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
damage or death due to toxins is a critical function of an
organism, as the stem cells need to remain intact for the
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
been known for over 25 years to also play a role in
multidrug resistance of tumor cells. An exciting outcome of
the concept of the cancer stem cell is that the tumor
initiating cell may be innately resistant to many standard
therapies. This provides one mechanism in which cancer
stem cells could survive cytotoxic or targeted therapies and
lead to tumor regrowth or relapse. Gaining a better insight
into the mechanisms of stem cell resistance to chemother-
apy might therefore lead to new therapeutic targets and
better anti-cancer strategies.
Keywords ATP-binding cassette transporters .
Cancer stem cell . Multidrug resistance . Estrogen .
Aromatase . EGF receptor . Transport inhibitors
Abbreviations
ABC ATP-binding cassette
EGF epidermal growth factor
TM transmembrane
NBFs nucleotide binding folds
MDR multidrug resistant
SP side population
M. Dean (*)
Laboratory of Experimental Immunology,
Cancer and Inflammation Program, National Cancer Institute,
Frederick, MD 21702, USA
e-mail: dean@ncifcrf.gov
CML chronic myelogenous leukemia
ALL acute lymphoblastic leukemia
TKI tyrosine kinase inhibitor
ATP-Binding Cassette Transporter Function
ABC transporters bind to and/or hydrolyze a pair of ATP
molecules and use the binding and/or hydrolysis energy to
either efflux specific compounds across the membrane or to
flip them from the inner to the outer leaf of the membranes
[1–3]. The classification of ABC transporters is based on
the sequence analysis of the amino acids in the ATP-
binding (ATP) domain(s), also known as nucleotide binding
folds (NBFs) [3, 4]. The NBFs contain domains originally
recognized in other ATP-binding proteins, the Walker A
and B motifs. ABC proteins contain an additional element,
the signature (C) motif, found just upstream of the Walker
B domain [5]. The complete transporter protein contains
two transmembrane (TM) domains with of 6–11 membrane
spanning α-helices, and two NBFs. These four domains can
be present in one protein (a full transporter) or as a single
TM and single NBF domain in the case of a half-
transporter. Half transporters need to form either homo- or
heterodimers to compose a functional molecule.
Structural analysis of either isolated NBFs or complete
bacterial ABC transporters have been accomplished [6–9].
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
of the ATP molecules is envisioned to cause a structural
conformation change that can be reversed by ATP hydro-
lysis and release; although not all transporters hydrolyze
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
of all non-plant eukaryotic organisms sequenced to date
(plants have greatly expanded the number of transporters,
presumably to deal with a wider array of toxins, and the
lack of filtering organs, such as the liver) [3, 10, 11].
Phylogenetic analysis groups these ABC proteins into
8 different subfamilies: ABCA-ABCH [12]. There are 48
ABC genes in the human genome, and they carry out a
wide array of normal physiological functions in many
distinct tissues [4]. Most of what we know about the
function of ABC genes comes from genetic disorders, and
18 ABC genes are associated with Mendelian inherited
disease in human or other mammals [12]. These disorders
range from cystic fibrosis (caused by mutation in the
ABCC7/CFTR gene, a chloride ion channel) to adrenoleu-
kodystrophy (caused by mutations in ABCD1, a peroxi-
somal protein of uncertain function).
Drug Resistance in Cancer Cells
Cancer cells can become resistant to cytotoxic drugs through a
number of mechanisms, such as mutation or overexpression of
the drug’s target, drug inactivation, or efflux of the drug from
the cell [13]. Many tumors in patients suffering from relapse
to therapy are resistant to multiple drugs (multidrug resistant,
MDR) [14]. MDR tumors are a major barrier to effective
cancer therapy and along with metastasis are estimated to be
major contributors to death by cancer. Clearly understanding
the molecular basis of MDR and developing clinical reagents
or strategies to prevent the occurrence of resistance, or treat
resistant tumors is a high priority.
