Motesanib (AMG706), a potent multikinase inhibitor, antagonizes multidrug resistance by inhibiting the efflux activity of the ABCB1

Abstract

Cancer cells often become resistant to chemotherapy through a phenomenon known as multidrug resistance (MDR). Several factors are responsible for the development of MDR, preeminent among them being the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters. Some small molecule tyrosine kinase inhibitors (TKIs) were recently reported to modulate the activity of ABC transporters. Therefore, the purpose of this study was to determine if motesanib, a multikinase inhibitor, could reverse ABCB1-mediated MDR. The results showed that motesanib significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to its substrate anticancer drugs. Motesanib significantly increased the accumulation of [³H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant change in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 3 μM motesanib for 72 h. Moreover, motesanib stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, the docking studies indicated favorable binding of motesanib within the transmembrane region of homology modeled human ABCB1. Here, we report for the first time, motesanib, at clinically achievable plasma concentrations, antagonizes MDR by inhibiting the efflux activity of the ABCB1 transporter. These findings may be useful for cancer combination therapy with TKIs in the clinic.

Introduction

Cancer, known medically as malignant neoplasm or tumor, is the second most leading cause of death after cardiovascular disease in the United States and developing countries [1]. Cancer treatment is often consisted of surgery, radiation therapy, chemotherapy and combination therapy. Chemotherapy has been the first-line approach for the treatment of most cancers for several decades, and it typically involves systemic administration of combination of anticancer drugs from different chemical groups with different mechanism of action and depends on the capability of highly cytotoxic drugs to penetrate and selectively kill cancer cells [2]. However, cancer cells often exhibit either intrinsic or acquired resistance to chemotherapy through a phenomenon known as multidrug resistance (MDR) [3]. Many factors are responsible for the development of MDR, preeminent among them is the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters [4]. These ABC transporters serve as a defense mechanism by pumping harmful substrates out of normal cells. In a similar manner, cancer cells tend to overexpress these transporters in order to protect themselves from cytotoxic anticancer drugs. These transporters efflux drugs by consuming the energy produced via hydrolysis of ATP [5]. So far, 48 human ABC transporters have been identified and categorized into seven subfamilies, from ABCA to ABCG, based on structural and sequence similarities [6]. Among them, ABCB1, ABCG2, and ABCCs are the primary contributors of MDR in cancer cells [7].

ABCB1, also called P-glycoprotein encoded by MDR1 gene, was the first discovered mammalian ABC transporter [8], [9]. It is comprised of two homologous halves, each containing six transmembrane helices and an ATP binding/utilization domain, separated by a flexible linker. ABCB1 is a 170-kDa apical plasma membrane protein ubiquitously expressed in kidney, placenta, liver, adrenal glands, intestine and blood-brain barrier cells, where it functions to protect against xenobiotics and cellular toxicants [10], [11]. In addition, ABCB1 can transport a wide range of anticancer drugs such as doxorubicin, vincristine, paclitaxel, and epipodophyllotoxins out of the cancer cells [7], [12]. The overexpression of ABCB1 can be induced after repeated exposure to anticancer drugs, when the tumor becomes refractory to chemotherapy. ABCG2, a 72-kDa protein, is the first known half transporter with only one nucleotide binding domain and one transmembrane domain to mediate MDR [13]. The functional unit of ABCG2 is a homodimer or an oligomer [14]. The wide spectrum of chemotherapeutic agents transported by ABCG2 ranges from organic anion conjugates, nucleoside analogues, organic dyes, tyrosine kinase inhibitors (TKIs) to anthracyclines (such as doxorubicin, mitoxantrone), camptothecin-derived indolocarbazole topoisomerase I inhibitors, methotrexate, and flavopiridols [14]. ABCC1 (MRP1), which is a member of the C subfamily of ABC transporters can also transport various hydrophobic drugs; some anionic drugs and its drug conjugates, including antifolates, certain nucleotides, and also vinca alkaloids [15], [16]. ABCC10, also known as multidrug resistance protein 7 (MRP7), is a 171-kDa protein that can transport various anticancer drugs, including docetaxel, paclitaxel, vincristine, vinblastine, cytarabine, gemcitabine and epothilone B [17], [18].

