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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.

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A proposed model of EVs structure and function.Shown is an EV formed between two attached MCF-7/MR cells. The EV is structurally reinforced by an actin cytoskeleton. TJ proteins including occludin and ZO-1 (indicated by the dashed line surrounding the vesicles) seal the EV to the extracellular milieu and to the surrounding cells. ABCG2 is highly expressed on the EV membrane, but not on the cell membrane facing the medium or neighbor cells. ABCG2-rich EVs highly concentrate multiple cytotoxic agents including topotecan, methotrexate, imidazoacridinones, Hoechst 33342 and MR [8] as well as the B2-vitamin riboflavin [12]. Just like ABCG2, ABCB1 and ABCC2 are also differentially targeted to the EV membrane, whereas ABCC1, ABCC3 and PCFT are localized to the cell membrane. Na+/K+ ATPase is present at the cell membrane. ERM proteins, which are highly targeted to the EV surface, presumably anchor these ABC transporters to the actin cytoskeleton.
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pone-0016007-g009: A proposed model of EVs structure and function.Shown is an EV formed between two attached MCF-7/MR cells. The EV is structurally reinforced by an actin cytoskeleton. TJ proteins including occludin and ZO-1 (indicated by the dashed line surrounding the vesicles) seal the EV to the extracellular milieu and to the surrounding cells. ABCG2 is highly expressed on the EV membrane, but not on the cell membrane facing the medium or neighbor cells. ABCG2-rich EVs highly concentrate multiple cytotoxic agents including topotecan, methotrexate, imidazoacridinones, Hoechst 33342 and MR [8] as well as the B2-vitamin riboflavin [12]. Just like ABCG2, ABCB1 and ABCC2 are also differentially targeted to the EV membrane, whereas ABCC1, ABCC3 and PCFT are localized to the cell membrane. Na+/K+ ATPase is present at the cell membrane. ERM proteins, which are highly targeted to the EV surface, presumably anchor these ABC transporters to the actin cytoskeleton.

Mentions: 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). Two or more attached cells form a tightly sealed extracellular compartment termed EV. The oval-shape structure of this EV is reinforced by actin microfilament-based cytoskeleton, thus forming an actin-rich brush border at the apical side of MCF-7/MR cells. Moreover, as would be expected from a compartment that highly concentrates multiple chemotherapeutics, EVs are secluded from the extracellular milieu by TJ proteins including occludin and ZO-1, which localize at the border between EV-forming cells, in a belt-like pattern. Each EV-forming cell contributes its relative share of the vesicular structure; e.g. when three attached cells form a common EV, three distinct belt-like structures of TJ proteins are apparent. ABCG2 is overexpressed and selectively targeted to the EV membrane. This unique localization of ABCG2 mediates the efficient pumping and hence concentration of multiple cytotoxic agents of distinct structure and mode of action (as well as non toxic compounds including riboflavin) from the cytoplasm into the lumen of EVs. These cytotoxic agents include topotecan, imidazoacridinones, methotrexate, MR and Hoechst 33342, hence representing various families of anticancer drugs. Most importantly, we further discovered that apart from ABCG2, key MDR efflux transporters including ABCB1 and ABCC2 are also selectively targeted to the EV membrane. Moreover, this differential targeting to the EVs membrane cannot be considered a general default phenomenon for all transporters, since ABCC1, ABCC3 and PCFT were targeted to the cell membrane but not to the EVs membrane. Likewise, Na+/K+ ATPase was directed to the cell membrane. We also found here that ERM proteins displayed a remarkably differential sorting to, and high abundance in the EV membrane border, thereby suggesting a functional role for the anchoring of EV-targeted transporters to the actin cytoskeleton. Hence, the formation of sealed EVs shared by attached breast cancer cells and the specific targeting of a network of MDR efflux transporters to the EV membrane is a novel modality of anticancer drug compartmentalization and consequent MDR. In this respect, a variety of cytotoxic ABCG2 transport substrates that enter cells by diffusion or via carrier-mediated transport, are extracted from the cytoplasm and actively pumped into the vesicular lumen by ABCG2.


