טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRaz Shachar
SubjectMolecular Mechanisms Regulating the Expression and
Subcellular Localization of Folylpoly-Gamma-
Glutamate Synthetase (FPGS) in Folate
Metabolism and Antifolate...
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

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Folates are essential vitamins that serve as co-factors in a variety of key cellular processes such as de novo DNA biosynthesis, amino acid biosynthesis and DNA methylation. Since folates are crucial for DNA synthesis, folate antagonists (i.e. antifolates) were developed in order to block folate metabolism and induce apoptosis in rapidly dividing cells. Antifolates are currently used in the treatment of various diseases (i.e. various cancers as well as non-malignant disorders). The enzyme folylpoly-γ-glutamate synthetase (FPGS) catalyzes the addition of a polyglutamate tail to (anti)folates, which then display an increased affinity for their target enzymes and become unrecognized by efflux transporters. Since FPGS plays a key role in folate metabolism and in the cytotoxicity of antifolates, it is not surprising that decreased FPGS activity is an established determinant of antifolate resistance in cancer.

Herein we undertook experiments to decipher the molecular mechanisms underlying loss of FPGS function in antifolate resistance in cancer. We discovered allele-specific silencing of the wild type (WT) FPGS allele in MTA C-3 antifolate-resistant leukemia cells that harbor a heterozygous FPGS mutation. This leads to the exclusive expression of the mutant FPGS allele, which encodes an inactive enzyme. The silencing of the WT allele in MTA C-3 cells is mediated by the binding of a transcriptional repressive protein complex, comprised of the transcription factors Smad2, Smad4, Ets-1 and Sp1 as well as epigenetic modifiers, to a novel transcriptional regulatory element that we identified in exon12 of FPGS. Strikingly, a reverse correlation between the expression of FPGS and the binding of Smad4 and Ets-1 to exon12 of FPGS was also identified in blast specimens derived from leukemia patients. Additional repression of FPGS gene expression was achieved by exposure of the cells to 5-Aza-deoxycytidine and trichostatin A (TSA). This was correlated with elevated levels of Smad3 and Smad7 and equally affected both WT and mutant FPGS alleles, suggesting the existence of additional binding sites for these transcription factors in the FPGS gene.

The expression of FPGS was also down-regulated in cells under hypoxia. This was accompanied with decreased expression of multiple genes in the folate metabolism and nucleotide homeostasis pathway as well as with hypoxia-induced cell-cycle arrest. Antifolates are cell cycle-dependent drugs and were unable to induce DNA damage under cell cycle arrest, which resulted in complete antifolate resistance under hypoxia.

The human FPGS (hFPGS) has not been extensively studied regarding its physiological role in folate metabolism, but rather in regards to its role in antifolate drug resistance. In contrast to the dogma in the field, we recently discovered that the hFPGS is not a cytosolic protein, but rather associated with the plasma membrane and is apparently part of a large protein complex. This suggests the coupling of folate uptake and polyglutamylation, thus providing a better explanation for the intracellular metabolism of folates as well as the ability of cells to accumulate micromolar concentrations of intracellular folates despite the low, nanomolar, physiological folate levels in the blood. These findings have important implications for folate homeostasis, chemoresistance and targeted cancer therapeutics.