Multidrug-resistant protein 4 (MRP4), a member of the C subfamily of ATP-binding cassette transporters, is distributed in a variety of tissues and a number of cancers. As a drug transporter, MRP4 is responsible for the pharmacokinetics and pharmacodynamics of numerous drugs, especially antiviral drugs, antitumor drugs, and diuretics. In this regard, the functional role of MRP4 is affected by a number of factors, such as genetic mutations; tissue-specific transcriptional regulations; post-transcriptional regulations, including miRNAs and membrane internalization; and substrate competition. Unlike other C family members, MRP4 is in a pivotal position to transport cellular signaling molecules, through which it is tightly connected to the living activity and physiologic processes of cells and bodies. In the context of several cancers in which MRP4 is overexpressed, MRP4 inhibition shows striking effects against cancer progression and drug resistance. In this review, we describe the role of MRP4 more specifically in both healthy conditions and disease states, with an emphasis on its potential as a drug target.
The ATP-binding cassette (ABC) transporter family is the largest family of transmembrane proteins, comprising 49 transporters that are further subdivided into seven subfamilies from A to G, based on sequence homology (Dean et al., 2001). Of these, the C subset, also called the multidrug-resistant protein (MRP) subfamily, has attracted increasing attention. Nine of the MRP subfamily members (MRP1 to 8 and cystic fibrosis transmembrane conductance regulator [CFTR]) are localized to the plasma membrane of different cell types, and are capable of pumping out a wide variety of structurally diverse endogenous and xenobiotic organic anions, as well as many glutathione (GSH) conjugates (Borst et al., 2000; Sodani et al., 2012). Thus, MRPs are involved in the absorption, distribution, and elimination of numerous drugs. In addition, the high expression of MRPs in cancer cells contributes to the multidrug resistance to chemotherapy (Sodani et al., 2012).
MRP4, encoded by the ABCC4 gene, was first discovered as a homolog of MRP1 and 2 by Kool et al. (1997). ABCC4 is located on chromosome 13q32.1 and has three splicing variants, isoforms 1, 2, and 3 (http://www.uniprot.org/uniprot/O15439). Isoform 1, as the main splicing variant, encodes a protein of 1325 amino acids that form two membrane spanning domains, two nucleotide binding domains, and a PDZK motif at the C terminus (Fig. 1). The crystal structure of MRP4 is characterized by a unique inward or outward cavity, which pumps substrates outside by using the energy released from ATP hydrolysis (Sauna et al., 2004; El-Sheikh et al., 2008b; Ravna and Sager, 2008). Compared with other MRPs, MRP4 shows a wider tissue distribution (kidney, liver, intestine, platelet, smooth muscle, heart, brain blood barrier, etc.), and can transport more endogenous signaling molecules such as cAMP, cGMP, ADP, prostaglandins, leukotrienes, and folic acid (Russel et al., 2008). As a result, MRP4 plays a vital role in intra- and extracellular communication under both healthy conditions and diseased states. Recently, a variety of studies have shown that MRP4 contributes to platelet aggregation, cell migration, proliferation, angiogenesis, and cardiomyocyte contraction, with a particular role in caner progression and prognosis (Huynh et al., 2012). A number of clinical observations and modern pharmacogenomics studies also substantiate these roles of MRP4. Moreover, it has been shown that the drug interactions on, and genetic variations of, MRP4 may be linked to altered drug efficacy or toxicity.
Although the transport functions of MRP4 have been reviewed, this protein likely participates in additional, as-yet-unidentified functions. In this review, we emphasize the recent progress related to the pharmacological and physiologic functions of MRP4 and its regulatory pathway, and we examine its potential as a drug target.
MRP4 Substrates and Modulators
To test the potential substrates, modulators, and inhibitors of MRP4, methods such as inside-out membrane vesicles (MVs), transfected cell lines, MRP4 knockout (KO) mice, and ex vivo tissues and cells have been used. The vesicular transport method measures the ATP-dependent transport of substrates into inside-out vesicles, which are made from insect cells, HEK293 cells, and V79 cells. The vesicular transport approach is generally considered more informative because it allows for determination of the Km value, as well as the Vmax value for the transport of the respective substrate. Although the transporters expressed in insect cells are underglycosylated and the cholesterol content of insect cells is 4- to 10-fold lower than mammalian cells, the uptake feature is not much altered by using the two different cell types. MRP4-transfected NIH3T3, HEK293, V79, and CEM-R1 cells are also used as the vehicles to determine substrates transported by MRP4 after a loading phase for drug entry into cells. Moreover, comparing the resistance values between MRP4-transfected and parental cell lines can indirectly indicate the substrates of MRP4. In this regard, it is noteworthy that a short drug exposure to cells in culture medium is more indicative than long drug exposure.
Among the drug transporters, MRP4 stands out for transporting a range of endogenous molecules that have a key role in cellular communication and signaling, including cAMP, cGMP, ADP, eicosanoids, bile acids, urate, and conjugated steroid hormones (Table 1). MRP4 is also involved in the absorption, disposition, or excretion of a wide range of drugs, including antiviral [adefovir and tenofovir (TFV)], antibiotic (cephalosporins), diuretic (furosemide and hydrochlorothiazide), antihypertensive (olmesartan), and cytotoxic [methotrexate, 6-thioguanine, 6-mercaptopurine (6-MP), topotecan] agents (Table 1). Using zebrafish MRP4 transfected cells and MRP4 KO models, it was demonstrated that MRP4 participates in the efflux of organochlorine pesticides, dichloro diphenyl trichloroethane, and lindane (Lu et al., 2014). The increased expression of zebrafish MRP4 after exposure to methyl parathion, and the decrease in cell viability following exposure to methyl parathion and the MRP4 inhibitor verapamil, indicated that methyl parathion is the potential substrate of zebrafish MRP4 (Nornberg et al., 2015). Thus, it is worth investigating whether human MRP4 can also interact with these pesticides.
