multidrug efflux pumps and cancer stem cells: insights into multidrug resistance and therapeutic...

CliniCal pharmaCology & TherapeuTiCs 1

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The unexpected discovery of cancer stem cells (CSCs) in solid tumors has radically changed our outlook on carcinogenesis and chemotherapy. The existence of a self-renewing stem cell population in a tumor would imply that the stem cell repre-sents the cell of origin for a tumor. These CSC populations are intrinsically resistant to chemotherapy, which kills most other cells in a tumor but leaves behind the CSCs. Surviving CSCs can subsequently propagate and give rise to tumors.

However, in order to understand the basic biology of CSCs it is necessary to first elaborate on the properties of normal stem cells.

Normal stem cellsThe Russian biologist Alexander Maximow first introduced the term “stem cell” in 1909 with relation to hematopoietic stem cells (HSCs).1 Stem cells have intrinsic properties that set them apart from normal, or “generic,” cells. The first property is that of “self-renewal”—the potential to propagate and divide (with-out differentiating into different cell types). This property allows stem cells to produce at least one daughter cell with a similar developmental potential (i.e., the potential to divide indefinitely without differentiation). The second property is that of differen-tiation—the potential to divide into cells that will form specific cell types.2 Therefore, when a stem cell divides, each new cell

retains the ability to either remain a stem cell or become a cell that is committed to differentiate into another cell type with a specialized function. This hierarchy is illustrated in Figure 1a, which shows that a stem cell can self-renew to give rise to a new stem cell but can also divide to form a progenitor cell that may subsequently give rise to differentiated cells (progeny cells) after rapid amplification. Normal tissues would thus have a pool of stem cells that would function in the maintenance of a particular tissue for the lifetime of an organism. Without this pool, the organ would eventually degenerate. However, expansion of this stem cell pool would have to be restricted in normal tissues so as to prevent “uncontrolled” growth.3

cscs and the csc modelThe CSC model does not dwell on the cellular origin of cancer; rather, it elaborates on the process by which cancers that have been already established are able to propagate.4 According to the CSC theory, some elements of cellular hierarchy that are seen in normal tissues are preserved in many tumors. The theory proposes that a highly defined population of cancer cells known as cancer stem cells have the exclusive property of driving the growth and spread of a tumor. CSCs can give rise to progeny that have limited ability to divide, thereby drawing a conceptual parallel to the development of normal stem cells2 (Figure 1b).

1Laboratory of Experimental Immunology, Human Genetics Section, Cancer and Inflammation Program, National Cancer Institute at Frederick, Frederick, Maryland, USA;2Human Genetics Section, Basic Science Program, SAIC-Frederick, Frederick, Maryland, USA. Correspondence: M Dean ([email protected])

Received 22 December 2010; accepted 7 January 2011;advance online publication 2 March 2011. doi:10.1038/clpt.2011.14

Multidrug Efflux Pumps and Cancer Stem Cells: Insights Into Multidrug Resistance and Therapeutic DevelopmentK Moitra1, H Lou2 and M Dean1

stem cells possess the dual properties of self-renewal and pluripotency. self-renewal affords these populations the luxury of self-propagation, whereas pluripotency allows them to produce the multitude of cell types found in the body. protection of the stem cell population from damage or death is critical because these cells need to remain intact throughout the life of an organism. The principal mechanism of protection is through expression of multifunctional efflux transporters—the adenosine triphosphate–binding cassette (aBC) transporters that are the “guardians” of the stem cell population. ironically, it has been shown that these aBC efflux pumps also afford protection to cancer stem cells (CsCs), shielding them from the adverse effects of chemotherapeutic insult. it is therefore imperative to gain a better understanding of the mechanisms involved in the resistance of stem cells to chemotherapy, which could lead to the discovery of new therapeutic targets and improvement of current anticancer strategies.

2 www.nature.com/cpt

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However, in contrast to normal stem cells, in which stem cell proliferation is tightly regulated and genomic integrity is main-tained, tumorigenic cells frequently lack some of these control mechanisms.

If the hypothesis that tumors contain stem cells is assumed to be correct, can we interpret the accumulation of mutations in these stem cells as the basic “multistep” process of carcinogen-esis? Among a myriad of other enigmatic questions that remain to be answered, there are some major conceptual issues that researchers need to solve. For instance, do CSCs arise from nor-mal stem cells or from differentiated cells that acquire the capac-ity for self-renewal? It is not necessary that these two concepts be mutually exclusive; it is equally possible that CSCs arise from both these sources. Regarding the uncanny ability of CSCs to resist chemo- and radiotherapy, the following question might be asked: does the innate resistance of normal stem cells to radia-tion and toxins contribute to the failure of some cancer thera-pies? Finally, if we are to make any kind of progress concerning the provocative issue of CSCs, we need to ask ourselves: how can we exploit our knowledge of stem cell biology to specifically target CSCs and improve therapeutic outcomes in the treatment of cancerous tumors?

