Structure and Function of ABCG2-Rich Extracellular Vesicles Mediating Multidrug Resistance

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.

多药耐药（Multidrug resistance, MDR）是癌症根治性化疗的主要障碍。ATP结合盒转运蛋白（ATP-Binding Cassette transporters）家族成员ABCG2、ABCB1与ABCC2构成了独特的防御网络，可抵御多种结构与功能迥异的化疗药物，进而介导多药耐药的产生。因此，解析多药耐药的全新机制并探索其逆转策略，是癌症研究的核心目标之一。近期本团队的研究证实，乳腺癌细胞来源的新型细胞外囊泡（extracellular vesicles, EVs）膜表面过表达ABCG2，可通过将米托蒽醌（mitoxantrone）大量隔离于囊泡内，导致肿瘤细胞对该药物产生耐药性。然而，目前学界对细胞外囊泡的结构、生物发生过程以及其富集多种抗肿瘤药物的能力仍知之甚少。为此，本研究发现，细胞外囊泡与胆小管（bile canaliculi）具有结构与功能上的同源性，其呈顶侧定位，为封闭性结构，由肌动蛋白细胞骨架（actin-based cytoskeleton）加固，并通过紧密连接蛋白occludin与ZO-1与细胞外环境隔绝。除ABCG2外，ABCB1与ABCC2同样被选择性靶向至细胞外囊泡的膜表面。此外，埃兹蛋白-根蛋白-膜突蛋白复合物（Ezrin-Radixin-Moesin protein complex）选择性定位于细胞外囊泡膜的边界区域，提示多药耐药转运蛋白与肌动蛋白细胞骨架的锚定过程发挥关键作用。本研究同时探究了细胞外囊泡富集并隔离不同抗肿瘤药物的能力。借助抗肿瘤药物的内源性荧光特性，本团队发现形成细胞外囊泡的乳腺癌细胞可通过高效的囊泡内药物富集过程，对拓扑替康（topotecan）、咪唑并吖啶酮类（imidazoacridinones）以及甲氨蝶呤（methotrexate）产生高水平耐药，从而将药物隔离于其细胞靶点之外。综上，本研究揭示了一种全新的抗肿瘤药物区室化与耐药机制：即通过靶向至细胞外囊泡膜的多药耐药转运蛋白网络，将多种化疗药物主动从细胞质泵出并高度富集于细胞外囊泡的管腔中。本团队提出了癌症细胞中富含多药耐药转运蛋白的细胞外囊泡的结构与功能复合模型，以及其介导多药抗癌药物耐药的潜在机制。