PI3-kinase and mTOR inhibitors differently modulate the function of the ABCG2 multidrug transporter

https://doi.org/10.1016/j.bbrc.2012.03.090Get rights and content

Abstract

The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.

Highlights

LY294002, a pharmacological PI3-kinase inhibitor directly inhibits ABCG2 function. ► Rapamycin, a pharmacological mTOR inhibitor directly inhibits ABCG2 function. ► LY294002 and rapamycin reverse ABCG2-mediated multidrug resistance. ► This phenomenon is independent from their effect on PI3K/Akt/mTOR signaling. ► Wortmannin, a pharmacological PI3-kinase inhibitor does not interact with ABCG2.

Introduction

The ABCG2 membrane glycoprotein belongs to the ATP-binding cassette transporter superfamily. ABCG2 eliminates various endogenous and environmental toxins from the cells by active efflux, and it plays a pivotal role in physiologic tissue protection against xenobiotics [1]. Exploiting the protective function of the transporter, cancer cells overexpressing ABCG2 become cross-resistant against a wide spectrum of chemically and target-wise unrelated drugs, a phenomenon known as multidrug resistance (MDR) [1], [2]. Being a key multidrug transporter involved in both physiologic and pathological processes, identification of cellular factors regulating expression and function of ABCG2 is intensively studied.

The phosphatidylinositol 3-kinase/Akt signaling axis has been implicated in the regulation of a number of cellular functions, including that of ABCG2. Upon growth factor receptor phosphorylation, phosphatidylinositol 3-kinase (PI3-kinase, PI3K) is activated and generates phosphatidylinositol (3,4,5) trisphosphate, which results in subsequent plasma membrane recruitment and activation of Akt kinase [3]. Downstream targets of activated Akt include the mammalian target of rapamycin (mTOR) kinase [4]. Inhibition of Akt signaling has been shown to provoke rapid translocation of ABCG2 from the plasma membrane to intracellular compartments in mouse bone marrow cells [5], in polarized ABCG2-overexpressing porcine LLC-PK1 cells [6], in glioma-derived cells [7], in human gallbladder epithelial cells [8], in hepatocellular carcinoma-derived cells [9], and also in ABCG2-expressing extracellular vesicles formed between MCF-7/MR breast cancer cells [10]. Nevertheless, opposing results have also been published, showing that inhibition of Akt signaling in human leukemia cells down-regulated overall ABCG2 protein levels rather than affecting only its plasma membrane localization [11]. Involvement of the downstream mTOR kinase in regulating plasma membrane insertion of ABCG2 is also controversial [7], [12]. The PI3K/Akt-mediated decrease in ABCG2 plasma membrane levels has also been associated with attenuated transporter function [5], [7], [9], [10] resulting in reversal of ABCG2-mediated drug resistance [9], [10].

In these studies, pharmacological inhibitors of the PI3K/Akt/mTOR pathway, such as the PI3K inhibitors wortmannin and LY294002 [13] and the mTOR inhibitor rapamycin [13] were also applied to manipulate cellular signal transduction. Given the multispecific drug recognition capability of ABCG2 [1], however, it is reasonable to assume that these compounds might also directly interact with the transporter. Such a direct drug-transporter interaction would also allow for different interpretation of the experimental results, especially those concerning ABCG2 function. Indeed, data regarding interaction of ABCG2 and rapamycin have already been published [12], [14], however the issue of susceptibility of rapamycin to ABCG2-mediated transport was conflicting. LY294002 has been shown to be a competitive inhibitor of another multidrug transporter MRP1 [15], but interaction of this compound and the other PI3K inhibitor wortmannin with ABCG2 has not yet been characterized. In this work, we aimed to give a detailed biochemical characterization regarding the interaction between the aforementioned pharmacological PI3K/Akt/mTOR inhibitors and ABCG2. Additionally, we also studied the presently controversial role of PI3K/Akt/mTOR signaling in the rapid regulation of the subcellular distribution of ABCG2.

Section snippets

Materials

Unless otherwise stated, all reagents were obtained from Sigma–Aldrich. Alexa-Fluor-conjugated antibodies, wheat germ agglutinin (WGA), TOPRO-3 and DAPI were obtained from Invitrogen. Ko143 was obtained from Tocris Bioscience. Phycoerythrin-conjugated secondary antibodies were purchased from Beckman Coulter.

Cell lines

A431 and PLB985 (PLB) cells stably expressing human wild-type ABCG2 were generated previously [16], [17]. MDCKII cells stably expressing the N-terminally GFP-tagged wild-type ABCG2

Effect of wortmannin, LY294002 and rapamycin on membrane ABCG2-ATPase

In order to pre-screen potential interaction between the pharmacological PI3K/Akt/mTOR inhibitors and ABCG2, we measured whether the inhibitors can influence the basal and the stimulated ATPase activity of the transporter (Fig. 1). Since GFP-tagged ABCG2 was also applied in further experiments, we also characterized the potential interaction between this protein and the pharmacological PI3K/Akt/mTOR inhibitors. Human wild-type ABCG2 or its GFP-tagged variant was expressed in Sf9 insect cells

Acknowledgments

The technical help of Gyöngyi Bézsenyi, Éva Krizsán and Zsuzsanna Andrási is greatly appreciated. The authors thank Drs. George Scheffer and Rik Scheper for the BXP-21 antibody and Brian P. Sorrentino for the 5D3 antibody. This work has been supported by grants from OTKA NK83533, KMOP-1.1.2-07/1-2008-0003, and NKTH-Stemkill. Csilla Özvegy-Laczka and Tamás I. Orbán are recipients of the János Bolyai Scholarship of the Hungarian Academy of Sciences. The authors declare no potential conflicts of

References (28)

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    But how does CD147 affect to ABCG2’s dimerization and subcellular location is still to be answered. Recent studies demonstrated that PI3K-Akt signaling pathway, which is involved in proliferation, differentiation, apoptosis and so on, modulates the function of ABCG2 and its subcellular localization translocation [48–51]. Mogi et al. and Vicky et al. showed that Akt inhibition could result in cytoplasmic internalization of ABCG2 [49,50].

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  • Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: Implications for the emergence and reversal of cancer drug resistance

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    As documented, A431/ABCG2 cells displayed resistance toward gefitinib and also toward pelitinib, both of which could be fully reversed by addition of the specific ABCG2 inhibitor FTC (5 μM). ( FTC alone does not significantly reduce viability of the cells [37]). A431/ABCG2 cells also showed a slightly increased resistance to neratinib, however, this could not be reverted by FTC.

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1

These authors contributed equally to this work.

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