The P-glycoprotein encoded by the ABCB1 gene was the
first ABC transporter identified to be amplified and/or over-
expressed in MDR tumor cell lines [15–17]. This is also the
best characterized ABC transporter both biochemically and
through mutational analysis. In addition, there is a wide
variety of clinical agents that inhibit ABCB1 that have been
attempted in clinical studies against cancer going back to 1994
[18]. None of these clinical trials were very positive and this
strategy is in currently in disfavor. However there are two
other ABC proteins widely overexpressed in tumor cells,
ABCC1/MRP1 [19] and ABCG2 [20–23]. And at least
seven other transporters have been shown to cause
resistance to at least one agent (Table 1) [18]. Therefore,
one roadblock to inhibiting only a single ABC transporter,
is the knowledge that there are multiple potential efflux
pumps that the cell can employ. The ABC transporters
also have important roles in drug distribution as they play
key roles in compound access across the blood-brain
barrier, intestine, and other tissues compartments [24].
ABC Transporters in Normal Stem Cells
An exciting development of stem cell biology was the
discovery that many stem cells express high levels of
specific ABC transporters. Hematopoietic stem cells ex-
press high levels of ABCG2 and/or ABCB1 and that
expression is often turned off during differentiation to
progenitor and mature blood cells [14, 20, 25]. Gene
knockouts for either Abcg2, Abcb1 or Abcc1 or combina-
tions of these genes yield mice that are viable, fertile, and
have normal stem cell compartments [26–28]. Therefore
none of these genes are required for stem cell growth or
maintenance. However, these mice are more sensitive to
certain compounds such as vinblastine, ivermectin, top-
otecan, and mitoxantrone, demonstrating a role for these
transporters in protecting the organism, and the stem cells,
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
An early method for stem cell isolation used the drug efflux
property of stem cells. Most cells accumulate fluorescent dyes
such as Hoechst 33342 and rhodamine, but a subset of ‘dull
cells’ is often found and termed the ‘side population’ (SP
phenotype). A large fraction of hematopoietic stem and tissue
stem cells are in the SP fraction, and most of the cells in the SP
fraction are stem cells [29–37]. Abcg2-null mice lack a
detectable SP population not because these cells are absent,
but because the lack of Abcg2 expression allows these cells
to accumulate dye [38]. This has provided an assay to
quantitate and isolate ABCG2 overexpressing cells, which is
crucial as antibodies useful in cell sorting are not widely
available for ABCG2 (Fig. 1).
ABC Transporters in the Breast
Given the role of many ABC transporters in the excretion
of diverse compounds, it is not surprising that a number of
these proteins are expressed in the breast, particularly
during lactation. The ABCG2 promoter contains a sterol
regulatory element [39]. Furthermore, ABCG2 is highly
expressed in the mammary gland during lactation and has
been documented to play a role in the secretion of
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,
42]. Several other ABC transporters involved in lipid and
cholesterol transport are also differentially expressed during
lactation [43]. However, apart from resistance to some first-
line chemotherapy agents, such as paclitaxel, ABC trans-
porters have not been shown to play a major role in the
Figure 1 Diagram of an ABC transporter. a A prototypical ABC
transporter is shown with two sets of transmembrane domains (TM,
blue) and two nucleotide binding domains (NBF, red), free ATP
5
resistance of breast cancer cells. For example, there is no
evidence for involvement of ABC transporters in resistance
to aromatase inhibiters or trastuzumab [44, 45].
Targeting breast cancer stem cells would be obviously be
desirable, and it has been shown that HER2 expression
drives the pool of breast tumor initiating cells [46].
Therefore agents that target HER2, such as trastuzumab may
function in part by suppressing and eliminating these cells.
However the major targeted breast cancer therapies are inhibitors
of estrogen production, receptor concentration, or action. At least
for some breast tumors, and in the normal mammary gland, the
stem cells appear to be estrogen unresponsive, and acquire this
trait upon differentiation [47, 48].
Therefore, like in CML, targeted breast cancer therapies
are primarily affecting the non-stem cells in the tumor.
While this can lead to remission, and if the stem cells
require contact or the production of compounds from non-
stem cells, this approach can lead to cures. However, as is
all too apparent, remission occurs in many patients,
particularly those that present with advanced, metastatic
disease. It follows logically from the cancer stem cell
hypothesis that metastatic lesions must be initiated by stem
cells. Therefore agents that target stem cells are vital to a
complete solution for breast cancer.
