Drug resistance molecules lessons from oncology
George L. Scheffer and Rik J. Scheper1
Department of Pathology, Free University Medical Center, de Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands
Abstract. Tumour cell insensitivity to anticancer drugs frequently appears as multidrug resistance (MDR), associated with overexpression of one or more of a set of at least 10 different molecules, causing reduced drug levels at the intracellular target sites. They include transmembrane transporter proteins such as P glycoprotein, MRP1—9 and BCRP. In addition, the lung-resistance protein, recently identified as the major vault protein, has been associated with MDR. We have generated monoclonal antibodies that specifically recognize most of these proteins, which we are using to try to identify their roles in clinical drug resistance, and also to explore their occurrence in normal human tissues and physiology. Both types of studies will also provide further insights into the molecular features of drugs associated with distinct MDR transporters.
2002 Mechanisms of drug resistance in epilepsy: lessons from oncology. Wiley, Chichester (Novartis Foundation Symposium 243) p 19-37
Drug resistance
Drug resistance has recently received attention as an important impediment in the treatment of a range of diseases from rheumatoid arthritis to epilepsy. The phenomenon of multidrug resistance (MDR; reviewed in Moscow et al 1997) has been acknowledged for many years as a major obstacle in cancer therapies and is characterized by resistance to a broad range of structurally and functionally unrelated cytotoxic agents. Our immunology research group at the Free University Medical Centre in Amsterdam became involved in cancer MDR through the development of monoclonal antibodies detecting the causative molecules. In the late 1970s, Victor Ling presented his exciting data on the MDR1-encoded P glycoprotein (Pgp) at a scientific meeting. This inspired Bob Pinedo, chairman of our medical oncology department, to approach us about whether we could develop antibodies that would detect MDR1 Pgp in patient
'This paper was presented at the symposium by Rik J. Scheper, to whom correspondence should be addressed.
tumour samples. Then, after the successful development of the JSB-1 antibody, now widely used as a marker for Pgp, we began a broad range of studies on the mechanisms of MDR.
ABC transporters in MDR
Energy-dependent, active, transport processes neutralizing the therapeutic toxic effect of cytotoxic agents operate in MDR cells. Drugs are prevented from entering cells, or are exported outward from the cytoplasm, reducing levels at their intracellular targets. The prototypic transporter protein involved in MDR is MDR1 Pgp (symbol ABCB1, reviewed in Ambudkar et al 1999), discovered in 1976 by Victor Ling (Juliano & Ling 1976). This heavily glycosylated 170 kDa protein is a member of the ATP binding cassette (ABC) transporters (Higgins 1992) and transports a broad range of substrates, including several anticancer drugs (Gottesman & Pastan 1993). The ABC superfamily also comprises several subfamilies with members that are involved in MDR, in particular the ABCB (MDR/TAP subfamily), ABCC (CFTR/MRP subfamily) and ABCG (White subfamily) subfamilies (see Table 1).
As mentioned, the ABCB subfamily includes the classical MDR transporter MDR1 Pgp, but the contribution of the other members in this subfamily to MDR is much less clearly defined. The MDR3 Pgp protein (ABCB4) and sister of Pgp (sPgp; also known as the bile salt efflux pump [BSEP], ABCB11) are highly related to MDR1 Pgp but although transport of some cytotoxic agents has been reported for both of these proteins (Childs et al 1998, Smith et al 2000), no clear involvement in MDR has been shown. The two TAP molecules (ABCB2 and ABCB3) are very important molecules in the field of immunology as they act in concert as a heterodimer in transporting peptides for antigen recognition over the cell membrane. Interestingly, we have previously reported a (moderate) contribution to MDR for the TAP molecule (Izquierdo et al 1996). For the other members of the ABCB subfamily no involvement in MDR is known.
