Reduced Folate Carrier Gene Silencing in Multiple Antifolate-resistant Tumor Cell Lines Is Due to a Simultaneous Loss of Function of Multiple Transcription Factors but Not Promoter Methylation

Lilah Rothem,Michal Stark,Yotam Kaufman,Lior Mayo,Yehuda G Assaraf

doi:10.1074/jbc.m309092200

Lilah Rothem, Michal Stark + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m309092200

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Abstract

The human reduced folate carrier (hRFC) is the major uptake route for antifolates used in cancer chemotherapy. Here we explored the molecular basis for the decrease or loss of hRFC gene expression in seventeen tumor cell lines with resistance to multiple antifolates due to impaired antifolate transport. We studied the role of various cis-acting elements including CRE/AP-1-like element and GC-box in hRFC promoters A and B, respectively, as well as AP-2, Mzf-1 and E-box that are contained within or near four tandemly repeated sequences upstream of promoter A. Decreased or abolished binding either to [32P]GC-box, Mzf-1, AP-1, E-box, or CRE oligonucleotides was detected in approximately 50-80% of antifolate-resistant cell lines. Strikingly, approximately 80% of the cell lines displayed a simultaneously decreased binding to three or more of these hRFC promoter elements, whereas normal AP-2 binding was retained. The possible contribution of promoter methylation to hRFC gene silencing was also explored. None of the antifolate-resistant cell lines, except for MDA-MB-231 cells, showed hRFC promoter methylation; consistently, MDA-MB-231 was the only cell line that retained binding to all six cis-acting elements. Western blot analysis demonstrated decreased expression of transcriptional activators (pCREB-1, pATF-1, USF-1, c-Fos, c-Jun, Sp1, and Sp3) and/or increased expression of repressors (short Sp3 isoforms), whereas normal AP2alpha levels were retained. Transient expression of the relevant transcription factors restored, at least partially, both promoter binding and hRFC gene expression. This is the first report that transcriptional silencing of the hRFC gene in multiple tumor cell lines with resistance to various novel antifolates is a result of a simultaneous loss of function of multiple transcription factors but not promoter methylation.

Highlights

Reduced folates are essential cofactors involved in various one-carbon transfer reactions resulting in the biosynthesis of nucleotides and amino acids essential for cell growth and DNA replication [1]
We have recently shown that loss of human reduced folate carrier (hRFC) gene expression in two drug-resistant cell lines with impaired antifolate transport is associated with alterations in the expression and binding of transcription factors to the inducible cAMP response element (CRE)/AP-1 and constitutive GC-box in hRFC promoters A and B, respectively [25]
We strikingly found that the majority of these cell lines displayed loss of binding to E-box, AP-1, and myeloid zinc finger 1 (Mzf-1), cis-acting elements that were not previously associated with decreased hRFC gene expression

Summary

Introduction

Reduced folates are essential cofactors involved in various one-carbon transfer reactions resulting in the biosynthesis of nucleotides and amino acids essential for cell growth and DNA replication [1]. We recently initiated studies that focus on the elucidation of the molecular basis of the loss of hRFC gene expression in antifolate-resistant tumor cells To this end, we recently found that alterations in the expression and binding of transcription factors to cAMP response element (CRE)/AP-1-like element and GC-box in hRFC promoters A and B, respectively, underlie the loss of. The markedly decreased binding to these cis-acting elements was associated with decreased expression of various transactivators This is the first demonstration that simultaneous alterations in the expression and function of multiple transcription factors that bind to various cis-acting elements are a major mechanism of loss of hRFC gene expression in tumor cell lines that display resistance to various novel antifolates due to impaired drug uptake. We further show that while hRFC promoter silencing via methylation is rare in antifolate-resistant cell lines, it appears to alleviate the stress that would otherwise lead to alterations in the expression and function of transcription factors

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