ABCC10, ABCC11, and ABCC12 Kruh, Gary D; Guo, Yanping; Hopper-Borge, Elizabeth ...
Pflügers Archiv,
02/2007, Volume:
453, Issue:
5
Journal Article
Peer reviewed
Open access
Multidrug resistance protein (MRP)7, MRP8, and MRP9 (gene symbols ABCC10, ABCC11, and ABCC12) are recently identified members of the MRP family that are at relatively early stages of investigation. ...Of these proteins, a physiological function has only been established for MRP8, for which a single nucleotide polymorphism determines wet vs dry earwax type. MRP7 and MRP8 are lipophilic anion pumps that are able to confer resistance to chemotherapeutic agents. MRP7 is competent in the transport of the glucuronide E(2)17betaG, and its resistance profile, which includes several natural product anticancer agents, is distinguished by the taxane docetaxel. MRP8 is able to transport a diverse range of lipophilic anions, including cyclic nucleotides, E(2)17betaG, steroid sulfates such as dehydroepiandrosterone (DHEAS) and E(1)S, glutathione conjugates such as leukotriene C4 and dinitrophenyl-S-glutathione, and monoanionic bile acids. However, the constituent of earwax that is susceptible to transport by MRP8 has not been identified. MRP8 has complex interactions with its substrates, as indicated by the nonreciprocal ability of DHEAS to stimulate E(2)17betaG transport. Similar to the case for other MRPs that possess only two membrane spanning domains (MRP4 and MRP5), MRP8 is a cyclic nucleotide efflux pump that is able to confer resistance to nucleoside-based agents, such as PMEA and 5FU. The functional characteristics of MRP9 are currently unknown.
The majority of gastrointestinal stromal tumor (GIST) patients develop resistance to the first-line KIT inhibitor, imatinib mesylate (IM), through acquisition of secondary mutations in KIT or bypass ...signaling pathway activation. In addition to KIT, AKT is a relevant target for inhibition, since the PI3K/AKT pathway is crucial for IM-resistant GIST survival. We evaluated the activity of a novel pan-AKT inhibitor, MK-4440 (formerly ARQ 751), as monotherapy and in combination with IM in GIST cell lines and preclinical models with varying IM sensitivities. Dual inhibition of KIT and AKT demonstrated synergistic effects in IM-sensitive and -resistant GIST cell lines. Proteomic analyses revealed upregulation of the tumor suppressor, PDCD4, in combination treated cells. Enhanced PDCD4 expression correlated to increased cell death. In vivo studies revealed superior efficacy of MK-4440/IM combination in an IM-sensitive preclinical model of GIST compared with either single agent. The combination demonstrated limited efficacy in two IM-resistant models, including a GIST patient-derived xenograft model possessing an exon 9 KIT mutation. These studies provide strong rationale for further use of AKT inhibition in combination with IM in primary GIST; however, alternative agents will need to be tested in combination with AKT inhibition in the resistant setting.
Mutations in human multidrug resistance protein 6 (MRP6, ABCC6), a member of the MRP family of drug efflux pumps, are the genetic basis of Pseudoxanthoma elasticum, a disease that affects elastin ...fibers in the skin, retina, and blood vessels. However, little is known about the functional characteristics of the protein, including its potential activity as a resistance factor for anticancer agents. Here, we report the results of investigations of the in vitro transport properties and drug resistance activity of MRP6. Using membrane vesicles prepared from Chinese hamster ovary cells transfected with MRP6 expression vector, it is shown that expression of MRP6 is specifically associated with the MgATP-dependent transport of the glutathione S-conjugates leukotriene C(4) and S-(2, 4-dinitrophenyl)glutathione and the cyclopentapeptide BQ123 but not glucuronate conjugates such as 17beta-estradiol 17-(beta-D-glucuronide). Analysis of the drug sensitivity of MRP6-transfected cells revealed low levels of resistance to several natural product agents, including etoposide, teniposide, doxorubicin, and daunorubicin. These results indicate that MRP6 is a glutathione conjugate pump that is able to confer low levels of resistance to certain anticancer agents.
