Folate Transport and One-Carbon Metabolism in Targeted Therapies of Epithelial Ovarian Cancer

New therapies are urgently needed for epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. To identify new approaches for targeting EOC, metabolic vulnerabilities must be discovered and strategies for the selective delivery of therapeutic agents must be established. Folate receptor (FR) α and the proton-coupled folate transporter (PCFT) are expressed in the majority of EOCs. FRβ is expressed on tumor-associated macrophages, a major infiltrating immune population in EOC. One-carbon (C1) metabolism is partitioned between the cytosol and mitochondria and is important for the synthesis of nucleotides, amino acids, glutathione, and other critical metabolites. Novel inhibitors are being developed with the potential for therapeutic targeting of tumors via FRs and the PCFT, as well as for inhibiting C1 metabolism. In this review, we summarize these exciting new developments in targeted therapies for both tumors and the tumor microenvironment in EOC.

Alpers- and MNGIE-like disease with disturbed CSF folate transport and an unusual mode of genetic transmission of POLG mutations: a case report

We report about a family with three of five siblings affected by a variable remitting-relapsing disease with epileptic seizures, coma and abdominal crises, and lethal outcome in all. In the youngest son and in one of his deceased brothers we identified two disease causing compound heterozygous POLG mutations. One of these was inherited from the mother, but the other was absent in the father`s blood, saliva, buccal swab and hair bulbs although his paternity was proven genetically. Thus, we assume germline mosaicism for this mutation in the father. Very low 5-methyltetrahydrofolate (5-MTHF) and absence of folate receptor-alpha was repeatedly found in the CSF of the youngest brother indicating a secondary cerebral folate transport deficiency. Folinic acid supplementation over 18 months resulted in some improvement of the neurological condition; however, it did not prevent progression of the systemic disease.

Folate Receptor Alpha Autoantibodies in Autism Spectrum Disorders: Diagnosis, Treatment and Prevention

Folate deficiency and folate receptor autoimmune disorder are major contributors to infertility, pregnancy related complications and abnormal fetal development including structural and functional abnormalities of the brain. Food fortification and prenatal folic acid supplementation has reduced the incidence of neural tube defect (NTD) pregnancies but is unlikely to prevent pregnancy-related complications in the presence of folate receptor autoantibodies (FRAb). In pregnancy, these autoantibodies can block folate transport to the fetus and in young children, folate transport to the brain. These antibodies are prevalent in neural tube defect pregnancies and in developmental disorders such as cerebral folate deficiency (CFD) syndrome and autism spectrum disorder (ASD). In the latter conditions, folinic acid treatment has shown clinical improvement in some of the core ASD deficits. Early testing for folate receptor autoantibodies and intervention is likely to result in a positive outcome. This review discusses the first identification of FRAb in women with a history of neural tube defect pregnancy and FRAb’s association with sub-fertility and preterm birth. Autoantibodies against folate receptor alpha (FRα) are present in about 70% of the children with a diagnosis of ASD, and a significant number of these children respond to oral folinic acid with overall improvements in speech, language and social interaction. The diagnosis of folate receptor autoimmune disorder by measuring autoantibodies against FRα in the serum provides a marker with the potential for treatment and perhaps preventing the pathologic consequences of folate receptor autoimmune disorder.

Systematic Review of Pharmacogenetic Factors That Influence High-Dose Methotrexate Pharmacokinetics in Pediatric Malignancies

Methotrexate (MTX) is a mainstay therapeutic agent administered at high doses for the treatment of pediatric and adult malignancies, such as acute lymphoblastic leukemia, osteosarcoma, and lymphoma. Despite the vast evidence for clinical efficacy, high-dose MTX displays significant inter-individual pharmacokinetic variability. Delayed MTX clearance can lead to prolonged, elevated exposure, causing increased risks for nephrotoxicity, mucositis, seizures, and neutropenia. Numerous pharmacogenetic studies have investigated the effects of several genes and polymorphisms on MTX clearance in an attempt to better understand the pharmacokinetic variability and improve patient outcomes. To date, several genes and polymorphisms that affect MTX clearance have been identified. However, evidence for select genes have conflicting results or lack the necessary replication and validation needed to confirm their effects on MTX clearance. Therefore, we performed a systematic review to identify and then summarize the pharmacogenetic factors that influence high-dose MTX pharmacokinetics in pediatric malignancies. Using the PRISMA guidelines, we analyzed 58 articles and 24 different genes that were associated with transporter pharmacology or the folate transport pathway. We conclude that there is only one gene that reliably demonstrates an effect on MTX pharmacokinetics: SLCO1B1.

