Receptor tyrosine kinase Axl is a member of

Receptor tyrosine kinase Axl is a member of the TAM (Tyro3/Axl/Mer) family, and has reported been associated with a spectrum of human cancers [8]. Numerous studies have revealed that the oncogenic potential of Axl is attributed to the anti-apoptotic and proliferative signaling pathways triggered by activation of the tyrosine kinase domain [9], [10]. Ligands that can induce Axl auto-phosphorylation have been described, such as GAS6 and protein S, potentially resulting in the activation of several canonical oncogenic signaling pathways [11]. Recent studies have shown that activation of Axl mediates resistance to chemotherapeutic drugs [12], as well as targeted therapy based on small-molecule inhibitor, such as erlotinib and lapatinib [13], [14]. However, the expression and functional consequences of Axl expression in neuroblastoma and the role in targeted therapy against neuroblastoma by ALK-TKIs are poorly understood.
Materials and methods
Results
Discussion Activating mutations in the oncogene ALK have been considered as the leading cause for most cases of hereditary neuroblastoma, providing the first tractable molecular target in this disease [2]. The development of targeted therapy based on selective small-molecule inhibitors of ALK in neuroblastoma has advanced rapidly in preclinical and early phase clinical. Although tyrosine kinase inhibitors (TKIs) targeting ALK, including crizotinib and ceritinib, have showed excellent treatment effect in preclinical models, their clinical efficacy will ultimately be hindered by drug resistance [15]. It has been generally considered that the appearance of resistance to targeted therapy against diverse tumors is now considered largely inevitable that it offers much of a barrier for patients to benefit from such treatment [16]. In this study, we found that ALK was co-expressed with Axl tyrosine kinase in both NB tissues and cell lines. Various lines of evidence have demonstrated that Axl is expressed at abnormally high levels in a variety of malignancies, including various tumors [17], [18]. Since activation of Axl by its ligands, such as GAS6, could result in strong upregulation of anti-apoptotic signaling pathways thereby promoting survival of tumor AP1903 [11], further analysis was performed to determine the role of Axl activation in ALK-targeted therapy. As expected, Axl can be dramatically activated by GAS6 in the Axl-expressing NB cells (Fig. 2A). While crizotinib suppressed the growth of ALK-mutated NB cells, addition of GAS6 greatly rescued such suppression (Fig. 2B and C), indicating that ligand-activation of Axl may mediate insensitivity to ALK-TKIs. Further study showed that inhibition of Axl by gene-silencing obviously increased the sensitivity to ALK-TKIs, crizotinib and ceritinib, in NB cells co-expressing Axl and ALK. Similar effect was also found in H2228 cells, a NSCLC cell lines carrying EML4-ALK rearrangement, implying the effectiveness of such strategy in other ALK-driving tumors. The cell apoptosis was further examined in NB cells after dual inhibition of ALK and Axl with inhibitors. BGB324 (R428), an inhibitor of Axl [19], could also elicit a certain degree of apoptosis and a delay in tumor growth, suggesting the intrinsic effects of Axl in regulation of survival signaling in ALK-mutated NB. While ALK inhibitors partly triggered cell apoptosis, a higher degree of apoptosis was induced when in combined with inhibitor of Axl, consistent with the results as elucidated by Western blot that dual inhibition lead to inhibition of both ALK and Axl activation as well as induction of activated-caspase3. Furthermore, the initial experiment in vivo verified the antitumor effects of the combination treatment regimen based on inhibitors of ALK and Axl.
Acknowledgment
Introduction Axl is a receptor tyrosine kinase (RTK) that is a member of the TAM RTK family comprising Tyro3, Axl and MerTK, which has important functions in vascular biology, immunology and tumorigenesis [1], [2]. Axl affects tumor growth, epithelial-to-mesenchymal transition (EMT), angiogenesis, metastasis and development of chemoresistance to targeted therapy. Axl overexpression is linked to adverse outcome and poor survival in many cancers and Axl inhibition in several cancer models results in decreased tumorigenesis [3]. The Axl-ligand Gas6 is a vitamin K-dependent protein and contains a y-carboxyglutamic acid-rich domain that binds calcium and negatively charged phospholipids. The y-carboxylation is shown to be necessary for proper Gas6 function [4]. Gas6-ligation of Axl drives conventional receptor dimerization and subsequent activation of conserved signaling pathways [5]. Axl is also a target for atypical ligand-independent activation, such as crosstalk between Axl and VEGFR, EGFR and HGFR signaling, detected in cancers. These novel crosstalk mechanisms broaden the repertoire of Gas6-Axl signaling [6].