abc traNsporters aNd stem cellsAdenosine triphosphate–binding cassette (ABC) transport-ers can power the translocation of substrates across biological membranes against a concentration gradient through hydroly-sis of ATP. These transporters are intricate molecular pumps, most of which catalyze the vectorial transport of a wide array of substrates across biological membranes. The human genome

encodes 48 ABC transporter genes, which are categorized into seven subfamilies ranging from A to G (Table 1).5 Proteins are classified as ABC transporters on the basis of the sequence and organization of their ATP-binding domain(s). ATP-binding domains (also known as nucleotide-binding folds) contain specific motifs—the Walker A and Walker B found in all ATP-binding proteins. However, ABC genes contain an additional element, the signature motif (C-loop), located upstream of the Walker B. The functional transporter usually contains two transmembrane domains and two nucleotide-binding folds. Transmembrane domains contain 6–12 membrane-spanning α-helixes (Figure 2a), which are typically responsible for deter-mining substrate specificity. The two nucleotide-binding folds bind and hydrolyze ATP, thereby providing the power for the pump (Figure 2b). Specific ABC transporters exhibit high expression levels in both normal stem cells and CSCs. ABC transporters are expressed in stem/progenitor cells derived from several types of normal tissue and also in hematopoietic cells.

HSCs were found to express high levels of certain specific ABC transporters such as ABCG2 and/or ABCB1.6 Mouse knockouts for ABCG2, ABCB1, or ABCC1 revealed that the animals were especially sensitive to some compounds such as mitoxantrone, vinblastine, ivermectin, and topotecan, thereby suggesting that these transporters may have a role in protecting the stem cells from certain toxic substances.7 In a more recent study, ABC transporters were found to be expressed in normal stem cells such as HSCs, unrestricted somatic stem cells, mesenchymal stem cells, and multipotent adult progenitor cells, using the aid of Taqman low-density arrays.8 The gene signatures for ABC trans-porters were found to be radically different between HSCs and

Stem cell Stem cell

Self-renewal

Progenitor cell

Cell division and differentiation

Progeny cells

Normal stem cell

Becomesmalignant

Committed cell

Acquires self-renewalcapacity

Self-renewal

Cancer stem cell Cancer stem cell

Progenitor cell

Cell division and differentiation

Malignant progeny cells

a b

Figure 1 Asymmetric division in stem cells. (a) Asymmetric division in a normal stem cell. A stem cell can self-renew to give rise to another stem cell (green) but can also divide to form a progenitor cell (pink). (b) Asymmetric division in a cancer stem cell. A cancer stem cell (orange) can also asymmetrically divide to form another cancer stem cell (orange) or give rise to a progenitor cell (brown).


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table 1 Human abc genes, subfamilies, chromosomal locations, and functions

symbol subfamily alias location Function anticancer drug transport

ABCA1 A ABC1 09q31.1 Cholesterol efflux onto high-density lipoprotein

ABCA2 A ABC2 09q34.3 Drug transport Yes

ABCA3 A ABC3 16p13.3 Surfactant secretion? Drug resistance Yes

ABCA4 A ABCR 01p21.3 N-retinylidene-phosphatidylethanolamine efflux

ABCA5 A ABC13 17q24.3

ABCA6 A 17q24.3

ABCA7 A ABCX 19p13.3

ABCA8 A 17q24.3

ABCA9 A 17q24.3

ABCA10 A 17q24.3

ABCA12 A 02q34

ABCA13 A 07p12.3

ABCB1 B MDR1 07q21.12 Multidrug resistance Yes

ABCB2 B TAP1 06p21 Peptide transport

ABCB3 B TAP2 06p21 Peptide transport

ABCB4 B PGY3 07q21.12 Phosphotidyl choline and drug transport Yes

ABCB5 B ABC19 07p21.1 Drug transport Yes

ABCB6 B ABC14 02q35 Iron transport

ABCB7 B ABC7 Xq21-22 Fe/S cluster transport

ABCB8 B ABC22 07q36.1

ABCB9 B ABC23 12q24.31

ABCB10 B 01q42.13

ABCB11 B SPGP 02q24.3 Bile salt transport, drug transport Yes

ABCC1 C MRP1 16p13.12 Drug resistance Yes

ABCC2 C MRP2 10q24.2 Organic anion efflux, drug transport Yes

ABCC3 C MRP3 17q21.33 Drug resistance Yes

ABCC4 C MRP4 13q32.1 Nucleoside transport, drug transport Yes

ABCC5 C MRP5 03q27.1 Nucleoside transport, drug transport Yes

ABCC6 C MRP6 16p13.12 Yes

ABCC7 C CFTR 07q31.31 Chloride ion channel

ABCC8 C SUR 11p15.1 Sulfonylurea receptor

ABCC9 C SUR2 12p12.1 K(ATP) channel regulation

ABCC10 C MRP7 06p21.1 Drug transport Yes

ABCC11 C MRP8 16q12.1 Drug transport Yes

ABCC12 C MRP9 16q12.1

ABCD1 D ALD Xq28 VLCFA transport regulation

ABCD2 D ALDL1 12q11

ABCD3 D PXMP1 01p22.1

ABCD4 D PMP69 14q24.3

ABCE1 E OABP 04q31.31 Oligoadenylate binding protein

ABCF1 F ABC50 06p21.1

ABCF2 F ABC28 07q36.1

ABCF3 F ABC25 03q27.1

ABCG1 G White 21q22.3 Cholesterol transport?

ABCG2 G ABCP 04q22 Toxin efflux, drug resistance Yes

ABCG4 G White2 11q23

ABCG5 G White3 02p21 Sterol transport

ABCG8 G White4 02p21 Sterol transport

Adapted from ref. 5.


