Chapter 4. Mechanisms of Multidrug Resistance in Cancer. Jean-Pierre Gillet and Michael M. Gottesman. Abstract. 1. Introduction

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1 Chapter 4 Mechanisms of Multidrug Resistance in Cancer Jean-Pierre Gillet and Michael M. Gottesman Abstract The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Resistance exists against every effective anticancer drug and can develop by numerous mechanisms including decreased drug uptake, increased drug efflux, activation of detoxifying systems, activation of DNA repair mechanisms, evasion of drug-induced apoptosis, etc. In the first part of this chapter, we briefly summarize the current knowledge on individual cellular mechanisms responsible for MDR, with a special emphasis on ATP-binding cassette transporters, perhaps the main theme of this textbook. Although extensive work has been done to characterize MDR mechanisms in vitro, the translation of this knowledge to the clinic has not been crowned with success. Therefore, identifying genes and mechanisms critical to the development of MDR in vivo and establishing a reliable method for analyzing clinical samples could help to predict the development of resistance and lead to treatments designed to circumvent it. Our thoughts about translational research needed to achieve significant progress in the understanding of this complex phenomenon are therefore discussed in a third section. The pleotropic response of cancer cells to chemotherapy is summarized in a concluding diagram. Key words: Multidrug resistance, Uptake transport, ABC transporters, Drug metabolism, DNA repair, Vaults, Microenvironment, Translational research 1. Introduction Chemotherapy is the treatment of choice for patients diagnosed in the late stages of locally advanced and metastatic cancers. The main challenge is then to administer a drug dosage that maximizes the efficacy and minimizes the toxicity of the treatment. Unfortunately, in a significant number of patients, the tumor does not respond to the therapeutic agents. This impediment to the clinical cure of cancers results from known and yet-to-be determined mechanisms of resistance to chemotherapy. Resistance, either inherent or acquired, exists against every effective anticancer drug and can develop by multiple mechanisms. The overall mechanisms were recently J. Zhou (ed.), Multi-Drug Resistance in Cancer, Methods in Molecular Biology, vol. 596, DOI / _4, Humana Press, a part of Springer Science + Business Media, LLC

2 48 Gillet and Gottesman reviewed in part by Lage (1), Mellor et al. (2), and Mimeault et al. (3). These mechanisms can act individually or synergistically, leading to multidrug resistance (MDR), in which the cell becomes resistant to a variety of structurally and mechanistically unrelated drugs in addition to the drug initially administered. In this chapter, we will first summarize the current knowledge on individual cellular mechanisms responsible for MDR. Since a detailed discussion of all the mechanisms is beyond the scope of this chapter, our aim will be to briefly depict them and provide the reader with the most recent and relevant reviews on the subject. As ATP-binding cassette (ABC) transporters are perhaps the unifying theme of this book, we will emphasize their biology and their role in the MDR process to provide a fairly detailed idea of what we currently know about this family of proteins. Although extensive research has characterized some of the mechanisms involved in MDR in vitro, translating this to the clinic still represents a major challenge. We will therefore briefly summarize our thoughts about translational research that could be done to achieve significant progress in the understanding of this complex phenomenon. Finally, we provide a diagram to summarize the pleotropic response of cancer cells to chemotherapy. 2. Mechanisms Involved in the Resistance of Cells to Chemotherapy 2.1. Drug and Plasma Membrane Interactions Uptake Transport: Emerging Role of Solute Carriers Aside from pharmaceutical factors such as drug administration, distribution, metabolism, and excretion, the primary obstacle that prevents a drug from reaching the intracellular compartment is the plasma membrane. Therapeutic agents can react with various molecules, resulting in a complex speciation profile. These species can enter cells by either passive diffusion (4) or facilitated transport (5). Although the exact mechanisms of cellular uptake are poorly understood for most chemotherapeutic drugs, it has been well established that decreased expression of reduced-folate carrier (SLC19A1/hRFC1) and polymorphisms in its gene significantly hamper a patient s response to methotrexate, a common therapeutic agent (6). The solute carrier (SLC) family comprises approximately 360 uptake transporters classified into 45 gene families, outlined at slc/menu.asp. Genes of the SLC superfamily encode passive transporters, ion-coupled transporters, and exchangers (7). We can rationally speculate that observations concerning SLC19A1 could also apply to other members of this large family involved in uptake of anticancer drugs. Transporters of the SLC28 and 29 families mediate equilibrative diffusion of nucleosides across the plasma membrane, contributing to salvage pathways of

