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.
Highlights
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)
Results are presented for RFC transport in R1-11/Tet-on-RFC cells (Fig. 4C; left panel) which paralleled those for R1-11/Tet-on-RFC/PCFT cells treated with AGF94 (Fig. 4C; right panel)
Whereas PCFT is important for folate internalization at the acid pH of the upper GI9,17,30, its contribution to net folate accumulation versus FRα and RFC over a wide range of pH values [including those associated with the tumor microenvironment[39,40] or accompanying cell proliferation in culture41] is not established
Summary
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). Uptake of folates into mammalian cells is principally mediated by facilitative transporters, the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), and by folate receptors (FRs)[6,7,8]. These uptake systems are genetically distinct and functionally diverse, and each plays a unique role in mediating folate uptake across epithelia and into systemic tissues[6,7,8]. Key cytosolic C1 enzymes such as dihydrofolate reductase, thymidylate synthase, and the purine biosynthetic enzymes β-glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) are important targets for cancer therapy with these inhibitors[25,26]
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