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other types of stem cells, thereby suggesting that HSCs rely on a different repertoire of these transporters as compared with other tissue/cell types. A total of 16 transporters including ABCB1 and ABCG1 were found to be consistently expressed at higher levels in HSCs as compared with other transporters. However, ABCG2 overexpression was not detected; this was in agreement with a finding that there was no association of ABCG2 with side population (SP) activity or hematopoietic progenitor function in human cord blood.9 The transporters ABCA4, ABCA8, ABCC9, and ABCG4 were consistently detected in mesenchymal stem cells and unrestricted somatic stem cells.8

cHaracteristic properties oF cscsCSCs are cancer cells that possess a stem cell–like phenotype and have the unique property of evading radiotherapy and chemo-therapy. In comparison with differentiated tumor cells, CSCs are (i) relatively quiescent, (ii) have a slow cycling rate, and (iii) retain the ability to form tumors if they are injected into non-obese diabetic/severe combined immunodeficiency mice. The relative quiescence and slow cycling rate affords these cells pro-tection against chemotherapeutics that are effective in targeting rapidly dividing cells rather than a slow-cycling subset.10 The ability to induce tumors in nonobese diabetic/severe combined immunodeficiency mice affords an effective animal model for researchers for further investigation of the properties of these rather elusive cells. Additionally, accumulating scientific evi-dence indicates that (iv) CSCs express ABC transporters that confer chemoresistance on this subset of cells.11

ideNtiFicatioN oF stem cells/cscsStem cells may be identified by characteristic cell surface mark-ers as well as by their property to exclude certain fluorescent dyes.

cell surface markersSeveral cell surface markers have been used to detect CSCs. These include cell surface proteins such as CD133 (Prominin 1, a transmembrane glycoprotein) and CD166, both of which were already known to define stem and progenitor cells. Other markers include ABC transporters such as ABCB1, ABCG2, and ABCB5. Specific antibodies against these proteins need to be generated to enable detection, and usually flow cytometry can be used to isolate CSCs. However, it must be kept in mind that tumor-initiating cells are, for the most part, heterogeneous, and a specific marker or set of markers has not been found to identify CSCs in solid tumors.12

sps in stem cellsThe SP is a subset of stem cells that have a high capability for effluxing antimitotic drugs. These cells can be isolated by utiliz-ing their capacity to efflux the fluorescent dye Hoechst 33342 or rhodamine 123. Because of this property, these cells can be detected by a flow cytometer, sorted, and collected for analy-sis. This population of cells is termed “side population” because during flow cytometry analysis these cells are visualized as a negatively stained population off to the side of the main popu-lation of cells13 (Figure 3). The drug transporting capability of these cells is possibly conferred by certain ABC transport-ers, including ABCB1 (rhodamine 123) and ABCG2 (Hoechst 33342). This method of isolating SPs has become an invaluable technique in stem cell biology, allowing researchers to isolate SP cells enriched in stem cells, identified by their property of negative staining.

drug resistaNce iN caNcerThe nature of clinical drug resistance is multifactorial, involving alteration in drug targets, inactivation/detoxification of the drug, decreased drug uptake, increased drug efflux, and the dysregula-tion of apoptotic pathways.14 Several ABC transporters are now known to be associated with drug resistance (Table 1). However, ABC transporters are not the sole cause of drug resistance in CSCs; several other factors, such as the capacity of a stem cell for DNA repair and its quiescent state, may also have an impact on drug resistance in a tumor. Usually, tumors that recur after

TMD1 TMD2

NBF1 NBF2

2 TMDs + 2 NBFs = full transporter

TMD1

NBF1 NBF2

TMD2

ATP ADP + Pi

a

b

Figure 2 Organization of ABC transporters. (a) Structural organization of an ABC (full) transporter. A full transporter contains two TMDs and two NBFs. (b) ABC transporters are driven by ATP. ABC transporters efflux substrates with the power provided by ATP hydrolysis. ABC, ATP-binding cassette; NBF, nucleotide-binding fold; TMD, transmembrane domain.

Hoe

chst

blu

e flu

ores

cenc

e

Hoechst red fluorescence

Side population

Figure 3 Pictoral depiction of side population cells in a fluorescence-activated cell sorter (FACS) scan. Side population cells (outlined) are located off to the side of the main population of cells in a FACS readout of cells stained with Hoechst dye (cartoon).


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an initial response to chemotherapeutic drugs become resist-ant to multiple drugs. Several models have been put forward to explain the origin of multidrug resistance in this particular class of tumors:

1. Conventional model of tumor drug resistance2. CSC model of drug resistance3. Acquired-resistance stem cell model4. Intrinsic resistance model

conventional model of tumor drug resistanceIn this model, the tumor consists of generic tumor cells and a population of tumor cells that have acquired drug resistance as a result of genetic changes. After exposure to chemotherapy, only the drug-resistant cells survive and proliferate, giving rise to a tumor made up of the clonal progeny of the drug-resistant cells (Figure 4a).

csc model of drug resistanceThis model postulates that the original tumor contains a small population of tumor stem cells and their differentiated progeny. After exposure to the drug, only the tumor stem cells (express-ing drug transporters) survive. These stem cells then divide and repopulate the tumor with stem cells and with differentiated cells that originate from the stem cells (Figure 4b).

acquired-resistance stem cell modelThis model is similar to the CSC model in that the original tumor contains a small population of tumor stem cells and their differentiated progeny. However, after exposure to the drug, the tumor stem cells survive, with some of the cells acquiring muta-tions that result in a drug-resistant phenotype (Figure 4c).

