SEARCH

SEARCH BY CITATION

Keywords:

  • folate binding proteins;
  • folic acid;
  • gene regulation;
  • carcinogenesis

Abstract

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Folate receptor α (FRα) is a membrane-bound protein with high affinity for binding and transporting physiologic levels of folate into cells. Folate is a basic component of cell metabolism and DNA synthesis and repair, and rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis, an observation supported by the widespread use of antifolates in cancer chemotherapy. FRα levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression. It has been suggested that FRα might confer a growth advantage to the tumor by modulating folate uptake from serum or by generating regulatory signals. Indeed, cell culture studies show that expression of the FRα gene, FOLR1, is regulated by extracellular folate depletion, increased homocysteine accumulation, steroid hormone concentrations, interaction with specific transcription factors and cytosolic proteins, and possibly genetic mutations. Whether FRα in tumors decreases in vivo among individuals who are folate sufficient, or whether the tumor's machinery sustains FRα levels to meet the increased folate demands of the tumor, has not been studied. Consequently, the significance of carrying a FRα-positive tumor in the era of folic acid fortification and widespread vitamin supplement use in countries such as Canada and the United States is unknown. Epidemiologic and clinical studies using human tumor specimens are lacking and increasingly needed to understand the role of environmental and genetic influences on FOLR1 expression in tumor etiology and progression. This review summarizes the literature on the complex nature of FOLR1 gene regulation and expression, and suggests future research directions. © 2006 Wiley-Liss, Inc.

Tumor initiation and progression are intrinsically related to perturbation of gene expression and gene-product function.1 This is particularly relevant for genes encoding folate binding proteins whose altered expression may have pleiotropic consequences. Folate, a basic component of cell metabolism and DNA synthesis and repair,2 is an essential vitamin required by both normal and tumor cells, an observation supported by the widespread use of antifolates in cancer chemotherapy. Folate binding proteins can be classified as low-affinity binders consisting of the reduced folate carrier (RFC) and responsible for the majority of folate transport across cell membranes, cytoplasmic-binding proteins consisting of specific enzymes involved in one-carbon metabolism, and high-affinity binders consisting of the folate receptor (FR) that mediate folate uptake by endocytosis.3

Three FR isoforms have been described – referred to as FRα, FRβ, and FRγ – each with tissue-specific distribution and folate-binding potential.4 FRα, the most widely studied FR isoform, has restricted expression in normal cells but is highly expressed in various nonmucinous tumors of epithelial origin.5 In the most widely studied tumor epithelial ovarian cancer expression increases in concentration with tumor progression and grade,6 and is associated with decreased survival.7 The reasons for its expression and function in tumor relative to normal tissue are unknown. Possibly, FRα might confer a growth advantage to the tumor by modulating folate uptake from serum8 or by generating regulatory signals.4, 9 This review summarizes folate's role in cancer, compares folate transport systems and tissue distributions of the FR isoforms, and outlines the complex nature of regulation and expression of the FRα gene, FOLR1, and its potential role in the development and progression of epithelial cancers, as well as its exploitation as a promising detection and treatment modality. The specific aim is to highlight opportunities for epidemiologists and clinicians to investigate genetic and environmental influences on FOLR1 expression in cancer through transdisciplinary collaborations with basic scientists.

The role of folate in cancer

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Folate is the generic term for folic acid and structurally related compounds that have the biochemical activity of folic acid.10 It is involved in essential one-carbon transfer reactions that are important in DNA synthesis and replication, cell division, and growth and survival, particularly for rapidly dividing cells.2 Folate is also required for the remethylation of homocysteine to methionine, which is important for the biosynthesis of S-adenosyl methionine, an essential supplier of methyl groups for the methylation of many compounds including DNA, RNA, proteins, and phospholipids.2 Folate deficiency is associated with misincorporation of uracil instead of thymine into DNA leading to increased chromosomal strand breaking,11 and abnormal methylation reactions.12 Both pathways are implicated in cancer.13 Unless otherwise indicated, the use of the term “folate” throughout the manuscript refers to the generic form of the vitamin as well as the principal plasma folate, 5-methyltetrahydrofolate (5-mTHF), whereas the use of “folic acid” strictly refers to the vitamin in its oxidized form.

Epidemiologic studies show that folate intake above basal requirements reduces risk of developing various tumors by about 30–50% including breast cancer,14, 15 colon adenomas,16 and colon cancer,16 although not all studies show this.17, 18 The importance of folate analogues (e.g., methotrexate) to cancer treatment provides strong evidence that folate is also required by rapidly proliferating tumor cells. Indeed, “supraphysiologic” doses of folate (4–20 times basal needs) in mouse models of colon carcinoma and in clinical studies of pediatric populations with leukemia showed progressive worsening of cancer.19 Although mechanisms are proposed to explain the intracellular pathways by which folate lowers risk, few studies mechanistically address the cancer-progressing effects of folate in humans. Increased growth and folate accumulation by cancer cells with elevated FRα4, 8 suggests this may be one mechanism by which folate could progress carcinomas in humans, and may afford novel insights into cancer management.

Folate receptor vs. reduced folate carrier transport

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Two functionally different systems exist for cellular uptake of folates: (i) membrane-bound FR (FRα and FRβ), which is linked to cell surfaces via a glycosylphosphatydilinositol (GPI) anchor20 and internalizes folates by receptor-mediated endocytosis (described in detail below) and (ii) RFC, which uses a bidirectional anion-exchange mechanism to transport folates into the cytoplasm.21, 22 The remaining discussion is restricted to FRα, the most widely studied FR isoform. The transport kinetics and affinity for folates and antifolates differ significantly between systems (Table I), in part, because FRα does not bind and carry folate into the cell as does RFC, but binds and internalizes folate via endocytosis. Therefore, the dissociation constant, Kd, describes the binding affinity of folates by FRα, whereas the Michaelis constant, Km, describes the binding affinity plus transport of folates by RFC.23 FRα binds oxidized folate – folic acid – with high affinity at low, physiologic concentrations (Kd: <1 nM),21 has high affinity for reduced folates such as 5-mTHF (Kd: 1–10 nM), and relatively low affinity for methotrexate (Kd: >100 nM).21 Although folic acid is normally found in low concentrations in human serum, folic acid consumed in quantities generally found in multivitamin supplements can be transported in serum in the oxidized form.24, 25 FRα can bind folic acid with ∼10 times greater affinity than any of the reduced forms of the vitamin26 or methotrexate.27 The significance of increased serum folic acid in the presence of FOLR1-expressing tumors is unclear.

Table I. Comparison of Folate Receptor α (FRα) and Reduced Folate Carrier (RFC) Uptake of Folates
CharacteristicFRαRFC
  • 1

    5-methyltetrahydrofolate.