In the early 1990s Susan Cole in Canada (Cole et al 1992) cloned multidrug resistance protein 1 (MRP1; ABCC1) from a lung cancer cell line. This is a 190 kDa MDR-related ABC transporter from the ABCC subfamily. MRP1 was shown to confer a similar resistance phenotype to MDR1 Pgp, although these two proteins share only 14% amino acid identity (Grant et al 1994, Zaman et al 1994). Later on, several closely related family members were described (Borst et al 2000). Of this MRP subfamily, MRP1-3 (ABCC1-3) are particularly involved in the transport of chemotherapeutic agents in human cancer cells (Cole & Deeley 1998, Cui et al 1999, Kool et al 1999a), whereas for the other ABCC family members no clear involvement in MDR has yet been shown.
The latest (and, since the completion of the Human Genome Project has put a stop to discovering new ABC transporter proteins, most likely last) ABC transporter involved in MDR was cloned by Ross and Doyle in 1998 from a mitoxantrone-resistant subline of the breast cancer cell line MCF-7/Adr/Vp. This transporter was named breast cancer resistance protein (BCRP, MXR, ABCP; ABCG2) (Doyle et al 1998, Miyake et al 1999, Allikmets et al 1998) and belongs to the ABCG subfamily. The BCRP is a half-transporter that probably acts as a homo- or heterodimer in transporting cytotoxic agents. Transfection experiments with the BCRP cDNA result in a full blown MDR phenotype (Doyle et al 1998), and therefore it seems valid to conclude that the BCRP is capable of transporting MDR drugs as a homodimer. Also, as yet, no heterodimer partner for BCRP has been described. For the other members of the ABCG subfamily no involvement in MDR has been reported.
Structure, gene localization and disease linkage of the ABC transporters
All of the above mentioned transporters belong to the superfamily of ABC transporters. The schematic representation of several family members in Fig. 1 shows that these proteins look very similar. In general, they all are composed of two core halves, each containing a nucleotide binding domain characterized by the consensus sequences of ABC transporters; Walker A and B motifs and the ABC signature, and a membrane-spanning domain with six transmembrane segments (TMs). In the MRP subfamily, the MRP1, -2, -3, -6 and -7 proteins have an extra N-terminal domain (MSD0) which contains an extra set of five TMs.
Although some of the ABC transporter genes are localized in tandem per chromosome (e.g. MDR1 Pgp/MDR3 Pgp on chromosome 7, TAP1/ TAP2 on chromosome 6 and MRP1/MRP6 on chromosome 16), they generally seem to be localized quite randomly on diverse chromosomes (see Table 1).
For some transporters it has become clear that mutations in the gene or lack of expression can cause genetic diseases. Absence of the TAP2 molecule causes a bare lymphocyte syndrome (BLS) (de la Salle et al 1994), in which patients lack surface major histocompatibilty complex (MHC) class I molecules and are immunodeficient due to the resulting absence of CD8+ T cells. Lack of expression of MDR3 in the liver is responsible for type 3 progressive familial intrahepatic cholestasis (PFIC3) (de Vree et al 1998), whereas type 2 PFIC is caused by absence of sPgp. Mutations in the MRP2 gene cause another liver disease, the Dubin—Johnson syndrome (Paulusma et al 1996). Recently, it was found that mutations in the MRP6 gene are responsible for the connective tissue disorder pseudoxanthoma elasticum (PXE) (Ringpfeil et al 2000, Bergen et al 2000, Le Saux et al 2000).
Cystic fibrosis is the result of mutations in the CFTR gene. Mutations in the recently described MRP8 and/or MRP9 genes are positional candidates for paroxysmal kinesigenic choreoathetosis (Tammur et al 2001). None of the transporters in the ABCG subfamily has been linked to a genetic disease as yet.
For some of these transporters the contribution to the disease is direct, e.g. in PFIC3 patients the lack of translocation of bile salt neutralizing phosphatidylcholine across the canalicular membrane of the hepatocyte is the cause of the observed cholestasis in the liver. For other transporters, the exact role in the disease is unclear. Whether mutations in MRP6 are directly responsible for PXE or contribute to this disease indirectly through deficient transport in liver and or kidney is still unclear.