intrinsic resistance modelIn this model, both the tumor stem cells and the differentiated cells are intrinsically resistant to the drug, and therapies would have little effect on the tumor. This would result in continued tumor growth (Figure 4d).13

abc traNsporters aNd cscsCSCs retain the exclusive property of driving the growth and spread of a tumor. As mentioned earlier, the CSC model of drug resistance postulates that a tumor possesses a population of pluripotent drug-resistant cells that can survive chemotherapeu-tic insult and grow to form a drug-resistant tumor. It has been found that CSCs express several ABC transporters, including ABCB1, ABCG2, and ABCC1.

abcg2ABCG2 is a half-transporter that is thought to function as a homodimer. It has a molecular weight of 72 kDa and a broad substrate specificity. It is capable of transporting doxorubicin, mitoxantrone, topetecan, methotrexate, and tyrosine kinase inhibitors, among other substances. This transporter was first identified by three different labs independently and given three names: BCRP (breast cancer resistance protein), ABCP

(ABC transporter in placenta), and MXR (Mitoxantrone resist-ance–associated gene). However, the HUGO (Human Gene Nomenclature Committee) recommended that it be named ABCG2 because it is the second member of the ABC super-family, subgroup G.15

regulation of abcg2 expressionThe human ABCG2 gene maps to chromosome 4q22, spans over 66 kb, and consists of 16 exons and 15 introns. The cod-ing protein contains 655 amino acids (72 kDa). The first exon contains the majority of the 5′ untranslated region (UTR).16 To date, little is known about the regulation of ABCG2 expression. Like most TATA-less gene promoters, the ABCG2 promoter contains numerous SP1, AP1, and AP2 binding sites down-stream of a putative CpG island16 and a CCAAT box. Most

MDR cells

Therapy

Therapy

Therapy

Therapy

Tumor

Tumor

Tumor

Tumor stem cell

Tumor stem cell

Mutation

a

b

c

d

Figure 4 Models of tumor drug resistance. (a) Conventional model of tumor drug resistance. In this model, the tumor contains a population of tumor cells that have acquired resistance to the drug (orange). After exposure to chemotherapy, the drug-resistant cells survive and proliferate, giving rise to a tumor made up of the progeny of the drug-resistant cells. (b) Cancer stem cell model of drug resistance. The original tumor contains a small population of tumor stem cells (red) and their differentiated progeny (blue). After exposure to the drug, only the tumor stem cells survive. These stem cells divide and repopulate the tumor both with stem cells and with differentiated cells originating from the stem cells. (c) Acquired-resistance stem cell model. The original tumor contains a small population of tumor stem cells (red) and their differentiated progeny (blue). After exposure to the drug, the tumor stem cells survive, and some of them acquire mutations that confer a drug-resistant phenotype (orange). (d) Intrinsic resistance model. Tumor stem cells (red) and the differentiated cells (blue) are intrinsically resistant to the drug, and therapy will have little effect on the tumor, which will therefore continue to grow. Adapted from ref. 13.


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studies examining the regulation of ABCG2 have focused on the transcriptional level. Promoter activity characterized by a luciferase reporter assay showed a sequence 312 bp directly upstream of the ABCG2 transcriptional start site that conferred basal promoter activity. The 5′ region upstream of the basal pro-moter is characterized by both positive and negative regulatory domains.16 The 3′UTR of ABCG2 mRNA is ~2 kb in length, which is considerably longer than the average 770 bp observed for human mRNAs.17 This suggests that it may have one or more important roles in the regulation of gene expression.

Recently, an increasing number of studies have focused on unraveling the molecular regulation of ABCG2 because ABCG2 expression is highly sensitive to various developmental and envi-ronmental stimuli. The functional regulatory elements located in the ABCG2 promoter region, such as transcription factor–bind-ing sites, are important in the regulation of ABCG2 expression. Both an estrogen response element in the ABCG2 promoter and estrogen upregulation of the expression of ABCG2 mRNA in the estrogen receptor–positive T47D:A18 cells and PA-1 cells (which stably express estrogen receptor) have been identified.18 Promoter luciferase assays of the ABCG2 promoter showed that the region between −243 and −115 is essential for the estrogen response effect.18 In addition, three peroxisome proliferator–ac-tivated receptor-γ response elements were located in the ABCG2 promoter region.19 Upregulation of functional ABCG2 expres-sion can be achieved via a lipid-activated transcription factor, peroxisome proliferator–activated receptor-γ. Other steroid hor-mones, such as progesterone, human placental lactogen, and human prolactin, have been shown to have stimulatory effects on ABCG2 expression in human placental choriocarcinoma BeWo cells.20 ABCG2 expression is upregulated by hypoxia and injury via hypoxia-inducible transcription factor complex HIF-1α and HIF-2α signaling. The ABCG2 promoter is also directly activated by HIF-1, and the hypoxia response element at −116 bp is essen-tial for the transcriptional activation of the ABCG2 promoter.21 In addition, ABCG2 and Oct-4/POU5F1 are highly coexpressed in Lucena stem-like cells that display chemotherapy resistance selected from its parental cell line K562. The presence of Oct-4 binding sites in the ABCG2 promoter suggests that Oct-4 regu-lates ABCG2 mRNA expression.22 The ABCG2 and Oct-4 genes may also be involved in the multidrug-resistance phenotype in human chronic myeloid leukemia K562.22 In drug-resistant MCF7 cells, it has been observed that the 5′ promoter is put to alternative use because of the differential expression of splice variants at the 5′ UTR of ABCG2 mRNA.23 Furthermore, three microRNAs—miR-520h, miR-328, and miR-519c—have been identified that target the 3′UTR of ABCG2 and decrease ABCG2 expression.24–26