3 Mechanisms of Multidrug Resistance in Cancer 49 nucleotide synthesis (8, 9). It has also been demonstrated that these transporters mediate the cellular uptake of nucleoside analogs used in the treatment of cancers (10). Mackey et al. have highlighted the role of SLC29A1, A2 and SLC28A1 but not SLC28A2 in gemcitabine transport (11). More recently, the same group reported that SLC29A1 is a predictive marker for overall survival in patients with pancreatic cancer who received gemcitabine (12). One could therefore suggest that tumors with reduced expression of SLC29A1, A2, or SLC28A1 transporters or with mutant transporters might be resistant to this drug. Genetic polymorphisms of these transporters may alter drug pharmacokinetics, triggering interindividual differences in the safety and efficacy of drug therapy. SLCO1B3/OATP1B3 was shown to mediate uptake of paclitaxel (13). Investigation of the functional consequences of mutations in SLCO1B3 showed that paclitaxel pharmacokinetics were not associated with SLCO1B3-344T>G or 699G>A (14). The SLC22A1/OCT1, SLC22A2/OCT2, and SLC22A3/OCT3 transporters may mediate uptake of some platinum anticancer drugs (15). Interestingly, genetic variants of SLC22A1 and SLC22A2 showed altered transport of some of their substrates, such as metformin, which is used in therapy for type 2 diabetes mellitus (16, 17). However, the transport of platinum anticancer drugs by these variant SLC22A1 and A2 transporters has not yet been directly investigated. To date, SLCs have not been intensively focused on as candidate transporters for anticancer drugs. Therefore, the correlation of SLC genetic variants to treatment outcomes still needs to be clarified. An important step toward a better understanding of the role of SLCs in drug transport was made with a recent study released by Okabe et al. who used a bioinformatics approach to identify SLC substrates (18). In that study, mrna expression of 28 members of the SLCO and SLC22 families in the NCI-60 cell line panel (19) was profiled. By correlating expression profiles with growth inhibitory profiles of 1,429 compounds (including anticancer drugs and drug candidates) tested against the cells (20 23), it was confirmed that SLC22A4 confers sensitivity to doxorubicin in cancer cells (18) Efflux Transport: Role of ABC and Other Transporters Drug uptake can also be significantly reduced by ATP-dependent drug efflux pumps. These include ABC transporters (24, 25) such as the extensively studied ABCB1 (26), C1 (27), and G2 (28) transporters. As the main mechanism of resistance discussed in this book, ABC transporters will be thoroughly discussed in Subheading 4.2 of this chapter. ABC transporters are not the only actors in the process of ATP-dependent drug efflux, as other transporters such as RLIP76/RALBP1 (29) and ATP7A/B (30) have also been reported to mediate drug resistance. RLIP76 is a GTPase-activating protein that mediates the export of the GSH-conjugates of chemotherapeutic agents such as melphalan

4 50 Gillet and Gottesman (31) as well as doxorubicin, vincristine, and mitomycin-c (32). ATP7A/B are copper efflux pumps that appear to have a role in resistance to platinating agents, as supported by numerous studies reviewed by Hall et al. (15) and Kuo et al. (30) Lipid Metabolism Affects Biophysical Properties of the Lipid Bilayer as well as Signaling Pathways MDR Mechanisms Related to Drug Entry: A Pleiotropic Phenomenon Multidrug-resistant cells may have alterations in lipid metabolism (33, 34), which induce modifications in the biophysical properties of the lipid bilayer, consequently influencing drug uptake. Ceramide has been intensively studied concerning its role as the cellular messenger of apoptosis (35). Gouaze- Andersson et al. recently showed that this lipid has a similar role in the regulation of ABCB1 expression (36). The same group also reported that ceramide plays a major role in acquired resistance to doxorubicin (37). The mechanism they propose is the following: doxorubicin increases ceramide levels by either activation of sphingomyelinase or activation of enzymes of de novo ceramide synthesis, resulting in activation of apoptotic pathways. However, as the natural lipid substrate of glucosylceramide synthase (GCS), ceramide upregulates GCS expression through the Sp1 transcription factor. The authors postulate that this positive feedback cycle is antiapoptotic and drives cellular resistance to ceramide-generating types of chemotherapeutic drugs, speculating further that the characterization of this signal transduction pathway might reveal a way to prevent anthracycline resistance (37). The mechanisms mentioned earlier are closely interconnected, and this can be illustrated by the reduced uptake of platinum drugs in cisplatin-resistant cells. Our laboratory has studied extensively the causes underlying resistance to platinum drugs (recently reviewed in Hall et al. (15)). Following a first study reporting the establishment of cisplatin-resistant cell lines and their crossresistance to a wide array of structurally and mechanistically unrelated drugs (38), subsequent extensive work by our laboratory has clearly shown the pleiotropic defect in uptake of cisplatin and unrelated compounds found in cancer cells. Using selected cell lines for cisplatin resistance as models, we have observed decreased expression of SLC19A1 transporter and arsenic-binding proteins (38 40). We later demonstrated that this defective uptake results from DNA hypermethylation (41). This suggests a common mechanism that might also reduce the uptake of platinum drugs, as no ABC transporter efflux pumps have been found to be overexpressed in the models studied. This may be explained by the mislocalization of membrane proteins caused by a defect of plasma membrane protein recycling associated with a cytoskeletal defect (42, 43). Other alterations in the biophysical properties of the lipid bilayer, such as a defective fluid-phase endocytosis (44) and an increase in membrane fluidity have also been detected in cisplatin resistant cells (45). Whether or not this latter defect is a

5 Mechanisms of Multidrug Resistance in Cancer 51 primary cause leading to cisplatin resistance, or a secondary effect has yet to be determined. These studies clearly demonstrate the pleiotropic phenomenon underlying multidrug resistance mechanisms and their effect on cellular drug entry Drug Metabolism Phase I Metabolic Enzymes Once in the intracellular compartment, drug metabolism enzymes are the second line of cellular resistance. This process involves phase I and II enzymes. Phase I or oxidative metabolism is mediated mainly by cytochrome P450 enzymes (CYPs) and epoxide hydrolases. CYPs belong to a superfamily of hemoproteins comprising 57 genes classified in 18 families and 44 subfamilies based on their degree of sequence homology. An up-to-date database and nomenclature for these enzymes can be found at: CytochromeP450.html. CYPs are localized in mitochondria and the endoplasmic reticulum (ER) and catalyze the monooxygenase reaction by incorporating one atom of molecular oxygen into the substrate and one into water. This reaction also requires a source of electrons, provided by the NADPH cytochrome P450 reductase in the ER and ferredoxin in the mitochondria (46). While mitochondrial CYPs are involved in the metabolism of endogenous substrates, microsomal CYPs metabolize both endogenous and exogenous compounds. Therapeutic drugs are therefore metabolized by microsomal CYP and epoxide hydrolases, which convert highly mutagenic aromatic metabolites (epoxide) created from the CYP metabolism in a metabolite that can be conjugated by the phase II enzymes and then effluxed by transporters such as the members of the ABCC transporter family (46). Although mainly expressed in the liver, extrahepatic expression of CYPs has been shown in both normal and tumor tissues (47, 48). The CYP3A, 2D6, and 2C families metabolize most chemotherapeutic drugs. These enzymes are genetically highly polymorphic, and the expression of some of their variants has been found to predict treatment outcome (49). This is exemplified by the 516G>T polymorphism in CYP2B6, which is related to an increase in cyclophosphamide metabolism (50, 51). However, the same polymorphism was correlated with a threefold decrease in efavirenz metabolism (52 54). Phase I reactions result mainly in drug detoxification. However, these enzymes can be utilized in a prodrugbased strategy (55). Understanding the effect of polymorphisms on CYP enzyme activity is consequently particularly important. This is well illustrated by the data obtained for the 516G>T polymorphism in CYP2B6, which indicate clearly that the effect of polymorphisms can vary dramatically, depending on the drug administered. Studies have highlighted the synergism between CYP enzymes and ABC transporters that occurs when metabolites produced by CYP enzymes, especially CYP3A4, are better substrates for ABCB1 than the parent compound or when ABCB1 prevents the

