ABC transporters as mediators of drug resistance and contributors to cancer cell biology

Drug Resistance Updates 26 (2016) 1–9

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ABC transporters as mediators of drug resistance and contributors to cancer cell biology Jamie I. Fletcher, Rebekka T. Williams, Michelle J. Henderson, Murray D. Norris, Michelle Haber ∗ Children’s Cancer Institute Australia, Lowy Cancer Research Centre, Randwick, NSW, Australia

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Article history: Received 16 June 2015 Received in revised form 4 March 2016 Accepted 12 March 2016 Keywords: Multidrug resistance Tumorigenesis Ovarian cancer Neuroblastoma Endogenous substrates

a b s t r a c t The extrusion of anticancer drugs by members of the ATP-binding cassette (ABC) transporter family is one of the most widely recognized mechanisms of multidrug resistance, and can be considered a hijacking of their normal roles in the transport of xenobiotics, metabolites and signaling molecules across cell membranes. While roles in cancer multidrug resistance have been clearly demonstrated for P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP) and Multidrug Resistance Protein 1 (MRP1), direct evidence for a role in multidrug resistance in vivo is lacking for other family members. A less well understood but emerging theme is the drug efflux-independent contributions of ABC transporters to cancer biology, supported by a growing body of evidence that their loss or inhibition impacts on the malignant potential of cancer cells in vitro and in vivo. As with multidrug resistance, these contributions likely represent a hijacking of normal ABC transporter functions in the efflux of endogenous metabolites and signaling molecules, however they may expand the clinical relevance of ABC transporters beyond P-gp, BCRP and MRP1. This review summarizes established and emerging roles for ABC transporters in cancer, with a focus on neuroblastoma and ovarian cancer, and considers approaches to validate and better understand these roles. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction The human ATP-binding cassette (ABC) transporter superfamily is composed of 48 genes subdivided into 7 subfamilies, ABCA through ABCG (Dean, 2005). The majority of these genes encode for large membrane proteins that function in the energy dependent transport of xenobiotics, metabolites and signaling molecules across cell membranes, often against their concentration gradient. Amongst this superfamily at least twelve members have been implicated in the transport of cancer drugs in laboratory studies (Szakacs et al., 2006). Numerous clinical studies demonstrate associations with patient outcomes, however these are generally correlative and derived from retrospective studies. Compelling evidence currently supports roles for only three of these ATP-driven efflux transporters in chemoresistance in vivo: ABCB1 (P-glycoprotein/P-gp/MDR1), ABCG2 (Breast Cancer Resistance Protein/BCRP) and ABCC1 (Multidrug Resistance Protein 1/MRP1). P-gp

∗ Corresponding author at: Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW, PO Box 81, Randwick, NSW 2031, Australia. Tel.: +61 2 9385 2170; fax: +61 2 9662 6583. E-mail address: [email protected] (M. Haber). http://dx.doi.org/10.1016/j.drup.2016.03.001 1368-7646/© 2016 Elsevier Ltd. All rights reserved.

in particular has been the subject of extensive efforts to develop small molecule transport inhibitors over several decades, and while strategies to reverse drug resistance by targeting ABC transporters have not been as successful as was initially hoped, improvement in both inhibitor properties and clinical trial design may overcome many of these limitations (Amiri-Kordestani et al., 2012; Shaffer et al., 2012). The alternative strategy of avoiding efflux by ABC transporters through the development of agents that are not substrates may also be a feasible approach to gain therapeutic benefit in multidrug resistant tumors, and important gains have been made in this area (Nobili et al., 2012). While the role of ABC transporters in multidrug resistance is widely recognized, a less well studied but emerging theme is the drug efflux independent contributions of ABC transporters to cancer biology (Fletcher et al., 2010). The loss or inhibition or various ABC transporters has been observed to influence tumor cell phenotypes closely associated with malignant potential, including proliferation, differentiation, migration and invasion, and these observations have been made across multiple cancer types (Copsel et al., 2011; Hedditch et al., 2014; Henderson et al., 2011; Zhang et al., 2012). More compellingly, loss of ABC transporters in both human xenograft and transgenic mouse cancer models also impacts on tumorigenesis and tumor progression (Copsel et al., 2014;

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Henderson et al., 2011; Mochida et al., 2003; Yamada et al., 2003; Zander et al., 2012). These contributions seem likely to relate to the normal physiological functions of ABC transporters in the efflux of endogenous metabolites and signaling molecules (Huls et al., 2009; Nigam, 2015; van de Ven et al., 2009), however for the most part a mechanistic understanding of how these functions contribute to tumor biology is lacking. These emerging contributions to tumor biology now require careful validation, thorough examination of their underlying mechanisms and evaluation of their potential as therapeutic targets in cancer models.

