Overcoming multidrug resistance via photodestruction of ABCG2-rich extracellular vesicles sequestering photosensitive chemotherapeutics.

Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing lysosomal photodestruction of normal breast epithelial cells. Thus, MDR modalities including ABCG2-dependent drug sequestration within EVs can be rationally converted to a pharmacologically lethal Trojan horse to selectively eradicate MDR cancer cells.

Inhibition of the PI3K-Akt signaling pathway disrupts ABCG2-rich extracellular vesicles and overcomes multidrug resistance in breast cancer cells.

We have recently shown that ABCG2-rich extracellular vesicles (EVs) form between neighbor breast cancer cells and actively concentrate various chemotherapeutics, resulting in multidrug resistance (MDR). Here we studied the signaling pathway regulating ABCG2 targeting to EVs as its inhibition would overcome MDR. The PI3K-Akt signaling pathway was possibly implicated in subcellular localization of ABCG2; we accordingly show here that pharmacological inhibition of Akt signaling results in gradual re-localization of ABCG2 from the EVs membrane to the cytoplasm. Cytoskeletal markers including ß-actin and the tight junction protein ZO-1, along with the EVs markers ABCG2 and Ezrin-Radixin-Moesin revealed that this intracellular ABCG2 retention leads to gradual decrease in the size and number of EVs, resulting in EVs elimination and complete reversal of MDR. Inhibition of Akt signaling restored drug sensitivity to mitoxantrone and topotecan, bona fide ABCG2 transport substrates, hence being equivalent to MDR reversal achieved with the ABCG2 transport inhibitor Ko143. Remarkably, apart from loss of ABCG2 transport activity, treatment of MCF-7/MR cells with Ko143 resulted in cytoplasmic re-localization of ABCG2, similarly to the phenotype observed after Akt inhibition. We conclude that the PI3K-Akt signaling pathway is a key regulator of subcellular localization of ABCG2, EVs biogenesis and functional MDR. Furthermore, proper folding of ABCG2 and its targeting to the EVs membrane are crucial components of the biogenesis of EVs and their MDR function. We propose that Akt signaling inhibitors which disrupt ABCG2 targeting and EVs biogenesis may readily overcome MDR thus warranting in vivo studies with these promising drug combinations.

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.

Riboflavin concentration within ABCG2-rich extracellular vesicles is a novel marker for multidrug resistance in malignant cells.

We have previously shown that overexpression of the multidrug resistance (MDR) efflux transporter ABCG2 in the membrane of novel extracellular vesicles that are confined to breast cancer cell-cell attachment zones confers mitoxantrone resistance and mediates a marked intravesicular concentration of an unknown endogenous green fluorescent compound (I. Ifergan, G.L. Scheffer, Y.G. Assaraf, Novel extracellular vesicles mediate an ABCG2-dependent anticancer drug sequestration and resistance, Cancer Res. 65 (2005) 10952-10958). Here we identified the latter as riboflavin (vitamin B2) and further demonstrated that the marked intravesicular concentration of riboflavin in ABCG2-overexpressing breast and lung cancer cells tightly correlates with the extent of ABCG2 overexpression and its differential localization to the vesicular membrane and not to the plasma membrane surrounded by growth medium. We hence propose that the ABCG2-dependent concentration of riboflavin in these intercellular compartments may serve as a novel, sensitive, and non-cytotoxic (i.e. based on vitamin accumulation) functional marker for the quantification of the levels of MDR mediated by ABCG2-rich extracellular vesicles in multiple malignant cells.

Transcription in the nucleus and mRNA decay in the cytoplasm are coupled processes.

Maintaining appropriate mRNAs levels is vital for any living cell. mRNA synthesis in the nucleus by RNA polymerase II core enzyme (Pol II) and mRNA decay by cytoplasmic machineries determine these levels. Yet, little is known about possible cross-talk between these processes. The yeast Rpb4/7 is a nucleo-cytoplasmic shuttling heterodimer that interacts with Pol II and with mRNAs and is required for mRNA decay in the cytoplasm. Here we show that interaction of Rpb4/7 with mRNAs and eventual decay of these mRNAs in the cytoplasm depends on association of Rpb4/7 with Pol II in the nucleus. We propose that, following its interaction with Pol II, Rpb4/7 functions in transcription, interacts with the transcript cotranscriptionally and travels with it to the cytoplasm to stimulate mRNA decay. Hence, by recruiting Rpb4/7, Pol II governs not only transcription but also mRNA decay.

The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms.

The steady-state level of mRNAs is determined by the balance between their synthesis by RNA polymerase II (Pol II) and their decay. In the cytoplasm, mRNAs are degraded by two major pathways; one requires decapping and 5' to 3' exonuclease activity and the other involves 3' to 5' degradation. Rpb7p is a Pol II subunit that shuttles between the nucleus and the cytoplasm. Here, we show that Rpb7p is involved in the two mRNA decay pathways and possibly couples them. Rpb7p stimulates the deadenylation stage required for execution of both pathways. Additionally, Rpb7p is both an active component of the P bodies, where decapping and 5' to 3' degradation occur, and is capable of affecting the P bodies function. Moreover, Rpb7p interacts with the decapping regulator Pat1p in a manner important for the mRNA decay machinery. Rpb7p is also involved in the second pathway, as it stimulates 3' to 5' degradation. Our genetic analyses suggest that Rpb7p plays two distinct roles in mRNA decay, which can both be uncoupled from Rpb7p's role in transcription. Thus, Rpb7p plays pivotal roles in determining mRNA levels.