Abstract 4546: Expression profiling of the major folate facilitative transporters in human tumors and normal tissues

Membrane transport of folates into mammalian cells involves major facilitative transporters including the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). Classical antifolates such as methotrexate (Mtx), pemetrexed, or raltitrexed are all substrates for RFC and PCFT such that the cytotoxic effects of these drugs are determined by levels of these transporters in tumors and normal tissues. As PCFT is a folate-proton symporter with an acidic pH optimum, extracellular pH represents another key determinant of transport, such that PCFT may provide a mechanism for targeting cytotoxic antifolates to tumors, based on their acidic microenvironments. We recently identified novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that are selective transport substrates for PCFT over RFC and that potently inhibit cell proliferation by targeting glycinamide ribonucleotide formyltransferase in de novo purine nucleotide biosynthesis. To better exploit this approach for selectively targeting tumors versus normal tissues, we systematically determined expression profiles for PCFT and RFC in human malignancies and normal tissues. Real-time reverse transcription PCR (qRT-PCR) was used to determine transcript levels in 57 human solid tumor and 27 leukemia cell lines. Both RFC and PCFT transcripts were highly expressed in the majority of solid tumor cell lines whereas only RFC was expressed appreciably in the leukemia cell lines. For 10 solid tumor cell lines, PCFT and RFC transcripts were accompanied by corresponding levels of respective proteins on western blots. Functional validation of transporter expression in solid tumor cell lines was determined by transport of [ 3 H]Mtx at pH 5.5 for PCFT, and at pH 7.2 for RFC. Tumor uptake of [ 3 H]Mtx correlated closely with transcript and protein levels for both PCFT and RFC. qRT-PCR of cDNA arrays from 48 normal human tissues measured high levels of PCFT expression in the small intestine, kidney, liver, and the adrenal and pituitary glands. On cDNA arrays of human primary tumors, significant albeit variable levels of PCFT transcripts were detected in breast, colon, kidney, liver, lung, ovary, and prostate tumors. Patterns of transporter expression in primary tumors were confirmed by immunohistochemistry of tissue arrays probed with transporter-specific antibodies. In conclusion, we have established the expression patterns and pH-specific transport for PCFT and RFC in human solid tumor cell lines, primary tumors and select normal tissues. This study represents the first comprehensive analysis of PCFT expression in tumors and normal tissues and how this relates to RFC. Our finding of widespread PCFT expression in human solid tumors paired with our previous discovery of novel PCFT-specific antifolates offers exciting new therapeutic possibilities for selectively targeting chemotherapy to tumors based on their acidic microenvironments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4546.