Ilan Ifergan

Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines.

We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and single-step selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17- and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87-96%, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92-97% of parental [(3)H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two confined regions in exons 2 and 3. The majority of these mutations resulted in frame-shift and/or premature translation termination and lack of RFC protein expression. The remaining mutations involved single amino acid substitutions predominantly residing in the first transmembrane domain (TMD1). Some RFC-inactivating mutations emerged during the early stages of antifolate selection and were stably retained during further drug selection. Furthermore, some sublines displayed a markedly decreased or abolished RFC mRNA and/or protein expression. This constitutes the first demonstration of clustering of multiple human RFC mutations in TMD1, thereby suggesting that it plays a functional role in folate/antifolate binding and/or translocation. This is the first molecular characterization of human RFC-associated modalities of resistance to various novel antifolates in multiple leukaemia sublines.

Molecular mechanisms of adaptation to folate deficiency.

Folic acid is an essential vitamin for a wide spectrum of biochemical reactions; however, unlike bacteria and plants, mammals are devoid of folate biosynthesis and thus must obtain this cofactor from exogenous sources. Therefore, folate deficiency may impair the de novo biosynthesis of purines and thymidylate and thereby disrupt DNA and RNA metabolism, homocysteine remethylation, methionine biosynthesis, and subsequent formation of S-adenosylmethionine (the universal methyl donor) which in turn may lead to altered methylation reactions. This impaired folate-dependent intracellular metabolism can lead to several key pathologies including, for example, megaloblastic anemia, homocysteinemia, cardiovascular disease, embryonic defects, in particular neural tube defects (NTDs), congenital heart defects, and possibly cancer. The current review presents and evaluates the up-to-date knowledge regarding the molecular mechanisms underlying cellular survival and/or adaptation to folate deficiency or insufficiency. These mechanisms of adaptation to folate deficiency generally associated with folate uptake, intracellular folate retention, folate-dependent metabolism, and active folate efflux specifically include: (a) Up- or downregulation of various folate-dependent enzymes like dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (b) Cellular retention of folates via polyglutamylation by the enzyme folylpoly-gamma-glutamate synthetase (FPGS), (c) Overexpression of folate influx systems including the reduced folate carrier (RFC), folate receptor (FR) as well as the proton-coupled folate transporter (PCFT), a recently identified intestinal folate influx transporter optimally functioning at the acidic microclimate of the upper intestinal epithelium, (d) Downregulation of ATP-driven folate efflux transporters of the multidrug resistance protein (MRP; ABCC) family and breast cancer resistance protein (BCRP; ABCG2) that belong to the multidrug resistance (MDR) efflux transporters of the ATP-binding cassette (ABC) superfamily. Moreover, the intricate interplay between various components of the adaptive response to folate deprivation is also discussed.

Novel Extracellular Vesicles Mediate an ABCG2-Dependent Anticancer Drug Sequestration and Resistance

Overexpression of the multidrug efflux transporter ABCG2 in the plasma membrane of cancer cells confers resistance to various anticancer drugs, including mitoxantrone. Here, we explored the mechanism underlying drug resistance in the MCF-7 breast cancer sublines MCF-7/MR and MCF-7/FLV1000 cells in which wild-type (R482) ABCG2 overexpression is highly confined to cell-cell attachment zones. The latter comprised the membrane of novel extracellular vesicles in which mitoxantrone was rapidly and dramatically sequestered. After 12 hours of incubation with mitoxantrone, the estimated intravesicular drug concentration was approximately 1,000-fold higher than in the culture medium. This drug compartmentalization was prevented by the specific and potent ABCG2 transport inhibitors Ko143 and fumitremorgin C, thereby resulting in restoration of drug sensitivity. Consistently, this intravesicular drug concentration was abrogated by energy deprivation and was restored upon provision of energy substrates. Fine-structure studies corroborated the presence of numerous large extracellular vesicles that were highly confined to cell-cell attachment zones between neighbor cells. Furthermore, high-resolution electron microscopy revealed that the membrane of these extracellular vesicles contained microvilli-like invaginations protruding into the intravesicular lumen. It is likely that these microvilli-like projections increase the vesicular membrane surface, thereby allowing for a more efficient ABCG2-dependent intravesicular anticancer drug concentration. Hence, these novel extracellular vesicles mediate the ABCG2-dependent extraction of intracellular drug, thereby serving as cytotoxic drug disposal chambers shared by multiple neighbor cancer cells. This constitutes a novel modality of anticancer drug resistance.

