Prostaglandins are involved in a wide variety of physiological and pathophysiological processes, but the mechanism of prostaglandin release from cells is not completely understood. Although poorly membrane permeable, prostaglandins are believed to exit cells by passive diffusion. We have investigated the interaction between prostaglandins and members of the ATP-binding cassette (ABC) transporter ABCC [multidrug resistance protein (MRP)] family of membrane export pumps. In inside-out membrane vesicles derived from insect cells or HEK293 cells, MRP4 catalyzed the time-and ATP-dependent uptake of prostaglandin E 1 (PGE1) and PGE2. In contrast, MRP1, MRP2, MRP3, and MRP5 did not transport PGE 1 or PGE 2. The MRP4-mediated transport of PGE1 and PGE2 displayed saturation kinetics, with K m values of 2.1 and 3.4 M, respectively. Further studies showed that PGF 1␣, PGF2␣, PGA1, and thromboxane B 2 were high-affinity inhibitors (and therefore presumably substrates) of MRP4. Furthermore, several nonsteroidal antiinflammatory drugs were potent inhibitors of MRP4 at concentrations that did not inhibit MRP1. In cells expressing the prostaglandin transporter PGT, the steady-state accumulation of PGE 1 and PGE2 was reduced proportional to MRP4 expression. Inhibition of MRP4 by an MRP4-specific RNA interference construct or by indomethacin reversed this accumulation deficit. Together, these data suggest that MRP4 can release prostaglandins from cells, and that, in addition to inhibiting prostaglandin synthesis, some nonsteroidal antiinflammatory drugs might also act by inhibiting this release. P rostaglandins are key mediators in the regulation of many physiological processes. They are involved in inflammatory responses and tumorigenesis, and their synthesis and metabolism are tightly regulated (1). The first step in prostaglandin synthesis is the production of arachidonic acid, which is released from membrane lipid primarily by cytosolic phospholipase A 2 (1). Arachidonic acid is then oxidized to the intermediate prostaglandin H 2 (PGH 2 ) by PGH synthases, also known as cyclooxygenase (COX)-1 and -2, and the recently identified COX-3 (2). These enzymes are known clinically as the targets of aspirin and other nonsteroidal antiinf lammatory drugs (NSAIDs) (3). Moreover, several recent studies have shown a link between COX-2 expression and carcinogenesis. Prostaglandins are overproduced by a variety of tumors, leading to the suggested prophylactic use of COX-2 inhibitors to decrease the incidence of colon cancer (4, 5). After COX-mediated synthesis, PGH 2 is further converted by tissue-specific prostaglandin synthases into PGE 2 , PGF 2␣ , PGD 2 , prostacyclin, or thromboxane B 2 , the biologically active molecules (1).Prostaglandins are formed and secreted by most cells, and act as autocrine-or paracrine-signaling molecules. In many cases they exert their effects extracellularly via interaction with a family of G protein-coupled membrane receptors (reviewed in ref. 6), although some prostaglandins interact with the nuclear horm...
We have studied in vivo responses of ''spontaneous'' Brca1-and p53-deficient mammary tumors arising in conditional mouse mutants to treatment with doxorubicin, docetaxel, or cisplatin. Like human tumors, the response of individual mouse tumors varies, but eventually they all become resistant to the maximum tolerable dose of doxorubicin or docetaxel. The tumors also respond well to cisplatin but do not become resistant, even after multiple treatments in which tumors appear to regrow from a small fraction of surviving cells. Classical biochemical resistance mechanisms, such as up-regulated drug transporters, appear to be responsible for doxorubicin resistance, rather than alterations in drug-damage effector pathways. Our results underline the promise of these mouse tumors for the study of tumor-initiating cells and of drug therapy of human cancer.multidrug resistance ͉ P-glycoprotein ͉ cancer stem cells
Cancer-associated systemic inflammation is strongly linked with poor disease outcome in cancer patients 1,2. For most human epithelial tumour types, high systemic neutrophil-tolymphocyte ratios are associated with poor overall survival 3 , and experimental studies have demonstrated a causal relationship between neutrophils and metastasis 4,5. However, the cancer cell-intrinsic mechanisms dictating the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Using a panel of 16 distinct genetically engineered mouse models (GEMMs) for breast cancer, we have uncovered a novel role for cancer cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, p53 loss in cancer cells induced secretion of Wnt ligands that stimulate IL-1β production by tumour-associated macrophages, which drives systemic inflammation. Pharmacological and genetic blockade of Wnt secretion in p53-null cancer cells reverses IL-1β expression by macrophages and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a novel mechanistic link between loss of p53 in cancer cells, Wnt ligand secretion and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for cancer patients. 4 Main text To determine how pro-metastatic systemic inflammation is influenced by genetic aberrations in tumours, we studied 16 GEMMs for breast cancer carrying different tissue-specific mutations. These GEMMs represent most subtypes of human breast cancer, including ductal and lobular carcinoma, oestrogen receptor-positive (luminal A), HER2 + , triple-negative and basal-like breast cancer. Because we and others have demonstrated that neutrophils expand systemically and promote metastasis 5-10 , we evaluated circulating neutrophil levels as a marker for systemic inflammation in mammary tumour-bearing mice with end-stage disease. As expected, most tumour-bearing mice displayed an increase in circulating neutrophils as compared to non-tumour-bearing animals (wild-type [WT]) (Fig. 1a). Like the inter-patient heterogeneity in systemic inflammation in human breast cancer 11 , we observed a striking variability in the extent of neutrophilia between the different tumour-bearing GEMMs (Fig. 1a, Extended Data Fig. 1a). We found that the models exhibiting high neutrophil expansion displayed a subset of neutrophils expressing the stem cell marker cKIT (Fig. 1b), indicative of an immature neutrophil phenotype 5. We subsequently searched for commonalities and differences among the 16 GEMMs with regards to high versus low systemic neutrophil levels. Strikingly, mice bearing tumours with a p53 deletion exhibited the most pronounced circulating neutrophil levels (Fig. 1a). The difference in magnitude of systemic inflammation between p53proficient and p...