2. ABC transporters in drug resistance and drug disposition 2.1. P-glycoprotein (P-gp, ABCB1) P-gp was the first human ABC transporter identified, having been described as a glycoprotein modulating drug permeability in 1976 (Juliano and Ling, 1976) and then shown to be an energy-dependent exporter of anticancer drugs a decade later (Assaraf et al., 1989a,b; Chen et al., 1986; Gerlach et al., 1986; Gros et al., 1986; Ueda et al., 1987). P-gp transports unmodified neutral or positively charged hydrophobic compounds, and has been demonstrated to efflux a wide range of clinically indispensable cancer chemotherapeutics including taxol, vincristine, etoposide, daunorubicin and irinotecan amongst many others (Ambudkar et al., 2003; Borgnia et al., 1996; Eytan et al., 1994; Szakacs et al., 2006). P-gp has similar expression in human and mouse tissues, with particularly prominent expression in the apical membrane of epithelial cells at physiological barriers and in excretory tissues, including the blood–brain barrier, gastrointestinal tract, kidney and liver (Cordon-Cardo et al., 1989; Croop et al., 1989; Schinkel et al., 1994; Thiebaut et al., 1987). Mice with targeted deletion of both ABCB1 homologs (Abcb1a, Abcb1b) are viable and fertile, indicating that P-gp is dispensable for development, and that transiently targeting P-gp may be safely achievable (Schinkel et al., 1997). However, loss of P-gp has significant effects on both drug distribution and elimination in mice, with profound increases in the penetration of P-gp substrate drugs to the brain, substantial increases in plasma drug levels and increased penetrance into heart, muscle, adrenal gland, ovary and hematopoietic progenitors (Schinkel et al., 1994, 1997). Loss of P-gp also substantially impacts oral drug availability (Sparreboom et al., 1997). These studies highlight the key role that P-gp plays in drug pharmacokinetics, as well as its prominent role in the blood–brain barrier and the intestinal mucosal epithelium. While human and mouse P-gp share high sequence identity (87%) and similar tissue distribution, there are notable differences in the susceptibility of some substrates to transport by the human and mouse P-gp proteins (Yamazaki et al., 2001). Numerous studies have demonstrated correlations between tumor P-gp expression and patient response rates or survival rates, or have reported observations of elevated P-gp expression following chemotherapy. For example, an extensive meta-analysis of studies examining P-gp in breast cancer concluded that patients with P-gp expressing tumors were 3-fold more likely not to respond to chemotherapy than those without and that chemotherapy increased the proportion of patients with P-gp expressing tumors (Trock et al., 1997). Similarly, P-gp expression has been reported to negatively correlate with response to chemotherapy in small cell lung cancer (Triller et al., 2006) and to correlate with lower rates of complete remission and higher rates of resistant disease (Leith et al., 1999). The potential clinical application of P-gp transport inhibitors has been an active area of research since the early clinical trials of first generation inhibitors in the early 1990s, however results have been