To extend these studies, we performed an in silico analysis to identify transcriptional regulation of the ABCG2 gene in humans and mice. The putative transcription factor binding sites, NFκB, HIF1A, ERE, EGR, SORY, CAAT, SP1, and STAT, as well as several Pu.1, HOXA9, C-myb, CREB, SP1, EGR, GATA, STAT, and HOXC sites were identified within the ABCG2 upstream regions −312/+362 in humans and −360/+328 in mice, respectively (Figure 5a,b). The alignment of predicted ABCG2

promoter regions in humans and mice demonstrates a low extent of similarity and conservation. This suggests that tran-scription of ABCG2 may be regulated by different mechanisms across mammalian species. These transcription factor binding sites are likely to function as key controlling elements in the regulation of the ABCG2 gene. These findings provide a platform for further defining the precise function of each transcription factor in ABCG2 regulation and revealing the molecular basis of ABCG2 expression.

abcb1ABCB1 is also known as P-glycoprotein (P-gp). It has been found to be expressed in >50% of all drug-resistant tumors. Human ABCB1 is the product of the MDR1 gene and acts as an ATP-dependent pump for a multitude of structurally unrelated hydrophobic compounds, including numerous anticancer and antimicrobial drugs.27 It is a large transmembrane glycopro-tein with a molecular weight of ~170 kDa and consists of 1,280 amino acids. It has two homologous halves (amino terminal and carboxy terminal) connected by a short, flexible linker region of 80 amino acids. Each half of the protein consists of a hydropho-bic region with six putative α-helixes and a nucleotide-binding domain.28 Isolation of SP cells from several cancer cell lines has shown that the expression of ABCB1 is upregulated in the SP as compared with the normal population. SP cells isolated from the pancreatic cancer cell line PANC1 have been found to express both ABCB1 and ABCG2.29 The isolated SP cells constituted ~2.1–8.7% of the total population of viable cells as identified by Hoechst 33342 staining. This population was found to have an enhanced capacity for the efflux of Hoechst dye, postulated as being caused by ABCB1 and ABCG2 expression. Targeting these SP cells may therefore provide an alternative approach to cancer therapy. However, a study carried out a few years ago provided evidence to show that, although ABCB1 was highly expressed in both normal and leukemic stem cells, the efflux of mitoxantrone from the leukemic stem cells could not be efficiently inhibited by the ABCB1 inhibitors verapamil and PSC833 (in 15 patients with leukemia). The researchers proposed the hypothesis that differences between the leukemic and normal stem cells may be attributable to the presence of additional transport mechanisms in the leukemic cells.30

transcriptional regulation of ABCB1The activation or repression of a gene in a particular cell type, under a given physiological condition, is determined by the complement of transcription factors as well as the response elements present in the promoter region of that gene. These protein–DNA interactions are also influenced by the dynamic chromatin ultrastructure. Coregulators—proteins that interact with transcription factors to mediate the transcriptional signal from the promoter-bound proteins to the basal transcriptional machinery—have also been identified. The majority of these interact with chromatin to activate or repress transcription.31 The MDR1 gene lacks a consensus TATA box in the proximal promoter region. In this case, basal transcription is directed by a sequence called the Inr, or the initiator sequence, which contains


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the transcription start site (+1). This initiator element was first identified from in vitro studies showing that sequences from −132 to +82 were needed for accurate initiation.32 This site was further narrowed down to −6 to +11 as determined by transient transfection experiments.33

The MDR1 promoter encompasses within it an inverted CCAAT box (−82 to −73) and also a GC-rich element that has been documented to interact with members of the Sp family of transcription factors containing a highly conserved DNA-binding region that consists of three zinc fingers in proximity to the C-terminus.34 A second GC-rich element is present at −110 to −103; however, this does not interact with Sp1.34 Immediately downstream, and overlapping the GC-rich region, there is an inverted MED-1 element (multiple start site element down-stream 1). Overexpression of MDR1 in drug-resistant cells may

result both from gene amplification and transcriptional over-expression. MEF1 (MDR1 promoter-enhancing factor 1) can interact with a promoter element within MDR1 to upregulate expression. In the drug-sensitive MCF7 cells, but not in the drug-resistant MCF7/ADR cells, this element binds to an inhibitory complex that contains NF-κB and c-Fos.35 Several investigators have studied the role of p53 in the regulation of the MDR1 gene. It was found that wild-type p53 can repress the transcription of both endogenous MDR1 gene and MDR1 reporter constructs36 through direct DNA binding at the HT (a novel p53 DNA bind-ing element) site. Other p53 family members such as p63 and p73 can activate MDR1 transcription through an indirect interaction with the APE site (the alternative p63/p73 element), thereby sug-gesting that p53 DNA-binding domains can differentially regu-late transcription, through both mechanisms that are dependent

