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

Abstract

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.

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

“…The PCFT is constitutively expressed in EOC [ 14 ] and has a modest expression in most normal tissues [ 8 , 29 , 39 ]. Further, PCFT is active under the acidic pH conditions associated with the tumor microenvironment, although significant PCFT transport is detectable up to pH 7 [ 154 ]. Above pH 7, PCFT transport activity is low [ 30 ].…”

The evolving biology of the proton‐coupled folate transporter: New insights into regulation, structure, and mechanism

“…In tissues which express PCFT, RFC is invariably present. A dynamic interplay has been reported between these major folate transport systems that depends on transporter levels and extracellular pH 38 . In HEK293 (human embryonic kidney) cells, hPCFT transport is maximal at pH 4.5, 39 with significant activity up to pH 6.5 16 .…”

“…For hPCFT, protein–protein homo and hetero interactions might explain anomalies such as the basis for an intracellular PCFT fraction reported in multiple studies, 57,126 the negative impact of increasing hRFC on hPCFT activity, 38 and the “disconnect” between levels of endogenous hPCFT proteins and hPCFT transport activity described in human tumor cell lines 127 …”

“…A dynamic interplay has been reported between these major folate transport systems that depends on transporter levels and extracellular pH. 38 In HEK293 (human embryonic kidney) cells, hPCFT transport is maximal at pH 4.5, 39 with significant activity up to pH 6.5. 16 As extracellular pH is further increased, hPCFT transport decreases precipitously.…”

Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes

“… 17 , 18 AMT and MTX enter cells primarily through the proton-coupled folate transporter in the upper gastrointestinal tract (at acidic pH optimum) and through the reduced folate carrier in normal tissues (at neutral pH). 16 , 19 For MTX’s mechanism in RA, multiple hypotheses beyond folate antagonism exist, including adenosine signaling, cytokine modulation, generation of reactive oxygen species, and downregulation of some adhesion molecules. 20 Compared with MTX, AMT is retained more efficiently in the cell because of its higher affinity for folylpolyglutamate synthase, but AMT itself has negligible binding affinity toward FRβ.…”