Localization of the Folate Transport Systems in the Murine Central Nervous System

Objectives Folates are critical for normal neurodevelopment, and folate transport in the brain is primarily mediated by folate receptor alpha (FRα) at the blood-cerebrospinal fluid barrier (BCSFB). However, studies have reported folate transporter/receptor expression in other brain compartments, which may significantly contribute to overall brain folate uptake. The objective of this study is to characterize the localization of the folate transport systems i.e., reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and FRα in the mouse central nervous system, which will provide insight on novel routes of brain folate transport. In particular, folate transporter/receptor localization is examined at brain barriers [blood-brain barrier (BBB), BCSFB, arachnoid barrier (AB)] and in brain parenchyma (astrocytes, microglia, neurons). Methods The localization of RFC, PCFT and FRα was observed in the brains of C57BL6/N wildtype mice by applying immunohistochemistry (IHC). Mouse brains were isolated, and IHC was performed on frozen coronal sections. Transporter/receptor localization was examined at brain barriers (BBB, BCSFB, AB) and in brain parenchyma (astrocytes, neurons, microglia) using specific antibodies. Standard IHC markers were utilized to identify various brain compartments, with confocal microscopy used for imaging. Results At the mouse BBB and BCSFB, localization of RFC, PCFT and FRα was observed, which is consistent with previous reported data and our own work. At the AB, in astrocytes and neurons localization of RFC and PCFT (but not FRα) was observed. In microglia, no expression of the folate transporters or receptor was detected. Conclusions RFC and PCFT localization at the AB may represent a novel route of folate transport into the CSF, with transporter expression in neurons and astrocytes facilitating folate uptake into brain parenchyma cellular targets. Modulating folate transport at these brain compartments may provide a novel strategy in increasing brain folate uptake in disorders associated with defective FRα and impaired brain folate transport at the BCSFB. Funding Sources This work is supported by the Natural Sciences and Engineering Research Council of Canada (RB). VS is a recipient of several graduate scholarships.

Folate transporter dynamics and therapy with classic and tumor-targeted antifolates

There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.

Polymorphism of folate cycle genes as a risk factor of hyperhomocysteinemia

Hyperhomocysteinemia (HHc) is a new factor being considered at the moment that can cause damage to vessel walls. Its occurrence depends on genetic peculiarities of a body. Our research goal was to estimate frequency of genetic polymorphisms (SNP) in folate cycle genes among people living in Perm region and its influence on homocysteine (Hc) concentration in blood serum. We examined 189 women (32.2±5.25). Hc concentration in blood serum was determined with immune chemiluminescent procedure. We examined frequency of SNP in folate cycle genes with pyrosequencing. Homozygote state as per minor alleles in methylene tetrahydrofolate reductase (MTHFR) gene (rs 1801133 и rs 1801131) and MTR gene (rs 1805087) was registered 7.5, 5.4, and 13.75 times less frequently than homozygote state as per neutral alleles. Heterozygote state prevailed for genes of methionine synthase reductase and folate transport protein among examined SNP. Homozygotes as per minor allele SNP in MTHFR gene (Ala222Val; rs 1801133) had higher Hc concentration in blood serum that amounted to 8.476 ± 3.193 mmol/L and was 1.276 times higher than the same parameter in homozygotes as per neutral allele (р=0.0036). We didn’t establish any influence on Hc contents in blood serum for the remaining 4 SNP in folate cycle genes (р> 0.1). Examined SNP in MTHFR and MTR genes tended to have neutral alleles more frequently than minor ones. SNP in genes of other examined proteins belonging to folate cycle didn’t have any differences in frequency of examined alleles. We didn’t detect a combination of homozygote state as per two SNP in MTHFR gene or homozygote state as per one SNP and heterozygote state as per another one in a genome. Only SNP in MTHFR gene (Ala222Val, rs 1801133) authentically causes increase in homocysteine concentration out of all the examined SNP in genes of folate cycle enzymes and proteins.