6 52 Gillet and Gottesman saturation of CYP enzymes by necessitating a subsequent entry of the drug into the cell (56). This process increases exposure to CYP enzymes and can dramatically decrease the efficacy of chemotherapeutic treatment Phase II Enzymes 2.3. Drug Sequestration Phase II enzymes are involved in conjugation reactions including glutathionylation (57), glucuronidation (58), and sulfation (59). These enzymes include glutathione-s-transferase (GST) (60), UDPglucuronosyltransferases (UGT) (61), sulfotransferases (62), and arylamine N-acetyltransferases (NAT) (63), which transform the reactive species into hydrophilic nontoxic metabolite conjugates. These conjugated metabolites are then effluxed by members of the ABCC family of ABC transporters (27). Genetic polymorphisms in these families of genes have also been associated with overall survival in cancer patients. Ekhart et al. (64) review the influence of genetic polymorphisms not only in these enzymes but also in phase I enzymes and ABC transporters on survival after chemotherapy. Recent evidence has led us to consider intracellular drug sequestration as an important mechanism of MDR. Abnormalities in lysosomal function, protein trafficking and secretion have now been clearly identified. It has been shown that cisplatin is sequestered into lysosome, golgi, and secretory compartments and then effluxed from the cell (44, 65 67). A complete profile of the mediators of the intravesicular transport has certainly not yet been developed. We do know that the copper efflux transporters ATP7A/B are colocalized with fluorescent cisplatin analogs in vesicles of the secretory pathway (65, 67, 68). A recent study carried out in our laboratory highlighted the role of melanosomes in cisplatin resistance (69). We showed that cisplatin is sequestered in melanosomes, altering the melanogenic pathway and accelerating extracellular transport of melonosomes that contain cisplatin (69). No transporter responsible for this intracellular transport has yet been identified. However, one could suggest a role of the ABC transporter ABCB5, found to be preferentially expressed in pigment-producing cells (70). ABCA3-mediated intracellular drug sequestration has recently been associated with poor overall survival in acute myeloid leukemia (71, 72). ABCA3 is first localized in lysosomal (73) and late endosomal membranes and then colocalized with daunorubicin (72). Its role in MDR was initially indirectly suggested by Steinbach et al. when they showed a significant effect on cell viability after a combination of ABCA3 gene expression silencing and doxorubicin treatment (71). Direct evidence of its role as mediator of MDR was reported very recently in a study carried out by Chapuy et al., in which the authors demonstrated that ABCA3 mediates resistance to daunorubicin, mitoxantrone, etoposide, Ara-C, and vincristine (72).

7 Mechanisms of Multidrug Resistance in Cancer 53 In addition to drug sequestration in intracellular vesicles, scavenger metallothioneins (MTs) play a role in ensnaring drugs within a cell. These molecules are cysteine-rich and have high affinity for metal ions. This specificity, along with their ability to ensnare reactive oxygen species strongly suggests them as significant players in resistance to metal-based therapeutic agents and radiation treatment. However, the disparate literature on their role in cancer drug resistance does not allow unequivocal corroboration of this assumption (reviewed by Theocharis et al. (74) and Thirumoorthy et al. (75)). Although important as part of the pleiotropic phenomena underlying MDR, intracellular drug sequestration does not appear to induce resistance to the same extent as that produced by typical efflux transporters DNA-Damage Response Network A complex network of interacting pathways has evolved to monitor the integrity of a cell s DNA and govern proper responses to any genetic damage (76). This network includes sensor complexes that detect DNA breaks. It has been shown that Mre11-Rad50-Nbs1 (MRN) recognizes or senses DNA doublestrand breaks (DSBs), while the RPA-ATRIP complex binds to single strand breaks (SSBs). Kinases such as ATM and ATR are then recruited by MRN and RPA-ATRIP, respectively, and phosphorylate/activate a myriad of other proteins including the checkpoint kinases Chk1 and 2, initiating a cascade that results in cell-cycle arrest and DNA repair (Fig. 4.1) (77). However, if the damage is too extensive, rather than repair itself, the cell will enter one of these states: (1) senescence, which is characterized by an irreversible growth arrest, (2) apoptosis, or (3) necrosis (see Subheading 4.2.4). Many chemotherapeutic drugs have been employed to kill proliferating cells, causing extensive DNA damage that ultimately leads to cell cycle arrest and cell death. However, the efficacy of these therapeutic agents such as platinum drugs (78) and alkylating agents (79) can be significantly reduced by the ability of cells to repair DNA. DNA repair involves an intricate network of repair systems that each target a specific subset of lesions. These pathways include (1) the direct reversal pathway (MGMT, ABH2, ABH3), (2) the mismatch repair (MMR) pathway, (3) the nucleotide excision repair (NER) pathway, (4) the base excision repair (BER) pathway, (5) the homologous recombination (HR) pathway, and (6) the nonhomologous end joining (NHEJ) pathway (80). Figure 4.2 summarizes the role of these pathways in the repair of lesions induced by some chemotherapeutic agents (reproduced from Helleday et al. (81)). Studies have reported an inverse correlation of ERCC1 (NER pathways) with either response to platinum therapy or survival in ovarian (82, 83), non-small cell lung cancer (84), and colorectal cancers (85). It has also been shown that MMR deficiency is

