Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1.

Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94 Progressive deletions of the hPCFT promoter construct (-2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions -50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.

Expression of Folate Pathway Genes in Stage III Colorectal Cancer Correlates with Recurrence Status Following Adjuvant Bolus 5-FU-Based Chemotherapy.

Colorectal cancer is commonly treated with 5-fluorouracil and 5-formyltetrahydrofolate (leucovorin). Metabolic action of leucovorin requires several enzymatic steps that are dependent on expression of corresponding coding genes. To identify folate pathway genes with possible impact on leucovorin metabolism, a retrospective study was performed on 193 patients with stage III colorectal cancer. Relative expression of 22 genes putatively involved in leucovorin transport, polyglutamation and metabolism was determined in tumor and mucosa samples using quantitative real-time polymerase chain reaction. After surgery, patients received adjuvant 5-fluorouracil-based bolus chemotherapy with leucovorin during six months, and were followed for 3 to 5 years. Cox regression analysis showed that high tumoral expression of the genes SLC46A1/PCFT (proton-coupled folate transporter) and SLC19A1/RFC-1 (reduced folate carrier 1) correlated significantly (p < 0.001 and p < 0.01, respectively) with a decreased risk of recurrent disease, measured as disease-free survival (DFS). These two genes are involved in the transport of folates into the cells and each functions optimally at a different pH. We conclude that SLC46A1/PCFT and SLC19A1/RFC-1 are associated with DFS of patients with colorectal cancer and hypothesize that poor response to 5-fluorouracil plus leucovorin therapy in some patients may be linked to low expression of these genes. Such patients might need a more intensified therapeutic approach than those with high gene expression. Future prospective studies will determine if the expression of any of these genes can be used to predict response to leucovorin.

DNA methylation and expression of the folate transporter genes in colorectal cancer.

Folate has a central role in the cell metabolism. This study aims to explore the DNA methylation pattern of the folate transporter genes FOLR1, PCFT, and RFC1 as well as the corresponding protein expressions in colorectal cancer (CRC) tissue and adjacent non-cancerous mucosa (ANCM). Our results showed statistically significant differences in the DNA-methylated fraction of all three genes at several gene regions; we identified three differentially methylated CpG sites in the FOLR1 gene, five CpG sites in the PCFT gene, and six CpG sites in the RFC1 gene. There was a pronounced expression of the FRα and RFC proteins in both the CRC and ANCM tissues, though the expression was attenuated in cancer compared to the paired ANCM tissues. The PCFT protein was undetectable or expressed at a very low level in both tissue types. Higher methylated fractions of the CpG sites 3-5 in the RFC1 gene were associated with a lower protein expression, suggestive of epigenetic regulation by DNA methylation of the RFC1 gene in the colorectal cancer. Our results did not show any association between the RFC and FRα protein expression and tumor stage, TNM classification, or tumor location. In conclusion, this is the first study to simultaneously evaluate both DNA methylation and protein expression of all three folate transporter genes, FOLR1, PCFT, and RFC1, in colorectal cancer. The results encourage further investigation into the possible prognostic implications of folate transporter expression and DNA methylation.