Human multidrug-resistance protein (MRP) 4 transports cyclic nucleotides and when overproduced in mammalian cells mediates resistance to some nucleoside analogues. Recently, it has been shown that Mrp4 is induced in the livers of Fxr ((-/-)) mice, which have increased levels of serum bile acids. Since MRP4, like MRP1-3, also mediates transport of a model steroid conjugate substrate, oestradiol 17-beta-D-glucuronide (E(2)17betaG), we tested whether MRP4 may be involved in the transport of steroid and bile acid conjugates. Bile salts, especially sulphated derivatives, and cholestatic oestrogens inhibited the MRP4-mediated transport of E(2)17betaG. Inhibition by oestradiol 3,17-disulphate and taurolithocholate 3-sulphate was competitive, suggesting that these compounds are MRP4 substrates. Furthermore, we found that MRP4 transports dehydroepiandrosterone 3-sulphate (DHEAS), the most abundant circulating steroid in humans, which is made in the adrenal gland. The ATP-dependent transport of DHEAS by MRP4 showed saturable kinetics with K (m) and V (max) values of 2 microM and 45 pmol/mg per min, respectively (at 27 degrees C). We further studied the possible involvement of other members of the MRP family of transporters in the transport of DHEAS. We found that MRP1 transports DHEAS in a glutathione-dependent manner and exhibits K (m) and V (max) values of 5 microM and 73 pmol/mg per min, respectively (at 27 degrees C). No transport of DHEAS was observed in membrane vesicles containing MRP2 or MRP3. Our findings suggest a physiological role for MRP1 and MRP4 in DHEAS transport and an involvement of MRP4 in transport of conjugated steroids and bile acids.
Hereditary breast cancers are frequently caused by germline BRCA1 mutations. The BRCA1(C61G) mutation in the BRCA1 RING domain is a common pathogenic missense variant, which reduces BRCA1/BARD1 heterodimerization and abrogates its ubiquitin ligase activity. To investigate the role of BRCA1 RING function in tumor suppression and therapy response, we introduced the Brca1(C61G) mutation in a conditional mouse model for BRCA1-associated breast cancer. In contrast to BRCA1-deficient mammary carcinomas, tumors carrying the Brca1(C61G) mutation responded poorly to platinum drugs and PARP inhibition and rapidly developed resistance while retaining the Brca1(C61G) mutation. These findings point to hypomorphic activity of the BRCA1-C61G protein that, although unable to prevent tumor development, affects response to therapy.
Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo. Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance.
Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3,5-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4-and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20 -600 M, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A 1 and E 1 , the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterasemediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.The cyclic nucleotides cAMP and cGMP are major second messengers in cells. cGMP is formed from GTP by the action of guanylate cyclase (GC) 1 and cAMP from ATP by adenylate cyclase (AC). Both GC and AC can be activated by a variety of intracellular and extracellular stimuli. GC is stimulated by nitric oxide (NO), which leads to a build up of cGMP in the cell, whereas AC is activated by the binding of ligands to membrane receptors (1). Phosphodiesterases (PDEs) hydrolyze cGMP and cAMP to 5Ј-GMP and 5Ј-AMP, respectively, returning cyclic nucleotide concentrations to basal levels and making the cell susceptible to a new stimulus. The efflux of cyclic nucleotides, which has been observed in tissues, might also influence intracellular concentrations (2-10). Efflux is most prominent when cells or tissues are incubated with a GC stimulator, such as the nitric oxide (NO) releasing compound sodium nitroprusside (SNP), in combination with a PDE inhibitor, such as 3-isobutyl-1-methylxanthine (IBMX). These results suggest that efflux may serve as an alternative mechanism of cyclic nucleotide reduction under conditions where PDE activity is insufficient, for instance under conditions of PDE saturation or inhibition. Tight regulation of intracellular cyclic nucleotide concen...
Mercaptopurines have been used as anticancer agents for more than 40 years, and most acute lymphoblastic leukemias are treated with 6-mercaptopurine (6MP) or 6-thioguanine (TG). Overexpression of the two related multidrug resistance proteins MRP4 and MRP5 has been shown to confer some resistance against mercaptopurines, which has been attributed to extrusion of mercaptopurine metabolites by these transporters. We have analyzed the mercaptopurine metabolites formed in human embryonic kidney cells and determined which metabolites are extruded by MRP4 and MRP5. Incubation with 6MP led to the formation of thioinosine and thioxanthosine metabolites and we found that thio-IMP was transported by both MRP4 and MRP5; MRP5 showed the highest transport rate. In contrast, only MRP5 transported thioxanthosine monophosphate (tXMP). During incubation with TG, the monophosphorylated form of thioguanosine was transported by both MRP4 and MRP5; the highest transport rate was for MRP4. Similarly, only 6-methylthio-IMP was formed during incubation with 6-methyl mercaptopurine riboside. This compound was a substrate for both MRP4 and MRP5; MRP4 showed the highest transport rate. Our results show that all major thiopurine monophosphates important in the efficacy of mercaptopurine treatment are transported by MRP4 and MRP5, although the substrate specificity of the two transporters differs in detail.
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