An allosteric interaction between substrates has been suggested for several MRPs. Sulfasalazine, furosemide, indomethacin, and benzbromarone can stimulate the transport of MRP2 substrates (Jemnitz et al., 2010). For MRP4, it was also reported that urate inhibited methotrexate transport but stimulated cGMP transport (Van Aubel et al., 2005). Allopurinol and oxypurinol both markedly stimulated urate transport by MRP4 in MVs (El-Sheikh et al., 2008a), whereas furosemide and hydrochlorothiazide inhibit urate transport (El-Sheikh et al., 2008a). Moreover, GSH is also considered to be a modulator that stimulates the transport of MRP4, as indicated by the multiple binding sites on MRP4. In particular, it was demonstrated that human MPR4 mediated the ATP-dependent efflux of leukotriene B4 (LTB4) and leukotriene D4 in the presence of reduced GSH and S-methyl GSH, whereas leukotriene C4, although structurally very close to LTB4, was not stimulated by GSH (Rius et al., 2008). GSH also stimulates the elimination of bile acids and E217βG (Rius et al., 2006; Zwaan et al., 2013). Alternatively, the transport of GSH can be stimulated by bile acids (Rius et al., 2003, 2006). One study demonstrated that S-methyl GSH stimulated the transport of cholyltaurine much more than GSH, while dithiothretiol (an antioxidant) further elevated the transport rate of cholyltaurine stimulated by GSH (Rius et al., 2003). This study concluded that GSH stimulated MRP4-mediated monoanionic bile salts extrusion by cotransport rather than in GSH conjugates. In addition to the cotransport feature of this modulator, it is also likely to stimulate substrate transport by GSH conjugates. The GSH conjugate of monomethylarsonous acid, MMA(GS)2, was transported by MPR4 MVs, and its concentration-velocity curve fit exactly to that for an allosteric interaction (Banerjee et al., 2014). Interestingly, the MRP4 MV transported monomethylarsonous acid in the presence of GSH at a similar speed (Banerjee et al., 2014). Although some evidence noted the allosteric modulation of MRP4, the exact mechanism including the binding sites, conformation changes, and modulator specificity is largely unknown. In addition to transporting stimulation, MRP4 was also inhibited by a range of drugs or compounds (Table 2). It is important to note that many nonsteroidal anti-inflammatory drugs (NSAIDs) strongly suppress the transport of MRP4 substrates (Reid et al., 2003a,b).
MRP4 Distribution and Organ Toxicity
As a drug transporter, MRP4 has broad tissue localization. To date, MRP4 was found expressed in blood cells, smooth muscle cells, cardiomyocytes, bone cells, fibroblasts, and cancer cells such as leukemia cell lines, lung cancer, pancreatic cancer, and neuroblastoma. Additionally, MRP4 is characterized by dual membrane localization in polarized cell types. In prostate tubuloacinar cells, hepatocytes, gastric epithelium, and the brain blood barrier, MRP4 is localized to the basolateral membrane. However, it is expressed at the apical side of renal proximal tubule cells and the luminal side of brain capillary endothelium. With respect to enterocyte epithelium, MRP4 is localized to both the basolateral and apical membranes. This different membrane localization plays a significant role in drug distribution. First, MRP4 localization at the brain blood barrier can prevent the penetration of oxins and xenobiotics into the brain. As demonstrated in the brain of MRP4 KO mice, the topotecan and adefovir concentrations were much higher than in the wild-type (WT) animals (Leggas et al., 2004; Belinsky et al., 2007). Because MRP4 is localized to the apical side of renal tubules, it can export both endogenous and exogenous substrates into the urine. Under certain conditions, the decline of MRP4 function is tightly connected to renal toxicity. Moreover, in the gastrointestinal tract, MRP4 may participate in oral drug absorption, such as cefadroxil, adefovir, and dasatinib (de Waart et al., 2012). However, its role is much different in the basolateral membrane of hepatocytes, where MRP4 transports drugs and bile acids into the systemic circulation.
MRP4 expressed at physiologic barriers is vital for the pharmacokinetics, pharmacodynamics, tissue accumulation, and toxicities of its substrates. Recently, it was found that MRP4 mediated the transport of toxic arsenic metabolites into hepatic sinusoidal blood (Banerjee et al., 2014). The fact that arsenic elimination occurs predominantly in urine (60%–80%) (Loffredo et al., 2003) also suggests that renal MRP4 mediates the excretion of these exogenous compounds. Due to its role in the elimination of bile acids, the impaired function of MRP4 is detrimental (Rius et al., 2003; Zelcer et al., 2003). Under the condition of impaired canalicular bile salt excretion, hepatic and renal MRP4 can function as an alternative elimination pathway for cholate. However, it is estimated that drugs with a 1% increase in MRP4 inhibition are associated with a 3.1% increased probability of being cholestatic (Köck et al., 2014). Indeed, the inhibition of MRP4 by prescription drugs is a risk factor for the development of cholestatic drug-induced liver injury (Welch et al., 2015).
The role of MRP4 as a transporter of adefovir and TFV, which contain a purine base like cAMP and cGMP, is linked to their renal pharmacokinetics (Imaoka et al., 2007). An increasing number of case reports and investigations have indicated the relationship of these drugs to acute kidney injury, especially proximal tubule dysfunction (Izzedine et al., 2009; Fernandez-Fernandez et al., 2011; Monteiro et al., 2014). One recent in vitro study also showed that the intracellular accumulation of adefovir and its induced cell toxicity were both increased under hypoxic conditions, which caused a decrease in MRP4, MRP5, Na+/H+ exchanger regulatory factor (NHERF)1, and NHERF3 levels (Crean et al., 2014). Interestingly, it has been demonstrated that the coadministration of lopinavir/ritonavir decreased TFV renal clearance by 17.5%, which is supported in the case report of a 51-year-old man with TFV treatment who developed acute tubular necrosis when given a lopinavir/ritonavir dose (James et al., 2004; Kiser et al., 2008b). Moreover, in another case report, a patient under long-term TFV treatment developed severe acute tubular necrosis, precipitated by the sudden start of diclofenac (Morelle et al., 2009). A retrospective analysis further demonstrated that more than half of patients given TFV developed proximal tubular damage shortly after initiating diclofenac treatment (Bickel et al., 2013). In addition, it is known that diclofenac, as well as other NSAIDs, can strongly inhibit MRP4-driven transport (Tian et al., 2005; El-Sheikh et al., 2007). Thus, the available evidence strongly suggests that NSAIDs should be avoided in patients under TFV treatment.