8 54 Gillet and Gottesman Fig Chk1 and Chk2 kinases are serine/threonine kinases that are activated by the ATM and ATR kinases in response to DNA damage. The checkpoint kinases are transducers of the DNA damage signal and both phosphorylate a number of substrates involved in the DNA damage response. Chk1 and Chk2 share a number of overlapping substrates, although it is clear that they have distinct roles in directing the response of the cell to DNA damage. Current understanding is that the checkpoint kinases are involved not only in cell cycle regulation but also in other aspects of the cellular response to DNA damage. The G1checkpoint is modulated primarily by the ATM-Chk2-p53 pathway, as expression of ATR, Chk1, and Cdc25a is limited until the cell passes this restriction point. At this point, levels of ATR, Chk1, and Cdc25a all increase. If DNA damage is detected, Chk1/Chk2 are activated, Cdc25a is phosphorylated, and thus, destabilized, resulting in a p53-independent S arrest. In S phase, the same cascade can result in an intra-s arrest in response to stalled replication forks. The G2-M checkpoint prevents entry into mitosis with unrepaired DNA lesions. Initiation of this checkpoint is mediated by the ATM/ATR/Chk1/Chk2 cascades as shown, which ultimately suppresses the promitotic activity of cyclin B/cdc2. Along with their pivotal roles in the modulation of the cell cycle checkpoints, Chk1 and Chk2 are also involved in other aspects of the DNA damage response, including DNA repair, induction of apoptosis, and chromatin remodeling. Reproduced from (77), by permission of the American Association for Cancer Research. associated with cisplatin resistance (86, 87). The MMR mechanism removes the newly inserted intact base instead of the damaged base, triggering subsequent rounds of futile repairs, which can lead to cell death (88). Furthermore, a role in triggering checkpoint signaling and apoptosis was also suggested (89). Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) has been extensively studied and is considered to be a significant barrier to the successful treatment of patients with malignant glioma (90). There is no doubt that other proteins involved in those mechanisms will be revealed in the future through the characterization of MDR tumors.

9 Mechanisms of Multidrug Resistance in Cancer 55 Fig Overview of DNA repair pathways involved in repairing toxic DNA lesions formed by cancer treatments. The DNA-damaging agents that are used in cancer treatment induce a diverse spectrum of toxic DNA lesions. These lesions are recognized by a variety of DNA repair pathways that are lesion-specific but are complementary in some respects. (a) Ionizing radiation and radiomimetic drugs induce double-strand breaks (DSBs) that are predominantly repaired by nonhomologous end joining (NHEJ). (b, c) Monofunctional alkylators (b) and bifunctional alkylators (c) induce DNA base modifications, which interfere with DNA synthesis. Lesions produced by some alkylators are processed into toxic lesions in a mismatch repair-dependent manner. The base excision repair (BER) and nucleotide-excision repair (NER) pathways are, together with alkyltransferases (ATs), major repair pathways, whereas other repair pathways repair toxic replication lesions, such as those produced by interstrand crosslinks. (d) Antimetabolites interfere with nucleotide metabolism and DNA synthesis, causing replication lesions that have not yet been characterized. Mismatch repair mediates the toxicity of some antimetabolites (for example, thiopurines). The repair pathways involved in repair of antimetabolite-induced lesions are, apart from BER, poorly characterized. (e) Topoisomerase poisons trap topoisomerase I or II in transient cleavage complexes with DNA, thus creating DNA breaks and interfering with replication. (f) Replication inhibitors induce replication fork stalling and collapse, resulting in indirect DSBs. The relative contributions of the major repair pathways to the respective types of DNA damage outlined are indicated by the sizes of the boxes. This is based on the extent of sensitivity of repair-deficient cells to anticancer drugs in each category. ENDO endonucleasemediated repair, FA Fanconi anaemia repair pathway, HR homologous recombination, O2G DNA dioxygenases, RecQ RecQ-mediated repair, SSBR DNA single-strand break repair, TLS translesion synthesis. Reprinted by permission of Macmillan Publishers Ltd., (81), copyright 2008.

10 56 Gillet and Gottesman 2.5. Evasion of Drug-Induced Apoptosis Cellular death is the underlying pharmacological purpose for chemotherapy. Disruption of apoptotic pathways, the hallmark of cancer, is a major obstacle in the success of chemotherapy. Both apoptotic and nonapoptotic mechanisms can lead to the cell death (91). Nonapoptotic mechanisms include autophagy, mitotic catastrophe, necrosis, and senescence (92) (Table 4.1). The apoptotic cascade can be initiated via two routes, involving either the release of cytochrome c from the mitochondria (intrinsic/ mitochondrial pathway), or activation of death receptors (TNF-tumor necrosis factor) in response to ligand binding (93). The activation of these pathways leads to the activation of a family of cysteine proteases, the caspases, that mediate the cleavage of cellular substrates leading to morphological and biochemical changes that characterize apoptosis. Increasing evidence, however, suggests that caspase-independent pathways also exist (94). Although in vitro studies have shown that nuclear translocation of the mitochondrial flavoprotein apoptosis-inducing factor (AIF) might Table 4.1 Characteristics of different types of cell death Morphological changes Type of cell death Nucleus Cell membrane Cytoplasm Biochemical features Apoptosis Autophagy Mitotic catastrophe Necrosis Senescence Chromatin condensation; nuclear fragmentation; DNA laddering Partial chromatin condensation; no DNA laddering Multiple micronuclei; nuclear fragmentation Clumping and random degradation of nuclear DNA Distinct heterochromatic structure (senescenceassociated heterochromatic foci) Blebbing Blebbing Fragmentation (formation of apoptotic bodies) Increased number of autophagic vesicles Caspase-dependent Caspase-independent; increased lysosomal activity Caspase-independent (at early stage) abnormal CDK1/ cyclin B activation Swelling; rupture Increased vacuolation; organelle degeneration; mitochondrial swelling Flattening and increased granularity SA-b-gal activity CDK1 cycline-dependent kinase 1, MDC monodansylcadaverine, MPM2 mitotic phosphoprotein 2, SA-b-gal senescence-associated b-galactosidase, RB retinoblastoma protein Reprinted by permission of Macmillan Publishers Ltd., (91), copyright 2004