Multidrug resistance protein 3 (MRP3) is an ATP-binding cassette transporter that is able to confer resistance to anticancer agents such as etoposide and to transport lipophilic anions such as bile ...acids and glucuronides. These capabilities, along with the induction of the MRP3 protein on hepatocyte sinusoidal membranes in cholestasis and the expression of MRP3 in enterocytes, have led to the hypotheses that MRP3 may function in the body to protect normal tissues from etoposide, to protect cholestatic hepatocytes from endobiotics, and to facilitate bile-acid reclamation from the gut. To elucidate the role of Mrp3 in these processes, the Mrp3 gene (Abcc3) was disrupted by homologous recombination. Homozygous null animals were healthy and physically indistinguishable from wild-type mice. Mrp3(-/-) mice did not exhibit enhanced lethality to etoposide phosphate, although an analysis of transfected human embryonic kidney 293 cells indicated that the potency of murine Mrp3 toward etoposide ( approximately 2.0- to 2.5-fold) is comparable with that of human MRP3. After induction of cholestasis by bile duct ligation, Mrp3(-/-) mice had 1.5-fold higher levels of liver bile acids and 3.1-fold lower levels of serum bilirubin glucuronide compared with ligated wild-type mice, whereas significant differences were not observed between the respective sham-operated mice. Bile acid excretion, pool size, and fractional turnover rates were similar in Mrp3(-/-) and wild-type mice. We conclude that Mrp3 functions as an alternative route for the export of bile acids and glucuronides from cholestatic hepatocytes, that the pump does not play a major role in the enterohepatic circulation of bile acids and that the lack of chemosensitivity is probably attributable to functional redundancy with other pumps.
We previously determined that expression of human multidrug resistance protein (MRP) 8, a recently described member of the MRP family of ATP-binding cassette transporters, enhances cellular extrusion ...of cyclic nucleotides and confers resistance to nucleotide analogs (J Biol Chem 278:29509-29514, 2003). However, the in vitro transport characteristics of the pump have not been determined. In this study, the substrate selectivity and biochemical activity of MRP8 is investigated using membrane vesicles prepared from LLC-PK1 cells transfected with MRP8 expression vector. Expression of MRP8 is shown to stimulate the ATP-dependent uptake of a range of physiological and synthetic lipophilic anions, including the glutathione S-conjugates leukotriene C4 and dinitrophenyl S-glutathione, steroid sulfates such as dehydroepiandrosterone 3-sulfate (DHEAS) and estrone 3-sulfate, glucuronides such as estradiol 17-beta-D-glucuronide (E(2)17betaG), the monoanionic bile acids glycocholate and taurocholate, and methotrexate. In addition, MRP8 is competent in the in vitro transport of cAMP and cGMP, in accord with the results of our previously reported cellular studies. DHEAS, E(2)17betaG, and methotrexate were transported with K(m) and V(max) values of 13.0 +/- 0.8 microM and 34.9 +/- 9.5 pmol/mg/min, 62.9 +/- 12 microM and 62.0 +/- 5.2 pmol/mg/min, and 957 +/- 28 microM and 317 +/- 17 pmol/mg/min, respectively. Based upon the stimulatory action of DHEAS on uptake of E(2)17betaG, the attenuation of this effect at high DHEAS concentrations and the lack of reciprocal promotion of DHEAS uptake by E(2)17betaG, a model involving nonreciprocal constructive interactions between some transport substrates is invoked. These results suggest that MRP8 participates in physiological processes involving bile acids, conjugated steroids, and cyclic nucleotides and indicate that the pump has complex interactions with its substrates.
The MRP family is composed of nine transporters, at least eight of which are lipophilic anion transporters that are capable of conferring resistance to various anticancer agents. Recently, mice with ...gene disruptions in Mrp2, Mrp3 and Mrp4 have been developed. This review will discuss insights into the physiological and pharmacological functions of Mrp2, Mrp3 and Mrp4 afforded by investigations of these new mouse models.
Recently, we reported that the ATP-binding cassette transporter 10 (ABCC10), also known as multidrug resistance protein 7 (MRP7), is able to confer resistance to a variety of anticancer agents, ...including taxanes. However, the in vivo functions of the pump have not been determined to any extent. In this study, we generated and analyzed Abcc10(-/-) mice to investigate the ability of Abcc10 to function as an endogenous resistance factor. Mouse embryo fibroblasts derived from Abcc10(-/-) mice were hypersensitive to docetaxel, paclitaxel, vincristine, and cytarabine (Ara-C) and exhibited increased cellular drug accumulation, relative to wild-type controls. Abcc10(-/-) null mice treated with paclitaxel exhibited increased lethality associated with neutropenia and marked bone marrow toxicity. In addition, toxicity in spleen and thymus was evident. These findings indicate that Abcc10 is dispensable for health and viability and that it is an endogenous resistance factor for taxanes, other natural product agents, and nucleoside analogues. This is the first demonstration that an ATP-binding cassette transporter other than P-glycoprotein can affect in vivo tissue sensitivity toward taxanes.