The above data strongly suggest that drug interactions with MRP4 can increase the incidence rate of hepatic or renal toxicity. Moreover, the ABCC4 gene mutations may also contribute to a detrimental outcome in patients.
Pharmacogenomics of MRP4
ABCC4 is a highly polymorphic gene, with more than 800 missense variants discovered. Although the mRNA and protein levels of ABCC4 were highly variable in the human liver, no correlation between the genetic variations and its expression levels was found in an early study (Gradhand et al., 2008). However, the result is inconclusive because of the small sample size and rare frequency of these mutations. Recently, one in vitro study of ABCC4 coding variants demonstrated that two nonsynonymous variants, 559G>T (187G>W) and 1640A>G (487G>E), were significantly linked to the reduced transport ability compared with its WT, as evidenced by the higher intracellular accumulation of azidothymidine and adefovir dipivoxil (Abla et al., 2008). Notably, the 187G>W variant was also associated with a decreased protein level (Abla et al., 2008). Besides these nonsynonymous variants, 2269G>A (757Y>C) polymorphism is recently highlighted because of its importance in thiopurine therapy. This sodium nitroprusside (SNP) causes amino acid substitution from tyrosine to cysteine in the fourth transmembrane helix, reduces the plasma membrane location of its protein, and correlates with increased 6-MP–induced toxicity (Krishnamurthy et al., 2008). It was also observed that patients with the AA or AG genotype had higher intracellular 6-thioguanine nucleotides concentration and were at higher risk of leukopenia (Ban et al., 2010). Moreover, it was shown in the Japanese childhood acute lymphoblastic leukemia (ALL) population that the homozygous variant allele in any of the ABCC4 2269G>A, 912C>A, and 559G>T variants was in correlation with reduced 6-MP dose (Tanaka et al., 2014). More recently, Gao et al. (2015) reported that 559G>T was significantly associated with decreased intraocular pressure in response to latanoprost. However, whether these SNPs reduce protein location or expression and impair the binding ability of MRP4 substrates and the mechanism responsible need further investigation. In addition, many other nonsynonymous polymorphisms, validated by in vitro studies, have substantial influence on MRP4 transport (Fig. 1), whereas the low frequency makes it difficult to verify their clinical significance.
In addition to nonsynonymous mutations, synonymous mutations also have impacts on MRP4 function. 3463A>G variant carriers had an intracellular TFV concentration that was 35% higher than WT patients, although the association between ABCC4 variants and TFV-induced kidney injury was not observed (Kiser et al., 2008a). The 3463G variant has an increased probability of altered mRNA splicing and may potentially alter MRP4 protein expression based on exonic splicing enhancer analyses (Anderson et al., 2006). In accordance with this, slower TVF renal elimination and higher plasma TFV were observed in 3463 G variant carriers than in those with WT alleles. In another study of Thai human immunodeficiency virus–infected patients, it was found that patients carrying the ABCC4 4131 TG or GG genotype had, on average, a 30% higher mean TVF plasma concentration than patients carrying the TT genotype (Rungtivasuwan et al., 2015). However, because of the small number of ABCC4 3463 GG carriers in this study, it may be that this polymorphism was not significantly correlated with TVF plasma concentration, as was also shown in a study of efavirenz (Sánchez et al., 2011). Moreover, ABCC4 1497C>T, as well as the CYP2B6*6 genotype, was identified as the major factors influencing the apparent efavirenz oral clearance (Sánchez et al., 2011). Since several studies have demonstrated the substantial correlation between plasma drug concentrations and efficacy or toxicity in antiretroviral therapy, it is strongly suggested that the genotypes of antiretroviral transporters, including MRP4, are taken into consideration in relation to individual dosage.
Moreover, the nonsynonymous polymorphisms located in the gene promoter and 3′-untranslated region can affect the binding ability of transcriptional factors and miRNAs, which may be involved in gene transcriptional and post-transcriptional regulation. For instance, Ansari et al. (2009) first investigated the promoter haplotype of ABCC4 in vitro and found a higher promoter activity for haplotype *C (containing -1393T>C), which also correlated with a lower methotrexate plasma level and higher event-free survival in children with ALL. Although -1393T>C did not significantly alter MRP4 mRNA levels in lymphoblastoid cell lines, it cannot be excluded that this allele enforces MRP4 expression in kidney, and therefore increases drug extrusion and decreases plasma concentration. However, in a replica of this association study, Brüggemann et al. (2009) did not find any association between -1393T>C and reduced event-free survival in adults with ALL, but did find a nonsignificant oppositional trend compared with that in children with ALL. This discrepancy can probably be attributed to the lower initial methotrexate dose intensity in adults with ALL than in children with ALL. To investigate the function of the polymorphisms in MRP4 3′-untranslated region, Markova and Kroetz (2014) constructed six common MRP4 3′-untranslated region haplotypes of Caucasians, African Americans, and Asians in vitro and compared the luciferase activity of each haplotype. However, none were found to be significantly associated with gene expression level (Markova and Kroetz, 2014).
In addition to single nucleotide polymorphism, copy number variation was also found in the ABCC4 gene through a genome-wide analysis of 1048 northern Chinese Han subjects. This copy number variation caused increased MRP4 level and significantly correlated with the risk of esophageal squamous cell carcinoma, with an odds ratio of 3.36. A subsequent in vitro study demonstrated that MRP4 inhibition in esophageal squamous cell carcinoma cells decreased cell proliferation and motility mainly through COX2–PGE2, cAMP-PKA (protein kinase A), and β-catenin pathways (Sun et al., 2014).
