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J Mammary Gland Biol Neoplasia (2009) 14:3–9

DOI 10.1007/s10911-009-9109-9

ABC Transporters, Drug Resistance, and Cancer Stem Cells

Michael Dean

Received: 7 December 2008 / Accepted: 3 February 2009 / Published online: 18 February 2009
# Springer Science + Business Media, LLC 2009

Abstract The protection of the body’s stem cells from CML chronic myelogenous leukemia
damage or death due to toxins is a critical function of an ALL acute lymphoblastic leukemia
organism, as the stem cells need to remain intact for the TKI tyrosine kinase inhibitor
entire life of the organism. One of the principal mechanisms
for protecting stem cells is through the expression of
multifunctional efflux transporters from the ATP-binding
cassette (ABC) gene family. These same transporters have ATP-Binding Cassette Transporter Function
been known for over 25 years to also play a role in
multidrug resistance of tumor cells. An exciting outcome of ABC transporters bind to and/or hydrolyze a pair of ATP
the concept of the cancer stem cell is that the tumor molecules and use the binding and/or hydrolysis energy to
initiating cell may be innately resistant to many standard either efflux specific compounds across the membrane or to
therapies. This provides one mechanism in which cancer flip them from the inner to the outer leaf of the membranes
stem cells could survive cytotoxic or targeted therapies and [1–3]. The classification of ABC transporters is based on
lead to tumor regrowth or relapse. Gaining a better insight the sequence analysis of the amino acids in the ATP-
into the mechanisms of stem cell resistance to chemother- binding (ATP) domain(s), also known as nucleotide binding
apy might therefore lead to new therapeutic targets and folds (NBFs) [3, 4]. The NBFs contain domains originally
better anti-cancer strategies. recognized in other ATP-binding proteins, the Walker A
and B motifs. ABC proteins contain an additional element,
Keywords ATP-binding cassette transporters . the signature (C) motif, found just upstream of the Walker
Cancer stem cell . Multidrug resistance . Estrogen . B domain [5]. The complete transporter protein contains
Aromatase . EGF receptor . Transport inhibitors two transmembrane (TM) domains with of 6–11 membrane
spanning α-helices, and two NBFs. These four domains can
Abbreviations be present in one protein (a full transporter) or as a single
ABC ATP-binding cassette TM and single NBF domain in the case of a half-
EGF epidermal growth factor transporter. Half transporters need to form either homo- or
TM transmembrane heterodimers to compose a functional molecule.
NBFs nucleotide binding folds Structural analysis of either isolated NBFs or complete
MDR multidrug resistant bacterial ABC transporters have been accomplished [6–9].
SP side population These studies show that the two NBFs are joined together
by binding to ATP. Thus a single ATP molecule binds
portions of both ATP-binding domains. Therefore binding
M. Dean (*) of the ATP molecules is envisioned to cause a structural
Laboratory of Experimental Immunology,
conformation change that can be reversed by ATP hydro-
Cancer and Inflammation Program, National Cancer Institute,
Frederick, MD 21702, USA lysis and release; although not all transporters hydrolyze
e-mail: both of the ATP molecules.
4 J Mammary Gland Biol Neoplasia (2009) 14:3–9

