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

Abstract

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 β-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.

Introduction

The phosphatidyl-inositol 3-kinase (PI3K)-Akt signaling pathway integrates a plethora of extracellular signals to generate diverse physiological outcomes including cell proliferation, motility, glucose homeostasis, survival and cell death. Activation of the PI3K-Akt pathway is thought to play a pivotal role in both the initiation and progression of human breast cancer [1], [2], [3]. There are three principal components of the Akt pathway: PI3K, its antagonist PTEN and the serine/threonine kinase Akt, which is expressed as three structurally similar isoforms that differ in their expression pattern and function (recently reviewed in [1], [2], [3]). Receptor-mediated activation of the PI3K-Akt pathway occurs through Akt phosphorylation at threonine 308 and serine 473; upon activation, Akt translocates to the cytoplasm and the nucleus where it phosphorylates a variety of downstream targets. Two established isoform-unselective PI3K inhibitors are the fungal furanosteroid metabolite wortmannin which covalently binds to the conserved lysine 802 involved in the phosphate-binding reaction as well as LY294002, a reversible ATP-competitive PI3K inhibitor [2], [4].

The frequent emergence of multidrug resistance (MDR) to structurally and functionally unrelated anticancer drugs is a major impediment to curative cancer chemotherapy [5], [6], [7], [8], [9], [10]. ATP-driven MDR efflux transporters belong to the large ATP-binding cassette (ABC) superfamily of transporters that include ABCB1 (P-gp), ABCC1 (MRP1) and ABCG2 (BCRP). Overexpression of these efflux pumps results in the expulsion of a multitude of chemotherapeutic drugs, thereby leading to acquisition of a broad spectrum drug resistance known as MDR. We have recently identified [11] and characterized [12] a novel modality of MDR where neighbor breast cancer cells form extracellular vesicles (EVs) which overexpress ABCG2. These mitoxantrone (MR) resistant MCF-7/MR cells overexpress ABCG2 relatively to their parental cells and target ABCG2 specifically to the membrane of EVs where it mediates MDR. ABCG2-dependent sequestration of various cytotoxic agents including mitoxantrone [11], topotecan, methotrexate [12] and imidazoacridinones (unpublished data) within the lumen of EVs was abolished by the specific ABCG2 transport inhibitors Ko143 and fumitremorgin C (FTC) [13]. However, in spite of the important implications of these drug-concentrating EVs for cancer chemotherapy, nothing was known about the molecular mechanism by which ABCG2 is specifically targeted to the membrane of EVs. In this respect, recent studies suggested that the PI3K-Akt signaling pathway may regulate cellular localization of ABCG2. Moreover, Mogi et al. [14] and Bleau et al. [15] reported that exposure of in vivo isolated mouse hematopoietic stem cells known as side population (SP) as well as SP of glioma stem cells to the AKT inhibitor LY294002, resulted in translocation of ABCG2 from the plasma membrane to the cytoplasmic compartment. Consistently, Takada et al. [16], who examined ABCG2 localization in polarized porcine renal epithelial LLC-PK-1 cells that were stably transfected with the human ABCG2 found that Akt inhibition resulted in cytoplasmic internalization of ABCG2. However, when cells were incubated with epidermal growth factor, cell surface expression of ABCG2 increased. In contrast, Nakanishi et al. [17] reported that as opposed to the above studies, inhibition of the Akt signaling pathway in cultured chronic myelogenous leukemia cells induced down-regulation of ABCG2 expression rather than a shift in the sub-cellular localization of ABCG2 from the plasma membrane to the cytosol.

In the current study we explored the impact of the PI3K-Akt signaling pathway on ABCG2 protein expression and sub-cellular localization in the context of ABCG2-rich EVs formed in MR-resistant breast cancer (MCF-7/MR) cells [18]. We found that pharmacological inhibition of the PI3K-Akt signaling pathway results in a gradual retraction of ABCG2 from the EVs membrane to the cytoplasmic compartment, hence abolishing the ability of EVs to mediate anticancer drug sequestration. Simultaneously, we also detected a gradual disappearance of EVs, hence overcoming the MDR phenotype displayed by MCF-7/MR cells to the ABCG2 substrates MR and topotecan. Treatment of MCF-7/MR cells with the ABCG2-specific inhibitors Ko143 and FTC resulted not only in the expected abolishment of drug transport activity but also in cytoplasmic retention of ABCG2 and a time-dependent decrease in the number of EVs, similarly to the effect observed after PI3K-Akt signaling inhibition. In contrast, no effect of Akt signaling inhibition was found on ABCG2 protein levels. Taken altogether, these findings reveal that the PI3K-Akt signaling pathway is a key regulator of subcellular localization of ABCG2. We further conclude that ABCG2 is essential for the biogenesis of EVs and their MDR function.