Regulation of MRP4
MRP4 is expressed in numerous tissues but is regulated in a tissue-dependent manner (Fig. 2). In the liver, the expression level of MRP4 is highly variable (Gradhand et al., 2008) and transcriptionally regulated by the constitutive androstane receptor (CAR) (Assem et al., 2004), peroxisome proliferator-activated receptor (PPAR)α (Moffit et al., 2006), NFE2-related factor 2 [nuclear factor (erythroid-derived 2)] (Xu et al., 2010), and aryl hydrocarbon receptor (Xu et al., 2010) via binding with its promoter region. The nuclear receptor CAR translocates into the nucleus after its activation and binds to the ER6 responsive element at the ABCC4 promoter, whereas the heterodimers formed by the activated farnesoid xenobiotic receptor and retinoid X receptor bind to the ER8 element that overlaps with ER6 (Renga et al., 2011). Thus, activation of the farnesoid xenobiotic receptor causes displacement of the CAR and reverses the MRP4 induction of the CAR agonist (Renga et al., 2011). Concordantly, MRP4 induction during cholestasis and primary biliary cirrhosis, presumably via the bilirubin-activated CAR, was even more pronounced in farnesoid xenobiotic receptor KO mice (Schuetz et al., 2001). Also highly expressed in renal cell lines and tissues, PPARα, NFE2-related factor, and aryl hydrocarbon receptor can participate in renal MRP4 regulation. Interestingly, MRP4 is more extensively expressed in the kidneys of female mice (Maher et al., 2005, 2006) and MRP4 expression in gonadectomized mice is downregulated, but upregulated by estrogen stimulation, indicating that renal MRP4 may be controlled by sex hormones and estrogen receptors (Maher et al., 2006). However, the regulation of MRP4 by sex hormones and estrogen receptors has not yet been validated with a promoter activity assay. Interestingly, Gori et al. (2013) demonstrated that endogenous anti-inflammatory lipid lipoxin A4 could suppress MRP4 expression in endometriotic epithelial cells through estrogen receptors and inhibit extracellular prostaglandin E2 (PGE2) release.
As a member of the ABC transporter family, MRP4 is mainly located in polar cell membrane and regulated by internalization. Similar to some other ABC family members (e.g., MRP2 and CFTR), the COOH terminus of MRP4 contains a -ETAL- amino acid sequence, which belongs to the class I PDZ interaction motif [X-(S/T)-X-A, where X denotes unspecified and A denotes hydrophobic residue] and can interact with PDZ-based adapter proteins (Russel et al., 2008). Through this PDZ-PDZ interaction, NHERF1 (Hoque and Cole, 2008) and sorting nexin 27 (SNX27) (Hayashi et al., 2012) promote MRP4 internalization and thereby negatively regulate its cell surface expression, whereas NHERF3 (Park et al., 2014) stabilizes cell surface MRP4 primarily by reducing its internalization. The results from both immunohistochemistry and immunoblotting showed that MRP4 expression was significantly decreased in NHERF3 KO mice. With the ablation of renal MRP4, the efflux of the MRP4 substrate adefovir from renal tubules was also significantly reduced in NHERF3 KO mice (Park et al., 2014). Showing high abundance in the kidneys, NHERF1/3 and SNX27 may compete with each other for binding to the same PDZ sequence and thereby modulate the internalization and recycling of membrane MRP4. Furthermore, NHERF1 is a major determinant of MRP4 trafficking to the apical membranes of mammalian kidney cells, although no in vivo data are yet available (Hoque et al., 2009). Considering the fact that NHERF3 can bind to MRP4 and regulate its location in the tubular brush border membrane, it is very likely that NHERF3 can participate in MRP4 membrane trafficking.
The regulation of MRP4 in cancer cells is much different from that in normal tissues. Indeed, it was reported in prostate cancer that the expression level of MRP4 is 3-fold greater than in normal prostate tissue (Ho et al., 2008). Dihydrotestosterone induced MRP4 expression in both androgen-dependent and -independent LNCaP cells. Similarly, MRP4 levels were significantly decreased in prostate cancers treated with neoadjuvant androgen ablation therapy compared with cancers exposed to normal testosterone levels. However, dihydrotestosterone did not alter the activation of the ABCC4 promotor in luciferase reporter assays (Cai et al., 2007; Ho et al., 2008). c-Myc is a major member of the proto-oncogene Myc family that globally controls multiple cell functions through transcriptional regulation of a target gene network. Several other ABC transporter genes have been demonstrated to be the targets of c-Myc in neuroblastomas, myelogenous leukemia, and breast epithelial cells (Norris et al., 1996, 1997; Kang et al., 2009; Porro et al., 2011). Similarly, MRP4 was demonstrated to be the direct transcriptional target of c-Myc in neuroblastomas (Porro et al., 2010). Recently, Wang et al. (2015) found that multiwalled carbon nanotubes could downregulate MRP4 and P-gp in colon carcinoma through the inhibition of c-Myc. Interestingly, it was also demonstrated that cAMP itself can stimulate the expression of MRP4 through a novel pathway that mediated by exchange proteins activated by cAMP in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells (Bröderdorf et al., 2014). In support of this finding, Carozzo et al. (2015) reported that intracellular cAMP upregulated MRP4 through exchange proteins activated by cAMP 2–mediated and Rap1-mediated mechanisms in pancreatic adenocarcinoma cell lines, whereas extracellular cAMP upregulated MRP4 mainly through a mitogen-activated protein kinase kinase–extracellular signal regulated kinase (MEK/ERK)–mediated pathway. Thus, it is proposed that intracellular cAMP, in order to maintain its cellular homeostasis, can upregulate cAMP-transporting protein MRP4 through this autoregulatory negative feedback.
Additionally, a broad range of ABC family transporters are epigenetically controlled by miRNAs, especially in cancers. For instance, miR-125a, miR-125b, and miR-143 have been found to downregulate MRP4 in hepatocellular carcinoma (Borel et al., 2012). Recently, an in vivo study showed that the MRP4 protein level was also downregulated by miR-124a and miR-506, both of which were negatively correlated with the renal MRP4 level in human kidneys (Markova and Kroetz, 2014).
The expression of MRP4 is regulated at the genetic, transcriptional, and post-transcriptional levels. More importantly, the regulation pathway is distinct among different tissues and diseases, although the specific processes remain largely unclear. Nonetheless, these different regulatory systems provide insights into the modulation of MRP4-mediated transport in different tissues and pathophysiological conditions.
The Action of MRP4 in Pathophysiological Processes
cAMP, cGMP, and prostaglandins are the pivotal endogenous signals that control a variety of physiologic processes. Accordingly, the alterations of these molecules may be responsible for pathophysiological change or disease improvements. Because MRP4 controls the export of the signals and their cellular concentration, it also plays a vital role in many pathophysiological processes.