−500

−443 −379

−847−821 −697

−680−649 −562

−543 −497−360 SP1

HOXA9C-mybCREB PBX1 Pu.1 Pu.1

−515−462 −374 −316

−292 −207 −127

−43 −13+141

+111 +253

+258 +328

+333Pu.1 Pu.1

−334

−885

−366

100 bp

100 bp

−283 −167 −30−12

−5 +88+140

+163

+191 +376 +465

+361 +413 +440−36

−54−176 −119

−283

−259

−189

−123

SORY TSS

SORYERE

TSS

ERESTAT

STATPu.1

AP1 EGR

SP1SP1

HOXC

STATSTAT

CREB

EGR GATASTAT HOXCPu.1 SP1SORY

Nanog CREB MYBNOLF

XBBF CAAT EGRHIF1A

EBOXSP1

NRSFMTEN NFKB

EGRMTEN CAAT

XBBF MTEN ATGSTATNFKB

SP1 miR−520h

miR−328

miR−519c

1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

5´-UTR

1

5´-UTR

68.64 kb Reverse strand

Forward strand

3´-UTR ABCG2

Chr 4 (Homo)

ABCG2

Chr 6 (Mus)

UTR Exon

95.78 kb

ATG

UTR Exon

a

b

Figure 5 Transcription factors binding to ABCG2. (a) Transcription factors binding to the human ABCG2 promoter region. Binding of transcription factors to the human ABCG2 5′UTR promoter region and miRNA binding to the 3′UTR. (b) Transcription factors binding to the mouse ABCG2 promoter region. The figure depicts the binding of transcription factors to the mouse ABCG2 promoter region (5′UTR). UTR, untranslated region.


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on DNA binding and mechanisms that are independent of such binding.37 Recently, it was discovered that C-terminal-binding protein 1 can act as an activator of MDR1 gene transcription and could potentially serve as a new target for inhibiting MDR1-mediated drug resistance.38 MDR1 gene expression may be activated by various means such as the use of UV irradiation, sodium butyrate, retinoic acid, histone deacetylase inhibitors, and certain chemotherapeutics. Signals from different stimuli may converge on a region of the MDR1 promoter that has been referred to as the MDR1 enhanceosome.31 The enhanceosome encompasses binding sites for a variety of transcription factors, including those of the NF-Y and Sp family. These DNA-binding proteins can cooperate and recruit P/CAF (a histone acetyl transferase) to the MDR1 promoter region. The outcome of P/CAF recruitment would be acetylation of promoter-proximal histones, followed by transcriptional activity. The regulation of transcriptional activity by the enhanceosome would make it an effective therapeutic target for MDR1-mediated drug resistance. Traditionally, functional modulators or reversal agents have been developed to combat MDR1-mediated drug resistance; however, if the complex mechanistic process of transcription regulation can be understood, there is potential to develop agents that pre-vent the transcriptional activity of drug transporters.

abcb5ABCB5 is evolutionarily closely related to ABCB1 and ABCB4 and has evolved as a full transporter for most of its evolu-tionary history (our unpublished data). The ABCB5β form

(half-transporter) has been found to be expressed in a number of cancer subtypes, including malignant melanoma and breast cancer.39 More importantly, it was shown that this transporter may be expressed in melanoma stem cells/stem-like cells that possess the CD133+ phenotype, and it was found to mediate doxorubicin resistance.39,40 There is evidence to suggest that ABCB5+ cells make up 2–20% of clinical melanoma tumors and that these ABCB5+ cell populations can recapitulate the tumor after being injected into immunodeficient mice. This subset of cells was also capable of reestablishing the tumor’s heterozygosity; however, these cells could not regenerate the ABCB5+ cell population, thereby raising a doubt about whether these cells actually possess a stem cell phenotype.41 Recently, it was demonstrated that knockdown of Tenascin-C (a secreted extracellular matrix protein) could reduce the SP cell ratio in melanoma spheres and reduce their resistance to doxorubicin, probably because of downregulation of ABC transporters such as ABCB5.42

transcriptional regulation of abcb5The ABCB5β gene, located on chromosome 7p21, spans 19 exons and 108.2 kb of genomic DNA. Using Genomatix soft-ware, we found a large number (232) of potential regulatory transcription factor binding sites in the promoter region of ABCB5β (some transcription factors could potentially bind to more than one site), including CREB, PAX6, CEBP, and OCT1 (Figure 6). However, to our knowledge, functional validation of these transcription factors has not yet been carried out.

3´-UTR5´-UTR

108.2 kb Forward strand

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

ABCB5 beta

100 bp

PTBPBNCFCIZFFASTHBOXKLFSMZF1PAX5SALLTEAF

YTBPBRNFCREBGATAHMTBLHXFNF1FPAXHSIX3YY1F

ABDBCAATDLXFGCMFHNF1MAZFNKX1PDX1SIXFZF03

AP1FCARTDMRTGREFHNF6MEF2NKX6PEROSNAPZF05

AP1RCDXFE2FFGRHLHOMFMITFNKXHPIT1SORYZICF

ARIDCEBPEBOXGZF1HOXCMTF1NR2FPLZFSP1FATBF

CHOPEREFHAMLHOXFMYBLOCT1PRDFSREBBCDFCHRE

ETSFHANDHZIPMYODPARFRORASRFFBCL6CHRFEVI1

HASFIRFFMYT1PAX3RXRFSTAT

VTBPBRN5CLOXFKHDHEATLEFFNBREPAX6SATBXBBF

UTR

Exon

Figure 6 Transcription factors binding to the human ABCB5β promoter region. Binding of transcription factors to the human ABCB5β promoter region (5′ untranslated region).


