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In addition to their role in drug resistance, the ATP-binding cassette (ABC) transporters ABCG2 and ABCB1 have been suggested to protect cells from a broad range of substances that may foster tumorigenesis. Phytoestrogens or their metabolites are substrates of these transporters and the influence of these compounds on breast cancer development is controversial. Estrogen-like properties might accelerate tumorigenesis on the one hand, whereas their proposed health-protective properties might antagonize tumorigenesis on the other. To address this issue, we used a newer generation mouse model of BRCA1-mutated breast cancer and examined tumor latency in K14cre;Brca1F/F; p53F/F, Abcb1a/b−/−;K14cre;Brca1F/F; p53F/F, or Abcg2−/−;K14cre;Brca1F/F; p53F/F animals, fed with genistein- or resveratrol-supplemented diets. Ovariectomized K14cre;Brca1F/F; p53F/F animals were included to evaluate whether any estrogen-mimicking effects can restore mammary tumor development in the absence of endogenous estrogens. Compared with the ABC transporter proficient model, ABCG2-deficient animals showed a reduced median tumor latency of 17.5 days (P < 0.001), whereas no significant difference was observed for ABCB1-deficient animals. Neither genistein nor resveratrol altered this latency reduction in Abcg2−/−;K14cre;Brca1F/F; p53F/F animals. Ovariectomy resulted in nearly complete loss of mammary tumor development, which was not restored by genistein or resveratrol. Our results show that ABCG2 contributes to the protection of genetically instable epithelial cells against carcinogenesis. Diets containing high levels of genistein or resveratrol had no effect on mammary tumorigenesis, whether mice were lacking ABCG2 or not. Because genistein and resveratrol only delayed skin tumor development of ovariectomized animals, we conclude that these phytoestrogens are no effective modulators of mammary tumor development in our mouse model. Cancer Prev Res; 5(8); 1053–60. ©2012 AACR.

PDF file - 28K, Influence of ovariectomy on skin tumor incidence of K14cre;Brca1F/F;p53F/F females, fed with three special diets

Overview of the sequenced tumor samples, including sample IDs, treatment information and the type of transplanted material.

Supplementary Data from Impact of Abcc2 (Mrp2) and Abcc3 (Mrp3) on the In vivo Elimination of Methotrexate and its Main Toxic Metabolite 7-hydroxymethotrexate

Supplementary Table 3 from Abcc2 (Mrp2), Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo

Supplementary Table 1 from Abcc2 (Mrp2), Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo

The multidrug transporters ABCC2, ABCC3, and ABCG2 can eliminate potentially toxic compounds from the body and have overlapping substrate specificities. To investigate the overlapping functions of Abcc2, Abcc3, and Abcg2 in vivo, we generated and characterized Abcc3;Abcg2−/− and Abcc2;Abcc3;Abcg2−/− mice. We subsequently analyzed the relative impact of these transport proteins on the pharmacokinetics of the anticancer drug methotrexate (MTX) and its main, toxic, metabolite 7-hydroxymethotrexate (7OH-MTX) after i.v. administration of MTX (50 mg/kg). Whereas in single and double knockout mice, the plasma and liver concentrations of MTX and 7OH-MTX decreased rapidly after MTX administration, in the Abcc2;Abcc3;Abcg2−/− mice, they remained very high. One hour after administration, 67% of the MTX dose was still present in livers of Abcc2;Abcc3;Abcg2−/− mice as MTX or 7OH-MTX versus 7% in wild-type, showing dramatic liver accumulation of these toxic compounds when Abcc2, Abcc3, and Abcg2 were all absent. Furthermore, the urinary and fecal excretion of the nephrotoxic metabolite 7OH-MTX were increased 27- and 7-fold, respectively, in Abcc2;Abcc3;Abcg2−/− mice. Thus, Abcc2, Abcc3, and Abcg2 together mediate the rapid elimination of MTX and 7OH-MTX after i.v. administration and can to a large extent compensate for each other's absence. This may explain why it is still comparatively safe to use a toxic drug such as MTX in the clinic, as the risk of highly increased toxicity due to dysfunctioning of ABCC2, ABCC3, or ABCG2 alone is limited. Nevertheless, cotreatment with possible inhibitors of ABCC2, ABCC3, and ABCG2 should be done with utmost caution when treating patients with methotrexate. [Mol Cancer Ther 2009;8(12):3350–9]