DistributionRestrictedNearly all cells
Preferred substrateFolic Acid5-mTHF1
Other substrates5-mTHF1, antifolates to a lesser extentDoes not bind folic acid
Substrate binding concentrationPhysiologic (nM)Pharmacologic (μM)
Preferred substrate plasma levelsLow, unless folic acid intake >300 μgMain circulating form
Binding affinity for folic acidHigh: FRα binds folic acid ∼ 10 times that of reduced folatesLow: folic acid binds FRα 100-200 times greater than it binds RFC
Kd: <1 nM21, 111Km: 200–400 μM21
Binding affinity for 5-mTHF and other reduced folatesHigh: Kd: 1–10 nM21High: Km: 1–10 μM21
Binding affinity for methotrexateLow: Kd: >100 nM21High: Km: 1–5 μM111

In contrast, the RFC is a 591 amino acid transporter with 12 transmembrane domains.22 It is a ubiquitous resident of most cells, and mediates the uptake of reduced folates and methotrexate with relatively high affinity and at higher, micromolar concentrations (Km: 1–10 μM); it has relatively poor affinity for oxidized folic acid (Km: 200–400 μM).21, 27, 28 The potential binding capacity of the RFC for reduced folates is several hundred times greater than the amount of folate in serum.28 Thus, the high selectivity of these transport mechanisms resides in the low affinity of folic acid for the RFC, and the high affinity of folic acid for the FRα, respectively.4, 29

Mechanism of folate uptake by FRα

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Unlike the RFC, endocytosis is the general mechanism of FRα-mediated folate uptake, although the precise pathway(s) has not been delineated. One specialized route, potocytosis, was proposed from the observation that FRα recycled between an acid-resistant (intracellular) and acid-sensitive (extracellular) pool.30 Rothberg et al.31 proposed that FRα was concentrated in clusters in invaginations of the cell membrane surface called caveolae, whereby the membrane would transiently close and internalize the folate-bound receptor complex. Increased acidification of the internal compartment would dissociate folate from the receptor and move it across the membrane into the cytoplasm of the cell using the energy generated by the acidic gradient. The cell surface membrane would then unseal and expose the receptors for the next cycle.31 Subsequent studies refuted the caveolar hypothesis of folate uptake and demonstrated that GPI-anchored proteins are diffusely distributed at the cell surface, that cross-linking of FRα was a necessary precondition for clustering into caveolae, and that cross-linking was not accomplished by folate binding.32 Some investigators, however, still argue for a role of caveloae.33

More recently, the lipid raft-dependent endocytosis theory proposed that membrane microdomains rich in lipids, GPI-anchored proteins, and signalling proteins can cluster into large platforms that can segregate membrane components, and may regulate various processes including lipid sorting, protein trafficking, cell polarization, and signal transduction.34, 35 Several lines of evidence point to a role of lipid raft-dependent endocytosis in FRα-mediated folate uptake,36, 37, 38 though the details of GPI-anchored protein recycling between cell interior and cell surface continue to be elucidated.35, 37 The distinct differences in FRα-mediated folate uptake compared to RFC-mediated transport motivate continued investigation into specialized functions of GPI-anchored proteins beyond that of folate uptake.

Folate transport via FRα in the presence of RFC

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Uncertainty surrounds the relevance of FRα-mediated uptake of folates when RFC is also present. Corona et al.39 reported that FOLR1 mRNA was 3 to 50-fold higher than SLC19A1 mRNA (or solute carrier family 19, the gene encoding RFC) in 16 nonmucinous ovarian carcinoma tumors, but varied greatly. In 5 ovarian carcinoma cell lines grown at physiologic 5-mTHF (20 nM), selective targeting of FRα activity with inhibitors reduced cellular 5-mTHF uptake by only 20% compared to the 70% reduction when RFC activity was inhibited. Only one cell line (IGROV-1) showed inhibition of both proteins to a similar extent. Spinella et al.40 studied a leukemia cell line with a nonfunctional RFC and which expressed the FOLR1 homolog found in the human nasopharyngeal epidermoid (KB) carcinoma cells, a cell line that constitutively expresses FOLR1. FRα in the leukemia cells allowed for growth in low levels of folic acid and 5-formyl THF even in the absence of a functional RFC, consistent with similar reports by other laboratories.41, 42 These in vitro studies refuted earlier suggestions of a functional coordination between FRα and RFC transport43 but do suggest that, when present at a sufficient level, FRα may be a significant uptake route for folates at physiologic concentrations, but only a minor contributor to uptake at higher levels of folates, when the RFC dominates.40

In the few animal studies to examine this, Ma and colleagues44 showed that mice homozygous or heterozygous for the targeted deletion of the folate binding protein 1 gene, Folbp1 (the murine equivalent to human FOLR1), had significantly lower colonic and plasma total folate concentration compared to wildtype (Folbp1+/+). In contrast, partial deletion of murine SLC19A1 (Slc19a1+/−) did not alter colonic and plasma total folate concentration compared to wildtype (Slc19a1+/+). This study's findings are complicated by the inability to study mice homozygous for the Slc19a1 deletion: Slc19a1−/− offspring died in utero and, unlike the Folbp1−/− mice, could not be rescued with folate supplementation of dams throughout pregnancy in this study. This may explain why folate measures were not altered in the Slc19a1-targeted mice. An interesting area of future study would be to investigate whether functional polymorphisms in the human SLC19A1 gene45, 46 in tissues that coexpress FOLR1 alter serum and tissue folate distributions in human cancers.

The folate receptor family

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

FRα belongs to a family of single-chain GPI-anchored membrane proteins.20 The FOLR multigene family (FOLR1, FOLR2 and FOLR3) is localized to chromosome 11q13.3–q14.1, and encodes the gene products FRα, β and γ, respectively.47 The 3 FR isoforms share highly conserved sequences (71–79%) in the open reading frame encoded by exons 4 through 7 in the 3′ region of the gene but differ in amino acid residues in the 5′ untranslated region (UTR) encoded by exons 1 through 4.48 As a result, the isoforms can differ in tissue expression (Table II), function, and biochemical properties.3 For example, FRα and FRβ are both membrane-bound GPI-anchored proteins, unlike the FRγ secretory protein that lacks an efficient signal for GPI modification.49 The FOLR3 gene is polymorphic due to a nonsense mutation resulting in a truncated protein; FRγ, but not the truncated protein, can bind folic acid.50 Also, although all FR isoforms can bind folic acid, FRα binds the physiologic (6S) diastereoisomer of 5-mTHF and 5-formylTHF with affinities as great or greater than folic acid, whereas FRβ binds these folates with >50-fold lower affinity.4, 51 In contrast, FRβ binds (6R) forms up to 12 times more tightly than (6S) forms.4 Even within isoforms, tissue specificity dictates functional differences. FOLR2 is predominantly expressed in normal hematopoietic and in placental cells, yet only FOLR2 expressed in placenta binds folate.51 When expressed in human leukemic cells, however, the receptor appears functional, possibly attributable to differences in post-translational modification between the normal and malignant cells.51 FRβ is also a neutrophilic lineage marker, and levels increase with neutrophil maturation.52 For a detailed discussion of the distribution and function of FRβ and FRγ, readers are referred to the excellent review by Elnakat and Ratnam.51

Table II. Expression of Folate Receptor Isoforms (FRα, FRβ, FRγ) in Normal and Malignant Human Tissues1
SystemFRαFRβFRγ
  • 1

    Adapted from Folate Receptors.4

Normal tissues
 GenitourinaryPlacenta49, 112Placenta112 
Kidney5
Bladder5
Testes113
 Central nervousChoroid plexus114  
Cerebrospinal fluid27
 Hematopoetic Spleen112Hematopoetic50
Thymus112
Neutrophils51
 GastrointestinalSubmandibular salivary glands4  
Colorectum5
 RespiratoryLung5  
 EndocrineHuman milk51, 115  
Malignant tissues
 High expressionNasopharyngeal epidermoid carcinoma20, 49Leukemia51Leukemia51
Non-mucinous ovarian carcinoma5Lymphoma5
Cervical carcinoma63
Uterine carcinoma56
Metastatic endometrial carcinoma5
Primary renal cell carcinoma5
Metastatic pancreatic carcinoma5
 Low or negligible expressionMucinous ovarian carcinoma5  
Primary endometrial carcinoma5
Metastatic renal cell carcinoma5
Lung carcinoma and adenocarcinoma5
Primary breast carcinoma5
Primary bladder carcinoma5
Primary pancreatic carcinoma5
Colorectal carcinoma5, 64
Primary prostate carcinoma5
Primary brain carcinoma4, 58
Primary liver carcinoma5
Primary head and neck carcinoma5

FRα distribution

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

FOLR1 exhibits restricted expression in normal adult epithelial tissues (Table II); however, FRα attachment at the apical surface of cells situates it away from, and out of direct contact with, folate in the circulation.51 For example, in normal intestinal and kidney cells, it functions in the absorption/re-absorption of folate from the luminal “exterior” cavities to the absorbed “interior” environment.53, 54 In placenta, both FRα and FRβ are important for the maternal-to-fetal transport of folate and guard against the consequences of maternal folate deficiency during critical stages in fetal growth and development.4 The central nervous system also depends on normal folate homeostasis, and FRα in choroid plexus is critical for the transfer of folate from the plasma to the cerebrospinal fluid and brain.55 Its function, however, in the apical membrane of lung epithelium has not yet been elucidated.4, 5

In contrast, FRα in cancer is accessible to folate in the circulation.5, 51 Recently, Parker et al.5 measured FR in various normal and tumor tissues using a radiolabeled-folic acid binding assay (Table II). Unlike immunohistochemistry, radioligand binding assays quantitatively determine FRα in tissues by assessing the receptor's ability to functionally bind folic acid.5 The authors corroborated previous reports6 that the highest FRα levels are found in nonmucinous epithelial ovarian cancers, and noted that approximately half of both pancreatic and bladder malignant tissues are FRα-positive. Overall expression in primary breast carcinomas is moderate,5 though Kelley et al.56 confirmed earlier observations57 of higher expression among estrogen receptor (ER)-negative primary breast cancers. Expression is elevated in clinically nonfunctional pituitary adenomas,58 endometrial adenocarcinomas (primary and metastatic),5, 59, 60 cervical carcinoma cell lines,56, 61, 62 and in cervical squamous carcinomas,59, 63 although FOLR1 mRNA was undetected among 8 women with this cancer in another study that did not examine protein levels.60 The capacity to bind folate may distinguish FRα-positive tumor cells from tumors with lower receptor expression possibly because of, or resulting in, different regulatory patterns.

A soluble form of the receptor with the folate-binding site intact arises from proteolytic cleavage of the membrane-associated FRα precursor8 by the combined action of a membrane-associated protease and GPI-specific phospholipases C and D, which are abundant in plasma.64, 65 The release of the soluble FRα into the circulation may provide a means of controlling the expression of FRα at the surface of the cells,64 and may also be a potential serum marker for FRα-positive tumors, since soluble FRα is reported to be low or undetected in normal human serum.66 The differences in the specificity of reduced folate diastereoisomers to FR isoforms could be advantageous in the design of such assays.

FOLR1 gene organization

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

The FOLR1 gene is composed of 7 exons and 6 introns, spans 6.8 kb, and is flanked by consensus splice site sequences20, 47 (the nucleotide sequence is available from Genbank, accession number U20391). Both the organization and transcription of FOLR1 are complex. Tissue-specific expression of the receptor is due to multiple transcripts arising from at least 2 promoter regions, one located upstream and within exon 1 (named P1) and the second upstream of exon 4 (named P4), and alternative splicing of exons 1 to 4.20, 47 The 2 promoters encode FOLR1 transcripts with different 5′ ends but identical amino acid sequences and 3′ ends.47 The identification of a steroid receptor-binding element upstream of P4 suggests steroid hormones may regulate gene expression (discussed below).47 In contrast, the FOLR2 and FOLR3 genes each consist of 5 exons, 4 introns and 1 promoter that encodes a single transcript.20, 51, 67, 68

Regulation of FOLR1 gene expression and protein

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Extracellular folate concentration

Earlier in vitro studies consistently showed that the FRα protein was regulated by the folate concentration in the culture medium,8, 27, 69, 70, 71 and that human KB cells grown at limited concentrations of 5-mTHF or 5-formylTHF had 4-fold higher levels of FRα than cells grown in standard tissue culture media replete with folic acid.8 Elevated FRα was followed by an increase in mRNA,72 growth rate and cellular proliferation of KB cells as measured by cell size, cell doubling time, and [3H]thymidine incorporation.8, 40, 69, 70, 73 The authors concluded that KB cells were regulated by extracellular folate through changes in intracellular folate concentration, and that FRα was important in cellular folate acquisition and cellular survival.8

Intracellular homocysteine concentration

Using human cervical carcinoma cells propagated in folic acid-replete culture medium (2.3 μM), Antony et al.62 showed that elevated FRα was accompanied by a progressive accumulation within the cells of homocysteine, a sensitive marker of intracellular folate deficiency, after stably adapted to grow in low 5-mTHF-containing medium (9 nM). Higher homocysteine appeared to stimulate the interaction of an 18-base cis-regulatory element in the 5′ end of FOLR1 mRNA with a cytosolic protein identified as heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), resulting in translational upregulation of FOLR1 in a dose-dependent manner.62, 74 In contrast, total cysteine, cystathionine, and methionine concentrations were not significantly changed from baseline values. The authors reported that the lowest concentrations of homocysteine that could stimulate interaction between the cis-element and hnRNP E1 were 20–25 μM, values consistent with physiologic folate deficiency.62 Possibly, imbalances in folate availability may lead to downstream alterations in cytosolic proteins to stimulate FOLR1 translation.

Other reports of transcriptional and translational regulation

Roberts et al.75 observed that the longer length of the 5′ leader sequence of the KB P1 promoter mRNA compared to the 5′ leader of the KB P4 promoter mRNA increased the potential to form a secondary structure that could influence translational efficiency. However, others observed that the interaction of various proteins within this region transcriptionally increased P1 promoter activity76 or inhibited FOLR1 gene expression73 in ovarian carcinoma. Sadasivan et al.77 showed that elevated FRα was mediated by both a 4.5-fold increase in the initial transcription rate of KB cells and a 2.4-fold prolongation of FOLR1 mRNA half-life following transfer from a folic acid-replete to a folate-deplete culture medium. An RNA gel-shift assay of the FOLR1 5′ UTR and a portion of the 3′ coding region revealed unique complexes with cytosolic proteins from KB cells grown in folic acid-replete cultures that were not observed with the cytosol from KB cells grown in folate-deplete cultures. Although the proteins were not identified, the authors speculated that their presence in a folic acid-replete environment functioned to de-stabilize the FOLR1 mRNA by binding to both 5′ and 3′ regions, a model similar to that of the coordinated regulation between ferritin and transferrin for iron regulation. Roberts et al.75 hypothesized that some cell types might preferentially express a transcript that is less efficiently translated in order to regulate cell growth, thus allowing for rapid increase in FR levels when the cells have a higher demand for folate.

Epigenetic regulation

Several authors proposed that elevated FRα might be the result of hypomethylation of the FOLR1 gene.20, 47, 71 Methylation of DNA in regions rich with CpG bases can serve to silence gene transcription in many eukaryotic genes. Folate deficiency and elevated plasma homocysteine and S-adenosyl homocysteine are associated with decreased DNA methylation.2, 78 Although earlier studies suggested a potential role of epigenetic regulation in FOLR1 expression, the evidence is inconclusive. The report by Elwood et al.20 that promoter regions upstream from, and including, exons 1–4 in the KB FOLR1 gene were relatively rich in CpG bases compared to the remainder of the downstream nucleotide sequence47 was not verified in a recent publication.79 Exposure of KB cells to DNA methylation inhibitors did not result in elevated FOLR1 mRNA levels.71 In a subsequent study by the same authors,80 exposure to DNA methylation inhibitors increased FRα levels by 2- to 3-fold in a methotrexate-resistant KB cell subline; the authors, however, were unable to identify which sites of the gene harbored the methylation changes and could not exclude hypomethylation of other genes. Epigenetic changes offer a plausible explanation for elevated FRα in some tumor types compared to normal cells,5 and advances in methylation assay design and construction may prove useful to extend earlier investigations.

Hormonal regulation

Reports that various steroid hormones, such as estrogen 17β-estradiol,56, 57 tamoxifen,56 the glucocorticoid receptor,66 and retinoic acid81 alter FRα levels support the need to employ epidemiologic study designs to investigate the relevance of these associations at a population level. For example, the effect of endogenous estrogen production on FOLR1 expression in human tumors, such as breast,56 in an increasingly overweight society is unknown.

Genetic mutations

Ablation of the FOLR gene in fetal mice results in embryonic death.82 Mutations in the human FOLR1 gene appear to be infrequent. Orr and Kamen83 reported a rare mutation at amino acid 67 of the FOLR1 gene in a squamous cell carcinoma cell line of the head and neck that blocked folate binding and transport but otherwise appeared to be processed correctly. Mangiarotti et al.84 reported the existence of several point mutations in the 5′ UTR region in the CABA I serous ovarian carcinoma cell line that were not present in IGROV1 cell lines. The FRα in CABA I cells bound folic acid with lower affinity although its distribution and interaction with cellular regulatory molecules were comparable to that of IGROV1 cells.

Because studies of human tumors are few, it is informative to review those studies that identified mutations in the FOLR1 gene in neural tube defects (NTDs), a folate-related disorder. Trembath et al.85 detected a single de novo silent mutation in the sequence coding for the stop codon in exon 6 (TGA>TAA), which corresponds to exon 7 according to the nomenclature used in this review (Genbank U20391), out of a sample of 154 children affected with NTD compared to healthy control children in Iowa, Minnesota, and Nebraska using mutation detection enhancement gels. The authors postulated that the mutation, though silent, could interfere with effective stopping of translation, tying up or inhibiting protein release from the translational machinery. Analysis of the 326 population-based control children without NTDs identified a phenotypically normal child who possessed an amino acid substitution of serine to asparagine within exon 6 (corresponds to exon 7, Genbank U20391) of the FOLR1 gene.85 Barber et al.86 did not detect any polymorphisms or mutations in the exons and intron–exon boundaries in DNA specimens from 1,688 individuals participating in 2 population-based case-control studies of NTDs conducted by the California Birth Defects Monitoring Program using 3 different screening methods (single-stranded conformational polymorphism [SSCP] analysis, DNA sequencing, and dideoxy fingerprinting). In an Italian population, De Marco et al.87 identified 4 unrelated NTD patients out of 50 with de novo missense mutations in exon 7 (Genbank U20391) and the 3′ UTR of the FOLR1 gene, and one out of 150 control children with a gene conversion within the coding region using SSCP and Southern blot analysis. The authors reported that all 4 of the mutations in exon 7 and the 3′ UTR affect the carboxy-terminal amino acid membrane tail, or the GPI anchor region of the receptor. Ablation of the GPI anchor would inhibit binding of the receptor to the membrane surface and inhibit folate internalization. Nilsson et al.88 identified 2 novel insertion and deletion mutations in a 323 bp region in the 5′ UTR of the FOLR1 gene among 6 of 778 adult outpatients in Sweden using SSCP and DNA sequencing. The regions appeared to house binding sites for transcription factors. Interestingly, 2 patients with the novel insertion had plasma homocysteine concentrations of 27 and 32 μmol/l. The authors posited that mutations in the promoter region might have subtle implications that affect FRα level and activity to varying degrees; such mild defects might manifest only later in life and may be important in the pathogenesis of certain diseases such as cancer.

FRα function

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

The above experiments suggested that FRα might confer a growth advantage to the tumor by modulating folate uptake from serum, which may facilitate rapid cellular growth and division. Others proposed that FRα might affect cell proliferation via cell signalling pathways similar to other cellular membrane proteins with a GPI anchor.4, 9 Indeed, loss of caveolin expression, a potential tumor suppressor that is inversely related to FRα levels,73 has been documented to interact with cell signalling proteins and may, thus, implicate FRα in intracellular signalling events,89 and a number of lymphocytic proteins that use GPI anchors have been shown to be capable of mediating mitogenic responses.90 Possibly, FRα may be elevated to increase folate uptake in order to stimulate cells to repair DNA damage in transcription factors or other proteins during the early stages of carcinogenesis.76 The inability to repair these proteins may result in continued FRα expression, which may eventually support the transition to a cellular environment that favors tumor progression and increased tumor folate requirements for rapid growth.

FRα and prognosis

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Few studies have examined human tumor levels of FRα and patient survival. In the most widely investigated and FRα-positive tumor, nonmucinous epithelial ovarian carcinoma, protein expression is associated with tumor progression,6 and also with ovarian cancers of high grade, platinum-therapy resistance, and poor prognosis,7 suggesting that metabolic defects related to its upregulation occur early in carcinogenesis. Toffoli et al.7 investigated whether FRα in epithelial ovarian cancer specimens is a predictor of response to chemotherapy and survival. Among 58 patients with residual epithelial ovarian cancer after primary surgery, failure to respond to chemotherapy (complete or partial remission) was about 15-fold higher (95% confidence interval, 2.96–77.43) when tumors had elevated (>median) FRα levels following multivariable adjustment, although this estimate was based on 8 cases. Further, patient survival at 3 years was 30% among patients with high FRα (n = 30) compared to 62% among patients with tumors with low FRα levels (n = 28; p = 0.06). The authors provided possible hypotheses to explain their findings including that FRα may increase folate uptake which could stimulate cells to repair DNA damage caused by platinum, FRα involvement in signal transduction could help cells progress through the cell cycle phases compared to cells with low levels of FRα, or FRα could predispose cells to overcome drug injury, as observed for genes involved in cellular signalling or apoptosis.91, 92 There is a significant dearth of systematic clinical studies that examined response to therapy and survival among individuals with FRα-positive tumors and, in particular, among various tissue types.93

Exploitation of FRα for therapeutic and diagnostic potential

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

The restricted tissue-specific expression of FR isoforms enables exploitation of FRα for the selective delivery of cytotoxic agents into malignant cells with reduced toxic side effects on nontarget tissues. The basis for FRα-targeted drug delivery lies in the substrate specificity of folic acid to FRα. In a series of experiments, Leamon et al.94 showed that covalent conjugation of folic acid with various pharmaceuticals such as horseradish peroxidase, IgG serum albumin and ribonuclease, resulted in the nondestructive intracellular delivery of these molecules via FRα. In a subsequent study, the authors showed that cellular protein synthesis was inhibited in a time and dose dependent manner by the uptake of the folic acid-toxin conjugate into the cell, providing direct evidence that folic acid conjugates not only reach the cytoplasm but do so in a functionally active form.95, 96 Unlike other drug delivery systems that are internalized and trafficked to lysosomes for destruction, the notable advantage of FRα-mediated endocytosis is the recognition of folic acid by the cell as essential and, consequently, most of the toxin attached to folic acid is not delivered to lysosomes, but rather is retained in endocytic compartments or released into the cytoplasm.97

The arsenal of folic acid-conjugated therapeutic agents delivered successfully to FRα-positive cancer cells advanced rapidly over the past decade. These include, but are not limited to, low molecular weight chemotherapeutic agents, liposomes with entrapped drugs, antisense oligonucleotides, and immunotherapeutic agents.98 Cytotoxic agents encapsulated by folic acid-conjugated liposomes offer the advantage of delivering a drug cargo in the order of 103–104 molecules per vesicle.99 Optimal targeting of liposomes to tumor cells relies on prolonged liposomal circulation time to ensure tumor accumulation, extravasation of the permeable tumor microvasculature to gain entry to the tumor interstitial/ascitic fluid, binding of folic acid-conjugated liposomes to FRα-expressing tumor cells for internalization and release of liposomal contents via endocytosis.99 FRα-targeted liposomes have also been evaluated for the delivery of antisense oligodeoxyribonucleotides (ODNs) as a method of cancer gene therapy.100 Delivery of antisense ODNs complementary to various gene sequences including epidermal growth factor receptor in cultured KB cells, and c-fos in Chinese hamster ovary cells, not only inhibited growth of these cells but also sensitized HER-2-expressing breast cancer cells to chemotherapeutic agents both in culture and in a murine xenograph.100 Nonviral gene delivery techniques have also employed folic acid conjugates of various polymers or polycationic vectors, such as polylysine and polyethylenimine with polyethyleneglycol.101 The electrostatic attraction between the polycations and their polyanionic oligonucleotide cargo promotes condensed packaging of nucleotides that facilitates endocytic entry into FRα-expressing cells.98 An alternative to polylysine vectors are nonlinear polycationic cascade polymers.102 Also known as dendrimers, these polycations are spheroidal, can amplify the amount of therapeutic or diagnostic agent reaching the tumor cells,103 and mediate higher transfection efficiency of therapeutic agents into cells than their linear counterparts.102 Immune-mediated targeting of FRα-positive tumors, founded on the historical observation that antibodies are raised against FRα,104 offer sophisticated strategies to deliver cytotoxic agents via bispecific antibody targeted T cell therapy (characterized by cytotoxic T lymphocyte-mediated tumor cell lysis), adoptive transfer of tumor-specific T lymphocytes (characterized by T cell activation and cytokine release), and immunocytokines (e.g., targeting IL-2/MOv19 fusion protein to FRα-expressing cells to activate migrating T cells).105

The exploitation of FRα-mediated binding of folic acid-conjugated imaging agents to tumor cells is also a rapidly advancing area of cancer diagnostics.106 For example, folic acid conjugated to various radiopharmaceutical agents such as 99mTechnetium (99mTc) showed promising in vivo tumor uptake qualities.107 Magnetic resonance (MR) imaging contrast agents based on dendrimers conjugated to folic acid and targeted to FRα-positive tumor cells have also shown improved enhancement of MR images in mice.103 These newer conjugates are capable of concentrating at pathologic sites as non-invasive probes for diagnostic imaging and do not suffer from problems associated with ligands such as antibodies, which among other factors, can be immunogenic and expensive to produce.98 Finally, the sensitivity of GPI links to specific phospholipases, such as phospholipase C and D, which are abundant in plasma, may also provide a means to control FRα level at the cell surface64 or to develop sensitive biomarker assays of the soluble protein for early detection,66, 108 as has recently been described for 2 folate-related disorders.109, 110

Implications and future directions

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References

Given the data, it is unknown whether perturbations in FOLR1 gene expression initiate neoplasia, or if neoplasia is a precondition for elevated FOLR1 expression. The positive association of FRα with tumor grade and stage suggests a role in tumor progression, but whether this role consists of active participant or sideline observer is unclear. Clinical trials of FRα-mediated uptake of folic acid-conjugated label or toxin offer much promise for diagnostic and therapeutic potential. These data also suggest that elevated serum folic acid, possibly resulting from consumption of folic acid-containing multivitamins, could plausibly bind to, and be endocytosed by, FRα-positive tumors, the implications of which are unknown. Epidemiologic and clinical studies using human tumor specimens are lacking and increasingly needed to understand the role of FRα in tumor etiology, progression, and patient survival. Suggested high priority areas of future research are warranted to investigate (i) etiologic associations between environmental (dietary, hormonal, obesity) and genetic (polymorphic, epigenetic) influences on FOLR1 expression in human cancers, particularly in the era of folic acid fortification and widespread vitamin supplement use in countries such as Canada and the United States; (ii) additional studies of prognostic (survival) outcomes of various FRα-positive human cancers and (iii) the utility of developing assays for the early detection of FRα-positive tumors using serum markers such as the soluble FR. Future studies are needed to assess and monitor any potentially untoward effects of environmental influences on FRα-positive tumors.

References

  1. Top of page
  2. Abstract
  3. The role of folate in cancer
  4. Folate receptor vs. reduced folate carrier transport
  5. Mechanism of folate uptake by FRα
  6. Folate transport via FRα in the presence of RFC
  7. The folate receptor family
  8. FRα distribution
  9. FOLR1 gene organization
  10. Regulation of FOLR1 gene expression and protein
  11. FRα function
  12. FRα and prognosis
  13. Exploitation of FRα for therapeutic and diagnostic potential
  14. Implications and future directions
  15. Acknowledgements
  16. References
  • 1
    Hanahan D, Weinberg R. The hallmarks of cancer. Cell 2000; 100: 5770.
  • 2
    Choi SW, Mason JB. Folate and carcinogenesis: an integrated scheme. J Nutr 2000; 130: 12932.
  • 3
    Henderson GB. Folate-binding proteins. Ann Rev Nutr 1990; 10: 31935.
  • 4
    Antony AC. Folate receptors. Ann Rev Nutr 1996; 16: 50121.
  • 5
    Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon C. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Anal Biochem 2005; 338: 28493.
  • 6
    Toffoli G, Cernigoi C, Russo A, Gallo A, Bagnoli M, Boicchi M. Overexpression of folate binding protein in ovarian cancers. Int J Cancer 1997; 74: 1938.
  • 7
    Toffoli G, Russo A, Gallo A, Cernigoi C, Miotti S, Sorio R, Tumolo S, Boicchi M. Expression of folate binding protein as a prognostic factor for response to platinum-containing chemotherapy and survival in human ovarian cancer. Int J Cancer (Pred Oncol) 1998; 79: 1216.
  • 8
    Kane MA, Elwood PC, Portillo RM, Antony AC, Najfeld V, Finley A, Waxman S, Kolhouse J. Influence on immunoreactive folate-binding proteins of extracellular folate concentration in cultured human cells. J Clin Invest 1988; 81: 1398406.
  • 9
    Miotti S, Bagnoli M, Tomassetti A, Colnaghi MI, Canevari S. Interaction of folate receptor with signaling molecules lyn and Ga13 in detergent-resistant complexes from the ovary carcinoma cell lines. J Cell Sci 2000; 113: 34957.
  • 10
    Mayes PA. Structure and function of the water-soluble vitamins. In: MurrayRK, GrannerDK, MayesPA, RodwellV, eds. Harper's biochemistry, 25th ed. New York: McGraw-Hill, 2000. 62741.
  • 11
    Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, Wickramasinghe SN, Everson RB, Ames B. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc Natl Acad Sci USA 1997; 94: 32905.
  • 12
    Stern LL, Mason JB, Selhub J, Choi S. Genomic DNA hypomethylation, a characteristic of most cancers, is present in peripheral leukocytes of individuals who are homozygous for the C677T polymorphism in the methylenetetrahydrofolate reductase gene. Cancer Epidemiol Biomark Prev 2000; 9: 84953.
  • 13
    Strathdee G, Appleton K, Illand M, Millan DW, Sargent J, Paul J, Brown R. Primary ovarian carcinomas display multiple methylator phenotypes involving known tumor suppressor genes. Am J Pathol 2001; 158: 11217.
  • 14
    Zhang SM, Willett WC, Selhub J, Hunter DJ, Giovannucci EL, Holmes MD, Colditz GA, Hankinson S. Plasma folate, vitamin B6, vitamin B12, homocysteine, and risk of breast cancer. J Natl Cancer Inst 2003; 95: 37380.
  • 15
    Shrubsole MJ, Jin F, Dai Q, Shu XO, Potter JD, Hebert JR, Gao YT, Zheng W. Dietary folate intake and breast cancer risk: results from the Shanghai Breast Cancer Study. Cancer Res 2001; 61: 713641.
  • 16
    Giovannucci E. Epidemiologic studies of folate and colorectal neoplasia: a review. J Nutr 2002; 132: 2350S55S.
  • 17
    Feigelson HS, Jonas CR, Robertson AS, Mc Cullough ML, Thun MJ, Calle E. Alcohol, folate, methionine, and risk of incident breast cancer in the American Cancer Society Cancer Prevention Study II Nutrition Cohort. Cancer Epidemiol Biomark Prev 2003; 12: 1614.
  • 18
    Slattery ML, Schaffer D, Edwards SL, Ma KN, Potter J. Are dietary factors involved in DNA methylation associated with colon cancer? Nutr Cancer 1997; 28: 5262.
  • 19
    Kim Y-I. Role of folate in colon cancer development and progression. J Nutr 2003; 133: 3731S9S.
  • 20
    Elwood PC, Nachmanoff K, Saikawa Y, Page ST, Pacheco P, Roberts S, Chung K-N. The divergent 5 termini of the human folate receptor (hFR) mRNAs originate from two tissue-specific promoters and alternative splicing: characterization of the hFR gene structure. Biochemistry 1997; 36: 146778.
  • 21
    Rijnboutt S, Jansen G, Posthuma G, Hynes JB, Schornagel JH, Strous G. Endocytosis of GPI-linked membrane folate receptor-α. J Cell Biol 1996; 132: 3547.
  • 22
    Kaufman Y, Drori S, Cole PD, Kamen BA, Sirota J, Ifergan I, Arush MW, Elhasid R, Sahar D, Kaspers GJ, Jansen G, Matherly LH, et al. Reduced folate carrier mutations are not the mechanism underlying methotrexate resistance in childhood acute lymphoblastic leukemia. Cancer 2004; 100: 77382.
  • 23
    Atkins P. Physical chemistry, 6th ed. New York: WH Freeman and Company, 1997.
  • 24
    Kelly P, McPartlin J, Goggins M, Weir DG, Scott J. Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements. Am J Clin Nutr 1997; 65: 17905.
  • 25
    Lucock M, Yates Z. Folic acid—vitamin and panacea or genetic time bomb? Nat Rev Genet 2005; 6: 23540.
  • 26
    Wang X, Shen F, Freisheim JH, Gentry LE, Ratnam M. Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates. Biochem Pharmacol 1992; 44: 1898901.
  • 27
    Kane MA, Waxman S. Biology of disease. Role of folate binding proteins in folate metabolism. Lab Invest 1989; 60: 73746.
  • 28
    Herbert VD, Colman N. Folic acid and vitamin B12. In: ShilsME, YoungV, eds. Modern nutrition in health and disease, 7th ed. Philadelphia: Lea and Febiger, 1988. 388416.
  • 29
    Kamen BA, Smith A. A review of folate receptor α cycling and 5-methyltetrahydrofolate accumulation with an emphasis on cell models in vitro. Adv Drug Deliv Rev 2004; 56: 108597.
  • 30
    Kamen BA, Wang MT, Streckfuss AJ, Peryea X, Anderson R. Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles. J Biol Chem 1988; 263: 136029.
  • 31
    Rothberg KG, Ying YS, Kolhouse JF, Kamen BA, Anderson R. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol 1990; 110: 63749.
  • 32
    Mayor S, Rothberg KG, Maxfield F. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 1994; 264: 194851.
  • 33
    Ritter TE, Fajardo O, Matsue H, Anderson RGW, Lacey S. Folate receptors targeted to clathrin-coated pits cannot regulate vitamin uptake. Proc Natl Acad Sci USA 1995; 92: 38248.
  • 34
    Simons K, Ikonen E. Functional rafts in cell membranes. Nature 1997; 387: 56972.
  • 35
    Le Roy C, Wrana J. Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat Rev Mol Cell Biol 2005; 6: 11226.
  • 36
    Sabharanjak S, Mayor S. Folate receptor endocytosis and trafficking. Adv Drug Deliv Rev 2004; 56: 1099109.
  • 37
    Mayor S, Sabharanjak S, Maxfield F. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998; 17: 462638.
  • 38
    Chatterjee S, Smith ER, Hanada K, Stevens VL, Mayor S. GPI anchoring leads to sphingolipid-dependent retention of endocytosed proteins in the recycling endosomal compartment. EMBO J 2001; 20: 158392.
  • 39
    Corona G, Giannini F, Fabris M, Toffoli G, Boiocchi M. Role of folate receptor and reduced folate carrier in the transport of 5-methyltetrahydrofolic acid in human ovarian carcinoma cells. Int J Cancer 1998; 75: 12533.
  • 40
    Spinella MJ, Brigle KE, Sierra EE, Goldman I. Distinguishing between folate receptor-α-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells. J Biol Chem 1995; 270: 78429.
  • 41
    Dixon KH, Mulligan T, Chung KN, Elwood PC, Cowan K. Effects of folate receptor expression following stable transfection into wild type and methotrexate transport-deficient ZR-75–1 human breast cancer cells. J Biol Chem 1992; 267: 241407.
  • 42
    Brigle KE, Seither RL, Westin EH, Goldman I. Increased expression and genomic organization of a folate-binding protein homologous to the human placental isoform in L1210 murine leukemia cell lines with a defective reduced folate carrier. J Biol Chem 1994; 269: 426772.
  • 43
    Kamen BA, Smith AK, Anderson R. The folate receptor works in tandem with a probenecid-sensitive carrier in MA104 cells in vitro. J Clin Invest 1991; 87: 14429.
  • 44
    Ma DW, Finnell RH, Davidson LA, Callaway ES, Spiegelstein O, Piedrahita JA, Salbaum JM, Kappen C, Weeks BR, James J, Bozinov D, Lupton JR, et al. Folate transport gene inactivation in mice increases sensitivity to colon carcinogenesis. Cancer Res 2005; 65: 88797.
  • 45
    Shaw GM, Lammer EJ, Zhu H, Baker MW, Neri E, Finnell RH. Maternal periconceptional vitamin use, genetic variation of infant reduced folate carrier (A80G), and risk of spina bifida. Am J Med Genet 2002; 108: 16.
  • 46
    Shaw GM, Zhu H, Lammer EJ, Yang W, Finnell R. Genetic variation of infant reduced folate carrier (A80G) and risk of orofacial and conotruncal heart defects. Am J Epidemiol 2003; 158: 74752.
  • 47
    Saikawa Y, Price D, Hance KW, Chen TY, Elwood P. Structural and functional analysis of the human KB cell folate receptor gene P4 promoter: cooperation of three clustered Sp1-binding sites with initiator region for basal promoter activity. Biochemistry 1995; 34: 995161.
  • 48
    Antony AC. The biological chemistry of folate receptors. Blood 1992; 79: 280720.
  • 49
    Antony AC, Kane MA, Portillo RM, Elwood PC, Kolhouse J. Studies of the role of a particulate folate-binding protein in the uptake of 5-methyltetrahydrofolate by cultured human KB cells. J Biol Chem 1985; 260: 149117.
  • 50
    Shen F, Wu M, Ross JF, Miller D, Ratnam M. Folate receptor type γ is primarily a secretory protein due to lack of an efficient signal for glycosylphosphatidylinositol modification: protein characterization and cell type specificity. Biochemistry 1995; 34: 56605.
  • 51
    Elnakat H, Ratnam M. Distribution, functionality and gene regulation of folate receptor isoforms: implications in targeted therapy. Adv Drug Deliv Rev 2004; 56: 106784.
  • 52
    Ross JF, Wang H, Behm FG, Mathew P, Wu M, Booth R, Ratnam M. Folate receptor β is a neutrophilic lineage marker and is differentially expressed in myeloid leukemia. Cancer 1999; 85: 34857.
  • 53
    Selhub J, Emmanouel D, Stavropoulos T, Arnold R. Renal folate absorption and the kidney folate binding protein. I. Urinary clearance studies. Am J Physiol 1987; 252: F7506.
  • 54
    Selhub J, Nakamura S, Carone F. Renal folate absorption and the kidney folate binding protein. II. Microinfusion studies. Am J Physiol 1987; 252: F75760.
  • 55
    Ramaekers VT, Blau N. Cerebral folate deficiency. Dev Med Child Neurol 2004; 46: 84351.
  • 56
    Kelley KM, Rowan BG, Ratnam M. Modulation of the folate receptor α gene by the estrogen receptor: mechanism and implications in tumor targeting. Cancer Res 2003; 63: 28208.
  • 57
    Rochman H, Selhub J, Karrison T. Folate binding protein and the estrogen receptor in breast cancer. Cancer Detect Prev 1985; 8: 715.
  • 58
    Evans C-O, Reddy P, Brat DJ, O'Neill EB, Craige B, Stevens VL, Oyesiku N. Differential expression of folate receptor in pituitary adenomas. Cancer Res 2003; 63: 421824.
  • 59
    Veggian R, Fasolato S, Menard S, Minucci D, Pizzetti P, Regazzoni M, Tagliabue E, Colnaghi MI. Immunohistochemical reactivity of a monoclonal antibody prepared against human ovarian carcinoma on normal and pathological female genital tissues. Tumori 1989; 75: 5103.
  • 60
    Wu M, Gunning W, Ratnam M. Expression of folate receptor type α in relation to cell type, malignancy, and differentiation in ovary, uterus, and cervix. Cancer Epidemiol Biomark Prev 1999: 77582.
  • 61
    Sun XL, Murphy BR, Li QJ, Gullapalli S, Mackins J, Jayaram HN, Srivastava A, Antony AC. Transduction of folate receptor cDNA into cervical carcinoma cells using recombinant adeno-associated virions delays cell proliferation in vitro and in vivo. J Clin Invest 1995; 96: 153547.
  • 62
    Antony AC, Tang YS, Khan RA, Biju MP, Xiao X, Li QJ, Sun XL, Jayaram HN, Stabler SP. Translational upregulation of folate receptors is mediated by homocysteine via RNA-heterogeneous nuclear ribonucleoprotein E1 interactions. J Clin Invest 2004; 113: 285301.
  • 63
    Pillai MR, Chacko P, Kesari LA, Jayaprakash PG, Jayaram HN, Antony AC. Expression of folate receptors and heterogeneous nuclear ribonucleoprotein E1 in women with human papillomavirus mediated transformation of cervical tissue to cancer. J Clin Pathol 2003; 56: 56974.
  • 64
    Campbell IG, Jones TA, Foulkes WD, Trowsdale J. Folate-binding protein is a marker for ovarian cancer. Cancer Res 1991; 51: 532938.
  • 65
    Brown D, Waneck GL. Glycosyl-phosphatidylinositol-anchored membrane proteins. J Am Soc Nephrol 1992; 3: 895906.
  • 66
    Tran T, Shatnawi A, Zheng X, Kelley KM, Ratnam M. Enhancement of folate receptor α expression in tumor cells through the glucocorticoid receptor: a promising means to improved tumor detection and targeting. Cancer Res 2005; 65: 443141.
  • 67
    Page ST, Owen WC, Price K, Elwood P. Expression of the human placental folate receptor transcript is regulated in human tissues. Organization and full nucleotide sequence of the gene. J Mol Biol 1993; 229: 117583.
  • 68
    Sadasivan E, Cedeno MM, Rothenberg S. Characterization of the gene encoding a folate-binding protein expressed in human placenta. Identification of promoter activity in a G-rich SP1 site linked with the tandemly repeated GGAAG motif for the ets encoded GA-binding protein. J Biol Chem 1994; 269: 472535.
  • 69
    Matsue H, Rothberg KG, Takashima A, Kamen GA, Anderson RGW, Lacey S. Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate. Proc Natl Acad Sci USA 1992; 89: 60069.
  • 70
    Luhrs CA, Raskin CA, Durbin R, Wu B, Sadasivan E, McAllister W, Rothenberg S. Transfection of a glycosylated phosphatidylinositol-anchored folate binding protein completmentary DNA provides cells with the ability to survive in low folate medium. J Clin Invest 1992; 90: 8407.
  • 71
    Hsueh CT, Dolnick B. Altered folate binding protein mRNA stability in KB cells grown in folate-deficient medium. Biochem Pharmacol 1993; 45: 253745.
  • 72
    Sadasivan E, Rothenberg SP. The complete amino acid sequence of a human folate binding protein from KB cells determined from the cDNA. J Biol Chem 1989; 264: 580611.
  • 73
    Bagnoli M, Tomassetti A, Figini M, Flati S, Dolo V, Canevari S, Miotti S. Downmodulation of caveolin-1 expression in human ovarian carcinoma is directly related to folate receptor overexpression. Oncogene 2000; 19: 475463.
  • 74
    Sun XL, Antony AC. Evidence that a specific interaction between an 18-base cis-element in the 5′-untranslated region of human folate receptor-α mRNA and a 46-kDa cytosolic trans-factor is critical for translation. J Biol Chem 1996; 271: 2553947.
  • 75
    Roberts SJ, Chung KN, Nachmanoff K, Elwood P. Tissue-specific promoters of the α human folate receptor gene yield transcripts with divergent 5′ leader sequences and different translational efficiencies. Biochem J 1997; 326: 43947.
  • 76
    Tomassetti A, Mangiarotti F, Mazzi M, Sfoorzini S, Miotti S, Galmozzi E, Elwood PC, Canevari S. The variant hepatocyte nuclear factor 1 activates the P1 promoter of the human-folate receptor gene in ovarian carcinoma. Cancer Res 2003; 63: 696704.
  • 77
    Sadasivan E, Regec A, Rothenberg S. The half-life of the transcript encoding the folate receptor α in KB cells is reduced by cytosolic proteins expressed in folate-replete and not in folate-depleted cells. Gene 2002; 291: 14958.
  • 78
    Yi P, Melnyk S, Pogribna M, Pogribny IP, Hine RJ, James S. Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation. J Biol Chem 2000; 275: 2931823.
  • 79
    Jhaveri MS, Wagner C, Trepel J. Impact of extracellular folate levels on global gene expression. Mol Pharmacol 2001; 60: 128895.
  • 80
    Hsueh CT, Dolnick B. Regulation of folate binding protein gene expression by DNA methylation in methotrexate-resistant KB cells. Biochem Pharmacol 1994; 47: 101927.
  • 81
    Bolton JA, Wood SA, Kennedy D, Don RH, Mattick J. Retinoic acid-dependent upregulation of mouse folate receptor-α expression in embryonic stem cells, and conservation of alternative splicing patterns. Gene 1999; 230: 21524.
  • 82
    Piedrahita JA, Oetama B, Bennett GD, van Waes J, Kamen BA, Richardson J, Lacey SW, Anderson RGW, Finnell R. Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development. Nat Genet 1999; 23: 22832.
  • 83
    Orr RB, Kamen B. Identification of a point mutation in the folate receptor gene that confers a dominant negative phenotype. Cancer Res 1995; 55: 84752.
  • 84
    Mangiarotti F, Miotti S, Galmozzi E, Mazzi M, Sforzini S, Canevari S, Tomassetti A. Functional effect of point mutations in the α-folate receptor gene of CABA I ovarian carcinoma cells. J Cell Biochem 2001; 81: 48898.
  • 85
    Trembath D, Sherbondy AL, Vandyke DC, Shaw GM, Todoroff K, Lammer EJ, Finnell RH, Marker S, Lerner G, Murray J. Analysis of select folate pathway genes, PAX3, and human T in a midwestern neural tube defect population. Teratology 1999; 59: 33141.
  • 86
    Barber RC, Shaw GM, Lammer EJ, Greer KA, Biela TA, Lacey SW, Wasserman CR, Finnell R. Lack of association between mutations in the folate receptor-α gene and spina bifida. Am J Med Genet 1998; 76: 3107.
  • 87
    De Marco P, Moroni A, Merello E, de Franchis R, Andreussi L, Finnell RH, Barber RC, Cama A, Capra V. Folate pathway gene alterations in patients with neural tube defects. Am J Med Genet 2000; 95: 21623.
  • 88
    Nilsson TK, Borjel A. Novel insertion and deletion mutations in the 5′-UTR of the folate receptor-α gene: an additional contributor to hyperhomocysteinemia? Clin Biochem 2004; 37: 2249.
  • 89
    Bagnoli M, Canevari S, Figini M, Mezzanzanica D, Raspagliesi F, Tomassetti A, Miotti S. A step further in understanding the biology of the folate receptor in ovarian carcinoma. Gynecol Oncol 2003; 88: S1404.
  • 90
    Low MG. The glycosyl-phosphatidylinositol anchor of membrane proteins. Biochim Biophys Acta 1989; 988: 42754.
  • 91
    Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell 1994; 78: 53942.
  • 92
    Righetti SC, Della Torre G, Pilotti S, Menard S, Ottone F, Colnaghi MI, Pierotti MA, Lavarino C, Cornarotti M, Oriana S, Bohm S, Bresciani GL, et al. A comparative study of p53 gene mutations, protein accumulation, and response to cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer Res 1996; 56: 68993.
  • 93
    Antony AC. Folate receptors: reflections on a personal odyssey and a perspective on unfolding truth. Adv Drug Deliv Rev 2004; 56: 105966.
  • 94
    Leamon CP, Low P. Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis. Proc Natl Acad Sci USA 1991; 88: 55726.
  • 95
    Leamon CP, Low P. Cytotoxicity of momordin-folate conjugates in cultured human cells. J Biol Chem 1992; 267: 2496671.
  • 96
    Leamon CP, Pastan I, Low P. Cytotoxicity of folate-pseudomonas exotoxin conjugates toward tumor cells. J Biol Chem 1993; 268: 2484754.
  • 97
    Low PS. Folate receptor-targeted drugs for cancer and inflammatory diseases. Adv Drug Deliv Rev 2004; 56: 10558.
  • 98
    Leamon CP, Low P. Folate-mediated targeting: from diagnostics to drug and gene delivery. Drug Discov Today 2001; 6: 4451.
  • 99
    Gabizon A, Shmeeda H, Horowitz AT, Zalipsky S. Tumor cell targeting of liposome-entrapped drugs with phospholipids-anchored folic acid-PEG conjugates. Adv Drug Deliv Rev 2004; 56: 117792.
  • 100
    Zhao XB, Lee R. Tumor-selective targeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor. Adv Drug Deliv Rev 2004; 56: 1193204.
  • 101
    Ward CM, Pechar M, Oupicky D, Ulbrich K, Seymour L. Modification of pLL/DNA complexes with a multivalent hydrophilic polymer permits folate-mediated targeting in vitro and prolonged plasma circulation in vivo. J Gene Med 2002; 4: 53647.
  • 102
    Haensler J, Szoka FC,Jr. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug Chem 1993; 4: 3729.
  • 103
    Konda SD, Aref M, Brechbiel M, Wiener E. Development of a tumor-targeting MR contrast agent using the high-affinity folate receptor: work in progress. Invest Radiol 2000; 35: 507.
  • 104
    Coney LR, Tomassetti A, Carayannopoulos L, Frasca V, Kamen BA, Colnaghi MI, Aurawski VJ. Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein. Cancer Res 1991; 51: 612532.
  • 105
    Lu Y, Low PS. Immunotherapy of folate receptor-expressing tumors: review of recent advances and future prospects. J Control Release 2003; 91: 1729.
  • 106
    Ke C-Y, Mathias CJ, Green M. Folate-receptor-targeted radionuclide imaging agents. Adv Drug Deliv Rev 2004; 56: 114360.
  • 107
    Leamon CP, Parker MA, Vlahov IR, Xu LC, Reddy JA, Vetzel M, Douglas N. Synthesis and biological evaluation of EC20: a new folate-derived, (99m)Tc-based radiopharmaceutical. Bioconjug Chem 2002: 120010.
  • 108
    Mantovani LT, Miotti S, Menard S, Canevari S, Raspagliesi F, Bottini C, Bottero F, Colnaghi M. Folate binding protein distribution in normal tissues and biological fluids from ovarian carcinoma patients as detected by the monoclonal antibodies MOv18 and MOv19. Eur J Cancer 1994; 30A: 3639.
  • 109
    Rothenberg SP, da Costa MP, Sequeira JM, Cracco J, Roberts JL, Weedon J, Quadros E. Autoantibodies against folate receptors in women with a pregnancy complicated by a neural-tube defect. N Engl J Med 2004; 350: 13442.
  • 110
    Ramaekers VT, Rothenberg SP, Sequeira JM, Opladen T, Blau N, Quadros EV, Selhub J. Autoantibodies to folate receptors in the cerebral folate deficiency syndrome. N Engl J Med 2005; 352: 198591.
  • 111
    Jackman AL, Theti DS, Gibbs D. Antifolates targeted specifically to the folate receptor. Adv Drug Deliv Rev 2004; 56: 111125.
  • 112
    Ross JF, Chaudhuri PK, Ratnam M. Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer 1994; 73: 243243.
  • 113
    Holm J, Hansen SI, Hoier-Madsen M, Christensen TB, Nichols CW. Characterization of a high-affinity folate receptor in normal and malignant human testicular tissue. Biosci Rep 1999; 19: 57180.
  • 114
    Theti DS, Jackman A. The role of α-folate receptor-mediated transport in the antitumor activity of antifolate drugs. Clin Cancer Res 2004; 10: 10809.
  • 115
    Antony AC, Utley CS, Marcell PD, Kolhouse JF. Isolation, characterization, and comparison of the solubilized particulate and soluble folate binding proteins from human milk. J Biol Chem 1982; 257: 100819.