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

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

A proposed model of EVs structure and function.Shown is an EV formed between two attached MCF-7/MR cells. The EV is structurally reinforced by an actin cytoskeleton. TJ proteins including occludin and ZO-1 (indicated by the dashed line surrounding the vesicles) seal the EV to the extracellular milieu and to the surrounding cells. ABCG2 is highly expressed on the EV membrane, but not on the cell membrane facing the medium or neighbor cells. ABCG2-rich EVs highly concentrate multiple cytotoxic agents including topotecan, methotrexate, imidazoacridinones, Hoechst 33342 and MR [8] as well as the B2-vitamin riboflavin [12]. Just like ABCG2, ABCB1 and ABCC2 are also differentially targeted to the EV membrane, whereas ABCC1, ABCC3 and PCFT are localized to the cell membrane. Na+/K+ ATPase is present at the cell membrane. ERM proteins, which are highly targeted to the EV surface, presumably anchor these ABC transporters to the actin cytoskeleton.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3025911&req=5

pone-0016007-g009: A proposed model of EVs structure and function.Shown is an EV formed between two attached MCF-7/MR cells. The EV is structurally reinforced by an actin cytoskeleton. TJ proteins including occludin and ZO-1 (indicated by the dashed line surrounding the vesicles) seal the EV to the extracellular milieu and to the surrounding cells. ABCG2 is highly expressed on the EV membrane, but not on the cell membrane facing the medium or neighbor cells. ABCG2-rich EVs highly concentrate multiple cytotoxic agents including topotecan, methotrexate, imidazoacridinones, Hoechst 33342 and MR [8] as well as the B2-vitamin riboflavin [12]. Just like ABCG2, ABCB1 and ABCC2 are also differentially targeted to the EV membrane, whereas ABCC1, ABCC3 and PCFT are localized to the cell membrane. Na+/K+ ATPase is present at the cell membrane. ERM proteins, which are highly targeted to the EV surface, presumably anchor these ABC transporters to the actin cytoskeleton.
Mentions: 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). Two or more attached cells form a tightly sealed extracellular compartment termed EV. The oval-shape structure of this EV is reinforced by actin microfilament-based cytoskeleton, thus forming an actin-rich brush border at the apical side of MCF-7/MR cells. Moreover, as would be expected from a compartment that highly concentrates multiple chemotherapeutics, EVs are secluded from the extracellular milieu by TJ proteins including occludin and ZO-1, which localize at the border between EV-forming cells, in a belt-like pattern. Each EV-forming cell contributes its relative share of the vesicular structure; e.g. when three attached cells form a common EV, three distinct belt-like structures of TJ proteins are apparent. ABCG2 is overexpressed and selectively targeted to the EV membrane. This unique localization of ABCG2 mediates the efficient pumping and hence concentration of multiple cytotoxic agents of distinct structure and mode of action (as well as non toxic compounds including riboflavin) from the cytoplasm into the lumen of EVs. These cytotoxic agents include topotecan, imidazoacridinones, methotrexate, MR and Hoechst 33342, hence representing various families of anticancer drugs. Most importantly, we further discovered that apart from ABCG2, key MDR efflux transporters including ABCB1 and ABCC2 are also selectively targeted to the EV membrane. Moreover, this differential targeting to the EVs membrane cannot be considered a general default phenomenon for all transporters, since ABCC1, ABCC3 and PCFT were targeted to the cell membrane but not to the EVs membrane. Likewise, Na+/K+ ATPase was directed to the cell membrane. We also found here that ERM proteins displayed a remarkably differential sorting to, and high abundance in the EV membrane border, thereby suggesting a functional role for the anchoring of EV-targeted transporters to the actin cytoskeleton. Hence, the formation of sealed EVs shared by attached breast cancer cells and the specific targeting of a network of MDR efflux transporters to the EV membrane is a novel modality of anticancer drug compartmentalization and consequent MDR. In this respect, a variety of cytotoxic ABCG2 transport substrates that enter cells by diffusion or via carrier-mediated transport, are extracted from the cytoplasm and actively pumped into the vesicular lumen by ABCG2.

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