We have studied the potential contribution of ABCG2 (breast cancer resistance protein) to resistance to nucleoside analogues. In cells transfected with DNA constructs resulting in overexpression of human or mouse ABCG2, we found resistance against cladribine, clofarabine, fludarabine, 6-mercaptopurine, and 6-mercaptopurine riboside in both MDCKII and HEK293 cells and against gemcitabine only in HEK293 cells. With Transwell studies in MDCK cells and transport experiments with vesicles from Sf9 and HEK293 cells, we show that ABCG2 is able to transport not only the nucleotide CdAMP, like several other ATP-binding cassette transporters of the ABCC (multidrug resistance protein) family, but also the nucleoside cladribine itself. Expression of ABCG2 in cells results in a substantial decrease of intracellular CdATP, explaining the resistance against cladribine. The high transport rate of cladribine and clofarabine by ABCG2 deduced from Transwell experiments raises the possibility that this transporter could affect the disposition of nucleoside analogues in patients or cause resistance in tumors. [Mol Cancer Ther 2008;7(9):3092–102]

This file describes Supplementary experimental details regarding the cell viability, clonogenic and competition assays, preparation of membrane vesicles and vesicular transport assays, immunohistochemistry, immunoblotting, RNA sequencing and analysis, amplification of SB transposon insertions and analysis, validation of the Fgfr2-Tbc1d1 fusion, Met qPCR copy number analysis and statistical analysis.

Supplementary Fig. S2 from Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides

Supplementary Table 2 from Abcc2 (Mrp2), Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo

Supplementary Figures S1-5 show additional information regarding the Fgfr2-Tbc1d1 fusion, the effect of AZD4547 on established WESB and WESB-Fgfr2 tumors and tumor-derived cells, the causes of death observed with the different AZD4547 treatment schedules, differential gene expression analysis of multiple RTK family members and the overview of insertions in genes across all tumors used in this study.

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified Fgfr2 as a key driver of invasive lobular carcinoma (ILC) in an in vivo insertional mutagenesis screen using the Sleeping Beauty transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our in vivo approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK–ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from de novo transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies.Significance: These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis in vivo is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. Cancer Res; 78(19); 5668–79. ©2018 AACR.

Purpose: ATP-binding cassette sub-family C member 2 [ABCC2; multidrug resistance–associated protein 2 (MRP2)] and ABCC3 (MRP3) mediate the elimination of toxic compounds, such as drugs and carcinogens, and have a large overlap in substrate specificity. We investigated the roles of Abcc2 and Abcc3 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) in vivo.Experimental Design:Abcc2;Abcc3−/− mice were generated, characterized, and used to investigate possibly overlapping or complementary roles of Abcc2 and Abcc3 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX.Results:Abcc2;Abcc3−/− mice were viable and fertile. In Abcc2−/− mice, the plasma area under the curve (AUCi.v.) for MTX was 2.0-fold increased compared with wild type, leading to 1.6-fold increased urinary excretion, which was not seen in Abcc2;Abcc3−/− mice. Biliary excretion of MTX was 3.7-fold reduced in Abcc2−/− but unchanged in Abcc2;Abcc3−/− mice. The plasma AUCi.v.s of 7OH-MTX were 6.0-fold and 4.3-fold increased in Abcc2−/− and Abcc2;Abcc3−/− mice, respectively, leading to increased urinary excretion. The biliary excretion of 7OH-MTX was 5.8-fold reduced in Abcc2−/− but unchanged in Abcc2;Abcc3−/− mice. 7OH-MTX accumulated substantially in the liver of Abcc2−/− and especially Abcc2;Abcc3−/− mice.Conclusions: Abcc2 is important for (biliary) excretion of MTX and its toxic metabolite 7OH-MTX. When Abcc2 is absent, Abcc3 transports MTX and 7OH-MTX back from the liver into the circulation, leading to increased plasma levels and urinary excretion. Variation in ABCC2 and/or ABCC3 activity may therefore have profound effects on the elimination and severity of toxicity of MTX and 7OH-MTX after MTX treatment of patients.

