טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBram Airon (Eran)
SubjectCellular and Molecular Mechanisms Underlying
ABCG2-Dependent Multi-Drug Resistance in Cancer
DepartmentDepartment of Biology
Supervisor Professor Yehuda Assaraf
Full Thesis textFull thesis text - English Version


Abstract

ABCG2, a recently discovered ATP-Binding Cassette (ABC) transporter, is one of the most prominent Multidrug Resistance (MDR) efflux transporters known. The aim of the current research was to unravel the major mechanisms facilitating functional overexpression of ABCG2 in MDR tumor cell-lines and to gain insight into different aspects of ABCG2 structure-function relationship and transporter-drug interaction. Here we report that ABCG2 is transcriptionally upregulated in various drug-selected MDR cell-lines by two distinct mechanisms: a) Loss of epigenetic silencing in parental cells via abcg2 promoter demethylation and b). Mono-allelic gene-amplification of the abcg2 locus. We reveal here that drug selection of the abcg2-heterozygous (C421A) lung-cancer cell-line A549, with the triazoloacridinone anti-cancer agent C-1305, resulted in a C421-specific monoallelic gene-amplification of abcg2; hence, leading to overexpression of the functionally superior ABCG2 transport-variant at the plasma membrane of the MDR A549/K1.5 sub-line. Thus, we portray a unique model for clonal selection and expansion of ABCG2-overexpressing MDR cells, under chemotherapeutic drug selection, via selective monoallelic gene amplification. Additionally, we find here that the frequently emerging R482G/T-ABCG2 point-mutation alters ABCG2 substrate specificity, thus enabling ABCG2-dependent cellular resistance to lipophilic-antifolate chemotherapy. Moreover, we explored the impact of short-term chemotherapeutic drug treatment on ABCG2 gene expression. Remarkably, 12-24hr pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of anthracycline chemotherapeutics, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that chemotherapy-induced re-activation of ABCG2 gene expression in cancer cells is an early molecular event en route towards acquisition of MDR. Collectively, these above findings have important implications for the emergence of malignant cells harboring an ABCG2-dependent MDR phenotype during chemotherapeutic treatment.

Our work also bears insight into substrate recognition properties of ABCG2. Using a platform of Imidazoacridinone (IA) anti-cancer agents we established a functional assay for the detection of ABCG2-substrate recognition. We discovered that a hydroxyl group at one of the R1-R3 positions in the proximal IA ring was essential for ABCG2-mediated efflux and consequent acridinone resistance. Moreover, elongation of the common distal side chain attenuated ABCG2-dependent efflux, thereby resulting in the retention of parental cell sensitivity. Interestingly, our results demonstrated that these IA determinants mediating ABCG2-dependent drug efflux are precisely those that facilitate IAs cellular cytotoxicity. Hence, the current study offers molecular insight into structural determinants facilitating ABCG2-mediated substrate recognition and consequent MDR. The possible clinical implications for the future design of ABCG2-circumventing therapies are also discussed.