largely disappointing. The possible reasons for these failures are many and varied and have been covered at length in several excellent reviews (Amiri-Kordestani et al., 2012; Shaffer et al., 2012; Szakacs et al., 2006), hence will only be summarized briefly here. The affinity and specificity of the inhibitors has long been a significant limitation. First generation P-gp inhibitors were of relatively low affinity, and exhibited toxicity at the doses required for physiological P-gp inhibition, while second generation inhibitors designed to reduce these off-target toxicities were still limited by pharmacokinetic interactions, most notably off-target inhibition of drug metabolism mediated by Cytochrome P450 enzymes (CYPs), particularly CYP3A. Third generation inhibitors appear to have mitigated many of these issues by exhibiting substantially higher affinity for P-gp and being largely devoid of cross-reactivity with CYPs. Unfortunately however, these inhibitors have to date failed to show benefit in large, randomized clinical trials (Amiri-Kordestani et al., 2012). Possible reasons for the absence of benefit include: the lack of selection of patients with high P-gp expression; compensatory upregulation of alternative drug efflux transporters or a failure to account for their contributions; interpatient variability resulting from transporter polymorphisms; and the possibility that dysregulated drug uptake rather than drug efflux may be the limiting factor in drug accumulation in some cases (Shaffer et al., 2012). Until these issues are resolved, it remains unclear whether P-gp inhibitors can actually provide a therapeutic window over normal tissues, particularly given the fundamental role of P-gp in drug absorption, distribution and metabolism. In contrast to ABC transporter inhibition, circumvention of multidrug resistance by the substitution of drugs for alternatives that are not subject to efflux by the transporters overexpressed in a given tumor may be a more achievable approach. Although the emergence of molecular profiling as a tool to inform drug choices provides an opportunity to select or avoid agents based on transporter expression, many anticancer agents cannot be readily substituted with alternatives. The development of new agents not subject to efflux by transporters will therefore be essential. Prominent examples of alternative agents that avoid P-gp efflux include the microtubule-stabilizing epothilones (Bollag et al., 1995; Kowalski et al., 1997), third generation taxanes (Ojima et al., 2008), lipophilic camptothecins (Gounder et al., 2008), non-camptothecin topoisomerase I inhibitors (Ruchelman et al., 2005) and imidazoacrdinones and triazoloacridones that function as topoisomerase II inhibitors (Bram et al., 2009a,b). 2.2. Breast cancer resistance protein (BCRP, ABCG2) The “half-transporter” ABCG2 was first identified as the mediator of acquired resistance in an adriamycin (doxorubicin) selected breast cancer cell line (Doyle et al., 1998). ABCG2 is able to transport a particularly wide range of substrates, including important cancer chemotherapeutics such as mitoxantrone, methotrexate, the camptothecins topotecan and irinotecan, epipodophyllotoxins, imidazoacridinones and the anthracycline doxorubicin (although a mutant form of ABCG2 is required for anthracycline transport) (Bram et al., 2009a,b; Mao and Unadkat, 2015). In addition to conventional cytotoxic agents, several targeted agents are also ABCG2 substrates, including the tyrosine kinase inhibitors imatinib, gefitinib, and nilotinib (Dohse et al., 2010; Hegedus et al., 2012). ABCG2 is expressed in the gastrointestinal tract, in excretory tissues and blood–tissue barriers including the liver, placenta, kidney, and blood–brain barrier (Cooray et al., 2002; Maliepaard et al., 2001). ABCG2-deficient mice are born at a normal Mendelian ratio, are fertile and show no gross or histologic abnormalities, indicating that the protein is not required for normal development (Zhou et al., 2001). However, these mice do however show altered drug pharmacokinetics, in particular increased oral absorption and decreased

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2.3. Multidrug Resistance Protein 1 (MRP1, ABCC1) MRP1 was originally identified as the mediator of acquired drug resistance in a small cell lung cancer cell line selected by repeated exposure to doxorubicin (Cole et al., 1992). Despite limited sequence identity with P-gp (19% at amino acid level), MRP1 and P-gp have significant substrate overlap. Notably however, while both transporters efflux unmodified hydrophobic molecules, MRP1 also transports organic anion conjugates of a broad range of endo- and xenobiotics (particularly GSH and glucuronide conjugates) and is thus a major route for the efflux of conjugates generated in Phase II drug metabolism reactions (Deeley and Cole, 2006). Furthermore, the efflux of many of the more hydrophobic compounds by MRP1 is highly dependent on glutathione (GSH), which may either stimulate transport or be co-transported (Cole and Deeley, 2006; Loe et al., 1996, 1998). Important cancer chemotherapeutics effluxed by MRP1 include etoposide, doxorubicin, vincristine, irinotecan, arsenic trioxide, mitoxantrone and methotrexate (Deeley and Cole, 2006; Hooijberg et al., 1999; Szakacs et al., 2006). MRP1 is broadly expressed in healthy human and mouse tissues (Flens et al., 1996; Maher et al., 2005) and clearly plays important roles in the protection of various tissues from xenobiotics. Abcc1−/− mice are developmentally normal, but display hypersensitivity to the MRP1 substrate etoposide (Lorico et al., 1997; Wijnholds et al., 1997), with etoposide-induced damage to the mucosa of the oropharyngeal cavity and the seminiferous tubules of the testis, polyuria, and substantial accumulation of etoposide in the cerebrospinal fluid (CSF) (Wijnholds et al., 1998, 2000). These studies indicate the importance of MRP1 in physiological barriers including the testis–blood barrier, the oral epithelium, the kidney urinary collecting duct tubules and the blood–CSF barrier and also highlight tissues for which the side effects of MRP1 substrate drugs might be increased by the coadministration of MRP1 inhibitors (Wijnholds et al., 1998). Elevated MRP1 levels have been reported to be associated with poor patient outcome in breast cancer (Filipits et al., 2005), nonsmall cell lung cancer (Li et al., 2009, 2010), AML (Schaich et al., 2005) and ALL (Plasschaert et al., 2005). Of the cancers where elevated MRP1 has been associated with poor outcome, the most

Abcc1-/-, etoposide (n=26)