Substrates of the ABC transporters
In general, the substrates of these transporters are rather diverse and range from salts to anionic conjugates and peptides (Table 1). Some transporters seem highly specialized in transporting just one substrate, such as phosphatidylcholine transport by the MDR3 Pgp. However, most transporters are capable of transporting a range of (un)related substrates that share some common features. The TAP molecule has a preference for peptides of a certain size, but the exact sequence of the peptides is less critical, since the TAP molecule can process peptides digested from many different foreign proteins. Pgp and the MRP1 transporter seem to pump substrates predominantly based on charge. Pgp mainly transports organic neutrals, including steroids, but also phenytoin-like anti-epileptic drugs (Tishler et al 1995), whereas MRP1 transports conjugates of organic anions. Glutathione is a negatively charged tripeptide that may also be co-transported. Of course, for both these proteins the substrate list also includes many cytotoxic agents, such as doxorubicin and vincristine. The substrate specificity of MRP2 is very similar to that of MRP1 (Keppler et al 1998, Jedlitschky et al 1997), whereas the transport range of MRP3 is rather limited compared to these two proteins. The MRP3 transporter is mainly involved in resistance to etoposide (Kool et al 1999a) and vincristine (Zeng et al 1999). For MRP4 (ABCC4) and MRP5 (ABCC5), no major changes were found in mRNA levels in doxorubicin- or cisplatin-resistant cells (Kool et al 1997). However, it was recently found that these transporters are able to transport nucleoside analogues used for antiviral therapies (Scheutz et al 1999, Wijnholds et al 2000). In addition, some low-level resistance against CdCl2 and potassium antimonyl tartrate was found in MRP5-transfected cell lines (McAleer et al 1999). Overproduction of MRP6 (ABCC6) mRNA in tumour cell lines was found to be invariably associated with the amplification of the adjacent MRP1 gene, and MRP6 probably does not contribute to the resistance of these cell lines (Kool et al
1999b). However, a preliminary report has suggested that MRP6 may mediate modest resistance to anthracyclines and epipodophyllotoxins (Belinsky et al 2001).
The MRP7, -8 and -9 transporter proteins were only very recently discovered and not much is yet known about their substrate preferences. BCRP is capable of transporting many anticancer agents. Of these, mitoxantrone is probably the best substrate for BCRP. Thus BCRP is actually the long-sought mitoxantrone transporter.
Recently, the occurrence of single nucleotide polymorphisms (SNPs) in ABC transporters have attracted quite a bit of attention because they can be highly critical for the substrate specificity of the pump (Zhang et al 2001, Kerb et al 2001, Honjo et al 2001). The presence of a threonine or a glycine at position 482 in the BCRP transporter was found to be essential for rhodamine 123 efflux; cells with the wild-type arginine at that position were unable to transport this substrate (Honjo et al 2001).
Blockers: resistance-modifying agents
For several ofthe transporters, molecules have been identified that block transport activities. Most of these blocking agents, or resistance-modifying agents, are themselves substrates for the transporters. They block the activity of the transporter by competing with the toxic substrate. Some of these blockers specifically block one of the transporters, whereas others have a negative effect on the transport activities of two or more pumps.
More-or-less specific blockers for MDR1 Pgp are verapamil, bepridil and cyclosporin A. For MRP1, probenecid, indomethacin and MK571 may be considered to be specific blockers. PSC 833 has a blocking effect on both MDR1 Pgp and MRP1, whereas GF120918 blocks both MDR1 Pgp- and BCRP-mediated transport (Bart et a; 2000). Fumitremorgin C (FTC) is a BCRP-specific blocker (Rabindran et al 2000).