Supplementary Figure Legend from Abcc2 (Mrp2), Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo

Purpose: ABCC2 (MRP2) and ABCG2 (BCRP) transport various endogenous and exogenous compounds, including many anticancer drugs, into bile, feces, and urine. We investigated the possibly overlapping roles of Abcg2 and Abcc2 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX).Experimental Design: We generated and characterized Abcc2;Abcg2-/- mice, and used these to determine the overlapping roles of Abcc2 and Abcg2 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX.Results: Compared with wild-type, the plasma areas under the curve (AUC) for MTX were 1.6-fold and 2.0-fold higher in Abcg2-/- and Abcc2-/- mice, respectively, and 3.3-fold increased in Abcc2;Abcg2-/- mice. The biliary excretion of MTX was 23-fold reduced in Abcc2;Abcg2-/- mice, and the MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-MTX were not significantly altered in Abcg2-/- mice, but the areas under the curve were 6.2-fold and even 12.4-fold increased in Abcc2-/- and Abcc2;Abcg2-/- mice, respectively. This indicates that Abcc2 compensates for Abcg2 deficiency but that Abcg2 can only partly compensate for Abcc2 absence. Furthermore, 21-fold decreased biliary 7OH-MTX excretion in Abcc2;Abcg2-/- mice and substantial 7OH-MTX accumulation in the liver and kidney were seen. We additionally found that in the absence of Abcc2, Abcg2 mediated substantial urinary excretion of MTX and 7OH-MTX.Conclusions: Abcc2 and Abcg2 together are major determinants of MTX and 7OH-MTX pharmacokinetics. Variations in ABCC2 and/or ABCG2 activity due to polymorphisms or coadministered inhibitors may therefore substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.

Supplementary Figure Legends from Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides

Supplementary Data from Functionally Overlapping Roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2) in the Elimination of Methotrexate and Its Main Toxic Metabolite 7-Hydroxymethotrexate In vivo

Pathologic soft tissue calcification can occur in both genetic and acquired clinical conditions, causing significant morbidity and mortality. Although the pathomechanisms of pathologic calcification are poorly understood, major progress has been made in recent years in defining the underlying genetic defects in Mendelian disorders of ectopic calcification. This review presents an overview of the pathophysiology of five monogenic disorders of pathologic calcification: pseudoxanthoma elasticum, generalized arterial calcification of infancy, arterial calcification due to deficiency of CD73, ankylosis, and progeria. These hereditary disorders, caused by mutations in genes encoding ATP binding cassette subfamily C member 6, ectonucleotide pyrophosphatase/phosphodiesterase 1, CD73, progressive ankylosis protein, and lamin A/C proteins, respectively, are inorganic pyrophosphate (PPi) deficiency syndromes with reduced circulating levels of PPi, the principal physiologic inhibitor of calcium hydroxyapatite deposition in soft connective tissues. In addition to genetic diseases, PPi deficiency has been encountered in acquired clinical conditions accompanied by pathologic calcification. Because specific and effective treatments are lacking for pathologic calcification, the unifying finding of PPi deficiency suggests that PPi-targeted therapies may be beneficial to counteract pathologic soft tissue calcification in both genetic and acquired diseases.

Pathological mineralization of intervertebral disc is debilitating and painful and linked to disc degeneration in a subset of human patients. An adenosine triphosphate transporter, progressive ankylosis (ANK) is a regulator of extracellular inorganic pyrophosphate levels and plays an important role in tissue mineralization. However, the function of ANK in intervertebral disc has not been fully explored. Herein we analyzed the spinal phenotype of Ank mutant mice (ank/ank) with attenuated ANK function. Micro-computed tomography and histological analysis showed that loss of ANK function results in aberrant annulus fibrosus mineralization and peripheral disc fusions with cranial to caudal progression in the spine. Vertebrae in ank/ank mice exhibit elevated cortical bone mass and increased tissue non-specific alkaline phosphatase-positive endplate chondrocytes with decreased subchondral endplate porosity. The acellular dystrophic mineral inclusions in the annulus fibrosus were localized adjacent to apoptotic cells and cells that acquired osteoblast-like phenotype. Fourier transform infrared spectral imaging showed that the mineral in the outer annulus fibrosus had similar chemical composition to that of vertebral bone. Microarray-based transcriptomic analysis of annulus fibrosus and nucleus pulposus tissues showed changes in several pathways associated with mineralization including transforming growth factor β and mitogen-activated protein kinase signaling. The present study provides new insights into the role of ANK in the disc tissue compartments, and highlights the importance of local inorganic pyrophosphate metabolism in inhibiting mineralization of this important connective tissue.

ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.

Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe an PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory.In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.

Exercise confers protection against obesity, type 2 diabetes and other cardiometabolic diseases1-5. However, the molecular and cellular mechanisms that mediate the metabolic benefits of physical activity remain unclear6. Here we show that exercise stimulates the production of N-lactoyl-phenylalanine (Lac-Phe), a blood-borne signalling metabolite that suppresses feeding and obesity. The biosynthesis of Lac-Phe from lactate and phenylalanineoccurs in CNDP2+ cells, including macrophages,monocytes and otherimmune and epithelial cells localized to diverse organs. In diet-induced obese mice, pharmacological-mediated increases in Lac-Phe reduces food intake without affecting movement or energy expenditure. Chronic administration of Lac-Phe decreases adiposity and body weight and improves glucose homeostasis. Conversely, genetic ablation of Lac-Phe biosynthesis in mice increases food intake and obesity following exercise training. Last, large activity-inducible increases in circulating Lac-Phe are alsoobserved in humans and racehorses, establishing this metabolite as a molecular effector associated with physical activity across multiple activity modalities and mammalian species. These data define a conserved exercise-inducible metabolite that controls food intake and influences systemic energy balance.

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the ABCC6-/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. ABCC6-/- mice exhibited diminished trabecular bone quality parameters at 7-months which remained significantly lower than the wild-type mice at 18 months-of-age. ABCC6-/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling.Author SummaryInherited mutations in the ABCC6 transporter gene results in mineralization, often in the form of hydroxyapatite, of connective tissues throughout the body, predominantly affecting the skin, eyes, and blood vessels. Functional loss of ABCC6 causes reduced levels of the potent mineralization inhibitor pyrophosphate (PPi) in blood resulting in these pathologies. Pathological mineralization is also a prominent feature of intervertebral disc degeneration, but the role of ABCC6 and systemic PPi levels and its correlation to disc mineralization and vertebral bone health has remained unexplored. In this study, we show for the first time that loss of ABCC6 in mice results in significant decline in vertebral bone quality and mild age-related disc degeneration without increased incidence of abnormal mineralization. Importantly, treatment of ABCC6 deficient mice with K3Citrate resulted in restoration of early cellular changes which drive bone loss and mechanical function of the vertebrae. In summary, our data reveal that ABCC6 is dispensable for mineralization prevention in the intervertebral disc. Unexpectedly, we found that vertebral bone quality and bone cell activities are linked to ABCC6 function.

The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints, but we recently showed that ANKH releases large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. This ATP is converted extracellularly into PPi and AMP by the ectoenzyme Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANK. We now addressed this question by determining the effect of a complete absence of ENPP1 on ANKH-dependent extracellular PPi concentrations. Introduction of ANKH in ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1-proficient cells.Ank-activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained < 2.5% of the PPi found in bones of wild type mice, showing that Enpp1-activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60-70 % of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself.

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.