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hepatobiliary excretion (Merino et al., 2005; van Herwaarden et al., 2003; Yamagata et al., 2007). ABCG2 expression has been associated most consistently with poorer outcomes in acute myelogenous leukemia (AML) as well as a range of other cancers, including acute lymphoblastic leukemia (ALL), breast and lung cancer (reviewed in Mao and Unadkat, 2015; Meyer zu Schwabedissen and Kroemer, 2011; Natarajan et al., 2012). In many cases however, these associations are apparent despite the limited use or even absence of ABCG2 substrate drugs in the treatment of these patients, leading to the suggestion that ABCG2 may be a marker of chemoresistance rather than a direct contributor to this phenotype. In this regard, ABCG2 expression is frequently observed in tumor cell populations with increased self-renewal capacity and tumorigenic potential, suggesting that inhibition of ABCG2 may be an approach to targeting cancer stem cells (Dean et al., 2005; Natarajan et al., 2012). Despite the uncertain nature of these clinical correlations, direct evidence that ABCG2 contributes to chemoresistance is provided by observations made in a genetically engineered mouse model for BRCA1-associated breast cancer (Brca1−/− ;p53−/− mice), where overexpression of Abcg2 was identified as a mechanism of acquired topotecan resistance, and tumor-specific genetic ablation of Abcg2 significantly increased the survival of topotecan-treated animals (Zander et al., 2010). In contrast to the extensive clinical development of P-gp inhibitors, no small molecule inhibitors specific for ABCG2 have been tested in clinical trials to date.

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Abcc1+/+, etoposide (n=22) Abcc1+/+, saline (n=23) Abcc1-/-, saline (n=25)

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Days after treatment Fig. 1. Loss of MRP1 sensitizes mouse neuroblastoma tumors to etoposide. Tumors were harvested from Th-MYCN/Abcc1+/+ and Th-MYCN/Abcc1−/− mice and engrafted subcutaneously in Balb/c Nude mice. Mice were treated for 5 days with daily 12 mg/kg etoposide (intraperitoneal injection) or saline control and tumor volume determined at regular intervals (Burkhart et al., 2009). Tumors lacking Abcc1 responded better to etoposide administration.

compelling case for clinical relevance and for the application of MRP1 inhibitors is the pediatric solid tumor neuroblastoma. Several lines of evidence support this conclusion. Firstly, overexpression of ABCC1 expression is strongly predictive of poor outcome in multiple independent neuroblastoma patient cohorts (Haber et al., 2006; Henderson et al., 2011; Norris et al., 1996), and retains prognostic significance following multivariable analysis including the major prognostic indicators in this disease. Secondly, ABCC1 is transcriptionally regulated by the MYCN oncogene (Porro et al., 2010), a driver of tumorigenesis in neuroblastoma (Weiss et al., 1997). Amplification of the MYCN oncogene occurs in approximately 20–30% of primary neuroblastomas and is consistently associated with poor clinical outcome (Brodeur et al., 1984; Seeger et al., 1985). Thirdly, many of the frontline agents used in neuroblastoma therapy are MRP1 substrates, including etoposide, doxorubicin, vincristine and irinotecan. Interestingly, loss of MRP1 induced increased intracellular reduced folate pool, resulting in antifolate resistance (Stark et al., 2003). While patient data supporting the role of MRP1 in chemoresistance is necessarily correlative, experiments conducted in the Th-MYCN transgenic mouse neuroblastoma model indicate that Mrp1 contributes to chemoresistance in vivo. The Th-MYCN mouse model selectively expresses MYCN in the neural crest and spontaneously develops neuroblastoma closely recapitulating the human disease (Weiss et al., 1997). When the Th-MYCN mouse was crossed with an Abcc1 knockout mouse and tumors harvested from Th-MYCN/Abcc1+/+ or Th-MYCN/Abcc1−/− mice and engrafted into secondary recipients, loss of Mrp1 enhanced the response to vincristine and etoposide, significantly delaying tumor growth and providing direct evidence that Mrp1 mediates chemoresistance in vivo (Burkhart et al., 2009) (Fig. 1). While reversal of MRP1-mediated drug resistance appears attractive as a chemosensitization strategy in certain tumor types, no small molecule inhibitors specific for MRP1 have been tested in clinical trials to date. Several P-gp inhibitors that also target MRP1 have been evaluated in adult cancers, including Biricodar (VX-710) and CBT-1 (Yu et al., 2013), however these have often been assessed in combination with cytotoxic agents that are P-gp but not MRP1 substrates (e.g. taxanes). Promising lead compounds with a high degree of selectivity for MRP1 have however been developed, including cyclohexyl-linked tricyclic isoxazoles (Norman et al., 2005) and flavonoid derivatives (Obreque-Balboa et al., 2016), however these remain to be comprehensively assessed in preclinical cancer models.