Monoclonal antibodies specifically detecting the MDR transporters
As mentioned above, specific monoclonal antibodies are essential for facilitating studies on transporter proteins in clinical material, to reveal their physiological functions and their possible contributions to MDR. In collaborative studies with our colleagues from the Department of Medical Oncology (Bob Pinedo, Henk Broxterman), the Dutch Cancer Center (NKI; Piet Borst, Jan Schellens), the Academic Medical Center (AMC; Ronald Oude Elferink) and The Netherlands Ophthalmic Research Institute (NORI; Arthur Bergen, Jan Wijnholds) we have produced monoclonal antibodies specifically detecting MDR1 Pgp, MDR3 Pgp, MRP1, -2, -3, -4, -5 and -6, and BCRP (Scheper et al 1988, Scheffer et al 2000a,b,
|
Name |
Symbol |
Chromosome |
RNA |
AA |
Disease link |
|
MDR1 Pgp |
ABCB1 |
7q21 |
4.5 |
1279 |
n.k. |
|
TAPI |
ABCB2 |
6p21.3 |
2.5 |
808 |
n.k. |
|
TAP2 |
ABCB3 |
6p21.3 |
2.8 |
653 |
BLS |
|
MDR3 Pgp |
ABCB4 |
7q21 |
4.5 |
1279 |
PFIC3 |
|
Est422562 |
ABCB5 |
7pl4 |
7.5 |
n.k. |
n.k. |
|
ABCB6 |
ABCB6 |
2q33-q36 |
3.5 |
842 |
n.k. |
|
ABC7 |
ABCB7 |
Xql3.1- |
2.4 |
752 |
Anaemia |
|
ql3.3 | |||||
|
M-ABC1 |
ABCB8 |
7q35-q36 |
2.4 |
718 |
n.k. |
|
ABCB9 |
ABCB9 |
12q24 |
3.5 |
723 |
n.k. |
|
M-ABC2 |
ABCB10 |
lq42 |
4.1 |
738 |
n.k. |
|
BSEP/sPgp |
ABCB11 |
2q24 |
5.4 |
1321 |
PFIC2 |
|
MRP1 |
ABCC1 |
16pl3.1 |
6.5 |
1531 |
n.k. |
|
MRP2/ |
ABCC2 |
10q24 |
5.5 |
1545 |
Dubin- |
|
cMOAT |
Johnson | ||||
|
MRP3 |
ABCC3 |
17q21.3 |
6.5 |
1527 |
n.k. |
Normal tissue distribution Substrate role in MDR
Many tissues, apical membranes
Most cells, ER Most cells, ER Hepatocyte, apical membranes Ubiquitous Mitochondria Mitochondria
Mitochondria
Heart brain lysosomes
Mitochondria
Hepatocytes, apical membranes
Ubiquitus, lateral membranes
Liver kidney intestine apical membranes
Kidney intestine lateral membranes
Many neutral yes hydrophobic compounds
Peptides possibly
Peptides possibly
Phosphatidylcholine no n.k. no
Iron no
Peptides? no
Biles salts no
Anionic conjugates yes glutathione
Anionic conjugates yes glutathione, bilirubin
Anionic conjugates, bile yes salts
|
MRP4 |
ABCC4 |
13q32 |
6.5 |
1325 |
n.k. |
Many tissues |
Cyclic nucleotides |
no? |
|
MRP5 |
ABCC5 |
3q27 |
6.6 |
1437 |
n.k. |
Many tissues |
Cyclic nucleotides |
no? |
|
MRP6 |
ABCC6 |
16pl3.1 |
6.5 |
1503 |
PXE |
Liver kidney lateral membranes |
Peptides? |
no? |
|
CFTR |
ABCC7 |
7q31.2 |
6.0 |
1480 |
Cystic fibrosis |
Lung intestine cholangiocytes |
Organic anions? |
no |
|
SURI |
ABCC8 |
llplS.l |
5.0 |
1581 |
fPHHI |
Pancreas |
n.k. |
no |
|
SUR2 |
ABCC9 |
12pl2.1 |
5.0 |
1549 |
n.k. |
Skeletal muscle heart |
n.k. |
no |
|
MRP7 |
ABCC10 |
6p21 |
5.5 |
1513 |
n.k. |
Low in all tissues |
n.k. |
no? |
|
MRP8 |
ABCC11 |
16ql2.1 |
4.6 |
1382 |
PKC? |
Low in all tissues |
n.k. |
no? |
|
MRP9 |
ABCC12 |
16ql2 |
5.0 |
1359 |
PKC? |
Low in all tissues |
n.k. |
no? |
