Limits...
Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

Goler-Baron V, Assaraf YG - PLoS ONE (2011)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.

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.

Show MeSH

Related in: MedlinePlus

Involvement of microtubules in the formation of EVs in MCF-7/MR cells and inhibition of microtubule polymerization by nocodazole.MCF-7/MR cells were either untreated (A–C and G–I) or treated with nocodazole (33 µM) for 1 hr at 37°C (D–F and J–L). Microtubules were visualized using mouse anti β-tubulin antibody followed by incubation with FITC-conjugated donkey anti-mouse IgG (B, E, H and K). Cells were co-reacted with antibodies to ABCG2 (BXP-53, A and D) or ERM (G and J), processed and analyzed as in Fig. 5 legend. Quantification of ABCG2 and ERM protein expression on the surface of EVs prior to and following treatment with nocodazole (M); random fields stained as in upper panels were photographed using the same exposure conditions for untreated and nocodazole-treated cells. The surface area of EVs and its relative fluorescence intensity were estimated using the AxioVision program. The fluorescence at the EVs surface indicated protein levels at the EVs membrane. A total of 100 EVs were analyzed for each examined protein. Bars represent SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3025911&req=5

pone-0016007-g006: Involvement of microtubules in the formation of EVs in MCF-7/MR cells and inhibition of microtubule polymerization by nocodazole.MCF-7/MR cells were either untreated (A–C and G–I) or treated with nocodazole (33 µM) for 1 hr at 37°C (D–F and J–L). Microtubules were visualized using mouse anti β-tubulin antibody followed by incubation with FITC-conjugated donkey anti-mouse IgG (B, E, H and K). Cells were co-reacted with antibodies to ABCG2 (BXP-53, A and D) or ERM (G and J), processed and analyzed as in Fig. 5 legend. Quantification of ABCG2 and ERM protein expression on the surface of EVs prior to and following treatment with nocodazole (M); random fields stained as in upper panels were photographed using the same exposure conditions for untreated and nocodazole-treated cells. The surface area of EVs and its relative fluorescence intensity were estimated using the AxioVision program. The fluorescence at the EVs surface indicated protein levels at the EVs membrane. A total of 100 EVs were analyzed for each examined protein. Bars represent SD.

Mentions: Based on our previous observation that EVs are dynamic structures that are easily disrupted upon standard trypsinization, we theorized that inhibition of microtubule polymerization may interfere with EVs formation and/or with vesicular trafficking of transmembrane proteins including ABCG2. To test this hypothesis, we first examined the distribution pattern of microtubules in relation to EVs location. In contrast to the localization pattern of actin filaments, there was no significant accumulation of microtubules around EVs (Figure 6A–6C). Microtubules were highly concentrated in the vicinity of EVs and appeared as radiating out both towards the cell membrane and the nucleus. We therefore used nocodazole, an established inhibitor of microtubule polymerization, which abolishes apical trafficking of proteins [16], [28]. Treatment with nocodazole resulted in disruption of the fine microtubular network that was observed in untreated cells. However, localization of the EV biomarkers ABCG2 and ERM complex proteins was not significantly affected (Figure 6D–6F; 6J–6L respectively). We hence quantified the co-localization of ABCG2 and ERM proteins to the surface of EVs prior to, and following treatment with nocodazole (Figure 6 M). Mean fluorescence intensity of ABCG2 and ERM proteins that localize to EVs (normalized to vesicular area) was not affected by nocodazole treatment. The relatively large SD observed with ABCG2 analysis is due to the fact that MCF-7/MR cells were established by pulse exposure to MR, thus resulting in a heterogeneous cell population with a large Gaussian distribution of ABCG2 expression (Supplementary Figure S4) and function [19]. Indeed, flow cytometric analysis of surface expression of ABCG2 in viable cells confirmed the large Gaussian distribution of ABCG2 expression in MCF-7/MR cells and ABCG2-overexpressing A549/K1.5 non-small lung cancer cells (Supplementary Figure S4).