ABC Transporters in Cancer Stem Cells
The identification of cancer stem cells, or tumor initiating
cells, has focused renewed attention on the role of ABC
transporters in drug resistance, and as cancer therapy
targets.
molecules and substrate molecules in the inner membrane leaflet (light
blue). b Upon binding of ATP, the NBFs are joined, leading to a
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
innately resistant to chemotherapy through their relative
quiescence, their enhanced capacity for DNA repair,
decreased entry into apoptosis, and ABC transporter
expression. This provides a mechanism in which at least
some tumor stem cells survive therapy and can give rise to
relapse, even many years following therapy. There of
course can still be a process of clonal evolution in which
gene translocations, mutations, or amplification, in cancer
stem cells, give rise to tumors in which all the cells display
MDR [18]. It is also apparent that expression of critical
ABC transporters remains on in certain adult tissues such as
in the kidney. In renal cell cancer, ABCB1 is expressed in
all cells and these tumors rarely respond to primary
chemotherapy treatment. Therefore a cancer stem cell
model of drug resistance has been popularized. A parallel
with normal stem cells is well recognized in the recovery of
many normal tissues (bone marrow, hair, gut lining)
following chemotherapy (Fig. 2).
However it is clear that the situation in human tumors
is complex. One of the most successful targeted therapies
are tyrosine kinase inhibitors (TKIs) such as imatinib
(Gleevec) a drug inhibiting activated ABL in patients with
CML or with acute lymphoblastic leukemia (ALL)
Figure 2 Models of breast cancer evolution and drug resistance. a A
primary lesion is shown composed of stem (red, self-renewing) and
non-stem cells. b Either chemotherapy or anti-estrogen therapy
(tamoxiphen, aromatase inhibitors, fulvestrant) lead to toxicity and
elimination of the non-stem cells, and tumor shrinkage. Combined
therapy including the use of EGF-receptor antibodies (tratuximab)
associated with t(9;22)(q34;q11). Imatinib is both an
ABCG2 substrate and inhibitor, making it susceptible to
efflux by stem cells that expresses this transporter [49–
51]. However the leukemia stem cells are not dependent on
ABL for growth. Therefore, while Gleevec successfully
inhibits the non-stem cells, and induces remission, the
patient must remain on the drug. The development of
resistant leukemia cells caused by ‘acquired’ mutations in
ABL’s kinase domain can lead to disease recurrence.
Therefore other targeted agents are required to eliminate
the stem cells. One possible approach is the use of
Hedgehog (HH) or Smoothened (SMO) inhibitors, as
CML stem cells are dependent on the Hedgehog signaling
pathway [52].
SP Cells in Tumor Stem Cell Isolation
The identification of ABC gene expression in cancer stem
cells has led to attempts to use this property to isolate or
characterize the cells. Some studies have used cell sorting
to attempt to isolate tumor stem cells from tumors and cell
lines. SP cells were identified in over half of neuroblastoma
samples and in neuroblastoma, breast cancer, lung cancer,
and glioblastoma cell lines [53]. While some early papers
may lead to complete cures (c), if therapy is initiated before clonal
evolution of the cancer stem cells. Surviving stem cells can give rise
to recurrent primary or metastatic lesions by regrowth of the stem cells
(d). The acquisition of therapy resistance in the stem cells can lead to
resistant stem cells (purple). These resistant stem cells can give rise to
drug resistant primary or metastatic tumors (e).
J Mammary Gland Biol Neoplasia (2009) 14:3–9
showed promising results, this isolation approach is limited
as the SP compartment is composed of stem and non-stem
cells, and not all stem cells are in the SP fraction [26].
However a much larger limitation has been the recognition
that most tumor cell lines appear not to have a stable
population of cells with self-renewal capacity that can be
isolated for study. Growth on plastic away from stromal
cells, and the use of calf serum appears to select for a cell
population that does not reflect the state of the tumor in
vivo.
Overcoming Drug Resistance: ABC Transporters
and the Stem Cell Paradigm
Because cancer stem cells express drug transporters
rendering them resistant to many chemotherapy agents,
strategies to target these cells are required. While trials that
have attempted to reverse MDR through therapies in which
a cytotoxic drug is given along with an ABC transporter
inhibitor have not succeeded, it has been shown that in vivo
tumors do express functional ABCB1. ABC transport
inhibitors could be re-invented as ‘cancer stem cell
sensitizers’ targeting the most critical cells in the tumor.