11 Mechanisms of Multidrug Resistance in Cancer 57 be a key factor in this pathway (94), the mechanisms governing caspase-independent cell death are little known. Chemotherapy-induced resistance occurs primarily via the mitochondrial pathway, regulated by the Bcl2 family of genes. Therefore, alterations in Bcl2 genes, which are involved in maintenance of the homeostasis of pro- and antiapoptotic factors, may considerably hamper the success of treatment (95, 96). Many researchers have investigated the usefulness of Bcl2 family proteins in the prediction of chemotherapy outcome. Sjöström et al. found that none of the apoptosis-related proteins they investigated (Bcl2, Bax, Bcl-xl, Bag1, FAS, FASL) could predict response to drug treatment for breast cancer (97). This observation was confirmed in two other studies, one in which the Bcl-2 status was not predictive for paclitaxel treatment in patients with breast cancer (98) and another involving vinorelbine plus docetaxel treatment in patients with non-small cell lung cancer (NSCLC) (99). On the other hand, some studies did find a correlation between Bcl2 expression status and chemotherapy response in patients with breast cancer (100, 101). This exemplifies the conflicting literature which to date does not allow us to establish any clear connection between defective apoptotic pathways and treatment failure Vaults Vaults were first described in 1986 (102). They are the largest ribonucleoprotein particles ever described, with a size of ~42 75 nm and a mass of ~13 MDa (103). They were given this name because of their morphological resemblance to vaulted ceilings in gothic cathedrals (104). Vault particles are evolutionary highly conserved, although they are not found in Saccharomyces cerevisiae, Caenorhabditis elegans, or Drosophila melanogaster (105). They form a barrel-shaped structure composed of multiple copies of three proteins including the major vault protein (MVP), the vault poly-adp-ribose polymerase (VPARP), and the telomeraseassociated protein-1 (TEP1) plus an untranslated vault RNA (vrna) (106). Vaults, as detected by MVP expression, are ubiquitously expressed, with high levels in tissues that are chronically exposed to xenobiotics such as lung and epithelial cells of the intestine, and in macrophages and dendritic cells (107). They are localized in the cytoplasm, where up to 100,000 particles can be found per cell (108). For detailed information, see Mossink et al. (109) and Steiner et al. (110). Several cellular functions have been proposed for vaults (111). The detection of the MVP protein (initially named LRP) in a multidrug-resistant ABCB1 negative non-small cell lung cancer cell line implied a role for vaults in MDR (112). However, 15 years later, their role is still not clear. In vitro studies have yielded conflicting results; some reports show a direct role of vaults in MDR ( ), whereas others fail to substantiate such involvement ( ).

12 58 Gillet and Gottesman In the clinic, numerous studies have been undertaken to understand the role of vaults in multidrug resistance and whether they could be used as predictive markers of treatment response. Most of the work has been focused on hematological malignancies, but a few solid tumors have also been analyzed. This research is summarized in Table 4.2. As is the case for other MDR mechanisms, the role of vaults is still the subject of debate. Disappointingly, vault knockout mice do not show any phenotypic defects compared with wild-type mice (119). Moreover, no enhancement of chemosensitivity was observed and no compensation by increased activity of ABC transporters was found following drug treatment (120). It was concluded that at least in mice, vaults are not directly involved in drug resistance. However, analysis of both human and mice promoters seems to indicate that vault-associated MDR may be species specific. Indeed, the mouse promoter lacks a DNA segment found in the human sequence containing lentiviral elements and an inverted CCAAT box (Ybox) and therefore cannot bind to the NF-Y transcription factor. It has been shown that activation of the ABCB1 promoter is dependent on both elements (121). These substantial differences between human and mice promoters may explain these puzzling observations and indicate that mouse models may not be the ideal tool to study the role of vaults in MDR. Table 4.2 Conflicting clinical data on the association of MVP with response to therapy and patient prognosis MVP expression correlated to response to chemotherapy MVP expression correlated to prognosis Type of cancer Yes No Yes No AML ( ) ( ) ( ) ( ) ALL (203) ( ) ATL (206) MM ( ) a (207, 208) (209) Ovarian cancer ( ) a (213) ( ) a (213) Breast cancer ( ) (214) (217, 218) NSCLC (219, 220) (221, 222) (222, 223) Bladder cancer (224) Sarcoma (225) (226, 227) (225) (226, 227) Testicular germ-cell tumors (228) (228, 229) a Borderline