ABCC6 encodes a transmembrane transporter playing a primary role in the efflux of ATP from hepatocytes to the bloodstream. ATP is then cleaved to AMP and inorganic pyrophosphate, a major inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a multisystemic recessive ectopic calcification disease of variable severity. One of the mechanisms influencing the heterogeneity of a disorder is the penetrance of pathogenic variants. The penetrance of a sequence variant shows the proportion of individuals developing the expected phenotype in the presence of the variant. Incomplete penetrance indicates that the disease does not develop in all the cases when the pathogenic variant is present. Here, we investigated whether incomplete penetrance participates in the heterogeneity of pseudoxanthoma elasticum. By integrating the clinical and genetic data of 590 patients, we created the largest European pseudoxanthoma elasticum cohort. We identified two incomplete penetrant pathogenic variants, p.(V787I) and p.(R391G), based on their allele frequencies in our cohort and in the European reference population of gnomAD. The detailed characterization of the frequent p.(R391G) pathogenic variant suggested only 2% penetrance with an unaltered severity of the clinical phenotype. Based on our biochemical analysis, we hypothesize that the variant becomes deleterious only if an interacting partner is mutated simultaneously. These data point to new molecular mechanisms by revealing the potential existence of the first interacting partner of ABCC6. Our data are important for genetic counseling of pseudoxanthoma elasticum, suggesting a much lower disease heritability of these pathogenic variants.

ABCC6 mediates release of ATP from hepatocytes into the blood. Extracellularly, ATP is converted into the mineralization inhibitor pyrophosphate. Consequently, inactivating ABCC6 mutations give low plasma pyrophosphate underlying the ectopic mineralization disorder pseudoxanthoma elasticum. How ABCC6 mediates cellular ATP release is unknown. Mechanistic studies are hampered because fluorophores attached to ABCC6’s N- or C-terminus result in intracellular retention and degradation. Here we describe that intramolecular introduction of fluorophores yields fully functional ABCC6 fusion proteins. An ABCC6 variant with the catalytic glutamate of the second nucleotide-binding domain mutated, correctly routed to the plasma membrane, but was inactive. Finally, N-terminal His(10) or FLAG tags did not affect the activity of fusion proteins, allowing their purification for biochemical characterization. Hence, these fusion proteins provide excellent tools to study ABCC6 biology.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality., Author summary Biallelic inactivating mutations in the progressive ankylosis gene (ANKH/Ank, humans/mice) cause a severe form of arthritis, primarily affecting joints of hands, feet and the spine. Low extracellular concentrations of the mineralization inhibitor pyrophosphate underlie the joint calcification in mice and humans that are devoid of functional Ank or ANKH, respectively. It was previously thought that ANKH mediates the release of pyrophosphate from the cytosol to the extracellular environment. We here show, however, that PPi is formed in the extracellular environment by Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1), from ATP released via ANKH. Untargeted metabolomics further revealed that ANKH provides a pathway for the release of nucleoside triphosphates in general and, unexpectedly, the Krebs-cycle intermediate citrate. Mice lacking Ank activity (Ankank/ank mice), have substantially reduced concentrations of citrate in their plasma and urine. Citrate was even undetectable in urine of a human patient lacking functional ANKH. Bones of Ankank/ank mice have reduced mechanical strength and their bone matrix contains dramatically reduced levels of PPi and citrate. Our data demonstrate that ANKH/Ank crucially contributes to the disposition of citrate and PPi in vivo and profoundly affects bone quality. Extracellular citrate is ubiquitous, and additional phenotypes are therefore expected to be associated with absence of Ank activity.

Intervertebral disc degeneration presents a wide spectrum of clinically degenerative disc phenotypes; however, the contribution of genetic background to the degenerative outcomes has not been established. We characterized the spinal phenotype of 3 mouse strains with varying cartilage‐regenerative potential at 6 and 23 months: C57BL/6, LG/J and SM/J. All strains showed different aging phenotypes. Importantly, LG/J mice showed an increased prevalence of dystrophic disc calcification in caudal discs with aging. Quantitative‐histological analyses of LG/J and SM/J caudal discs evidenced accelerated degeneration compared to BL6, with cellular disorganization and cell loss together with fibrosis of the NP, respectively. Along with the higher grades of disc degeneration, SM/J, at 6M, also differed the most in terms of NP gene expression compared to other strains. Moreover, although we found common DEGs between BL6 and LG/J aging, most of them were divergent between the strains. Noteworthy, the common DEGs altered in both LG/J and BL6 aging were associated with inflammatory processes, response to stress, cell differentiation, cell metabolism and cell division. Results suggested that disc calcification in LG/J resulted from a dystrophic calcification process likely aggravated by cell death, matrix remodelling, changes in calcium/phosphate homeostasis and cell transformation. Lastly, we report 7 distinct phenotypes of human disc degeneration based on transcriptomic profiles, that presented similar pathways and DEGs found in aging mouse strains. Together, our results suggest that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease., This study investigates how genetic background influences the acquisition of specific morphological phenotypes in the intervertebral disc with aging. By uncovering the aging transcriptomic signature of three inbred mouse strains, and distinct transcriptomic profiles in human degenerated discs, our studies shed light on the wide spectrum of phenotypes presented during disc disease.

