Abstract
Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR pump-rich EVs in cancer cells and their ability to confer multiple anticancer drug resistance.
Highlights
The frequent emergence of drug resistance phenomena to structurally and functionally unrelated anticancer drugs known as multidrug resistance (MDR) continues to be a major impediment to curative cancer chemotherapy [1,2,3,4,5,6]
MCF-7/MR cells were co-stained with antibodies to ABCG2 which served as a specific biomarker for extracellular vesicles (EVs), as well as with ZO-1 (Figure 1A–1C) and occludin (Figure 1D–1F)
Based on our present findings and on our previous studies with EVs in MCF-7/MR cells [8,12], we propose a composite model for the structure and function of ABCG2-rich EVs, and their ability to confer resistance to multiple anticancer drugs (Figure 9)
Summary
The frequent emergence of drug resistance phenomena to structurally and functionally unrelated anticancer drugs known as multidrug resistance (MDR) continues to be a major impediment to curative cancer chemotherapy [1,2,3,4,5,6]. In spite of the important implications of these drug-concentrating EVs for cancer chemotherapy, nothing is known about their structure, biogenesis and ability to sequester multiple anticancer drugs Towards this end, we here explored the possible association of cytoskeletal components characteristic of polarized epithelia including tight junction (TJ) proteins, actin and microtubule filaments as well as Ezrin-Radixin-Moesin (ERM) complex proteins with the membrane of EVs. TJ proteins have three mutually exclusive functions; a fence function which differentiates between proteins of the apical and basolateral membranes, a gate function which controls the paracellular passage of ions and solutes in-between epithelial and endothelial cells, as well as a bridge function which facilitates the communication between neighboring cells [9,10]. A new modality of MDR is described here in which ABC transporters that are selectively sorted out to the membrane of EVs, actively pump and sequester multiple anticancer drugs within the EV lumen, thereby resulting in a marked chemoresistance
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