13 Mechanisms of Multidrug Resistance in Cancer Impact of the Microenvironment Solid tumors are heterogeneous structures composed of tumor and stromal cells that are embedded in the extracellular matrix and sustained by an abnormal vasculature network. This tumor microenvironment can influence the tumor cells propensity to metastasize (122, 123) and can dramatically affect treatment outcome ( ). For a general review on this, see Tredan et al. (127). Tumor responsiveness to chemotherapy is highly influenced both by the abnormal vasculature and an elevated interstitial fluid pressure, which dramatically impede the penetration of macromolecules into the tumor (125, 128). Tumor vasculature is characterized by a disorganized architecture composed of dilated and convoluted blood vessels. These vessels may also be compressed by tumor cells (129), leading to the disruption of blood flow (130). Furthermore, the lack of lymphatic vessels in the tumor vasculature network contributes to the increase of interstitial fluid pressure, which hinders the delivery of therapeutic agents by convection ( ). As a result, metabolites are not cleared and sustaining nutrients cannot be delivered, leading to hypoxia and acidification of the extracellular compartment (124). Tumor cells that lack oxygen (hypoxic cells) fulfill their energetic needs via the glycolytic pathway, which ultimately generates lactic acid, leading to intracellular acidification (124). The cells then maintain their ph homeostasis through the expression of proton (H + ) pumps, rendering the extracellular environment highly acidic. This feature may severely hamper chemotherapy. Indeed, according to the ion trapping theory, weakly basic drugs (e.g., doxorubicin, mitoxantrone, vincristine, etc.) are ionized in the acidic compartment, consequently hindering their entry into the cell. Conversely, an increased accumulation of weakly acidic drugs (e.g., chlorambucil, cyclophosphamide, camptothecin, etc.) in the cell will be observed as the plasma membrane is permeable to nonionized molecules (134, 135). Hypoxia plays an important role in MDR (124). It dramatically reduces the effectiveness of chemotherapeutic agents (e.g., bleomycin) that require oxidation to become cytotoxic or enhances the cytotoxicity of other agents (e.g., mitomycin C) that must undergo reduction to form active cytotoxic species (136). Hypoxic cells also display reduced rates of cell proliferation and can avoid apoptosis by triggering Bcl2 family genes, rendering them resistant to many therapeutic agents that target proliferative cells. It has been shown that hypoxia upregulates glutathione, GSH, and metallothionein (MT) protein levels, which reduces significantly the effectiveness of alkylating agents and platinum-based drugs (137, 138). The main mediator of the response to hypoxia is the HIF1 transcription factor, which was also shown to induce the expression of ABCB1, ABCC1, and ABCG2 ( ). Taken together, the tumor microenvironment not only leads to the development of the pleiotropic mechanisms underlying MDR, but also amplifies or intensifies them.

14 60 Gillet and Gottesman 2.8. The Cancer Stem Cell Paradigm Cancer-initiating cells, also known as cancer stem cells (CSCs), might underlie the intractable nature of many human cancers, explaining why conventional cancer therapy fails in many patients (144, 145). These cells have the capacity to initiate and sustain the growth of a heterogeneous cancer through self-renewal and differentiation. Moreover, they acquire most of the MDR mechanisms discussed in this section. CSCs can be isolated based on the expression of cell surface markers associated with immature cell types. However, no unique set of markers has been identified that distinguishes them from normal stem cells (144, 145). ABCB1 and ABCG2 are well-characterized ABC transporters expressed in both cancer and normal stem cells ( ). Besides these transporters, ABCA3 was also detected in neuroblastoma CSCs along with ABCG2 (149). ABCA3 is localized in membranes of lysosomes and endoplasmic reticulum, suggesting a role in drug sequestration (72). More recently, it was suggested that ABCB5 could be a marker of melanoma cancer-initiating cells (150). Although the concept is exciting, knowledge of ABCB5 from the genomic to the proteomic level is rudimentary and does not support this hypothesis (70, 151, 152). Although there has been a great deal of interest in the elegant CSC paradigm, its technological and experimental challenges are colossal. One of these challenges is the isolation of normal and cancer stem cell populations to identify differences in self-renewal mechanisms. If achieved, this could open new avenues to cancer research. 3. The ATP-Binding Cassette (ABC) Transporters The ATP-binding cassette (ABC) transporter proteins are a large superfamily of membrane proteins comprising 48 members divided into seven different families based on sequence similarities. The nomenclature for human ABC transporter genes is provided at: These proteins are evolutionary highly conserved and there is a high sequence homology among all the members, especially those within a particular family. The functional protein typically contains two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) encoded by a single polypeptide. ABC transporters may also be multicomponent units in which different genes encode each domain or half molecule. ABC transporters can have a wide array of cellular roles (24). They regulate local permeability by being expressed in the blood brain barrier, blood cerebrospinal fluid, blood testis barrier, and placenta (153). In the liver, gastrointestinal tract, and kidney,

15 Mechanisms of Multidrug Resistance in Cancer 61 ABC transporters excrete toxins, thereby protecting the organism (154). ABC transporters also play an active role in the immune system by transporting peptides into the endoplasmic reticulum that are identified as antigens by class I HLA molecules (e.g., ABCB2/TAP1, ABCB3/TAP2) (155, 156). Furthermore, they play physiological roles in cellular lipid transport and homeostasis (157). Finally, as expected from the diverse functional roles of ABC genes, the genetic deficiencies associated with their mutation also vary widely, as reviewed in Borst and Elferink (154). The first ABC transporter discovered was ABCB1 in 1976 (158). This transporter, called P-glycoprotein, was found to be expressed in Chinese hamster ovary cells selected for colchicine resistance. The authors also discovered that these cells displayed resistance to a variety of structurally and mechanistically unrelated drugs in addition to colchicine (158). Human P-glycoprotein, the product of the MDR1 or ABCB1 gene, was subsequently shown to confer MDR on drug-sensitive cells (159). More than 30 years later, 15 ABC transporters (ABCA2, ABCA3, ABCB1, ABCB4, ABCB5, ABCB11, ABCC1 6, ABCC11 12, and ABCG2) have been associated with drug resistance (Table 4.3). Of these, ABCB1 (160), ABCC1 (161), and ABCG2 (162) have been the most extensively studied. Yet attempts to translate these transporters into clinical targets have so far been unsuccessful (discussed further in Subheading 4 of this chapter). A lot of progress has been made in understanding the molecular mechanisms of ABCB1 through mutagenesis experiments and the resolution of structures of non-mammalian ABC proteins (163). A number of recent reports have addressed genetic polymorphisms in drug transporters; for an excellent review, see Cascorbi (164). Among the 48 ABC transporters, ABCB1 is one of the most thoroughly studied and characterized, with more than 50 SNPs reported ( ). The correlation of ABC transporter genetic variants to treatment outcomes is gradually being clarified, yet the overall picture is still puzzling, as much of the published data are conflicting. Nevertheless, the many studies reporting correlations between SNPs and clinical outcome indicate the necessity to pursue further investigations; for a detailed review on the role of polymorphisms in ABCB1 drug transport including the role of synonymous polymorphisms in altering ABCB1 transporter function (167), see Fung and Gottesman (168). The regulation of ABCB1 is poorly understood. It has been suggested that therapeutic agents (169, 170) and endogenous stimuli such as hypoxia, acidosis, free radical formation, or glucose deprivation could also induce its expression through multiple signal transduction pathways (26). Thus, the complex network of ABCB1 regulation ensures rapid emergence of pleiotropic resistance in cancer cells.