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified Fgfr2 as a key driver of invasive lobular carcinoma (ILC) in an in vivo insertional mutagenesis screen using the Sleeping Beauty transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our in vivo approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK–ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from de novo transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies. Significance: These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis in vivo is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. Cancer Res; 78(19); 5668–79. ©2018 AACR.

The membrane protein Ankylosis homologue (ANKH, mouse orthologue: ANK) prevents mineralization of joint-space and articular cartilage. The accepted view is that ANKH mediates cellular release of inorganic pyrophosphate (PPi), a strong physiological inhibitor of mineralization. Using global metabolite profiling, we identified citrate as the most prominent metabolite leaving HEK293 cells in an ANKH-dependent manner. Although PPi levels were increased in culture medium of HEK293-ANKH cells, PPi was formed extracellularly after release of ATP and other nucleoside triphosphates.Ankank/ankmice, which lack functional ANK, had substantially reduced concentrations of citrate in plasma and urine, while citrate was undetectable in urine of a human patient lacking functional ANKH. Bone hydroxyapatite ofAnkank/ankmice also contained markedly reduced levels of citrate and PPi and displayed diminished strength. Together, our data show that ANKH is a crucial factor in extracellular citrate and PPi homeostasis that is essential for normal bone development.

Pseudoxanthoma elasticum (PXE), a heritable ectopic mineralization disorder, is caused by mutations in the ABCC6 gene primarily expressed in the liver and the kidneys. The fundamental question on pathogenesis of PXE, whether lack of ABCC6 expression in liver or kidney is the primary site of molecular pathology in peripheral tissues, has not been addressed. We generated a series of Abcc6−/− rats as models of PXE depicting ectopic mineralization in the skin, eyes, and the arterial blood vessels. Plasma PPi level was reduced (

Ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (E-NPP1), encoded by ENPP1, is a plasma membrane protein that generates inorganic pyrophosphate (PPi ), a physiologic inhibitor of hydroxyapatite formation. In humans, variants in ENPP1 are associated with generalized arterial calcification of infancy, an autosomal-recessive condition causing premature onset of arterial calcification and intimal proliferation resulting in stenoses. ENPP1 variants also cause pseudoxanthoma elasticum characterized by ectopic calcification of soft connective tissues. To determine the functional impact of ENPP1 missense variants, we analyzed 13 putative pathogenic variants in vitro regarding their functional properties, that is, activity, localization, and PPi generation. Transfection of eight of the 13 variants led to complete loss of NPP activity, whereas four mutants (c.1412A > G, p.Tyr471Cys; c.1510A > C, p.Ser504Arg; c.1976A > G, p.Tyr659Cys; c.2330A > G, p.His777Arg) showed residual activity compared with wild-type E-NPP1. One putative pathologic variant (c.2462 G > A, p.Arg821His) showed normal activity. The five mutants with normal or residual E-NPP1 enzyme activity were still able to generate PPi and localized in the plasma membrane. In this study, we identified a functional ENPP1 polymorphism, which was expected to be pathogenic till now. Furthermore, we identified four mutants (p.Tyr471Cys, p.Ser504Arg, p.Tyr659Cys, p.His777Arg) with residual E-NPP1 function, which would be potential therapeutical targets for conformational-stabilizing agents.

Contains fulltext : 170847.pdf (Publisher’s version ) (Closed access) Multidrug resistance-associated proteins (MRP) of Plasmodium falciparum have been associated with altered drug sensitivity. Knowledge on MRP substrate specificity is indispensible for the characterization of resistance mechanisms and identifying its physiological roles. An untargeted metabolomics approach detected decreased folate concentrations in red blood cells infected with schizont stage parasites lacking expression of MRP1. Furthermore, a tenfold decrease in sensitivity toward the folate analog methotrexate was detected for parasites lacking MRP1. PfMRP1 is involved in the export of folate from parasites into red blood cells and is therefore a relevant factor for efficient malaria treatment through the folate pathway.