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2.4. Other Multidrug Resistance Protein family members (ABCC subfamily) ABC transporters other than P-gp, ABCG2 and MRP1 are also able to efflux chemotherapeutics in vitro, including multiple members of the ABCA (Albrecht and Viturro, 2007) and ABCB (Childs et al., 1998) subfamilies and the majority of the ABCC subfamily (Keppler, 2011; Slot et al., 2011). Several of these transporters have also been demonstrated to contribute to the pharmacokinetics of chemotherapeutics in animal models, including Mrp2, loss of which leads to elevated plasma methotrexate (Vlaming et al., 2006), Mrp4, which contributes to the distribution and excretion of topotecan (Leggas et al., 2004) and Mrp7, loss of which sensitizes mice to paclitaxel-induced toxicities (Hopper-Borge et al., 2011). Whether these transporters contribute directly to multidrug resistance in tumors is currently unclear, however crosses of transporter-deficient mice with appropriate genetically modified mouse cancer models may provide important insights. As outlined above, previous studies have demonstrated a direct contribution to tumor chemo-responsiveness for Mrp1 in a model of MYCNamplified neuroblastoma (Burkhart et al., 2009) and for Abcg2 in a model of BRCA1-associated breast cancer (Zander et al., 2010), supporting clinical correlations for these tumors (Burger et al., 2003; Haber et al., 2006; Norris et al., 1996). Opportunities exist for comparable studies with other transporters and mice deficient in Abcc2 (Chu et al., 2006; Nezasa et al., 2006; Vlaming et al., 2006), Abcc3 (Belinsky et al., 2005; Zelcer et al., 2005), Abcc4 (Belinsky et al., 2007; Leggas et al., 2004; Lin et al., 2008), Abcc5 (de Wolf et al., 2007), Abcc6 (Gorgels et al., 2005; Klement et al., 2005) and Abcc10 (Hopper-Borge et al., 2011) have all been previously described. While these experiments have the potential to be highly informative, their interpretation requires care as the affinity of some ABC transporter substrates varies markedly between the mouse and human transporters. For example, human MRP1 confers resistance to anthracyclines and antifolates (Hooijberg et al., 1999), whereas mouse Mrp1 does not confer resistance (Stride et al., 1997), cGMP has a very low affinity for mouse Mrp4 compared to human MRP4 (de Wolf et al., 2007) and there are notable differences in substrate susceptibility between human and mouse P-gp (Yamazaki et al., 2001). 3. MDR-independent roles for ABC transporters in cancer We previously reviewed evidence that ABC transporters may play important roles in cancer biology beyond their established functions in multidrug efflux (Fletcher et al., 2010). Here we extend that review to more recent observations, including findings that strongly suggest causality in transgenic mouse cancer models, and provide illustrative examples taken from the neuroblastoma and ovarian cancer fields. 3.1. Loss of ABC transporters alters tumorigenesis in transgenic mouse cancer models While the knockout of individual ABC transporters in mice does not appear to influence viability, fertility and development, at least under the controlled conditions of animal research facilities, several examples have emerged where genetic ablation of an ABC transporter has altered tumorigenesis in mouse cancer models. In the pioneer study, disruption of the Abcb1 gene in the ApcMin/+ mouse model resulted in a reduction in intestinal polyps and tumor incidence associated with an increase in spontaneously occurring DNA damage, apoptosis, proliferation and migration in enterocytes (Mochida et al., 2003) and with morphological alterations to increased necrosis (Yamada et al., 2003). More recently, disruption

