Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type


Degree Name



Cancer Biology

First Advisor

Larry H. Matherly


Folates are essential cofactors of tumor cell proliferation and survival required for nucleotide biosynthesis and amino acid metabolism. In cancer therapy, inhibition of folate-dependent metabolic pathways has been achieved through the use of antifolates. Unfortunately, the efficacy of many clinically approved antifolates is limited by a lack of tumor selectivity. Facilitative transport of folates into mammalian cells is achieved by the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). As PCFT is a folate-proton symporter with an acidic pH optimum, PCFT may provide a mechanism for targeting cytotoxic antifolates to tumors, based on their acidic microenvironments. To establish the feasibility of this approach, we systematically determined the expression profiles for PCFT and RFC. In various human malignant cell lines and tissues PCFT was highly expressed and functional, while its expression in normal tissue was more limited compared to RFC.

Screening and characterization of multiple series of novel antifolates led to the identification of 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl and thienoyl antifolates, which were selective for PCFT-mediated uptake, but not RFC. Upon internalization, these novel antifolates were extensively polyglutamylated, as detected by high performance liquid chromatography (HPLC). Growth inhibition assay paired with nucleoside protection identified glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) in the de novo purine biosynthesis pathway as the principle drug target. This was confirmed by in situ measurement of [14C]glycine incorporation into [14C]formylGAR in treated cells. Furthermore, contraction of intracellular purine nucleotide triphosphate pools occurred in a dose- and time-dependent manner, as demonstrated by quantitative HPLC analysis. Further, drug treatment induced S-phase accumulation eventually leading to irreversible cell death. Drug treatment caused a significant delay in tumor growth in an in vivo efficacy trial of SCID mice implanted with subcutaneous human tumors where PCFT was the sole mechanism of drug uptake.

Although these compounds are not substrates for RFC, its expression does impact drug efficacy by influencing intracellular tetrahydrofolate (THF) cofactor pools. Loss of functional RFC leads to increased polyglutamylation of these analogs within the cell causing enhanced cytotoxicity. Similarly, these novel PCFT-selective antifolates displayed increased in vivo efficacy in subcutaneously implanted human tumors lacking RFC.

Our finding of widespread PCFT expression in human solid tumors paired with our discovery of novel antifolates internalized via PCFT offers exciting new therapeutic possibilities for selectively targeting tumors based on their acidic microenvironments. These compounds display immense therapeutic potential, especially in tumors with de novo or acquired resistance to classic antifolates caused by lack of RFC function.