In the last three decades, MDR research has mainly focused on developing inhibitors of ABC transporters, which have minimal toxicity in normal cells. It has been reported that some epidermal growth factor receptor (EGFR) TKI inhibitors including AG1478, erlotinib and lapatinib significantly reversed ABCB1- and ABCG2-mediated MDR, indicating that these TKIs might be modulators of ABCB1 and ABCG2 transporters [19]. In addition, BCR-Abl TKIs (imatinib and nilotinib) were also found to reverse ABCB1-and ABCG2-mediated MDR [20]. An in vivo study using the combination of gefitinib and a camptothecin derivative has shown a better pharmacokinetic profile and anti-tumor activity compared to camptothecin derivatives alone [21]. Our lab has also reported that the anti-tumor response to paclitaxel was enhanced by lapatinib in ABCB1 overexpressing nude mice tumor xenografts [22]. Moreover, erlotinib, lapatinib, imatinib, and nilotinib significantly reverse ABCC10-mediated MDR [23], [24]. Canertinib (CI-1033), a human epidermal receptor (HER) TKI, was found to reverse ABCG2-mediated MDR in cancer cells [25]. Some multikinase TKIs (such as sunitinib) have shown a reversal activity in both ABCB1- and ABCG2-mediated MDR [26], [27]. All these in vitro and in vivo studies reveal that the combination therapy of TKIs and conventional chemotherapeutic drugs could significantly sensitize MDR cells that overexpress diverse ABC transporters. Therefore, given the studies showing that TKIs play a significant role in reversing MDR in cancer cells, it is important to understand their mechanism of action.

Motesanib (AMG706), a novel nicotinamide derivative, was identified as a potent, orally bioavailable inhibitor of the vascular endothelial growth factor receptor 1 (VEGFR1/Flt1), VEGFR2/kinase domain receptor/Flk-1, VEGFR3/Flt4, platelet-derived growth factor receptor (PDGFR) and Kit receptors in preclinical models (Fig. 1A) [28]. In preclinical studies, motesanib induced significant tumor regression in xenograft models of human breast carcinoma [29], non-small cell lung cancer, medullary thyroid cancer, and epidermoid and colon carcinoma [30]. Motesanib is currently under Phase II and Phase III clinical trials for advanced gastrointestinal stromal tumor (GIST), fallopian tube cancer, ovarian cancer, thyroid cancer, non-small cell lung cancer (NSCLC) (www.clinicaltrials.gov). For instance, in the study of patients with imatinib-resistant GIST, motesanib treatment has shown acceptable tolerability and modest tumor control as evident in the proportion of patients who achieved stable disease and durable stable disease [31]. In addition, motesanib can induce partial responses in patients with advanced or metastatic differentiated thyroid cancer that is progressive. However, a broader applicability of motesanib treatment that inhibits angiogenesis in thyroid cancer needs to be established in further studies [32]. The most common side effects of motesanib treatment were diarrhea, fatigue, hypothyroidism, hypertension and anorexia [33]. The analysis of the phase III MONET1 study demonstrated that motesanib combined with carboplatin/paclitaxel could improve overall survival (OS), progression-free survival (PFS) and objective response rate (ORR) compared with chemotherapy alone, in a subset of Asian patients with nonsquamous NSCLC [34]. Importantly, the overexpression of ABCB1 has been associated with various cancers, such as GIST, NSCLC, fallopian tube, ovarian and thyroid cancer [35], [36], [37], [38], [39]. It is conceivable that motesanib probably could inhibit the function of ABC transporters by binding to their drug-binding sites as has been found for other TKIs [19]. This has triggered our efforts to determine if motesanib could reverse MDR, which is associated with the overexpression of ABCB1, ABCG2, ABCC1 and ABCC10.