טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSheban Daoud
SubjectMolecular Basis of Antifolate Resistance in Carcinoma
Cells under Hypoxia
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

Antifolates are folic acid antagonists which are widely used as antineoplastic agents for the treatment of multiple solid tumors and hematologic malignancies. Antifolates exert their cytotoxic activity by blocking essential enzymes in the folate metabolic pathway, therefore disrupting the biosynthesis of purines and thymidylate results in cell death. While hemato-lymphoid tumors are generally more responsive to antifolates, solid tumors frequently exhibit inherent or acquired resistance to these chemotherapeutic agents. The core of various solid tumors was shown to be highly hypoxic due to an imbalance between oxygen supply and consumption. These studies showed a heterogeneous oxygenation of solid tumors and identified the tumor core as the most hypoxic region within the tumor. It has been shown by several research groups that hypoxia invokes chemoresistance in cancer cells by different mechanisms such as reduced generation of free radicals, increased activity of DNA repair enzymes, cell cycle arrest and induction of anti-apoptosis. However, until recently, there has not been any investigation regarding the impact of hypoxia on the pharmacological activity of antifolates in solid tumors. Towards this end, we have shown that carcinoma cells display a complete resistance to various hydrophilic and lipophilic antifolates under severe hypoxia. Furthermore, we demonstrated that tumor cells retain sensitivity to the proteasome inhibitor Bortezomib and the topoisomerase II inhibitor Doxorubicin, which exert their cytotoxic activity in a manner that is independent of the cell cycle. We demonstrated that this antifolate resistance is attributable to the hypoxia-induced cell cycle arrest which results in the inability of antifolates to induce DNA damage. Remarkably, we have also shown for the first time that this cell cycle arrest is associated with repression of gene expression of ubiquitously expressed key enzymes and transporters in folate metabolism and nucleoside homeostasis. Whereas the normal expression of these genes is crucial for the de novo biosynthesis of purines and thymidylate, suppression of nucleoside biosynthesis has been previously proposed as a mechanism underlying cell cycle arrest under hypoxia. Therefore, revealing the molecular mechanism of the downregulation of folate-related genes is crucial for deciphering the molecular basis of the antifolates resistance under hypoxia which is provoked by hypoxia-induced cell cycle arrest. We provided evidence that these gene expression repression under hypoxia is likely to be dependent on epigenetic modifications. Moreover, whilst promoters of folate-related genes repressed under hypoxia were not methylated, Chromatin Immunoprecipitation analysis revealed that an increase in histone H3 lysine 9 di-methylation in the promoters might be the underlying mechanism of the downregulation. Nonetheless, histone deacetylases inhibitor Trichostatin A but not the demethylation agent 5-Aza-2′-deoxycytidine partially prevented the repression in gene expression of the proton-coupled folate transporter (PCFT) implying that histone deacetylation might mediate PCFT downregulation under hypoxia alongside with other regulatory factors. This study bears important implications for the molecular basis underlying antifolate resistance under hypoxia and its rational overcoming in solid tumors.