|
ABC8/White ABCG1 |
21q22.3 |
2.7 |
638 |
n.k. |
Brain spleen lung |
Sterols? lipids? |
no | |
|
BCRP/MXR ABCG2 |
4q22 |
2.4 |
655 |
n.k. |
Breast liver intestine |
Drugs |
yes | |
|
White 2 |
ABCG4 |
1 lq23 |
n.k. |
749? |
n.k. |
Liver |
n.k. |
no |
|
White 3 |
ABCG5 |
2p21 |
2.3 |
651 |
Sitosterolemia |
Liver small intestine |
Plant sterols |
no |
|
White 4 |
ABCG8 |
2p21 |
2.0 |
673 |
Sitosterolemia |
Liver small intestine |
Plant sterols |
no |
AA, amino acids; BLS, bare lymphocyte syndrome; PFIC2, type 2 progressive intrahepatic cholestasis; PFIC3, type 3 progressive intrahepatic cholestasis; PXE, pseudoxanthoma elasticum; fPHHI, familial persistent hyperinsulinemic hypoglycemia of infancy; PKC, paroxysmal kinesigenic choreoathetosis; n.k., not known.
AA, amino acids; BLS, bare lymphocyte syndrome; PFIC2, type 2 progressive intrahepatic cholestasis; PFIC3, type 3 progressive intrahepatic cholestasis; PXE, pseudoxanthoma elasticum; fPHHI, familial persistent hyperinsulinemic hypoglycemia of infancy; PKC, paroxysmal kinesigenic choreoathetosis; n.k., not known.
out tn c
Q Transmembrane segmenl (TM)
Putative glycosylate site O Nucleotide binding Domain (NBp)
FIG. 1. A schematic representation of the structure of some ABC transporters.
G. L. Scheffer, unpublished results). For the immunization and the selection of monoclonal antibodies (Mabs), most of the time we used fusion proteins of the Escherichia coli maltose binding protein (MBP) and fragments of the transporter protein of interest. Intriguingly, only for MDR1 Pgp have several Mabs been described that detect external epitopes (Mechetner & Roninson 1992, Hamada & Tsuruo 1986, Cianfriglia et al 1994). Mabs that detect external epitopes are widely used to detect the protein on viable cells that have not been altered by fixation. Those binding a functional epitope of the protein, as is the case with the UIC2 Mab (Mechetner & Roninson 1992), allow studies of the substrate specificity in blocking experiments. Despite many attempts by ourselves — employing methods ranging from peptide and DNA immunization to the use of knockout mice and the phage display method — and colleagues all over the world, it has not been possible to produce Mabs for any of the other MDR-related transporter molecules. Whether this is due to a lack of immunogenicity of the external regions or a limited exposure of protein at the external side, possibly because of glycosylation moieties, is unknown.
Normal tissue localization and function of the MDR transporters
The development of this broad panel of Mabs allowed us first to study the localization of several transporter molecules in normal tissues (Scheffer et al
- FIG. 2. Immunostaining of normal human liver tissue for MRP6 and MRP2. MRP6 is present at the basolateral membranes of the hepatocytes (a), whereas MRP2 is present at the apical site of the hepatocyte, the canalicular membrane (b).