MRP4 and Cancer.
Recently, a moderate MRP4 expression was found in blast cells of patients with acute myelocytic leukemia (AML), while higher levels of this protein were detected in the less differentiated FAB subtypes M0 and M1 (Guo et al., 2009; Oevermann et al., 2009). Interestingly, MRP4 expression levels decrease with leukocyte differentiation, indicating that the intracellular cAMP concentration contributes to stem cell differentiation (Guo et al., 2009). In support of this, dibutyryl cAMP was shown to induce monocytic differentiation of mouse myeloid leukemia cells and the human promonocytic U937 cell line (Shayo et al., 1997). Furthermore, Shayo et al. (2004) reported that the time course of cAMP signaling was also critical for leukemia U937 cell differentiation. However, U937 cells stimulated by H2 receptor agonists failed to mature despite a robust increase in cAMP, which may be attributed to the rapid desensitization of H2 receptor and phosphodiesterase activation (Lemos Legnazzi et al., 2000; Monczor et al., 2006). MRP4 inhibition induced the differentiation of leukemic stem cells, which are crucial for leukemia initiation, progression, metastasis, and relapse, and one approach to combatting leukemia is to force the cells into differentiation. Moreover, Copsel et al. (2011) reported that MRP4 extrusion is directly involved in the regulation of intracellular cAMP in leukemia cell lines. Pharmacological inhibition or small interfering RNA (siRNA) knockdown of MRP4 led to cell cycle arrest and cell differentiation. Blockade of MRP4 was also shown to play a role in tumor growth and apoptosis in a xenograft AML model (Copsel et al., 2014). However, direct evidence that the MRP4 expression level correlates with the clinical outcome of AML is not yet available.
Additionally, RNA interference (RNAi)–mediated inhibition of MRP4 significantly decreased in vitro proliferation and colony formation in pancreatic cancers (Zhang et al., 2012), lung cancer (Maeng et al., 2014), neuroblastoma (Norris et al., 2005; Henderson et al., 2011), and gastric cancer (Chen et al., 2014). In addition, MRP4 expression levels in cancers are significantly correlated with clinical outcome. Notably, the MRP4 expression level in primary neuroblastoma was strongly associated with reduced progression-free survival, although none of the antitumor agents used in neuroblastoma therapy are extruded by MRP4. Moreover, in ovarian cancer, high expression of MRP4 was also confirmed to play an unfavorable role (Bagnoli et al., 2013). With respect to the prognosis of locally advanced colorectal carcinoma after neoadjuvant radiotherapy, high expression of MRP4 is also considered an independent risk factor (Yu et al., 2014). Moreover, MRP4 KO colorectal carcinoma cells showed aggravated radiation-induced apoptosis (Yu et al., 2014). The role of MRP4 in resistance to irradiation may be related to the enhanced intracellular cAMP accumulation and G1/S phase checkpoint deficiency following irradiation.
Taken together, these results show that MRP4 plays an important role in cancer progression. Because of the transport capacity of both endogenous and exogenous substrates, three independent pathways are presumed to be involved in the role of MRP4 in cancer (Fig. 3):
The cAMP-PKA pathway is the most relevant mechanism to the outcome of MRP4 expression. cAMP, as an important endogenous regulator of cell activities, is extruded extracellularly by MRP4, as a result of its decreased concentration in tumor tissue. cAMP elevations were validated, by many observations, to inhibit cell proliferation in various established cell lines, mostly of tumoral origin, which has become the paradigm of cell cycle regulation (Pastan et al., 1975; Friedman, 1976). The general mechanism mainly involves the inhibition of the ERK pathway by inhibitory phosphorylations of c-Raf by the cAMP-PKA pathway (Dumaz and Marais, 2003; Boutros et al., 2008), which leads to transcriptional repression of proto-oncogenic transcription factors (Cowlen and Eling, 1992), repression of D-type cyclins (cyclin D1 and D3) (Ward et al., 1996; L’Allemain et al., 1997), and accumulation of the cell cycle inhibitor p27kip1 suppressing both CDK4 and CDK2 and thus inactivating phosphorylations of phosphorylation of pRb (Kato et al., 1994; Van Keymeulen et al., 2001; Kuiperij et al., 2005). In macrophages, cAMP elevation induced p27kip1, increased complexes between cyclin D1 and CDK4, impaired the activating T172-phosphorylation of CDK4 by CDK-activating kinase, and caused G1 phase arrest (Kato et al., 1994). Also, it was demonstrated that an increase in intracellular cAMP prevented cell cycle progression from the G1 phase to the S phase in breast cancer (Naviglio et al., 2009), vascular smooth muscle cells (Liu et al., 2014), and lymphoma cells (Zambon et al., 2005). Concordantly, MRP4 blockade led to cell cycle arrest at the G1 phase in cell lines of lung cancer, neuroblastoma, pancreatic cancer, and AML where MRP4 was overexpressed. Notably, downregulation of MRP4 in lung cancer cell lines inhibits pRb, which initiates the cell cycle transition from the G1 phase to the S phase (Maeng et al., 2014).