The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate was 2.4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs.

Ever since Garrod deduced the existence of inborn errors in 1901, a vast array of metabolic diseases has been identified and characterized in molecular terms. In 2018 it is difficult to imagine that there is any uncharted backyard left in the metabolic disease landscape. Nevertheless, it took until 2013 to identify the cause of a relatively frequent inborn error, pseudoxanthoma elasticum (PXE), a disorder resulting in aberrant calcification. The mechanism found was not only biochemically interesting but also points to possible new treatments for PXE, a disease that has remained untreatable. In this review we sketch the tortuous road that led to the biochemical understanding of PXE and to new ideas for treatment. We also discuss some of the controversies still haunting the field.

Ectopic mineralization is a global problem and leading cause of morbidity and mortality. The pathomechanisms of ectopic mineralization are poorly understood. Recent studies on heritable ectopic mineralization disorders with defined gene defects have been helpful in elucidation of the mechanisms of ectopic mineralization in general. The prototype of such disorders is pseudoxanthoma elasticum (PXE), a late-onset, slowly progressing disorder with multisystem clinical manifestations. Other conditions include generalized arterial calcification of infancy (GACI), characterized by severe, early-onset mineralization of the cardiovascular system, often with early postnatal demise. In addition, arterial calcification due to CD73 deficiency (ACDC) occurs late in life, mostly affecting arteries in the lower extremities in elderly individuals. These three conditions, PXE, GACI, and ACDC, caused by mutations in ABCC6, ENPP1, and NT5E, respectively, are characterized by reduced levels of inorganic pyrophosphate (PPi) in plasma. Because PPi is a powerful antimineralization factor, it has been postulated that reduced PPi is a major determinant for ectopic mineralization in these conditions. These and related observations on complementary mechanisms of ectopic mineralization have resulted in development of potential treatment modalities for PXE, including administration of bisphosphonates, stable PPi analogs with antimineralization activity. It is conceivable that efficient treatments may soon become available for heritable ectopic mineralization disorders with application to common calcification disorders.

Soft tissue calcification occurs in several common acquired pathologies, such as diabetes and hypercholesterolemia, or can result from genetic disorders. ABCC6, a transmembrane transporter primarily expressed in liver and kidneys, initiates a molecular pathway inhibiting ectopic calcification. ABCC6 facilitates the cellular efflux of ATP, which is rapidly converted into pyrophosphate (PPi), a major calcification inhibitor. Heritable mutations in ABCC6 underlie the incurable calcification disorder pseudoxanthoma elasticum and some cases of generalized arterial calcification of infancy. Herein, we determined that the administration of PPi and the bisphosphonate etidronate to Abcc6 −/− mice fully inhibited the acute dystrophic cardiac calcification phenotype, whereas alendronate had no significant effect. We also found that daily injection of PPi to Abcc6 −/− mice over several months prevented the development of pseudoxanthoma elasticum–like spontaneous calcification, but failed to reverse already established lesions. Furthermore, we found that the expression of low amounts of the human ABCC6 in liver of transgenic Abcc6 −/− mice, resulting in only a 27% increase in plasma PPi levels, led to a major reduction in acute and chronic calcification phenotypes. This proof-of-concept study shows that the development of both acute and chronic calcification associated with ABCC6 deficiency can be prevented by compensating PPi deficits, even partially. Our work indicates that PPi substitution represents a promising strategy to treat ABCC6-dependent calcification disorders.

Objective— Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PP i ). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PP i , and whether human PXE ptients have low plasma PP i concentrations. Approach and Results— Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PP i . The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PP i concentrations. Conclusions— Hepatic ABCC6-mediated ATP release is the main source of circulating PP i , revealing an unanticipated role of the liver in systemic PP i homeostasis. Patients with PXE have a strongly reduced plasma PP i level, explaining their mineralization disorder. Our results indicate that systemic PP i is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PP i supplementation therapy.