ABCG2 Harboring the Gly482 Mutation Confers High-Level Resistance to Various Hydrophilic Antifolates

ABCG2 is an ATP-binding cassette transporter that confers resistance to various chemotherapeutic agents. Recent studies have established that an Arg (wild-type) to Gly mutation at amino acid 482 in ABCG2 alters substrate specificity. Here, we explored the role of this G482 mutation in antifolate resistance using a clinically relevant 4-hour drug exposure. Stable transfectants overexpressing the mutant G482 transporter displayed 120-, 1,000-, and >6,250-fold resistance to the antifolates methotrexate, GW1843, and Tomudex, respectively, relative to parental human embryonic kidney cells. Moreover, although overexpressing equal transporter levels at the plasma membrane, G482-ABCG2 cells were 6-, 23-, and >521-fold more resistant to methotrexate, GW1843, and Tomudex, respectively, than R482-ABCG2 cells. In contrast, upon a continuous (72-hour) drug exposure, both the G482- and R482-ABCG2 cells lost almost all their antifolate resistance; this result was consistent with the inability of ABCG2 to extrude long-chain antifolate polyglutamates. Ko143, a specific and potent ABCG2 inhibitor reversed methotrexate resistance in both G482- and R482-ABCG2 cells. Consistently, whereas the pool of free methotrexate in parental human embryonic kidney cells was prominent after 4 hours of transport with 1 micromol/L [3H]methotrexate, in R482- and G482-ABCG2 cells, it was minimal. Furthermore, G482-ABCG2 cells contained marked decreases in the di- and triglutamate species of [3H]methotrexate at 4 hours of incubation with methotrexate and in the tetra- and pentaglutamates at 24 hours. These changes were not associated with any significant decrease in folylypoly-gamma-glutamate synthetase activity. These results provide the first evidence that the G482-ABCG2 mutation confers high-level resistance to various hydrophilic antifolates.

Reduced folate carrier protein expression in osteosarcoma: implications for the prediction of tumor chemosensitivity.

High‐dose methotrexate (MTX) is an important component of current protocols for the treatment of osteosarcoma. Although MTX uptake proceeds primarily through the reduced folate carrier (RFC) protein and efflux occurs via multidrug resistance protein 1 (MRP1), RFC protein expression in osteosarcoma remains unexamined.

The reduced folate carrier (RFC) is cytotoxic to cells under conditions of severe folate deprivation. RFC as a double edged sword in folate homeostasis

The reduced folate carrier (RFC), a bidirectional anion transporter, is the major uptake route of reduced folates essential for a spectrum of biochemical reactions and thus cellular proliferation. However, here we show that ectopic overexpression of the RFC, but not of folate receptor α, a high affinity unidirectional folate uptake route serving here as a negative control, resulted in an ∼15-fold decline in cellular viability in medium lacking folates but not in folate-containing medium. Moreover to explore possible mechanisms of adaptation to folate deficiency in various cell lines that express the endogenous RFC, we first determined the gene expression status of the following genes: (a) RFC, (b) ATP-driven folate exporters (i.e. MRP1, MRP5, and breast cancer resistance protein), and (c) folylpoly-γ-glutamate synthetase and γ-glutamate hydrolase (GGH), enzymes catalyzing folate polyglutamylation and hydrolysis, respectively. Upon 3–7 days of folate deprivation, semiquantitative reverse transcription-PCR analysis revealed a specific ∼2.5-fold decrease in RFC mRNA levels in both breast cancer and T-cell leukemia cell lines that was accompanied by a consistent fall in methotrexate influx, serving here as an RFC transport activity assay. Likewise a 2.4-fold decrease in GGH mRNA levels and ∼19% decreased GGH activity was documented for folate-deprived breast cancer cells. These results along with those of a novel mathematical biomodeling devised here suggest that upon severe short term (i.e. up to 7 days) folate deprivation RFC transport activity becomes detrimental as RFC, but not ATP-driven folate exporters, efficiently extrudes folate monoglutamates out of cells. Hence down-regulation of RFC and GGH may serve as a novel adaptive response to severe folate deficiency.

