I. David Goldman

Identification of an Intestinal Folate Transporter and the Molecular Basis for Hereditary Folate Malabsorption

Folates are essential nutrients that are required for one-carbon biosynthetic and epigenetic processes. While folates are absorbed in the acidic milieu of the upper small intestine, the underlying absorption mechanism has not been defined. We now report the identification of a human proton-coupled, high-affinity folate transporter that recapitulates properties of folate transport and absorption in intestine and in various cell types at low pH. We demonstrate that a loss-of-function mutation in this gene is the molecular basis for hereditary folate malabsorption in a family with this disease. This transporter was previously reported to be a lower-affinity, pH-independent heme carrier protein, HCP1. However, the current study establishes that a major function of this gene product is proton-coupled folate transport required for folate homeostasis in man, and we have thus amended the name to PCFT/HCP1.

Membrane transport of folates.

The chapter reviews the current understanding of the transport mechanisms for folates in mammalian cells--their molecular identities and organization, tissue expression, regulation, structures, and their kinetic and thermodynamic properties. This encompasses a variety of diverse processes. Best characterized is the reduced folate carrier, a member of the SLC19 family of facilitative carriers. But other facilitative organic anion carriers (SLC21), largely expressed in epithelial tissues, transport folates as well. In addition to these bi-directional carrier systems are the membrane-localized folate receptors alpha and beta, that mediate folate uptake unidirectionally into cells via an endocytotic process. There are also several transporters, typified by the family of multidrug resistance-associated proteins, that unidirectionally export folates from cells. There are transport activities for folates, that function optimally at low pH, related in part to the reduced folate carrier, with at least one activity that is independent of this carrier. The reduced folate carrier-associated low-pH route mediates intestinal folate transport. This review considers how these different transport processes contribute to the generation of transmembrane folate gradients and to vectorial flows of folates across epithelia. The role of folate transporters in mouse development, as assessed by homologous deletion of folate receptors and the reduced folate carrier, is described. Much of the focus is on antifolate cancer chemotherapeutic agents that are often model surrogates for natural folates in transport studies. In particular, antifolate transport mediated by the reduced folate carrier is a major determinant of the activity of, and resistance to, these agents. Finally, many of the key in vitro findings on the properties of antifolate transporters are now beginning to be extended to patient specimens, thus setting the stage for understanding response to these drugs in the clinical setting at the molecular level.

Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues.

Members of the family of B9 vitamins are commonly known as folates. They are derived entirely from dietary sources and are key one-carbon donors required for de novo nucleotide and methionine synthesis. These highly hydrophilic molecules use several genetically distinct and functionally diverse transport systems to enter cells: the reduced folate carrier, the proton-coupled folate transporter and the folate receptors. Each plays a unique role in mediating folate transport across epithelia and into systemic tissues. The mechanism of intestinal folate absorption was recently uncovered, revealing the genetic basis for the autosomal recessive disorder hereditary folate malabsorption, which results from loss-of-function mutations in the proton-coupled folate transporter gene. It is therefore now possible to piece together how these folate transporters contribute, both individually and collectively, to folate homeostasis in humans. This review focuses on the physiological roles of the major folate transporters, with a brief consideration of their impact on the pharmacological activities of antifolates.

Resistance to antifolates

The antifolates were the first class of antimetabolites to enter the clinics more than 50 years ago. Over the following decades, a full understanding of their mechanisms of action and chemotherapeutic potential evolved along with the mechanisms by which cells develop resistance to these drugs. These principals served as a basis for the subsequent exploration and understanding of the mechanisms of resistance to a variety of diverse antineoplastics with different cellular targets. This section describes the bases for intrinsic and acquired antifolate resistance within the context of the current understanding of the mechanisms of actions and cytotoxic determinants of these agents. This encompasses impaired drug transport into cells, augmented drug export, impaired activation of antifolates through polyglutamylation, augmented hydrolysis of antifolate polyglutamates, increased expression and mutation of target enzymes, and the augmentation of cellular tetrahydrofolate-cofactor pools in cells. This chapter also describes how these insights are being utilized to develop gene therapy approaches to protect normal bone marrow progenitor cells as a strategy to improve the efficacy of bone marrow transplantation. Finally, clinical studies are reviewed that correlate the cellular pharmacology of methotrexate with the clinical outcome in children with neoplastic diseases treated with this antifolate.

Mechanisms of Membrane Transport of Folates into Cells and Across Epithelia

Until recently, the transport of folates into cells and across epithelia has been interpreted primarily within the context of two transporters with high affinity and specificity for folates, the reduced folate carrier and the folate receptors. However, there were discrepancies between the properties of these transporters and characteristics of folate transport in many tissues, most notably the intestinal absorption of folates, in terms of pH dependency and substrate specificity. With the recent cloning of the proton-coupled folate transporter (PCFT) and the demonstration that this transporter is mutated in hereditary folate malabsorption, an autosomal recessive disorder, the molecular basis for this low-pH transport activity is now understood. This review focuses on the properties of PCFT and briefly addresses the two other folate-specific transporters along with other facilitative and ATP-binding cassette (ABC) transporters with folate transport activities. The role of these transporters in the vectorial transport of folates across epithelia is considered.

Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications

Pemetrexed is a new-generation antifolate, approved for the treatment of mesothelioma and non–small cell lung cancer, currently being evaluated for the treatment of a variety of other solid tumors. This review traces the history of antifolates that led to the development of pemetrexed and describes the unique properties of this agent that distinguish it from other antifolates. These include (a) its very rapid conversion to active polyglutamate derivatives in cells that build to high levels and are retained for long intervals to achieve prolonged and potent inhibition of its major target enzyme thymidylate synthase, (b) its high affinity for three folate transporters, and (c) its marked sensitivity to the level of physiologic folates in cells. The latter results in the unique and paradoxical finding that when transport mediated by the major folate transporter (the reduced folate carrier) is impaired, pemetrexed activity is preserved. This is due to concurrent contraction of competing cellular physiologic folates and utilization of a novel second transport carrier for which pemetrexed has high affinity, recently identified as the proton-coupled folate transporter (PCFT). Laboratory studies are reviewed that raise the possibility of new approaches to the use of folic acid supplementation in clinical regimens with pemetrexed. [Mol Cancer Ther 2007;6(2):404–17]

Schedule-Dependent Cytotoxic Synergism of Pemetrexed and Erlotinib in Human Non–Small Cell Lung Cancer Cells

Purpose: This study was undertaken to select the optimal combination schedule of erlotinib and pemetrexed for the treatment of relapsed non–small cell lung cancer (NSCLC) using a panel of human NSCLC lines. Experimental Design: Human NSCLC cell lines, with variable expression of the known molecular determinants of erlotinib sensitivity, were exposed to pemetrexed and erlotinib using different schedules. Antitumor effect was measured by growth inhibition by cell count and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell cycle distribution and apoptosis by flow cytometry, and expression of cell cycle mediators by immunoblots. The cytotoxic interaction between pemetrexed and erlotinib (i.e., synergistic, additive, or antagonistic) was determined by median effect analysis. Results: When cells were exposed to concurrent pemetrexed and erlotinib or sequential pemetrexed followed by erlotinib, cytotoxic synergism was observed in both erlotinib-sensitive and erlotinib-resistant human NSCLC cell lines. This was independent of the mutation status of epidermal growth factor receptor or K-Ras genes. Synergism was associated with a combination of cell cycle effects from both agents. In contrast, exposure of cells to erlotinib followed by pemetrexed was mostly antagonistic in erlotinib-sensitive cells and additive at best in erlotinib-resistant cells. Antagonism was associated with erlotinib-induced G1-phase blockade of erlotinib-sensitive cells, which protects cells from pemetrexed cytotoxicity. Pemetrexed induced an epidermal growth factor receptor–mediated activation of the phosphatidylinositol 3-kinase/AKT pathway, which was inhibited by erlotinib and a specific phosphatidylinositol 3-kinase inhibitor, LY294002. Conclusions: The combination of pemetrexed and erlotinib is synergistic in NSCLC in vitro if exposure to erlotinib before pemetrexed is avoided, particularly in tumors that are sensitive to erlotinib. Based on these findings, a randomized phase II study comparing the progression-free survival between an intermittent combination of erlotinib and pemetrexed (experimental arm) and pemetrexed alone (control arm) in patients with relapsing NSCLC has been initiated.

Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis

The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.

SLC19A3 encodes a second thiamine transporter ThTr2.

Recently, a new family of facilitative carriers has been cloned consisting of the reduced folate (SLC19A1) and the thiamine (SLC19A2) transporters. Despite a high level of sequence identity and similarity there is essentially no functional overlap between these carriers. The former transports folates and the latter thiamine. In this paper we describe the function of SLC19A3, another member of this transporter family most recently cloned, after transient transfection of the cDNA into HeLa cells. Uptake of [3H]thiamine, but not of methotrexate nor folic acid, was enhanced in SLC19A3 transfectants relative to vector control. Similarly, in the transfectants thiamine transport increased with an increase in pH with peak activity at pH approximately 7.5. While [3H]thiamine uptake was markedly inhibited by nonlabeled thiamine it was not inhibited by several organic cations in 100-fold excess. Hence this carrier has a high degree of specificity for vitamin B1. The data indicate that SLC19A3 has the characteristics of SLC19A2 (ThTr1) and represents a second thiamine transporter (ThTr2) in this family of facilitative carriers.

The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared with the Reduced Folate Carrier

The reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) are ubiquitously expressed in normal and malignant mammalian tissues and in human solid tumor cell lines. This article addresses the extent to which PCFT contributes to transport of pemetrexed and to the activities of this and other antifolates relative to RFC at physiological pH. Either RFC or PCFT cDNA was stably transfected into a transporter-null HeLa cell variant to achieve activities similar to their endogenous function in wild-type HeLa cells. PCFT and RFC produced comparable increases in pemetrexed activity in growth medium with 5-formyltetrahydrofolate. However, PCFT had little or no effect on the activities of methotrexate, N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-l-glutamic acid (raltitrexed, Tomudex; ZD1694), or Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine (PT523) in comparison with RFC irrespective of the folate growth source. PCFT, expressed at high levels in Xenopus laevis oocytes and in transporter-competent HepG2 cells, exhibited a high affinity for pemetrexed, with an influx Km value of 0.2 to 0.8 μM at pH 5.5. PCFT increased the growth inhibitory activity of pemetrexed, but not that of the other antifolates in HepG2 cells grown with 5-formyltetrahydrofolate at physiological pH. These findings illustrate the unique role that PCFT plays in the transport and pharmacological activity of pemetrexed. Because of the ubiquitous expression of PCFT in human tumors, and the ability of PCFT to sustain pemetrexed activity even in the absence of RFC, tumor cells are unlikely to become resistant to pemetrexed as a result of impaired transport because of the redundancy of these genetically distinct routes.