|Ph.D Student||Goler-Baron Vicky|
|Subject||ABCG2-Rich Extracellular Vesicles: a Determinant of MDR and|
Targeted Photodynamic Therapy of Cancer
|Department||Department of Biology||Supervisor||Professor Yehuda Assaraf|
|Full Thesis text|
Deciphering novel mechanisms of multidrug resistance (MDR) and their overcoming is a major goal of cancer research. Towards this end, we here characterized the structure and the function of a novel mechanism of MDR that was recently identified in our lab. This novel modality of MDR is based upon ABCG2-rich extracellular vesicles (EVs) that form between neighbor breast cancer cells and efficiently concentrate the anticancer drug mitoxantrone (MR) up to ~1000 fold higher than in the culture medium, thus resulting in MR resistance. We found that EVs are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by tight junction proteins. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin (ERM) protein complex selectively localized to the EVs membrane, suggesting a key role in the tethering of MDR pumps to the actin cytoskeleton. We found that apart from MR, EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones (IAs) and methotrexate via ABCG2-dependent intravesicular drug concentration. In addition to anticancer drugs, EVs efficiently concentrated the B2 vitamin riboflavin. Based on our findings we suggest that ABCG2-rich EVs mimic lactating breast epithelium and serve as a reliable model for studying ABCG2-mediated MDR in breast cancer cells.
We further found that the PI3K-AKT signaling pathway mediates the selective ABCG2 targeting to EVs. Thus, inhibition of Akt signaling, restored drug sensitivity to MR and topotecan, hence being equivalent to MDR reversal achieved with the specific ABCG2 transport inhibitor Ko143. Remarkably, treatment of MCF-7/MR cells with Ko143 resulted in cytoplasmic re-localization of ABCG2, similarly to the phenotype observed after Akt inhibition. We conclude that the PI3K-Akt signaling pathway is a key regulator of subcellular localization of ABCG2, EVs biogenesis and functional MDR. Moreover, proper folding of ABCG2 and its targeting to the EVs membrane are crucial components of the biogenesis of EVs and their MDR function. Finally, we developed a novel strategy to differentially target and kill MDR cancer cells. Specifically, we applied a photodynamic therapy approach demonstrating that illumination of EVs that accumulated photosensitive cytotoxic drugs including IAs and topotecan resulted in intravesicular formation of reactive oxygen species and severe damage to the EVs membrane that is shared by EVs-forming cells. Thus, we propose that EVs-based MDR modalities can be rationally converted to a pharmacologically lethal Trojan horse to selectively overcome and eradicate MDR cancer cells.