PGE2 and LTB4 may be involved in the role of MRP4 through an autocrine or paracrine process. COX2–PGE2 signaling is highly activated in numerous cancers, especially lung (Furugen et al., 2013) and colon cancer (Park et al., 2011), and in situations of abundant intracellular accumulation PGE2 can be extruded by MRP4. The continuous elevated extracellular PGE2 can shift the tumor microenvironment from antitumor to immunosuppressive responses, resulting in escape of tumor cells from effective immunosurveillance (Wang and DuBois, 2013). It has been shown that PGE2 can downregulate tumor immunity through inhibiting dendritic cell (DC) differentiation and switching the function of DCs from induction of immunity to T-cell tolerance (Chinen et al., 2011); inhibiting CD8+ T-cell proliferation and activity (Ganapathy et al., 2000); the inhibition of mature B-cell proliferation and induction of immature B-cell apoptosis (Shimozato and Kincade, 1999); downregulation of antitumor TH1 cytokines and upregulation of immunosuppressive TH2 cytokines (Fabricius et al., 2010); and inducing myeloid-derived suppressor cell differentiation (Sinha et al., 2007). More importantly, PGE2 can bind to the EP2/4 receptor and activate the downstream cascades, leading to epithelium stem cell transition, metastasis, antiapoptosis, and proliferation (Rao et al., 2007; Rasmuson et al., 2012; Charo et al., 2013). PGE2 induces the proliferation of colon and lung cancer cells by activating Ras-ERK and glycogen synthase kinase-3β (GSK3β)–β-catenin pathways (Krysan et al., 2005; Wang et al., 2005). In breast cancer, PGE2 can upregulate aromatase production in stromal fat cells, and concomitantly estrogen production, which stimulates neoplastic cell proliferation (Krysan et al., 2005). In addition, PGE2 promotes colon cancer cell survival by activating a PI3K (phosphoinositide 3-kinase)-Akt-PPARδ cascade (Wang et al., 2004) and upregulates Bcl-2, which attributes to PGE2-induced inhibition of apoptosis in colon cancer cells (Sheng et al., 1998). Interestingly, the activation of PI3K-Akt by PGE2 can further inhibit P21 and p27kip1, which have an inhibitory effect on cell cycle (Wang et al., 2009; Li et al., 2010). Leukotrienes are also responsible for carcinogenesis, but to a lesser extent. Although most cancers lack the enzymes necessary for leukotrienes biosynthesis, they can use LTA4 released from immune cells to synthesize LTB4, which is also a substrate of MRP4. For instance, it has been shown that LTB4 levels were increased in human colon and prostate cancers (Dreyling et al., 1986; Larré et al., 2008). The increasing LTB4 can then bind to the BLT1 receptor and activate ERK and PI3k-Akt signaling, resulting in proliferation and cell survival (Tong et al., 2005; Ihara et al., 2007). In particular, LTB4, PGE2 and other proinflammatory factors play a pivotal role in inducing angiogenesis (Wang and Dubois, 2010). They can directly act on epithelial, endothelial, and/or immune cells to induce angiogenic factors, such as vascular endothelial growth factor and fibroblast growth factor 2, and the chemokines CXCL1 and CCL2 (Nakayama et al., 2006; Wang et al., 2006; Battersby et al., 2007; Jain et al., 2008). In transformed epithelial cells, PGE2 induces vascular endothelial growth factor and CXCL1 secretion through an EP2 or EP4 epidermal growth factor/ERK cascade (Ding et al., 2005; Wallace et al., 2009). These secreting proangiogenic factors finally stimulate endothelial cell recruitment, proliferation, migrationm and tubule formation.
The extrusion of toxic substances is also an underlying mechanism responsible for drug resistance and poor clinical outcome. Because MRP4 mediates the efflux of xenobiotics such as methotrexate, topotecan, and the active drug active metabolites SN-38 [(4S)-4,11-diethyl-4,9-dihydroxy-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)dione] and 6-MP, the activity of MRP4 is proportional to the antitumor efficacy or toxicity of these drugs. Moreover, MRP4 expression is also related to the drug resistance of several other antitumor agents that have not been identified as MRP4 substrates. For instance, MRP4 was found upregulated in a cisplatin-resistant gastric cancer cell line, and MRP4 inhibition reversed the cisplatin resistance of these cells (Zhang et al., 2010). In support of this finding, an experiment using an oxaliplatin-resistant ovarian carcinoma cell line displayed a remarkably reduced accumulation of cisplatin and increased expression of MRP4 (Beretta et al., 2010). Similarly, in a doxorubicin-resistant osteosarcoma cell line, both the mRNA and protein levels of MRP4 were increased, which was correlated with a higher cellular doxorubicin concentration (He et al., 2015). A MV uptake experiment should be performed to further test if these drugs serve substrates of MRP4.
It has been shown that MRP4 is predominantly localized in δ-granules and, to a lesser extent, in the plasma membrane of platelets (Jedlitschky et al., 2004). However, in the platelets from δ-granules pool deficiency patients MRP4 localization in δ-granules was decreased, whereas the surface expression was normal or increased (Jedlitschky et al., 2010). In normal δ-granules, MRP4 pumping can trap ADP and other cyclic nucleotides, which are secreted extracellularly, and induce platelet aggregation during platelet activation (Jedlitschky et al., 2004). As a result, ADP-induced aggregation is impaired by MRP4 deficiency or inhibition, which leads to decreased ADP accumulation.
Aspirin is a long-used antiplatelet agent that inhibits the production of thromboxane. Using a dense granules-rich preparation from platelets, it was also shown that aspirin was transported into dense granules-rich preparation in an ATP-dependent manner, and its uptake was reduced by dipyridamole or MK571 preincubation (Mattiello et al., 2011), suggesting that MRP4 mediates aspirin efflux. Moreover, MRP4 expression increased in an aspirin-treated human megakacaryoblastic dami cell line in platelets from aspirin-treated healthy volunteers and in platelets from patients treated for five days with aspirin after coronary artery bypass grafting (Mattiello et al., 2011; Massimi et al., 2014). Intriguingly, the antiaggregation effects of aspirin were reduced in these aspirin-treated patients on the fifth day, at which time aspirin and cAMP were more extensively extruded from platelets, presumably owing to the elevated expression of MRP4 (Mattiello et al., 2011). Platelet MRP4 induction by aspirin itself possibly may lead to the decline in the cellular aspirin concentration and the decrease in the inhibitory effects of platelets during continuous exposure (Fig. 4) (Borgognone and Pulcinelli, 2012). Dipyridamole in combination with aspirin has also been demonstrated to have a favorable role in the secondary prevention of stroke (Rivey et al., 1984; Li et al., 2013), which may be partially because dipyridamole increases the intracellular concentration of aspirin in platelets via MRP4 inhibition (Fig. 4).