of Abcc1 was shown to decrease tumor incidence and increased tumor latency in the Th-MYCN transgenic mouse model of neuroblastoma (Henderson et al., 2011), consistent with the observation that high MRP1 expression correlates with poor patient outcome in this malignancy (Haber et al., 2006; Norris et al., 1996). While the mechanisms underlying this observation are unknown, the reduction in tumor incidence with MRP1 loss might imply that MRP1 contributes to the survival of neuroblasts within hyperplastic lesions in early postnatal sympathetic ganglia (Alam et al., 2009; Hansford et al., 2004). In contrast to the apparent pro-tumorigenic functions of P-gp and Mrp1, loss of Abcg2 in conditional (mammary and epithelial) K14cre;Brca1F/F ;p53F/F mice significantly reduced median tumor latency, indicating that Abcg2 contributes to the protection of epithelial cells against carcinogenesis (Zander et al., 2012). Increased exposure of BRCA1;p53-deficient epithelial cells to unidentified endogenous metabolites or dietary xenobiotics is a possible explanation for these findings, particularly in light of the established role of Abcg2 in protection from dietary toxins in mouse models (Jonker et al., 2002, 2005). 3.2. ABC transporters in neuroblastoma biology Some of the most compelling evidence for ABC transporters contributing to cancer biology stems from neuroblastoma. Several ABC transporters including ABCC1, ABCC3 and ABCC4 are under the direct transcriptional control of MYCN (Porro et al., 2010) and their expression is strongly correlated with patient outcome (Haber et al., 2006; Henderson et al., 2011; Norris et al., 1996, 2005) (Fig. 2). As mentioned above, deletion of Abcc1 decreases tumor incidence and increases tumor latency in the Th-MYCN mouse model, strongly implicating this transporter in neuroblastoma genesis. While the mechanisms underlying this observation are not clear, the findings are supported by a range of additional cell culture-based observations. First, MRP1 knockdown via antisense RNA led to an increased proportion of apoptotic cells and an increased proportion of cells undergoing neuronal differentiation, as measured by increases in synaptophysin and neurofilament expression (Peaston et al., 2001). A follow-up study found that MRP1 suppression inhibited colony formation and migration in MYCN-amplified neuroblastoma cells, and conversely, that overexpression of MRP1 in neuroblastoma cells enhanced migratory ability and colony formation (Henderson et al., 2011). The impact of MRP1 on cell growth and migration most likely depends on substrate efflux, since overexpression of MRP1 harboring a mutation, previously shown to abolish ATP-dependent transport (Payen et al., 2005), failed to enhance migration or colony formation (Henderson et al., 2011). In support of an alternative role for MRP4 in neuroblastoma biology, siRNA-mediated knockdown of MRP4 in the neuroblastoma cell lines reduced cell proliferation and clonogenicity in the absence of cytotoxic drug treatments (Henderson et al., 2011). Additional evidence suggests MRP4 is important in the biology of other cancers, although definitive studies are lacking. Downregulation or inhibition of MRP4 through shRNA or the MRP4 transport inhibitor probenecid in the human leukemia cell line U937 caused intracellular accumulation of cAMP and consequent leukemic cell maturation toward a more differentiated phenotype (Copsel et al., 2011), and inhibited the growth of U937 xenografts in mice (Copsel et al., 2014). These findings are consistent with the apparent role for MRP4 and cAMP-mediated signaling in normal hematopoietic cell development, where ABCC4 expression levels decreased during differentiation toward mature leukocytes (Oevermann et al., 2009). MRP4 may also have a vital role dendritic cell migration as inhibition of MRP4, either by virally transduced shRNA or pharmacologically with the transport inhibitor sildenafil, impaired the ability of interstitial dendritic cells to migrate toward lymph node chemokines (van de Ven et al., 2008). In pancreatic cancer, the

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Fig. 2. ABCC1 and ABCC4 are predictive of outcome in neuroblastoma. Expression of ABCC1 (left) and ABCC4 (right) is strongly correlated with outcome in neuroblastoma. Kaplan–Meier curves generated using publicly available microarray data (Molenaar et al., 2012) and the R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl).

levels of MRP4 protein in tumors were higher compared to adjacent non-cancerous pancreatic tissue and knockdown of MRP4 using shRNA in each of two pancreatic cancer cell lines, Panc1 and BxPC3, inhibited cell growth and clonogenicity through cell cycle arrest, albeit via unknown mechanism of action (Zhang et al., 2012). In contrast to ABCC1 and ABCC4, ABCC3 is negatively regulated by MYCN (Porro et al., 2010), and its expression in neuroblastoma is typically very low. Human BE(2)-C neuroblastoma cells, which have extremely low endogenous levels of MRP3, have significantly reduced cell migration and colony formation rates when MRP3 is constitutively expressed. As with MRP1, mutagenesis indicates that this inhibitory effect depends on ATP-dependent transport (Henderson et al., 2011). 3.3. ABC transporters in ovarian cancer biology Advanced epithelial ovarian cancer (EOC) is another cancer for which survival rates remain poor, often due to the emergence of drug resistance phenomena, and therefore many studies have examined the potential influence of ABC transporters in the context of drug efflux in this disease. However, reports are currently emerging where ABC transporters not known for their roles in drug efflux are being associated with poor patient outcome or tumor cell characteristics. In the examples presented herein, new roles are emerging for transporters with already well-established essential biological functions and well-known disease phenotypes associated with their loss of function, making their potential exploitation as targets challenging. ABCA1 is well-known for its key role in lipid homeostasis, being responsible for the export of cholesterol and phospholipids to Apolipoprotein A1 (APOA1), forming high density lipoproteins (HDL) (Tarling et al., 2013; Vedhachalam et al., 2007; Zhao et al., 2012). Mutations that impair ABCA1 function lead to Tangiers disease, which is associated with a severe reduction in circulating HDL (Brooks-Wilson et al., 1999; Rust et al., 1999). Other members of the A subfamily are less well-studied, but are generally thought to mediate transport of various lipids across the plasma membrane or intracellular membranes. In a recent study, high levels of ABCA1 expression in primary serous EOC were significantly correlated with poor patient outcome (Hedditch et al., 2014). Enrichment of ABCA1 mRNA expression in primary serous EOC tumors from a large Australian Ovarian Cancer Study (AOCS) cohort correlated significantly with decreased overall survival, particularly following optimal debulking surgery and with correction for tumor stage (Hedditch et al., 2014). This finding was then confirmed in a substantial cohort from The Cancer Genome Atlas (TCGA) that consists largely of high-grade, advanced ovarian cancer. Immunohistochemical staining of a serous EOC tumor

