracellular loop between transmembrane domains 1 and 2 and 5 and 6

5

Arg482

Substrate unbound Substrate bound

Linker region


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Figure 5

NH

O

N

N HN

HO

O

O

O

NH

NH

N

O

O

O

O

O

NH

N

NH

N

S

N

HN

O

S

OO

H

H

HHOO

HN

NH

O

NH

O

HO

HO OH

HN

OH

N

N

O

O

HO

OHO

N

Estrone-3-sulfate

Pheophorbide A

Cimetidine Elacridar

Topotecan

Mitoxantrone


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Figure 6

Methotrexate

MitoxantronePheophorbide a

Hoechst 3342FlavopiridolTopotecan

Tyrosine KinaseInhibitors

DoxorubicinEpirubicin

Rhodamine 123Daunorubicin

Wild-type R482G


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Figure 7

Flavanone, EC60

24.61 ± 5.21 μMFlavone, EC60

3.99 ± 1.78 μM

Boeravinone G, % maximal mitoxantrone accumulation 31 ± 4.2

Boeravinone C, % maximal mitoxantrone accumulation 92 ± 6.5

Rotenoids

Flavonoids

2

3

2

3

6

12

6

12

Camtothecin Analogs

SN-443, IC50

1.52 ± 0.04 μMSN-38, IC50 262 ± 5.70 μM

10

11

11

10


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Brain

Kidney

TestisPlacenta

IntestinesLiver

ABCG2 expressing cells

Direction of ABCG2transport

Blood flow

Lactating breast

Direction of ABCG2 substrate excretion

Figure 8


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Figure 9


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