16 62 Gillet and Gottesman Table 4.3 Established anticancer drugs as substrates of ABC transporters Subfamily Genes Established anticancer drugs (Nonexhaustive list) A/ABC1 B/MDR-TAP C/MRP ABC A2/ABC2/STGD ABCA3/ABC3 ABC B1/MDR1/P-gp/PGY1 ABC B4/MDR3/PFIC3/PGY3 ABCB5 ABC B11/BSEP/PFIC2/SPGP ABC C1/MRP1 ABC C2/MRP2/CMOAT ABC C3/MRP3/CMOAT2 ABC C4/MRP4/MOATB ABC C5/MRP5/MOATC ABC C6/MRP6/MOATE/PXE ABC C10/MRP7 ABC C11/MRP8 Estramustine, mitoxantrone (230, 231) Daunorubicin, Ara-C, mitoxantrone, etoposide (72) Anthracyclines, vinca alkaloids, taxanes, etoposide, teniposide, imatinib, irinotecan, SN-38, bisantrene, colchicine, methotrexate, mitoxantrone, saquinivir, actinomycin D, etc. (231, 232) Daunorubicin, doxorubicin, vincristine, etoposide, mitoxantrone (234) Doxorubicin, camptothecin, 10-OH camptothecin, and 5FU (151, 235) Paclitaxel (236) Anthracyclines, vinca alkaloids, methotrexate, antifolate antineoplastic agents, etoposide, imatinib, irinotecan, SN-38, arsenite, colchicine, mitoxantrone, saquinivir, etc. (27, 237) Vinca alkaloids, cisplatin, etoposide, doxorubicin, epirubicin, metotrexatetaxanes, irinotecan, SN-38, topotecan, arsenite, mitoxantrone, saquinivir ( ) Etoposide, tenoposide, metotrexate (242, 243) 6-Mercaptopurine, 6-thioguanine, irinotecan, SN-38, topotecan, AZT, metotrexate, PMEA ( ) 6-Mercaptopurine, 6-thioguanine, 5-FU, cisplatin, metotrexate, PMEA, AZT (245, 248) Etoposide, doxorubicin, daunorubicin, teniposide, cisplatin (249) Taxanes, vinca-alkaloids (250, 251) 6-Mercaptopurine, 5-FU, PMEA (252, 253) G/WHITE ABC G2/BCRP/MXR/ABCP Mitoxantrone, camptothecin, anthracycline, etoposide, teniposide imatinib, flavopiridol, bisantrene, methotrexate, AZT, etc. (171) Modified from Gillet et al. (24) 4. Translational Research: From the Lab to the Clinic Extensive research has characterized some of the mechanisms involved in multidrug resistance in vitro. However, translating this to the clinic still represents a major challenge, as evidenced by the failure of trials to modulate ABCB1 expression (25) and the disputed role in vivo of ABC transporters in MDR (24, 171). Briefly, meta-analyses exploring ABC transporter expression

17 Mechanisms of Multidrug Resistance in Cancer 63 profiles in leukemia have highlighted the disparity of the literature ( ). In contrast to adult acute myeloid leukemia (AML), for which the role of ABCB1 gene expression in the drug resistance of tumors and prognosis of patients is widely accepted (175, 176), the data for adult acute lymphoblastic leukemia (ALL) are conflicting ( ). In solid tumors, most attention has been directed to the roles played by ABCB1, ABCC1, and ABCG2 in MDR observed in breast cancer. However, it is difficult to decipher their exact role in the clinical drug resistance observed in this disease (179, 180). Various reasons have been suggested for the conflicting reports on the role of ABC transporters in the clinic. The most obvious one is that ABC transporters other than the three usual suspects (ABCB1, C1, and G2) can also influence treatment outcome. As mentioned in Subheading 4.2, 15 ABC transporters have been associated with drug resistance. Moreover, several recent studies suggest that more than 25 ABC transporters can be involved in chemotherapy-induced resistance (71, ). In the previous section, we discussed the pleiotropic response of cancer cells to treatment. Therefore, another explanation for the disparity of the data reported is that studies focus on only one particular mechanism and neglect to investigate the other MDR mechanisms involved. The presence of normal or inflammatory tissues in tumors can also dramatically influence the gene expression profiles generated. Besides these biological factors, there are emerging issues associated with the -omic technologies. The variety of experimental systems developed to characterize cancers at a molecular level, such as cdna expression profiling, comparative genomic hybridization (array CGH), promoter arrays, SNP arrays, etc., has increased dramatically in the last 5 years. In addition, the variety of platforms proposed for each of those analytical systems is remarkable, and numerous proposed normalization processes also render an integrative computational and analytical approach extremely challenging (185). The establishment of standard analytical methods and the development of systems biology/integrative approaches should help to produce a more unified picture of MDR. That could also lead to progress not only in understanding the mechanisms governing multidrug resistance but also in the translation of this knowledge to clinical practice, especially in personalized medicine. In this regard, the recent development of Oncomine, a bioinformatics initiative aimed at collecting, standardizing, analyzing, and delivering cancer transcriptome data to the scientific community is a step in the right direction ( ). The knowledge acquired these last three decades by our own laboratory on MDR mechanisms has recently been tentatively translated to the clinic. Annereau et al. developed a high-density microarray platform dedicated to multidrug resistance to address the roles of MDR-linked genes in camptothecin resistance using cancer cell lines (189). However, microarrays require relatively