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The development of candidate drugs that could potentially inhibit ABC transporters at the transcriptional level would be an effective alternative strategy for avoiding drug resistance and could also be an excellent approach to targeting CSCs that overexpress these efflux pumps. Efforts to combat drug resist-ance caused by ABC efflux pumps have focused on the use of functional modulators or reversal agents rather than on thera-peutic targeting of transcription. With the aid of prophylactic intervention, it may be possible to prevent transcriptional activa-tion. Ecteinascidin 743 (ET-743) is one such agent that is being developed as a natural-product therapeutic that can target P-gp (ABCB1/MDR1) transcriptional activation. This compound is a (tris)-tetrahydroisoquinoline related to the saframycin family of compounds isolated from the sea squirt Ecteinascidia tur-binata, ET-743. It is capable of interfering with the activation of MDR1 transcription, and studies have indicated that it may affect the MDR1 enhanceosome complex; however, its precise mechanism of action has not yet been elucidated.31 Even so, advances in the understanding of the transcriptional regulation of ABC transporters may pave the way toward the development of novel therapeutic agents.

microrNa iN tHe regulatioN oF abc traNsporters aNd stem cellsMicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs ranging in length from 19 to 25 nucleotides. They have the capability to regulate gene expression, usually by binding to the 3′UTR. No studies have been published to date describing miRNA expression profiling in CSCs; however, an “indirect” or “potential” model has been proposed to link miRNAs with the regulation of CSCs.43 DeSano and Xu (2009) have proposed that the aberrant expression of microRNA in the form of either oncogenic miRNA (oncomiRs) or tumor suppressor miRNA may result in the dysregulation of certain stem cell genes. The result may be an increase in self-renewal of CSCs and impaired differentiation of a subset of CSCs. The researchers propose that this dysregulation would subsequently result in carcinogenesis and oncogenesis. Molecular miRNA therapy involving the phar-macological use of miRNA antagonists to target oncomiRs or miRNA mimics can restore the capabilities of tumor supres-sor genes. This approach holds promise for addressing the issue of carcinogenesis linked to the dysregulation of CSCs. Several miRNAs have been found that regulate ABCB1 and ABCG2 and could mediate their expression in CSCs. It was found that hsa-miR-451 may regulate ABCB1 in MCF7 breast cancer cells.44 It was also discovered that both miR-451 and miR-27a may regu-late ABCB1 expression in multidrug-resistant A2780DX5 and KB-V1 cancer cell lines.45 These results suggest that miRNA therapeutics should be explored to target stem cells and the transporters that they express.

csc tHerapeuticsTargeting of CSCs holds great promise in treating aggressive, metastatic, and even recurrent cancers. A number of diverse pathways are involved in stem cell differentiation and renewal. These include oncogenic cascades such as EGFR, hedgehog

(HH), and WNT-β-catenin, and also a variety of oncogenic sig-naling elements including, but not limited to, NF-κB, AKT, PI3 Kinase, Cox 2, and ABC efflux pumps.46,47 These factors play a pivotal role in regulating the self-renewal, survival, differen-tiation, and drug resistance of CSCs and may be viable candi-dates for molecular targeting. The HH/WNT pathway controls several tumor suppressor genes as well as several oncogenes. This pathway is mediated by a receptor protein known as PTCH (patched), which is the receptor for HH molecules. The PTCH gene was found to be mutated in patients with nevoid basal cell carcinoma syndrome and has attracted interest as a pos-sible target molecule for the HH pathway. The mammalian HH genes are overexpressed in a variety of cancer cell lines including gastric, breast, and prostate cells. HH signals are picked up by the PTCH receptor and are transmitted through a GPCR-like protein, SMO (smoothened) (Figure 7a). The expression of the HH ligand (Figure 7b) or the mutational inactivation of PTCH (Figure 7c) can constitutively activate SMO, which may then dysregulate HH pathway genes such as GLI1, GLI2, and GLI3.46 The discovery that cyclopamine, a steroid-like compound natu-rally occurring in the corn lily plant (Veratrum californicum), could bind to and inhibit the SMO protein (Figure 7d) provided hope that targeted therapies could be developed against CSCs. The signaling mechanism of the HH pathway was deciphered a few years ago. It was found that vitamin D3 is a crucial signaling molecule between PTCH and SMO. PTCH secretes vitamin D3, which can inhibit SMO on that cell and also on surrounding cells. HH can inhibit this secretion and release cells from SMO repression. Cyclopamine can compete with vitamin D3 bind-ing to SMO and repress the signaling pathway even when HH is activated. In this manner, it is possible that cyclopamine can inhibit growth pathways in CSCs.