There are between 38 and 61 ABC genes in the genomes best characterized ABC transporter both biochemically and
of all non-plant eukaryotic organisms sequenced to date through mutational analysis. In addition, there is a wide
(plants have greatly expanded the number of transporters, variety of clinical agents that inhibit ABCB1 that have been
presumably to deal with a wider array of toxins, and the attempted in clinical studies against cancer going back to 1994
lack of filtering organs, such as the liver) [3, 10, 11]. [18]. None of these clinical trials were very positive and this
Phylogenetic analysis groups these ABC proteins into strategy is in currently in disfavor. However there are two
8 different subfamilies: ABCA-ABCH [12]. There are 48 other ABC proteins widely overexpressed in tumor cells,
ABC genes in the human genome, and they carry out a ABCC1/MRP1 [19] and ABCG2 [20–23]. And at least
wide array of normal physiological functions in many seven other transporters have been shown to cause
distinct tissues [4]. Most of what we know about the resistance to at least one agent (Table 1) [18]. Therefore,
function of ABC genes comes from genetic disorders, and one roadblock to inhibiting only a single ABC transporter,
18 ABC genes are associated with Mendelian inherited is the knowledge that there are multiple potential efflux
disease in human or other mammals [12]. These disorders pumps that the cell can employ. The ABC transporters
range from cystic fibrosis (caused by mutation in the also have important roles in drug distribution as they play
ABCC7/CFTR gene, a chloride ion channel) to adrenoleu- key roles in compound access across the blood-brain
kodystrophy (caused by mutations in ABCD1, a peroxi- barrier, intestine, and other tissues compartments [24].
somal protein of uncertain function).
ABC Transporters in Normal Stem Cells
Drug Resistance in Cancer Cells
An exciting development of stem cell biology was the
Cancer cells can become resistant to cytotoxic drugs through a discovery that many stem cells express high levels of
number of mechanisms, such as mutation or overexpression of specific ABC transporters. Hematopoietic stem cells ex-
the drug’s target, drug inactivation, or efflux of the drug from press high levels of ABCG2 and/or ABCB1 and that
the cell [13]. Many tumors in patients suffering from relapse expression is often turned off during differentiation to
to therapy are resistant to multiple drugs (multidrug resistant, progenitor and mature blood cells [14, 20, 25]. Gene
MDR) [14]. MDR tumors are a major barrier to effective knockouts for either Abcg2, Abcb1 or Abcc1 or combina-
cancer therapy and along with metastasis are estimated to be tions of these genes yield mice that are viable, fertile, and
major contributors to death by cancer. Clearly understanding have normal stem cell compartments [26–28]. Therefore
the molecular basis of MDR and developing clinical reagents none of these genes are required for stem cell growth or
or strategies to prevent the occurrence of resistance, or treat maintenance. However, these mice are more sensitive to
resistant tumors is a high priority. certain compounds such as vinblastine, ivermectin, top-
The P-glycoprotein encoded by the ABCB1 gene was the otecan, and mitoxantrone, demonstrating a role for these
first ABC transporter identified to be amplified and/or over- transporters in protecting the organism, and the stem cells,
expressed in MDR tumor cell lines [15–17]. This is also the from toxins [28].

Table 1 ABC transporters involved in drug resistance.

Gene Protein/alias Chemotherapeutic drugs effluxed by transporter Other drugs and substrates

ABCA2 ABCA2 Estramustine

ABCB1 PGP/MDR1 Colchicine, doxorubicin, etoposide, vinblastine, paclitaxel Digoxin, saquinivir,
ABCC1 MRP1 Doxorubicin, daunorubicin, vincristine, etoposide, colchicine, Rhodamine
camptothecins, methotrexate
ABCC2 MRP2 Vinblastine, cisplatin, doxorubicin, methotrexate Sulfinpyrazone
ABCC3 MRP3 Methotrexate, etoposide
ABCC4 MRP4 6-MP and 6-TG and metabolites, methotrexate PMEA, cAMP, cGMP
ABCC5 MRP5 6-MP and 6-TG and metabolites PMEA, cAMP, cGMP
ABCC6 MRP6 Etoposide
ABCC11 MRP8 5-fluorouracil PMEA, cAMP, cGMP
ABCG2 MXR/BCRP Mitoxantrone, topotecan, doxorubicin, daunorubicin, CPT-11, Pheophorbide A, Hoechst 33342, rhodamine
imatinib, methotrexate

6-MP 6-mercaptopurine, 6-TG 6-thioguanine, PMEA 9-[2-(phosphonomethoxy)ethyl]adenine, cAMP cyclic adenosine monophosphate, cGMP
cyclic guanine monophosphate, CPT-11 irinotecan
J Mammary Gland Biol Neoplasia (2009) 14:3–9 5

An early method for stem cell isolation used the drug efflux resistance of breast cancer cells. For example, there is no
property of stem cells. Most cells accumulate fluorescent dyes evidence for involvement of ABC transporters in resistance
such as Hoechst 33342 and rhodamine, but a subset of ‘dull to aromatase inhibiters or trastuzumab [44, 45].
cells’ is often found and termed the ‘side population’ (SP Targeting breast cancer stem cells would be obviously be
phenotype). A large fraction of hematopoietic stem and tissue desirable, and it has been shown that HER2 expression
stem cells are in the SP fraction, and most of the cells in the SP drives the pool of breast tumor initiating cells [46].
fraction are stem cells [29–37]. Abcg2-null mice lack a Therefore agents that target HER2, such as trastuzumab may
detectable SP population not because these cells are absent, function in part by suppressing and eliminating these cells.
but because the lack of Abcg2 expression allows these cells However the major targeted breast cancer therapies are inhibitors
to accumulate dye [38]. This has provided an assay to of estrogen production, receptor concentration, or action. At least
quantitate and isolate ABCG2 overexpressing cells, which is for some breast tumors, and in the normal mammary gland, the
crucial as antibodies useful in cell sorting are not widely stem cells appear to be estrogen unresponsive, and acquire this
available for ABCG2 (Fig. 1). trait upon differentiation [47, 48].
Therefore, like in CML, targeted breast cancer therapies
are primarily affecting the non-stem cells in the tumor.
ABC Transporters in the Breast While this can lead to remission, and if the stem cells
require contact or the production of compounds from non-
Given the role of many ABC transporters in the excretion stem cells, this approach can lead to cures. However, as is
of diverse compounds, it is not surprising that a number of all too apparent, remission occurs in many patients,
these proteins are expressed in the breast, particularly particularly those that present with advanced, metastatic
during lactation. The ABCG2 promoter contains a sterol disease. It follows logically from the cancer stem cell
regulatory element [39]. Furthermore, ABCG2 is highly hypothesis that metastatic lesions must be initiated by stem
expressed in the mammary gland during lactation and has cells. Therefore agents that target stem cells are vital to a
been documented to play a role in the secretion of complete solution for breast cancer.
riboflavin [40]. Genetic analysis identified a locus affecting
milk yield in cattle and implicated a missense allele in
ABCG2, although the mechanism is not understood [41, ABC Transporters in Cancer Stem Cells
42]. Several other ABC transporters involved in lipid and
cholesterol transport are also differentially expressed during The identification of cancer stem cells, or tumor initiating
lactation [43]. However, apart from resistance to some first- cells, has focused renewed attention on the role of ABC
line chemotherapy agents, such as paclitaxel, ABC trans- transporters in drug resistance, and as cancer therapy
porters have not been shown to play a major role in the targets.