We have recently determined that human multidrug resistance protein (MRP) 3, which confers resistance to certain natural product agents and methotrexate (MTX), is competent in the MgATP-energized ...transport of MTX and the monoanionic bile constituent glycocholate as well as several glutathione and glucuronate conjugates. Of these capabilities, the facility of MRP3 in conferring resistance to and mediating the transport of MTX is of particular interest because it raises the possibility that this pump is a component of the previously described cellular efflux system for this antimetabolite. However, if this is to be the case, a critical property of cellular MTX efflux that must be addressed is its ability to mediate the export of MTX but not that of its intracellular polyglutamylated derivatives. Here we examine the role of MRP3 in these and related processes by determining the selectivity of this transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, we show that MRP3 is not only active in the transport of MTX but is also active in the transport the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin) and that polyglutamylation of MTX abolishes transport. Both FA and leucovorin are subject to high-capacity (V(max(FA)), 1.71 +/- 0.05 nmol/mg/min; V(max(leucovorin)), 3.63 +/- 1.20 nmol/mg/min), low-affinity (K(m(FA)), 1.96 +/- 0.13 mM; K(m(leucovorin)), 1.74 +/- 0.65 mM) transport by MRP3. Addition of a single glutamyl residue to MTX is sufficient to diminish transport by >95%. We also show that polyglutamylation similarly affects the capacity of MRP1 to transport MTX and that physiological folates are also subject to MgATP-stimulated transport by MRP1. On the basis of the capacity to transport MTX but not MTX-Glu(2), it is concluded that MRP3 and MRP1 represent components of the previously described cellular efflux system for MTX. The capacity of MRP3 to transport folates indicates that it may reduce intracellular levels of these compounds and thereby indirectly influence antifolate cytotoxicity, and it also implies that this pump may play a role in the response to chemotherapeutic regimens in which leucovorin is a component.
Despite initial efficacy of imatinib mesylate in most gastrointestinal stromal tumor (GIST) patients, many experience primary/secondary drug resistance. Therefore, clinical management of GIST may ...benefit from further molecular characterization of tumors before and after imatinib mesylate treatment. As part of a recent phase II trial of neoadjuvant/adjuvant imatinib mesylate treatment for advanced primary and recurrent operable GISTs (Radiation Therapy Oncology Group S0132), gene expression profiling using oligonucleotide microarrays was done on tumor samples obtained before and after imatinib mesylate therapy. Patients were classified according to changes in tumor size after treatment based on computed tomography scan measurements. Gene profiling data were evaluated with Statistical Analysis of Microarrays to identify differentially expressed genes (in pretreatment GIST samples). Based on Statistical Analysis of Microarrays False Discovery Rate (FDR), 10%, 38 genes were expressed at significantly lower levels in the pretreatment biopsy samples from tumors that significantly responded to 8 to 12 weeks of imatinib mesylate, that is, >25% tumor reduction. Eighteen of these genes encoded Krüppel-associated box (KRAB) domain containing zinc finger (ZNF) transcriptional repressors. Importantly, 10 KRAB-ZNF genes mapped to a single locus on chromosome 19p, and a subset predicted likely response to imatinib mesylate-based therapy in a naïve panel of GIST. Furthermore, we found that modifying expression of genes within this predictive signature can enhance the sensitivity of GIST cells to imatinib mesylate. Using clinical pretreatment biopsy samples from a prospective neoadjuvant phase II trial, we have identified a gene signature that includes KRAB-ZNF 91 subfamily members that may be both predictive of and functionally associated with likely response to short-term imatinib mesylate treatment.
Management of gastrointestinal stromal tumors (GISTs) has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of RTK inhibitors in advanced ...disease. Stratification of GISTs into molecularly defined subsets provides insight into clinical behavior and response to approved targeted therapies. Although these RTK inhibitors are effective in most GISTs, resistance remains a significant clinical problem. Development of effective treatment strategies for refractory GISTs requires identification of novel targets to provide additional therapeutic options. Global kinome profiling has the potential to identify critical signaling networks and reveal protein kinases essential in GISTs. Using multiplexed inhibitor beads and mass spectrometry, we explored the majority of the kinome in GIST specimens from the 3 most common molecular subtypes (KIT mutant, PDGFRA mutant, and succinate dehydrogenase deficient) to identify kinase targets. Kinome profiling with loss-of-function assays identified an important role for G2/M tyrosine kinase, Wee1, in GIST cell survival. In vitro and in vivo studies revealed significant efficacy of MK-1775 (Wee1 inhibitor) in combination with avapritinib in KIT mutant and PDGFRA mutant GIST cell lines as well as notable efficacy of MK-1775 as a monotherapy in the engineered PDGFRA mutant line. These studies provide strong preclinical justification for the use of MK-1775 in GIST.
PDF