Another approach to the problem would be to inactivate ABC efflux pumps in order to reinstate the drug sensitivity of CSCs. A directed effort has been devoted to the development of inhibitors against these efflux pumps. First-generation compounds that were tested included drugs such as verapamil and cyclosporine, which were able to inhibit the ABCB1 multidrug efflux pump. However, the low efficacy of these drugs in the clinical setting necessitated the development of second-generation compounds rationally designed on a quantitative structural activity relation-ship (QSAR). PSC833 and Biricoder (VX-710) were developed on the basis of the QSAR approach. PSC833 is a nonimmu-nosuppressive cyclosporine derivative; however, despite being more potent than cyclosporin A (as shown in in vitro studies) it was found that patients developed serious complications when it was administered in tandem with anticancer drugs. It was later discovered that PSC833 can significantly reduce the clearance of chemotherapeutics and thereby elevate the toxicity of drug.48 The outcomes of clinical trials with these second-generation inhibitors were largely negative, possibly because of pharmacokinetic interactions between the chemotherapeutics and the P-gp inhibitor or because of the presence of additional transporters that were unaffected by the inhibitor.

Efforts are now under way to develop a third generation of inhibitors that may possibly overcome the shortcomings of the


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previous generation of inhibitors. XR9576 (Tariquidar) and Ly335979 (Zosuqidar) are two such drugs. XR9576 can inhibit both ABCB1 and ABCG2, whereas Ly335979 is a specific inhibi-tor of P-gp. Phase I and II clinical trials are being carried out with these drugs. Another promising inhibitor is Fumetrimorgin C (FTC), a fungal toxin proven to be a very potent inhibitor of ABCG2. However, even though FTC is highly potent, a major drawback is the undesirable neurotoxic effects. This necessitated the development of alternative compounds such as Kol143, a tetracycline analog of FTC. A variety of natural products have also been shown to have chemosensitizing effects on numer-ous ABC transporters, including ABCB1, ABCC1, and ABCG2. Polyphenols and curcumin have been observed to modulate the effects of all three (ABCB1, ABCC1, and ABCG2) major drug transporters.48

Another promising ABC transporter modulator on the hori-zon is NSC 73306, a thiosemicarbazone derivative. Its mecha-nism of action is still under investigation, but it has been shown to be selectively cytotoxic to cells that overexpress P-gp, and it can resensitize these cells to chemotherapeutics.49 It was also discovered that NSC73306 is a substrate for ABCG2 and

can effectively inhibit ABCG2-mediated resistance to both mitoxantrone and topetecan. On the basis of these results, it was postulated that NSC73306 has a dual mode of action—it can eliminate P-gp-expressing cells while also being a potent modulator that may resensitize ABCG2-overexpressing cells to chemotherapeutics.50

Future directioNsThe stem cell model of drug resistance offers an appealing expla-nation as to why cancers that show an apparent complete clini-cal response to chemotherapy can relapse months or even years later. However, this model cannot explain stem cell response to therapy in most drug-resistant cancers, such as kidney cancer or colon cancer, in which only a few cells die as a result of chemo-therapy rather than only a few cells surviving. To improve the current stem cell model, it is necessary to define stem cells by their long-term self-renewal potential and not merely by the existence of an SP. The model must also (i) allow for the fact that many normal tissues and well-differentiated tumors have high levels of the same ABC transporters, (ii) explain how, over time, repopulated tumors acquire increasing drug resistance,

SHH

SHH

SHH

PTCH

PTCH

SMOSMO

ci (GLI)

ci (GLI)

ci (GLI)

ci (GLI)

PTCH

PTCH

Cytoplasm

Cytoplasm

Nucleus Nucleus

Nucleus

Extracellular

SMO

SMO

Cyclopamine

a b

c d

Figure 7 The Sonic hedgehog pathway (a) Signal transduction in the Sonic hedgehog pathway. HH signals are picked up by the PTCH receptor and are transmitted through a GPCR-like protein, SMO. (b) Hedgehog overexpression in the SHH pathway. The expression of the HH ligand can constitutively activate SMO, which dysregulates HH signaling and leads to a downregulation of HH pathway genes such as SHH, PTCH, GLI2, and GLI3. (c) Patched inactivation leads to SMO activation. The mutational inactivation of PTCH can constitutively activate SMO, which can dysregulate HH signaling and lead to a downregulation of HH pathway genes such as SHH, PTCH, GLI2, and GLI3. (d) Cyclopamine is able to inhibit SMO. Cyclopamine can bind to and inhibit the SMO protein. GPCR, G protein–coupled receptor; HH, hedgehog; PTCH, patched; SHH, Sonic hedgehog; SMO, smoothened.


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and (iii) address the issue of “plasticity” in a tumor in which cells downstream of stem cells can acquire the property of self-renewal. The issue of intrinsic resistance (in which all the cells rather than a small population of cells are refractory to the drug) also needs to be tackled. The new model will also need to incorporate the possible added layer of regulation afforded by microRNA as applicable to stem cells. Fortunately, there are now many tools at hand, including the availability of Abcb1-, Abcg2-, and Abcc1-null mice, that can be used to delve into these exciting new lines of inquiry. One such line of inquiry is: if transporters encoded by these genes are required for the protection of stem cells, would mice that lack these genes show a higher susceptibility to tumorigenesis from certain agents? Such studies could lead to the development of interesting new cancer models and new avenues for research. The fact that we can now identify, purify, and propagate CSCs may go a long way in allowing the development of new strategies to improve targeted cancer therapy.

ackNowledgmeNtsWe acknowledge the researchers who have contributed to the advancements in stem cell and ABC transporter research and whose works have not been cited here because of space limitations. This research was supported in part by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute, and SAIC-Frederick under contract NO1-CO-12400.

coNFlict oF iNterestThe authors declared no conflict of interest.

© 2011 American Society for Clinical Pharmacology and Therapeutics

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