cohort further validated these findings, with strong ABCA1 protein staining associated with poor progression-free and overall survival. In support of these findings, siRNA-mediated suppression of ABCA1 expression in the human ovarian cancer cell lines A2780, 27/87 and SKOV-3 led to inhibition of cell proliferation and migration, suggesting that ABCA1 expression contributes to the malignant characteristics of ovarian cancer cells. ABCA1 may also have a pro-malignant role in prostate cancer, since prostate cancer cell lines expressing high levels of ABCA1 exhibited enhanced growth and migration capacity (Her et al., 2013; Sekine et al., 2010). By contrast, in colon cancer cells, it has been reported that ABCA1 acts in an anti-tumor manner by facilitating cytochrome-C release from the mitochondria (Smith and Land, 2012), suggesting some level of tissue selectivity underlying the impact of ABCA1. Whilst ABCA1 may present as a potential novel target in ovarian cancer, the development and clinical use of ABCA1 inhibitors requires caution due to the normal physiological functions of this transporter. ABCA1 is recognized to exhibit a protective role against cardiovascular disease and is required for lipid and cholesterol homeostasis indicating that treatments aimed at directly reducing ABCA1 may require tissue specific targeting. Interestingly, some evidence would suggest that various statins may in fact reduce ABCA1 expression, which may enable treatment of ovarian cancers expressing high ABCA1 levels with clinically tested, safe and established therapeutics (Dong et al., 2009; Niesor et al., 2015). However this research is largely limited in scope and would require expansion to a relevant cancer model. An alternative approach to directly inhibiting ABCA1 would be to identify and block pro-malignant pathways induced by ABCA1. Potential mechanisms of action for ABCA1 in ovarian cancer remain to be investigated, however evidence from other cancers as well as its normal physiological function, indicate involvement in several signaling pathways. In androgen-independent prostate cancer cell lines, treatment with either HDL or the transforming growth factor ␤1 (TGF␤1) protein lead to phosphorylation and subsequent activation of ERK1/2 and AKT through an ABCA1-dependent mechanism (Her et al., 2013; Sekine et al., 2010) (Fig. 3). Additionally, activation of this ABCA1-dependent signaling cascade, enhances migration and growth, both in vitro and in vivo (Her et al., 2013; Sekine et al., 2010). Interestingly, TGF␤1, a protein involved in growth inhibition under normal physiological conditions, was found to be redirected toward the pro-malignant pathway through a feedback loop involving upregulation of ABCA1 (Her et al., 2013). Similarly, ABCA1 has been shown in several studies to be involved in both directly binding and activating, through phosphorylation, the protein-tyrosine kinase, Janus Kinase 2 (JAK2), and the signal transducer and activator of transcription 3 (STAT3) in endothelial, fibrosarcoma and baby

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Fig. 3. Proposed mechanisms of action for ABCA1 in tumor biology. (a) Upregulation of ABCA1 protein through TGF␤1 signaling leads to stabilization of CAV1, followed by phosphorylation and activation of the AKT/ERK pathway (adapted from Her et al., 2013), or alternatively (b) HDL or APOA1 interaction with ABCA1 facilitates phosphorylation of either the AKT/ERK or JAK2/STAT3 pathways, subsequently promoting downstream tumorigenic properties. PPAR␦ – peroxisome proliferatoractivated receptor ␦, CAV1 – caveolin1.