Figure 1 Diagram of an ABC transporter. a A prototypical ABC molecules and substrate molecules in the inner membrane leaflet (light
transporter is shown with two sets of transmembrane domains (TM, blue). b Upon binding of ATP, the NBFs are joined, leading to a
blue) and two nucleotide binding domains (NBF, red), free ATP conformational change, and the movement of the substrate molecule.
6 J Mammary Gland Biol Neoplasia (2009) 14:3–9

Current views favor the model that cancer stem cells are associated with t(9;22)(q34;q11). Imatinib is both an
innately resistant to chemotherapy through their relative ABCG2 substrate and inhibitor, making it susceptible to
quiescence, their enhanced capacity for DNA repair, efflux by stem cells that expresses this transporter [49–
decreased entry into apoptosis, and ABC transporter 51]. However the leukemia stem cells are not dependent on
expression. This provides a mechanism in which at least ABL for growth. Therefore, while Gleevec successfully
some tumor stem cells survive therapy and can give rise to inhibits the non-stem cells, and induces remission, the
relapse, even many years following therapy. There of patient must remain on the drug. The development of
course can still be a process of clonal evolution in which resistant leukemia cells caused by ‘acquired’ mutations in
gene translocations, mutations, or amplification, in cancer ABL’s kinase domain can lead to disease recurrence.
stem cells, give rise to tumors in which all the cells display Therefore other targeted agents are required to eliminate
MDR [18]. It is also apparent that expression of critical the stem cells. One possible approach is the use of
ABC transporters remains on in certain adult tissues such as Hedgehog (HH) or Smoothened (SMO) inhibitors, as
in the kidney. In renal cell cancer, ABCB1 is expressed in CML stem cells are dependent on the Hedgehog signaling
all cells and these tumors rarely respond to primary pathway [52].
chemotherapy treatment. Therefore a cancer stem cell
model of drug resistance has been popularized. A parallel SP Cells in Tumor Stem Cell Isolation
with normal stem cells is well recognized in the recovery of
many normal tissues (bone marrow, hair, gut lining) The identification of ABC gene expression in cancer stem
following chemotherapy (Fig. 2). cells has led to attempts to use this property to isolate or
However it is clear that the situation in human tumors characterize the cells. Some studies have used cell sorting
is complex. One of the most successful targeted therapies to attempt to isolate tumor stem cells from tumors and cell
are tyrosine kinase inhibitors (TKIs) such as imatinib lines. SP cells were identified in over half of neuroblastoma
(Gleevec) a drug inhibiting activated ABL in patients with samples and in neuroblastoma, breast cancer, lung cancer,
CML or with acute lymphoblastic leukemia (ALL) and glioblastoma cell lines [53]. While some early papers

