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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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Specific targeting of transiently transfected ABCB1 and ABCC2 to the EVs membrane in MCF-7/MR cells.Cells were transfected with ABC transporters as described in Materials and Methods, and then studied by immunofluorescence microscopy. Shown are the subcellular localizations of ABCB1 (A–C), ABCC1 (J–L), ABCC2 (D–I), ABCC3 (M–O), and PCFT (P–R), when compared to that of ABCG2. ABCG2 was followed using the monoclonal antibodies BXP-21 (J) or BXP-53 (A, D, G, M and P). Ectopically expressed proteins are indicated on the top of each panel. Cells were examined using either a Leica (×400) or the Cell-Observer (×630) microscopes. Arrows denote the location of ABCG2-rich premature and mature EVs.
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pone-0016007-g003: Specific targeting of transiently transfected ABCB1 and ABCC2 to the EVs membrane in MCF-7/MR cells.Cells were transfected with ABC transporters as described in Materials and Methods, and then studied by immunofluorescence microscopy. Shown are the subcellular localizations of ABCB1 (A–C), ABCC1 (J–L), ABCC2 (D–I), ABCC3 (M–O), and PCFT (P–R), when compared to that of ABCG2. ABCG2 was followed using the monoclonal antibodies BXP-21 (J) or BXP-53 (A, D, G, M and P). Ectopically expressed proteins are indicated on the top of each panel. Cells were examined using either a Leica (×400) or the Cell-Observer (×630) microscopes. Arrows denote the location of ABCG2-rich premature and mature EVs.

Mentions: Endogenous ABCG2 is overexpressed in MCF-7/MR cells and is targeted differentially to the membrane of EVs [8]. However, in MCF-7/MR cells which are not part of a colony and/or that do not participate in the formation of EVs, ABCG2 typically localizes at the perinuclear ER area (Supplementary Figure S2 G–I, continuous arrows). Based on the localization pattern of TJ proteins to cell-cell contact sites of EVs-forming cells (Figure 1 and supplementary Figure S1), we hypothesized that there may be a physical separation of the EV membrane from the cell membrane. To examine this hypothesis we first stably overexpressed an ABCG2-EGFP construct in parental MCF-7 and MCF-7/MR cells. Ectopic overexpression of ABCG2-EGFP resulted in differential transporter targeting in parental and drug resistant cells; in parental MCF-7 cells, which infrequently form EVs, ABCG2-EGFP localized primarily at the cell membrane (Supplementary Figure S2 A–C). However, when EVs infrequently formed in MCF-7 cells, ABCG2-EGFP was targeted to the EVs membrane. In contrast, in MCF-7/MR cells, ABCG2-EGFP was targeted exclusively to the membrane of EVs and cell-cell attachment zones (Supplementary Figure S2 D–F). Thus, we focused our further studies on MCF-7/MR cells in an attempt to explore the possible differential sorting of various MDR efflux transporters of the ABC superfamily including ABCB1, ABCC1, ABCC2 and ABCC3 (i.e. P-gp, MRP1, MRP2 and MRP3, respectively) to the EV membrane. These ATP-driven MDR efflux transporters differ markedly in substrate specificity, tissue distribution and intracellular localization [1], [4], [5], [6]. Previously we have shown that ABCC2 and ABCC3 levels are undetectable in MCF-7/MR cells, whereas low levels of ABCC1 were present [19]. To determine the targeting specificity of these ABC transporters to the EV membrane, we introduced these MDR transporter genes into MCF-7/MR cells and determined their sub-cellular localization by immunofluorescence microscopy. ABCB1 and ABCC2 were specifically targeted to the EV membrane and to cell-cell attachment zones, hence perfectly co-localizing with ABCG2 (Figure 3A–3C and 3D–3I, respectively). Analysis of ABCB1- and ABCC2-transfectant cells revealed that some EVs displayed a unique transporter co-localization pattern with ABCG2 and the exogenously transfected transporter solely present in one half of the EV membrane (Figure 3A–3C and 3D–3F arrows), whereas other EVs exhibited an equally distributed co-localization pattern (Figure 3G–3I). In contrast, in ABCC1- (Figure 2J–3L) and ABCC3-transfected (Figure 3M–3O) MCF-7/MR cells, these MDR transporters were equally targeted to the cell membrane. The proton-coupled folate transporter (PCFT/SLC46A1), which is representative of various proton-coupled low pH carriers, that mediate intestinal absorption of various essential nutrients [20], [21], [22], was further examined. Consistent with previous findings [22], [23], PCFT, the dominant intestinal folate transporter [22], was targeted to the entire cell membrane of MCF-7/MR cells, thus no co-localization of PCFT and ABCG2 was found in the EVs membrane (Figure 3P–3R). The subcellular localization of Na+/K+ ATPase was also determined using immunohistochemistry and confocal laser microscopy, as this central ATP-driven cation pump is ubiquitously expressed at high levels and targeted to the basolateral membrane of secretory epithelial cells including the intestine, glands and kidney [24]. Na+/K+ ATPase was targeted to the cell membrane of MCF-7/MR cells, but not to the membrane of EVs (Supplementary Figure S3 and supplementary Video S2).