18 64 Gillet and Gottesman large amounts of sample and often have poor probe specificity for families of genes with a high degree of homology, such as those involved in MDR. At the same time, Szakacs et al. generated a database to identify lead compounds in the early stages of drug discovery that are not ABC transporter substrates (184). This repository is of great importance to help avoid the use of toxic drugs that ultimately will not provide any benefit to cancer patients. In that study, qrt-pcr using SYBR Green chemistry was used to profile the expression of ABC transporters. Although this platform is more reliable and accurate than microarrays, high-throughput analyses are tedious and require multiple pipetting steps, which can introduce variability. The availability of a reliable, sensitive and specific high-throughput platform that would allow the discrimination of highly homologous genes from small amounts of clinical tissue as opposed to in vitro models is fundamental to achieving any significant enhancement in the understanding of clinical multidrug resistance. In a recent study (190), our laboratory demonstrated the superiority of two platforms over established technologies in assessing ABC transporter expression profiles. This was demonstrated by an improved database that allows a more precise identification of compounds whose resistance is mediated by ABC transporters. In addition, it helps to pinpoint the compounds responsible for collateral sensitivity. We are now applying these platforms to identify genes critical to the development of MDR in cancer. 5. Conclusion For many years, MDR has been explained solely by an overexpression of ABCB1 in the tumor. Since its discovery in the late seventies, extensive research has characterized this ABC transporter-mediated MDR mechanism in vitro. These studies have also highlighted a multiplicity of additional mechanisms governing MDR. Although, these mechanisms can act individually, this concise review underlines the pleiotropic phenomenon of the cell response to drug treatment (Fig. 4.3). Beside the cellular response, we have mentioned the role of the microenvironment on the initiation and maintenance of MDR, which is now supported by strong data. These last years have seen the development of a new paradigm suggesting the role of cancer stem cells in the intractability of cancers. Whether or not cancer stem cells represent one of the mechanism(s) of MDR needs to be unraveled. In this chapter, we have discussed briefly the fundamental need for translational research to confirm MDR mechanisms in human

19 Mechanisms of Multidrug Resistance in Cancer 65 Fig Pleotropic mechanisms of multidrug resistance. (1) Drug entry. Aside from pharmaceutical factors, the primary obstacle that prevents a drug from reaching the intracellular compartment is the plasma membrane. Therapeutic agents can react with many molecules resulting in a complex speciation profile. These species represented uniformly in this schema as a hexagonal (D) can enter cells by passive diffusion, endocytosis, or facilitated transport (uptake transporters). However, drug uptake can be significantly reduced by ATP-dependent drug efflux pumps (such as the ABC transporters ABCB1 and G2) and alterations in lipid metabolism (ceramide pathway) usually found in multidrug-resistant cells, which induce modifications in the biophysical properties of the lipid bilayer. (2) Drug metabolism. Once in the intracellular compartment, drug metabolism enzymes are the second line of cellular resistance. This process involves three phases. Phase I, or oxidative metabolism is mediated mainly by cytochrome P450 enzymes (CYPs) and epoxide hydrolases. Drug species are metabolized and converted into highly mutagenic aromatic metabolites (epoxide) that can be conjugated by phase II enzymes including GSTs, UGTs, SULTs, and NATS. These conjugated metabolites are then effluxed by transporters, which can be considered as phase III of drug metabolism. (3) Drug sequestration. Drug species can be trapped in subcellular organelles such as lysosomes and endosomes through ATP7A/B, ABCA3, or ABCB5 influx and then expelled from the cell. Scavenger metallothioneins ensnare metal ions and reactive oxygen species, leading to resistance to metal-based therapy and radiation. (4) Mechanisms activated after nuclear entry. Drug species (newly activated in the case of a prodrug-based strategy) that evade the above mechanisms of resistance enter the nucleus, where they encounter several mechanisms of resistance. Drug species can be effluxed via vault proteins into the cytoplasm and be either sequestered in intracellular vesicles or effluxed from the cell via ATP-dependent transport. Some drug species remain in the nucleus and form damaging adducts with DNA. A complex network of interacting pathways is then initiated, leading either to cell-cycle arrest and DNA repair or if the damage is extensive, rather than repair itself, the cell will enter one of these states: (1) senescence, (2) apoptosis, or (3) necrosis. (5) Evasion of drug-induced apoptosis. Disruption of apoptotic pathways, the hallmark of cancer, is a major obstacle to the success of chemotherapy. Blockage of apoptosis can result through the inhibitory effect of glycosylceramide and a myriad of pathways. (6) Microenvironment. Hypoxia upregulates the expression of numerous MDR-linked genes such as ABC transporters, Bcl2 family genes, glutathione, MT, etc., mainly through the activation of the transcription factor HIF1. It also dramatically reduces the effectiveness of chemotherapeutic agents that require oxidation to become cytotoxic or enhances the cytotoxicity of other agents that must undergo reduction to form active cytotoxic species. The acidic extracellular compartment also has important effects on the success of chemotherapy. (7) Signal transduction pathways. Cancer cells have altered signal transduction pathways, governed via