In platelets, cAMP and cGMP are largely condensed in δ-granules or extruded outside in correlation with their potential physical function. Platelet aggregation induced by PAR peptides was inhibited by forskolin and nitric oxide–donor SNP, which, activate adenylate cyclase and guanylate cyclase, respectively, and elevated intracellular cAMP and cGMP. MK571 (3-[[[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propanoic acid) pretreatment enhanced these inhibitory effects triggered by forskolin or SNP and increased the intracellular cAMP and cGMP concentrations (Borgognone and Pulcinelli, 2012). The utility of the MRP4 blockade was further evidenced by a recent study showing that MRP4 inhibition suppressed platelet aggregation induced by collagen and prolonged the time required to form a thrombus in the irradiated venules of fuorescein sodium–pretreated mice (Lien et al., 2014). Furthermore, it was found that MRP4-mediated cAMP entrapment in δ-granules was significantly inhibited by the pretreatment with aspirin or salicylic acid, the MRP4 inhibitor (Mattiello et al., 2011). Thus, aspirin, dipyridamole, and other NSAIDs may increase cellular cAMP signals, which can represent a novel mechanism of action of NSAIDs in antiaggregation treatment.
MRP4 Involved in the Cardiovascular System.
It has long been observed that increased cAMP levels inhibit vascular smooth muscle cell proliferation in vitro and reduce neointimal lesion formation after arterial injury in vivo (Southgate and Newby, 1990; Indolfi et al., 1997). Furthermore, Sassi et al. (2008) revealed that MRP4 was upregulated during proliferation of isolated human coronary artery smooth muscle cells and in rat carotid arteries after injury. Moreover, these authors also found that MRP4 inhibition significantly increased cAMP and cGMP levels, sufficiently blocking the proliferation of human vascular smooth muscle cells in vitro and preventing neointimal growth in injured rat carotid arteries. Moreover, cAMP signaling is also vital for retinal neovascularization. Using human retinal microvascular endothelial cells, Tagami et al. (2010) demonstrated that MRP4 knockdown enhanced cell migration, decreased cell apoptosis, and produced a massive tube-like structure, suggesting a proangiogenic effect of MRP4 inhibition. Although the systemic KO of MRP4 in mice showed no overt abnormality in retinal vasculature development, this process was significantly suppressed by forskolin treatment in MRP4 KO mice, but not in WT mice (Matsumiya et al., 2012). It was also shown that continuous hyperoxia stimulation led to a significant increase in the unvascularized retinal area during the neonatal period in MRP4 KO mice.
In addition to angiogenesis, MRP4 also plays a role in cardiomyocyte contraction, which is presumably caused by the CTFR-mediated chloride current (Sellers et al., 2012). Intriguingly, the increase rate in contraction in response to the maximum cAMP level via the blockade of phosphodiesterase 4 is CTFR independent, whereas the effect of MRP4 inhibition on contraction rate is compartmentalized with CTFR, suggesting that the effect of MRP4 inhibition is localized (Sellers et al., 2012). MRP4-deficient mice also displayed enhanced cardiac myocyte cAMP formation and contractility, as well as cardiac hypertrophy (Sassi et al., 2012). Furthermore, MRP4 expression is 3.6-fold higher in senescent compared with young rat myocardium, which is related to the reduced positive inotropic response to β-adrenoceptor stimulation of the senescent heart (Carillion et al., 2015). Although the intracellular cAMP concentrations in young and senescent rat myocardium after β-adrenoceptor stimulation were not compared, the highlighted role of MRP4 in cAMP transport makes its position in the positive inotropic response more substantial.
MRP4-Mediated Other Physiologic Processes.
Over the past 10 years, accumulating evidence has suggested that the cAMP-stimulated pathway plays a beneficial role in acute pancreatitis through the enhanced secretion of activated enzymes from acinar cells (Chaudhuri et al., 2005). Furthermore, the pharmacological inhibition of soluble adenylyl cyclase showed a protective effect and reduced the pathologic activation of proteases during pancreatitis (Kolodecik et al., 2012). However, it was recently demonstrated that the blockade of cAMP extrusion by probenecid or MK571, prior to secretin stimulation, made the pancreas prone to injury in normal rats and aggravated the onset of acute pancreatitis (Ventimiglia et al., 2015). The study also showed that atrial natriuretic factor attenuated the onset of acute pancreatitis presumably by increasing cAMP extrusion through MRP4 (Ventimiglia et al., 2015). The discrepancy between these studies may be related to the time course of intracellular cAMP levels, which were elevated by the pretreatment with atrial natriuretic factor or pharmacological inhibition prior to the induction of acute pancreatitis.
Diarrhea is one of the most common adverse side effects of prescribed drugs and is observed in approximately 7% of treated individuals. Although the mechanism for this effect is not fully understood, the function of MRP4 may be involved in CFTR-mediated secretory diarrhea (Li et al., 2007). In vitro adenosine treatment can stimulate CFTR-mediated chloride currents, which are potentiated by the inhibition of the apical cAMP transporter MRP4. For irinotecan and azidothymidine, which can cause the side effect of diarrhea, it was revealed that they can inhibit the MRP4-mediated cAMP efflux and augment the formation of MRP4-CFTR–containing macromolecular complexes (Moon et al., 2015).
In addition to the aforementioned processes, the contraction of trabecular meshwork cells (Pattabiraman et al., 2013), wound healing (Sinha et al., 2013), and migration of DCs (van de Ven et al., 2008), which involve the function of MRP4, are more or less related to the regulation of cyclic nucleotide signals. There are two canonical pathways through which cAMP and cGMP activate PKA and PKG, respectively, which lead to phosphorylation of their downstream targets and regulation of a variety of cellular activities. With respect to the migration of fibroblast cells, the inhibition of MRP4 elevates cAMP and thereby activates more PKA at or near the leading edge. In the absence of MRP4-mediated cAMP efflux, the augmented PKA activity stimulates actin polymerization likely via VASP phosphorylation or small-GTPase activation and facilitates the formation of restricted and localized structured extensions that initiate cell migration (Sinha et al., 2015). However, there are still several mysteries regarding the role of MRP4 in this process. In one case, fibroblasts from MRP4 KO mice or MK571-treated fibroblasts migrated faster than WT cells, whereas the cAMP-elevating agents forskolin and isobutylmethylxanthine significantly led to a slower migration of mouse embryonic fibroblasts. In another case, pharmacological inhibition of MRP4 or downregulation through RNAi in DCs reduced their migration from human skin explants, as well as that of in vitro–generated Langerhans cells (van de Ven et al., 2008). A highly coordinated balance between these molecules is essential for cell migration: a moderate increase in these cyclic nucleotides stimulates cell migration, whereas a further increase leads to migration inhibition in fibroblasts (Sinha et al., 2013). With regard to different cell types, highly regulated PKA and PKG activities, and therefore a balanced intracellular cyclic nucleotide concentration, are essential for its specific physiologic function.