hamster kidney cells (Liu et al., 2011; Tang et al., 2004; Vaughan et al., 2009) (Fig. 3). The AKT/ERK and JAK2/STAT3 pathways are commonly deregulated in ovarian cancer, making them attractive possibilities for targeted therapy. Further research is required to determine whether or not ABCA1 promotes either of these pathways in ovarian cancer and whether targeted therapies against these pathways might be efficacious in cancers overexpressing ABCA1. ABCC7, better known as the cystic fibrosis transmembrane conductance regulator (CFTR), normally functions in transporting chloride ions across epithelial membranes, with mutations leading to cystic fibrosis. Somewhat surprisingly then, Xu and colleagues reported that ABCC7 protein expression was significantly higher in EOC specimens than in benign or normal samples, in addition to being associated with late stage, high grade and both the serous and clear cell histological subtypes (Xu et al., 2015). Furthermore, ABCC7 silencing in both Skov3 and A2780 human ovarian cancer cell lines resulted in reduced migration, invasion, proliferation and adhesion, which translated to decreased tumorigenicity in vivo for mice harboring Skov3 xenografts (Xu et al., 2015). Since CFTR interacts with various modulators of tyrosine kinase signaling, the authors propose that high levels of CFTR could activate signaling via c-src, another pathway activated in ovarian cancers (Brdickova et al., 2001; Kim et al., 2011; Moyer et al., 1999; Wiener et al., 1999, 2003). 4. Future perspectives While efforts to reverse clinical multidrug resistance through inhibition of P-gp have been frustrating to date, additional roles for ABC transporters in cancer biology are being uncovered that may eventually lead to new therapeutic opportunities. The reduction in malignant potential observed with the loss or inhibition or various ABC transporters in both cell culture systems and mouse models suggests that tumors benefit from ABC transporter expression in ways that are not currently well understood and that impact on fundamental cellular functions such as proliferation, differentiation and migration and invasion.

While the logical assumption is that these additional roles in tumor biology are mediated by the efflux of endogenous metabolites and signaling molecules, this remains to be proven in most cases, and alternative explanations such as effects mediated via protein–protein interactions involving ABC transporters cannot be excluded at this stage. One strategy to determine the role of ATPdependent transport in these phenomena is to assess the ability of transport-deficient mutants to rescue the observed phenotypes, an approach that has been used to demonstrate that MRP1 influences neuroblastoma cell migration in a transport-dependent manner, at least when overexpressed (Henderson et al., 2011; Payen et al., 2005). The development of relatively simple gene editing technologies such as CRISPR/Cas systems (Sander and Joung, 2014) means this should be achievable with proteins expressed at biologically meaningful (endogenous) levels, both in tissue culture and animal models. If, as expected, these additional roles for ABC transporters prove to be dependent on efflux capacity, what substrates mediate these roles? While candidate approaches to identifying effector substrates can be devised based on the ABC transporter substrates with established roles in tumor biology, such as cyclic AMP, eicosanoids and glutathione (Fletcher et al., 2010), our understanding of the full range of endogenous ABC transporter substrates remains limited. Targeted metabolomics studies have identified previously unrecognized endogenous substrates of MRP2 (Krumpochova et al., 2012), MRP3 (van de Wetering et al., 2009) and ABCG2 (van de Wetering and Sapthu, 2012), and an untargeted approach has recently been used to identify previously unrecognized ABCC5 substrates (Jansen et al., 2015). Understanding these additional roles for ABC transporters in cancer biology may therefore require combining approaches to novel substrate identification with phenotypic studies. If these putative additional roles for ABC transporters in cancer biology prove to be targetable, would the difficulties in reversing clinical multidrug resistance with P-gp inhibitors also bedevil approaches to targeting these functions in the clinic? Targeting endogenous ABC transporter functions differs from targeting multidrug resistance functions in several key respects that may prove advantageous. Firstly, pharmacokinetic interactions with co-administered chemotherapeutics, a major complication with targeting drug transport by P-gp, could be avoided. Secondly, direct targeting of an ABC transporter may not be essential if other components of a signaling pathway such as substrate biosynthesis or receptor binding can be targeted instead, thereby avoiding issues of functional redundancy or compensatory upregulation amongst alternative ABC transporter family members.

5. Concluding remarks Drug efflux-independent roles for ABC transporters in cancer biology are an emerging theme and will likely expand the clinical relevance of this family beyond P-gp, BCRP and MRP1. While these drug efflux-independent roles have been demonstrated in multiple mouse tumor models and cell culture systems, much remains to be learned about their underlying mechanisms, most crucially identification of the substrates that underlie these effects. This mechanistic understanding will be essential for evaluation of their significance for human cancers, and for the design of strategies for ABC transporter inhibition in this context.

Acknowledgements Children’s Cancer Institute Australia is affiliated with UNSW Australia and the Sydney Children’s Hospitals Network.

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