Structure and function of ABCG2-rich extracellular vesicles mediating multidrug resistance.

Goler-Baron V, Assaraf YG - PLoS ONE (2011)

Involvement of microtubules in the formation of EVs in MCF-7/MR cells and inhibition of microtubule polymerization by nocodazole.MCF-7/MR cells were either untreated (A–C and G–I) or treated with nocodazole (33 µM) for 1 hr at 37°C (D–F and J–L). Microtubules were visualized using mouse anti β-tubulin antibody followed by incubation with FITC-conjugated donkey anti-mouse IgG (B, E, H and K). Cells were co-reacted with antibodies to ABCG2 (BXP-53, A and D) or ERM (G and J), processed and analyzed as in Fig. 5 legend. Quantification of ABCG2 and ERM protein expression on the surface of EVs prior to and following treatment with nocodazole (M); random fields stained as in upper panels were photographed using the same exposure conditions for untreated and nocodazole-treated cells. The surface area of EVs and its relative fluorescence intensity were estimated using the AxioVision program. The fluorescence at the EVs surface indicated protein levels at the EVs membrane. A total of 100 EVs were analyzed for each examined protein. Bars represent SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3025911&req=5

pone-0016007-g006: Involvement of microtubules in the formation of EVs in MCF-7/MR cells and inhibition of microtubule polymerization by nocodazole.MCF-7/MR cells were either untreated (A–C and G–I) or treated with nocodazole (33 µM) for 1 hr at 37°C (D–F and J–L). Microtubules were visualized using mouse anti β-tubulin antibody followed by incubation with FITC-conjugated donkey anti-mouse IgG (B, E, H and K). Cells were co-reacted with antibodies to ABCG2 (BXP-53, A and D) or ERM (G and J), processed and analyzed as in Fig. 5 legend. Quantification of ABCG2 and ERM protein expression on the surface of EVs prior to and following treatment with nocodazole (M); random fields stained as in upper panels were photographed using the same exposure conditions for untreated and nocodazole-treated cells. The surface area of EVs and its relative fluorescence intensity were estimated using the AxioVision program. The fluorescence at the EVs surface indicated protein levels at the EVs membrane. A total of 100 EVs were analyzed for each examined protein. Bars represent SD.
Mentions: Based on our previous observation that EVs are dynamic structures that are easily disrupted upon standard trypsinization, we theorized that inhibition of microtubule polymerization may interfere with EVs formation and/or with vesicular trafficking of transmembrane proteins including ABCG2. To test this hypothesis, we first examined the distribution pattern of microtubules in relation to EVs location. In contrast to the localization pattern of actin filaments, there was no significant accumulation of microtubules around EVs (Figure 6A–6C). Microtubules were highly concentrated in the vicinity of EVs and appeared as radiating out both towards the cell membrane and the nucleus. We therefore used nocodazole, an established inhibitor of microtubule polymerization, which abolishes apical trafficking of proteins [16], [28]. Treatment with nocodazole resulted in disruption of the fine microtubular network that was observed in untreated cells. However, localization of the EV biomarkers ABCG2 and ERM complex proteins was not significantly affected (Figure 6D–6F; 6J–6L respectively). We hence quantified the co-localization of ABCG2 and ERM proteins to the surface of EVs prior to, and following treatment with nocodazole (Figure 6 M). Mean fluorescence intensity of ABCG2 and ERM proteins that localize to EVs (normalized to vesicular area) was not affected by nocodazole treatment. The relatively large SD observed with ABCG2 analysis is due to the fact that MCF-7/MR cells were established by pulse exposure to MR, thus resulting in a heterogeneous cell population with a large Gaussian distribution of ABCG2 expression (Supplementary Figure S4) and function [19]. Indeed, flow cytometric analysis of surface expression of ABCG2 in viable cells confirmed the large Gaussian distribution of ABCG2 expression in MCF-7/MR cells and ABCG2-overexpressing A549/K1.5 non-small lung cancer cells (Supplementary Figure S4).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.

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.

Show MeSH
Related in: MedlinePlus