ABC inhibitors might not reduce tumor burden immediate-
ly, and efficacy would need to be observed in alternative
endpoints, such as the frequency of relapse or the time to
relapse. However, it will be important to develop a panel of
ABC transporters, and the most important transporter for
stem cells may be ABCG2. While fumitremorgin C (FTC)
and Ko143 are specific ABCG2 inhibitors [54], FTC is
toxic to cells and mice. A screen for ABCG2 inhibitors has
yielded a set of new candidates, some of which are
amenable to structure modification to identify a potent
antagonist. Peptides mimicking TM domains of ABC
transporters have proved to be selective and specific
inhibitors, and could in principle be designed for any
transporter [55].
Interestingly the compound GF120918 is an ABCB1
inhibitor that also has activity against ABCG2 [56], and
perhaps other pan-ABC inhibitors could be developed.
Of course the complete elimination of cancer stem cells
by this approach may be difficult to accomplish in vivo
without toxicity and the elimination of normal stem cells.
Stem cell-driven tissue regrowth is also required for
normal tissue recovery including the bone marrow,
gastrointestinal tract, and hair follicles. The potential
for quiescence of cancer stem cells is also a potential
concern, and these cells may be resistant to drug even in
the absence of transporter expression or activity. There-
fore a ‘therapeutic window’ will have to be established
that destroys the cancer stem cells but not normal stem
cells.
7
Future Directions
As with all exciting and controversial areas of study, many
more questions than answers remain.
& Pure normal stem cells and cancer stem cells need to be
isolated from many tissues to determine the ABC gene
expression profile
& A panel of ABC protein inhibitors needs to be
developed. Pan-ABC inhibitors will be difficult as the
three major transporters, ABCB1, ABCC1, and ABCG2
come from three evolutionarily and presumably struc-
turally distinct subfamilies. Several essential ABC
transporters exist in the mitochondria and other tissues,
which would likely be deleterious to inhibit. However
the identification of TKIs that also inhibit ABCs
directly or indirectly suggests alternate approaches.
& Whether traditional small molecule inhibitors will be
sufficiently selective and effective for ABC transporters
or whether biological approaches such as antibody or
peptide inhibitors are best. There are currently no high
throughput screens for assaying inhibitors of the full set
of ABC transporters, although siRNA screens that have
worked for tyrosine kinases could be applied to ABCs.
& Methods to quantitate cancer stem cells in tumors need
to be established so that rapid endpoints can be studied
in early stage clinical trials, or in preclinical models.
& It is not established that all tumors have discrete stem or
initiating populations. For example liver regeneration
suggests that many or all cells in the liver can become
multipotent. These tissues may require alternative
approaches.
& Killing profiles of cancer stem cells need to be
established to determine if doses and drug combinations
can be established to eliminate them, without eliminat-
ing hematopoietic stem cells.
& For many drug resistant cancers, including kidney,
pancreatic, and colon cancers, few cells are eliminated
by even the initial therapy. Does the targeting of drug
transporters in stem cells have a role here?
& For the breast a focus on metastasis is in order. Does
longer term administration of lower doses of an ABC
reversal agent and a cytotoxic drug suppress metastasis
in the adjuvant setting?
The intersection of developmental biology and cancer
biology continues to prove to be very fruitful. Many of the
genes that are critical to stem cell growth and polarity, such
as Patched, Notch, and WNT are oncogenes or tumor
suppressors, and have become highly studied new targets
for cancer stem cell therapies [57, 58]. Stem cells have
evolved to have multiple non-redundant systems to protect
them from toxins in the environment. The detailed
8 J Mammary Gland Biol Neoplasia (2009) 14:3–9
understanding of this process will lead to new insights into the
cancer stem cells. It is interesting that many normal stem cells
have migratory and invasive properties. Therefore metastasis
can be viewed as a ‘normal’ function of an abnormal cell, and
further insight into the control of migration and invasion in the
normal developing embryo may illuminate pathways that lead
to more effective cancer therapies [59].
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