2000a, Maliepaard et al 2001, G. L. Scheffer, unpublished results). In general, the transporters can be found at locations and tissues that are heavily exposed to toxic materials, e.g. along the gastrointestinal tract and the lung mucosa. At these sites there is a real need to protect the inside against potentially dangerous exogenous compounds, toxic materials, microbial products, etc. Furthermore, the transporters can be found in organs which are involved in secretion processes such as liver and kidney. Some of the transporter molecules turned out to be (almost) ubiquitously expressed (MDR1 Pgp, MRP1), whereas others have a (much) more restricted normal tissue distribution. MDR3 Pgp is expressed exclusively at the hepatocyte membrane and MRP2 is mainly present at the canalicular membrane of the hepatocyte. Of the genuine MDR transporters, MDR1 Pgp, BCRP and MRP1, only the first two are present at the apical membrane sections of polarized cells. MRP1, like the other MRP family members (except for MRP2), localizes laterally instead. As an example, in Fig. 2 normal human liver tissue is immunostained for both MRP6 and MRP2. MRP6 is present at the basolateral membranes of the hepatocytes (Fig. 2a), whereas MRP2 is present at the apical site of the hepatocyte, the canalicular membrane (Fig. 2b). An important implication of the opposite localization of MRP1 is, that this transporter protects the surrounding tissue in a different way than MDR1 Pgp and BCRP. In drug transport, MRP1 is most likely involved in transport from the exposed (epithelial) tissue to the bloodstream, rather than preventing the entry of toxic compounds by re-effluxing these xenobiotics from the tissue to the lumen.
The presence of all three of the most prominent MDR transporters on endothelial cells is very interesting and deserves further attention. Of course, functionally blocking these transporters would cause increased uptake of therapeutic drugs from the bloodstream. This may be particularly important for the treatment of diseases which require the drugs to cross the blood—brain barrier.
Many of the physiological functions of the transporters are still unknown. Very likely, the MDR transporter molecules function normally as xenobiotic pumps. Still, other functions have been attributed to both MDR1 Pgp and MRP1. Recently, Gwendolyn Randolph reported particularly intriguing immunological functions for these transporter proteins (Randolph et al 1998, Robbiani et al 2000). She noted a role for MDR1 Pgp in the migration of antigen-presenting dendritic cells (DC) from explants of cultured human skin into the culture medium via dermal lymphatic vessels, since only anti-MDR1 Pgp Mabs or antagonists inhibited this process. Because of MRPl's role in transporting the important inflammatory mediator leukotriene LTC4, it has an essential role in DC migration from skin to lymph nodes. DC mobilization from the epidermis and trafficking into lymphatic vessels was greatly reduced in MRP17/7 mice, while migration was restored by exogenous cysteinyl leukotrienes LTC4 or LTD4. Indeed, experiments in our own laboratory have shown a marked up-regulation of the expression of both MDR1 Pgp and MRP1 during DC maturation. Interestingly, strong up-regulation of another MDR marker, the major vault protein (MVP), was also observed during this maturation process (Schroeijers et al 2000). Of note, the MVP was originally described by us as the lung resistance protein (LRP) and subsequently found to be unrelated to the ABC superfamily of transporter proteins (Scheffer et al 1995). Experiments to further elucidate the precise roles of these molecules during DC maturation and in antigen-presentation are ongoing.
Concluding remarks
At least three ABC transporter molecules (MDR1 Pgp, MRP1 and BCRP) have been identified that act like genuine MDR transporters adversely affecting chemotherapeutic approaches in cancer treatment. Several drugs used in the treatment of a broad variety of other diseases, e.g. phenytoin-like drugs in epilepsy and the anti-malarial drug chloroquine in rheumatoid arthritis, have also been identified as substrates for these transporter molecules. Therefore, efficacy of treatments of these diseases is most likely to be influenced by the presence or, after extended periods of exposure to these drugs, increased expression of these transporter molecules on target cells. Extended analyses of critical tissues in these patients for the presence of these molecules therefore seem warranted.
Ackowledgements
This work was supported by the Dutch Cancer Society, grants VU95-923 and VU96-1256.
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