Figure 2 Models of breast cancer evolution and drug resistance. a A may lead to complete cures (c), if therapy is initiated before clonal
primary lesion is shown composed of stem (red, self-renewing) and evolution of the cancer stem cells. Surviving stem cells can give rise
non-stem cells. b Either chemotherapy or anti-estrogen therapy to recurrent primary or metastatic lesions by regrowth of the stem cells
(tamoxiphen, aromatase inhibitors, fulvestrant) lead to toxicity and (d). The acquisition of therapy resistance in the stem cells can lead to
elimination of the non-stem cells, and tumor shrinkage. Combined resistant stem cells (purple). These resistant stem cells can give rise to
therapy including the use of EGF-receptor antibodies (tratuximab) drug resistant primary or metastatic tumors (e).
J Mammary Gland Biol Neoplasia (2009) 14:3–9 7

showed promising results, this isolation approach is limited Future Directions

as the SP compartment is composed of stem and non-stem
cells, and not all stem cells are in the SP fraction [26]. As with all exciting and controversial areas of study, many
However a much larger limitation has been the recognition more questions than answers remain.
that most tumor cell lines appear not to have a stable
population of cells with self-renewal capacity that can be & Pure normal stem cells and cancer stem cells need to be
isolated for study. Growth on plastic away from stromal isolated from many tissues to determine the ABC gene
cells, and the use of calf serum appears to select for a cell expression profile
population that does not reflect the state of the tumor in & A panel of ABC protein inhibitors needs to be
vivo. developed. Pan-ABC inhibitors will be difficult as the
three major transporters, ABCB1, ABCC1, and ABCG2
come from three evolutionarily and presumably struc-
Overcoming Drug Resistance: ABC Transporters turally distinct subfamilies. Several essential ABC
and the Stem Cell Paradigm transporters exist in the mitochondria and other tissues,
which would likely be deleterious to inhibit. However
Because cancer stem cells express drug transporters the identification of TKIs that also inhibit ABCs
rendering them resistant to many chemotherapy agents, directly or indirectly suggests alternate approaches.
strategies to target these cells are required. While trials that & Whether traditional small molecule inhibitors will be
have attempted to reverse MDR through therapies in which sufficiently selective and effective for ABC transporters
a cytotoxic drug is given along with an ABC transporter or whether biological approaches such as antibody or
inhibitor have not succeeded, it has been shown that in vivo peptide inhibitors are best. There are currently no high
tumors do express functional ABCB1. ABC transport throughput screens for assaying inhibitors of the full set
inhibitors could be re-invented as ‘cancer stem cell of ABC transporters, although siRNA screens that have
sensitizers’ targeting the most critical cells in the tumor. worked for tyrosine kinases could be applied to ABCs.
ABC inhibitors might not reduce tumor burden immediate- & Methods to quantitate cancer stem cells in tumors need
ly, and efficacy would need to be observed in alternative to be established so that rapid endpoints can be studied
endpoints, such as the frequency of relapse or the time to in early stage clinical trials, or in preclinical models.
relapse. However, it will be important to develop a panel of & It is not established that all tumors have discrete stem or
ABC transporters, and the most important transporter for initiating populations. For example liver regeneration
stem cells may be ABCG2. While fumitremorgin C (FTC) suggests that many or all cells in the liver can become
and Ko143 are specific ABCG2 inhibitors [54], FTC is multipotent. These tissues may require alternative
toxic to cells and mice. A screen for ABCG2 inhibitors has approaches.
yielded a set of new candidates, some of which are & Killing profiles of cancer stem cells need to be
amenable to structure modification to identify a potent established to determine if doses and drug combinations
antagonist. Peptides mimicking TM domains of ABC can be established to eliminate them, without eliminat-
transporters have proved to be selective and specific ing hematopoietic stem cells.
inhibitors, and could in principle be designed for any & For many drug resistant cancers, including kidney,
transporter [55]. pancreatic, and colon cancers, few cells are eliminated
Interestingly the compound GF120918 is an ABCB1 by even the initial therapy. Does the targeting of drug
inhibitor that also has activity against ABCG2 [56], and transporters in stem cells have a role here?
perhaps other pan-ABC inhibitors could be developed. & For the breast a focus on metastasis is in order. Does
Of course the complete elimination of cancer stem cells longer term administration of lower doses of an ABC
by this approach may be difficult to accomplish in vivo reversal agent and a cytotoxic drug suppress metastasis
without toxicity and the elimination of normal stem cells. in the adjuvant setting?
Stem cell-driven tissue regrowth is also required for
normal tissue recovery including the bone marrow, The intersection of developmental biology and cancer
gastrointestinal tract, and hair follicles. The potential biology continues to prove to be very fruitful. Many of the
for quiescence of cancer stem cells is also a potential genes that are critical to stem cell growth and polarity, such
concern, and these cells may be resistant to drug even in as Patched, Notch, and WNT are oncogenes or tumor
the absence of transporter expression or activity. There- suppressors, and have become highly studied new targets
fore a ‘therapeutic window’ will have to be established for cancer stem cell therapies [57, 58]. Stem cells have
that destroys the cancer stem cells but not normal stem evolved to have multiple non-redundant systems to protect
cells. them from toxins in the environment. The detailed
8 J Mammary Gland Biol Neoplasia (2009) 14:3–9

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