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

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

Specific targeting of transiently transfected ABCB1 and ABCC2 to the EVs membrane in MCF-7/MR cells.Cells were transfected with ABC transporters as described in Materials and Methods, and then studied by immunofluorescence microscopy. Shown are the subcellular localizations of ABCB1 (A–C), ABCC1 (J–L), ABCC2 (D–I), ABCC3 (M–O), and PCFT (P–R), when compared to that of ABCG2. ABCG2 was followed using the monoclonal antibodies BXP-21 (J) or BXP-53 (A, D, G, M and P). Ectopically expressed proteins are indicated on the top of each panel. Cells were examined using either a Leica (×400) or the Cell-Observer (×630) microscopes. Arrows denote the location of ABCG2-rich premature and mature EVs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3025911&req=5

pone-0016007-g003: Specific targeting of transiently transfected ABCB1 and ABCC2 to the EVs membrane in MCF-7/MR cells.Cells were transfected with ABC transporters as described in Materials and Methods, and then studied by immunofluorescence microscopy. Shown are the subcellular localizations of ABCB1 (A–C), ABCC1 (J–L), ABCC2 (D–I), ABCC3 (M–O), and PCFT (P–R), when compared to that of ABCG2. ABCG2 was followed using the monoclonal antibodies BXP-21 (J) or BXP-53 (A, D, G, M and P). Ectopically expressed proteins are indicated on the top of each panel. Cells were examined using either a Leica (×400) or the Cell-Observer (×630) microscopes. Arrows denote the location of ABCG2-rich premature and mature EVs.
Mentions: Endogenous ABCG2 is overexpressed in MCF-7/MR cells and is targeted differentially to the membrane of EVs [8]. However, in MCF-7/MR cells which are not part of a colony and/or that do not participate in the formation of EVs, ABCG2 typically localizes at the perinuclear ER area (Supplementary Figure S2 G–I, continuous arrows). Based on the localization pattern of TJ proteins to cell-cell contact sites of EVs-forming cells (Figure 1 and supplementary Figure S1), we hypothesized that there may be a physical separation of the EV membrane from the cell membrane. To examine this hypothesis we first stably overexpressed an ABCG2-EGFP construct in parental MCF-7 and MCF-7/MR cells. Ectopic overexpression of ABCG2-EGFP resulted in differential transporter targeting in parental and drug resistant cells; in parental MCF-7 cells, which infrequently form EVs, ABCG2-EGFP localized primarily at the cell membrane (Supplementary Figure S2 A–C). However, when EVs infrequently formed in MCF-7 cells, ABCG2-EGFP was targeted to the EVs membrane. In contrast, in MCF-7/MR cells, ABCG2-EGFP was targeted exclusively to the membrane of EVs and cell-cell attachment zones (Supplementary Figure S2 D–F). Thus, we focused our further studies on MCF-7/MR cells in an attempt to explore the possible differential sorting of various MDR efflux transporters of the ABC superfamily including ABCB1, ABCC1, ABCC2 and ABCC3 (i.e. P-gp, MRP1, MRP2 and MRP3, respectively) to the EV membrane. These ATP-driven MDR efflux transporters differ markedly in substrate specificity, tissue distribution and intracellular localization [1], [4], [5], [6]. Previously we have shown that ABCC2 and ABCC3 levels are undetectable in MCF-7/MR cells, whereas low levels of ABCC1 were present [19]. To determine the targeting specificity of these ABC transporters to the EV membrane, we introduced these MDR transporter genes into MCF-7/MR cells and determined their sub-cellular localization by immunofluorescence microscopy. ABCB1 and ABCC2 were specifically targeted to the EV membrane and to cell-cell attachment zones, hence perfectly co-localizing with ABCG2 (Figure 3A–3C and 3D–3I, respectively). Analysis of ABCB1- and ABCC2-transfectant cells revealed that some EVs displayed a unique transporter co-localization pattern with ABCG2 and the exogenously transfected transporter solely present in one half of the EV membrane (Figure 3A–3C and 3D–3F arrows), whereas other EVs exhibited an equally distributed co-localization pattern (Figure 3G–3I). In contrast, in ABCC1- (Figure 2J–3L) and ABCC3-transfected (Figure 3M–3O) MCF-7/MR cells, these MDR transporters were equally targeted to the cell membrane. The proton-coupled folate transporter (PCFT/SLC46A1), which is representative of various proton-coupled low pH carriers, that mediate intestinal absorption of various essential nutrients [20], [21], [22], was further examined. Consistent with previous findings [22], [23], PCFT, the dominant intestinal folate transporter [22], was targeted to the entire cell membrane of MCF-7/MR cells, thus no co-localization of PCFT and ABCG2 was found in the EVs membrane (Figure 3P–3R). The subcellular localization of Na+/K+ ATPase was also determined using immunohistochemistry and confocal laser microscopy, as this central ATP-driven cation pump is ubiquitously expressed at high levels and targeted to the basolateral membrane of secretory epithelial cells including the intestine, glands and kidney [24]. Na+/K+ ATPase was targeted to the cell membrane of MCF-7/MR cells, but not to the membrane of EVs (Supplementary Figure S3 and supplementary Video S2).

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