טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentIlan Ifergan
SubjectThe Role of Transporters in Folate Homeostasis and
Anticancer Drug Resistance
DepartmentDepartment of Biology
Supervisor Full Professor Assaraf Yehuda
Full Thesis textFull thesis text - English Version


Abstract

The role of plasma membrane transporters in drug resistance and homeostasis of folate vitamins is of great physiological and therapeutic importance. The reduced folate carrier (RFC) is the primary high-affinity bi-directional transporter for reduced folate cofactors essential for nucleotide biosynthesis and thus DNA replication. Here we show that upon folate deprivation, RFC activity results in a decreased rate of cellular proliferation. Moreover, we suggest that the cytotoxic folate efflux activity of RFC may be abrogated by a novel adaptive down-regulation of this transporter. In contrast to RFC, the breast cancer resistance protein (BCRP/ABCG2) is currently the only known transporter that exports both mono-, di-, and triglutamate conjugates of folate and the antifolate methotrexate (MTX)). We therefore explored the relationship between cellular folate status and BCRP expression as well as transport function. Toward this end, MCF-7 breast cancer cells, with low BCRP protein levels, and their MR (i.e.  mitoxantrone, an  anticancer drug which is a specific BCRP substrate)-resistant MCF-7/MR subline, with BCRP overexpression  were gradually deprived (three months) of folic acid from 2.3 µM to 3 nM resulting in the sublines MCF-7/LF and MCF-7/MR-LF, respectively. These low folate adapted sublines displayed only residual mRNA, protein levels and activity of BCRP. Additionally, the low folate adapted sublines displayed a ~2-fold increase in the 4h accumulation of [3H]folic acid along with a significant increase in folylpoly-γ-glutamate synthetase activity (FPGS), an enzyme that catalyzes the conversion of folate monoglutamates to polyglutamates that no longer serve as BCRP substrates. Moreover, we found that cellular adaptation of MCF7/MR cells to short-term (two weeks) folate deprivation is associated with a selective confinement of BCRP to the endoplasmic reticulum instead of the plasma membrane. Hence, consistent with the mono- and polyglutamate folate exporter function of BCRP, down-regulation of BCRP, increased FPGS activity and selective confinement of BCRP to the endoplasmic reticulum appear to be crucial components of cellular folate homeostasis. During these BCRP cellular confinement studies, we discovered that this transporter is highly confined to cell-cell attachment zones in the MCF-7 breast cancer sublines MCF-7/MR and MCF-7/FLV1000 in which wild type (R482) BCRP is overexpressed. The cell-cell attachment zones were found to be the membrane of novel extracellular vesicles in which mitoxantrone was rapidly, dramatically and specifically sequestered via BCRP. We suggested that the novel extracellular vesicles serve as cytotoxic drug disposal chambers shared by multiple neighbor cancer cells. This finding constitutes a novel modality of anticancer drug resistance.