Riboflavin concentration within ABCG2-rich extracellular vesicles is a novel marker for multidrug resistance in malignant cells

We have previously shown that overexpression of the multidrug resistance (MDR) efflux transporter ABCG2 in the membrane of novel extracellular vesicles that are confined to breast cancer cell-cell attachment zones confers mitoxantrone resistance and mediates a marked intravesicular concentration of an unknown endogenous green fluorescent compound (I. Ifergan, G.L. Scheffer, Y.G. Assaraf, Novel extracellular vesicles mediate an ABCG2-dependent anticancer drug sequestration and resistance, Cancer Res. 65 (2005) 10952-10958). Here we identified the latter as riboflavin (vitamin B2) and further demonstrated that the marked intravesicular concentration of riboflavin in ABCG2-overexpressing breast and lung cancer cells tightly correlates with the extent of ABCG2 overexpression and its differential localization to the vesicular membrane and not to the plasma membrane surrounded by growth medium. We hence propose that the ABCG2-dependent concentration of riboflavin in these intercellular compartments may serve as a novel, sensitive, and non-cytotoxic (i.e. based on vitamin accumulation) functional marker for the quantification of the levels of MDR mediated by ABCG2-rich extracellular vesicles in multiple malignant cells.

Sulfasalazine sensitises human monocytic/macrophage cells for glucocorticoids by upregulation of glucocorticoid receptor α and glucocorticoid induced apoptosis

Background: Glucocorticoids (GCs) are commonly used in the treatment of (chronic) inflammatory diseases and cancer, but inherent or acquired resistance to these drugs limits their optimal efficacy. The availability of drugs that could modulate GC resistance is therefore of potential clinical interest. Objective: To explore the molecular basis of GC sensitisation of GC resistant monocytic/macrophage cells after chronic exposure to sulfasalazine. Methods: Human monocytic/macrophage THP1 and U937 cells represent a cell line model system characterised by inherent resistance to the GCs dexamethasone and prednisolone. Both cell lines were chronically exposed in vitro to 0.3–0.6 mM sulfasalazine (SSZ) for approximately 3 months, after which they were characterised for GC sensitivity, expression levels of GC receptor and components of the nuclear factor kappa B (NFκB) signalling pathway, and their ability to undergo GC induced apoptosis. Results: Chronic exposure to SSZ markedly sensitised both U937 and THP1 cells to dexamethasone (781-fold and 1389-fold, respectively) and prednisolone (562-fold and 1220-fold, respectively). Restoration of GC sensitivity in cells exposed to SSZ was provoked via GC induced apoptosis, coinciding with inhibition of NFκB activation. Moreover, western blot analysis revealed a markedly increased expression of glucocorticoid receptor α (GRα) in cells exposed to SSZ. Since GRα mRNA levels were only marginally increased, these results suggest that an altered post-transcriptional mechanism was operable which conferred a stable GRα protein on SSZ exposed cells. Conclusion: These results suggest that chronic targeting of the NFκB signalling pathway by SSZ may be exploited as a novel strategy to stabilise GRα expression and thereby sensitise primary resistant cells to GCs.