The Potential of MRP4 as a Drug Target
MRP4 is overexpressed in a number of cancers and contributes to cancer progression and drug resistance as a result of enhanced endogenous signal efflux or cytotoxic drug pumping. In other pathophysiological processes, such as platelet aggregation, intraocular pressure, and wound repair, MRP4 inhibition also shows a desirable effect. Thus, the available evidence strongly suggests that MRP4 has the potential to serve as a drug target.
Most of the MRP4 pharmacological inhibitors have the same effect on other ABC family transport members, such as MRP1, 2, 3, and 5 (Huynh et al., 2012), and hence the use of these inhibitors would produce certain off-target effects. Early clinical trials using inhibitors of P-glycoprotein are in support of the need for selective inhibitors of target transporters (Szakács et al., 2006). In this regard, Cheung et al. (2014) recently identified two chemically distinct small molecules (ceefourin 1 and 2), which inhibit the transport of a broad range of MRP4 substrates and yet are highly selective for MRP4 over the other ABC transporters. Based on the platform of a high-throughput bioluminescence screen, Cheung et al. (2015) identified 36 compounds from a library of Food and Drug Administration–approved drugs that effectively inhibited MRP4. It is noteworthy that three of these compounds are also phosphodiesterase inhibitors.
Over the past decade, a large number of studies have revealed the pivotal role of RNAi in gene silencing, which has tremendous implications in biologic and medical research (Luo et al., 2007). In particular, RNAi therapy holds great promise for the treatment of a variety of diseases, including cancer (Battistella and Marsden, 2015). Since 2004, 26 different siRNA drugs have entered into clinical trials for the treatment of a variety of diseases involving macular degeneration, diabetic retinopathy, acute renal failure, Ebola virus infection, and cancers (Ozcan et al., 2015). Because substantial evidence has suggested that RNAi can block the function of MRP4 and produce promising pharmacotherapeutic effects, it is worth introducing MRP4 siRNA into clinical trials.
The systemic injection of MRP4 inhibitors or siRNAs inevitably leads to their accumulation in nontarget tissues and organs, generating off-target effects or side effects. Although the systemic KO of MRP4 does not show any obvious abnormality in mice, the side effects of human MRP4 inhibition should be carefully addressed, especially in populations with coagulation disorders, secretory diarrhea, and cholestasis, and those receiving MRP4 substrate drugs. However, rational design strategies, chemical modification, and a wise delivery system can substantially improve these problems including off-targets effects, side effects, and inferior pharmacokinetic characteristics. Recently, neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine–based nanoliposomes incorporating siRNAs against a target gene were shown to be active in orthotopic and subcutaneous xenograft models of various tumors without any detectable distress, toxicity, or immune response (Landen et al., 2005; Halder et al., 2006; Gray et al., 2008). Additionally, the delivery of siRNAs in vivo into tumors was 10- and 30-fold more effective than that using cationic liposomes and naked siRNAs, respectively (Landen et al., 2005; Halder et al., 2006). Moreover, locally delivered siRNA has also been successfully used in the eyes, respiratory system, and pancreatic cancer (Zamora et al., 2011; Zorde Khvalevsky et al., 2013; Martínez et al., 2014), suggesting further potential for MRP4 siRNA.
An increasing body of evidence points to the role of MRP4 in drug pharmacokinetics and pathophysiological processes. As a transporter expressed in multiple tissues, MRP4 participates in the distribution of various types of drugs. Drug interactions with MRP4 and its polymorphisms can alter the pharmacokinetics of MRP4 substrates and have an impact on drug responses. Furthermore, MRP4 is in a position to pump both endogenous signals and exogenous drugs, which can impact cancer progression and drug resistance. Other pathophysiological processes including platelet aggregation, retinal neovascularization, fibroblast migration, and CFTR-mediated secretory diarrhea are all partially related to the transport activity of MRP4.
Although in vitro studies have elucidated the insightful function of MRP4 in many physiologic processes, especially in cancer treatment, more definitive proof from both in vivo and clinical studies should be provided to substantiate the role of MRP4. Additionally, the distinctive regulation of MRP4 in different organs and disease circumstances merits further investigation. Subsequently, significant efforts should be made to develop unique drugs targeted to MRP4. Presently, MRP4 selective pharmacological inhibitors and siRNAs represent two drug forms with great promise. Moreover, tissue-specific control of MRP4 expression and function could serve as a novel strategy for targeted therapy.
The authors thank Prof. Zhaoqian Liu and Xiaoping Chen for the kind and careful guidance and education and offer special thanks to Dan Wang, Fazhong He, Lihua Zhang, Zhipeng Wen, Guojin Liu, and Meizi Zeng.
Wrote or contributed to the writing of the manuscript: Wen, Luo, Huang, Tang, Zhou, Zhang.
- Received May 3, 2015.
- Accepted June 30, 2015.
This study was supported by the National Natural Scientific Foundation of China [81273595, 81202594, and 81001445]; the “863” Project [2012AA02A518]; and the Natural Scientific Foundation of Hunan [11K073 and 10JJ4020].
- ATP-binding cassette
- acute lymphoblastic leukemia
- acute myelocytic leukemia
- constitutive androstane receptor
- cystic fibrosis transmembrane conductance regulator
- dendritic cell
- extracellular signal regulated kinase
- leukotriene B4
- MK571 (3-[[[3-[(1E)-2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propanoic acid
- multidrug-resistant protein
- membrane vesicle
- Na+/H+ exchanger regulatory factor
- nonsteroidal anti-inflammatory drug
- prostaglandin E2
- phosphoinositide 3-kinase
- protein kinase A
- peroxisome proliferator-activated receptor
- RNA interference
- small interfering RNA
- sodium nitroprusside
- wild type
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics