Abstract 873: Acute regulation of human reduced folate carrier by folates as a novel adaptive mechanism to folate deprivation.
Authors: Hou Zhanjun , Orr Steve , Etnyre Erika , Cherian Christina , Matherly Larry 
Published: August 2014

Abstract
The reduced folate carrier (RFC) is the major tissue folate transporter in mammalian cells. RFC is essential for providing folates for biosynthetic pathways leading to nucleotides and amino acids. Clinically relevant antifolates including methotrexate (Mtx), pemetrexed, and pralatrexate are all substrates for RFC and RFC levels and function are key determinants of antitumor activities. While recent reports have described the development of novel 6-pyrrolo[2,3-d]pyrimidine antifolates with selectivity for cellular uptake and tumor targeting by the proton-coupled folate transporter, RFC levels are nonetheless important determinants of antitumor activities for these agents, by impacting cellular folate pools which compete for polyglutamylation and binding to intracellular enzyme targets. Human RFC is subject to complex transcriptional and posttranscriptional controls. RFC forms homo-oligomers, providing another potential level of regulation, as a dominant-phenotype results with mutant-wild type RFC mixtures. This reflects impaired intracellular trafficking of oligomeric RFC from the endoplasmic reticulum to the plasma membrane surface. To explore the question of whether folates can acutely regulate RFC levels or activity, RFC-null HeLa cells were stably transfected with RFCMyc-his10 in pCDNA3 plasmid. Transfectants were cultured without folate (10 d), then with (6R,S)5-formyltetrahydrofolate (leucovorin (LCV)) from 0.5 to 200 nM (5 d). Cells were assayed for RFC transcripts and total cellular RFC proteins. Membrane surface RFC was measured by sulfo-N-hydroxysuccinimide-SS-biotin labeling and the formation of homo-oligomers was assessed by cross-linking with 1,1-methanediyl bismethanethiosulfonate. Finally, RFC transport was assayed with [3H]Mtx at neutral pH. Levels of RFC transcripts and total cell RFC protein progressively increased with increasing LCV up to 200 nM. Conversely, surface RFC and [3H]Mtx transport were maximal at ∼ 2 nM LCV, then deceased (∼2-3-fold) with increasing LCV. These effects were maximal at 20 nM LCV. RFC plasma membrane targeting was confirmed by immunofluorescence staining and confocal microscopy. RFC protein was completely localized to the plasma membrane at low (≤ 2 nM) LCV concentrations, however, at higher concentrations, significant intracellular RFC was detected. There were no changes in ratios of RFC homo-oligomer over monomer in response to changes in extracellular LCV. Our results suggest a novel acute regulation of RFC in response to changes in extracellular folate concentrations involving (i) increased RFC transcripts and proteins, possibly due to differences in transcript stability, and (ii) increased cytosolic RFC due to impaired intracellular trafficking and membrane targeting. The net effect would be enhanced RFC-mediated folate and antifolate accumulations under conditions of folate deprivation.
Citation Format: Zhanjun Hou, Steve Orr, Erika Etnyre, Christina Cherian, Larry Matherly. Acute regulation of human reduced folate carrier by folates as a novel adaptive mechanism to folate deprivation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 873. doi:10.1158/1538-7445.AM2013-873