Published OnlineFirst June 30, 2017; DOI: 10.1158/0008-5472.CAN-17-0392 Cancer Review Research AXL-Driven EMT State as a Targetable Conduit in Cancer Jane Antony1,2,3 and Ruby Yun-Ju Huang1,4,5 Abstract The receptor tyrosine kinase (RTK) AXL has been intrinsically further elucidation to define targetable conduits. Therapeuti- linked to epithelial–mesenchymal transition (EMT) and pro- cally, as AXL inhibition has shown EMT reversal and resensi- moting cell survival, anoikis resistance, invasion, and metas- tization to other tyrosine kinase inhibitors, mitotic inhibitors, tasis in several cancers. AXL signaling has been shown to and platinum-based therapy, there is a need to stratify patients directly affect the mesenchymal state and confer it with aggres- based on AXL dependence. This review elucidates the role of sive phenotype and drug resistance. Recently, the EMT gradient AXL in EMT-mediated oncogenesis and highlights the recipro- has also been shown to rewire the kinase signaling nodes that cal control between AXL signaling and the EMT state. In facilitate AXL–RTK cross-talk, protracted signaling, converging addition, we review the potential in inhibiting AXL for the on ERK, and PI3K axes. The molecular mechanisms under- development of different therapeutic strategies and inhibitors. playing the regulation between the kinome and EMT require Cancer Res; 77(14); 3725–32. Ó2017 AACR. Introduction was originally identified in 1991 as a transforming gene in chronic myeloid leukemia (CML; ref. 3). Under normal phys- AXL, which stems from the Greek word for uncontrolled, iologic conditions, it is ubiquitously expressed in several tissues "anexelekto," is a receptor tyrosine kinase (RTK) belonging to and organs, including but not limited to the hippocampus, the tumor-associated macrophage (TAM) family, comprising of cerebellum, macrophages, platelets, endothelial cells, heart, TYRO-3, AXL, and MER (Fig. 1). Structurally, the TAM receptors liver, kidney, and skeletal muscle. It was found to be over- comprise two Immunoglobulin-like (Ig) domains, two fibro- expressed in several cancers (6) such as breast, lung, liver, colon, nectin type III (FNIII) moieties in their extracellular domain, gastric, ovarian, pancreatic, and glioblastoma. and the conserved amino acid sequence KW(I/L)A(I/L)ES in their kinase domain (1). Among the RTK family, TIE and TEK are also known to contain both Ig and FNIII motifs on their AXL Signaling Pathways ectodomains. The Ig domains are common to the FGF, VEGF, Gas6 binds to the ectodomain of AXL, causing receptor and platelet-derived growth factor (PDGF) receptor families, dimerization with a 2:2 stoichiometry of Gas6 and AXL (7). whereas the FNIII is prevalent in the ephrin and insulin families. The proximity of the kinase domains of two AXL moieties in the The MET RTK family, comprising of cMET and RON, share high RTK–ligand complex enables trans-autophosphorylation of the degree of sequence similarity in the kinase domain (2). The residues on the cytoplasmic tails, where signaling molecules human AXL gene resides on chromosome 19q13.2 and is like phospholipase C-g (PLCg), PI3K, and growth factor recep- encoded by 20 exons to form the full-length protein comprising tor-bound protein 2 (Grb2) can dock (8, 9). The tyrosine of 894 amino acids. Though the expected molecular weight is 98 residues 698, 702, and 703 in the human sequence of AXL kDa, it is posttranslationally glycosylated to form either a 120 are conserved among the TAM receptors and are involved in the kDa (partially glycosylated form) or 140 kDa protein (complete functional activity of the kinase. The tyrosine residues 779, 821, glycosylation; ref. 3). It is activated by its ligand growth arrest and 866 are also potential autophosphorylation sites in the specific 6 (Gas6; ref. 4), which becomes biologically active upon C-terminal AXL domain (9). Gas6-independent AXL phos- vitamin K–dependent posttranslational modifications (5). AXL phorylation can also occur when the RTK is overexpressed, resulting in RTK dimerization (10, 11). – 1Cancer Science Institute of Singapore, National University of Singapore, In a cell type dependent context, Gas6/AXL signaling can Singapore. 2NUS Graduate School for Integrative Sciences and Engineering, mediate growth, survival, proliferation, motility, and invasion National University of Singapore, Singapore. 3Department of Surgery and by harnessing a diverse repertoire of signaling networks such Cancer, Imperial College London, London, United Kingdom. 4Department of as the Ras/Raf/MEK/ERK cascade, PI3K/Akt signaling path- Obstetrics and Gynecology, National University Health System, Singapore. ways. The MAPK/ERK cascade is usually involved in prolifer- 5 Department of Anatomy, Yong Loo Lin School of Medicine, National University ation, whereas PI3K activation signaling converges on cell of Singapore, Singapore. survival through the Akt/ribosomal s6 kinase (S6K) axis Corresponding Author: Ruby Yun-Ju Huang, National University Hospital, 1E (12). Gas6/AXL signaling also causes increased expression of Kent Ridge Road, NUHS Tower Block Level 12, Singapore 119228, Singapore. antiapoptotic proteins such as B-cell lymphoma 2 (Bcl-2) and Phone: 656-516-1148; Fax: 656-779-4753; E-mail: [email protected] B-cell lymphoma-extra large (Bcl-xL), phosphorylation and doi: 10.1158/0008-5472.CAN-17-0392 activation of NF-kB, phosphorylation and stabilization of Ó2017 American Association for Cancer Research. Bad, and inhibition of proapoptotic proteins such as caspase 3, www.aacrjournals.org 3725 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 30, 2017; DOI: 10.1158/0008-5472.CAN-17-0392 Antony and Huang VEGFR TIE InsulinR PDGFR EGFR α α FGFR HGFR EGFD IgD EPHR AXL α β CRD AB FNIII β β EGFR INSR PDGFR-α VEGFR1 FGFR1 MET EPHA1 AXL TIE ERBB2 IGF-1R PDGFR-β VEGFR2 FGFR2 RON EPHA2 MER TEK ERBB3+ IRR CSF-1R VEGFR3 FGFR3 EPHA3 TYRO3 ERBB4 KIT/SCFR EPHA4 FLK2/FLT3 EPHA5 EPHA6 EPHA7 EPHA8 EPHB1 EPHB2 EPHB3 EPHB4 EPHB5 EPHB6 © 2017 American Association for Cancer Research Figure 1. AXL belongs to the TAM family of RTKS. Legend for RTK subfamilies: InsulinR, insulin receptor; PDGFR, platelet-derived growth factor receptor; HGFR, hepatocyte growth factor receptor; EPHR, ephrin receptor; TIE, tyrosine kinase receptor in endothelial cells. CRD, cysteine-rich domain; FNIII, fibronectin type III domain; IgD, Immunoglobulin-like domain; AB, acidic box; EGFD, epidermal growth factor-like domain. a and b denote distinct RTK subunits. NOTE: RTKs in bold font are implicated in human cancers. The "þ" on ERBB3 signifies it is devoid of intrinsic kinase activity. to induce prosurvival signaling (13, 14). Signaling through (22), can interact with AXL (23). There is evidence to suggest the PI3K/Ras/Rac axis causes actin reorganization and enables that the suppressor of cytokine signaling (SOCS-1) could serve migration (15, 16). The adapter protein Nck2, which comprises to negatively modulate AXL signaling (24, 25). The E3-ubiqui- of three tandem SH3 and one SH1 domains, is involved tin ligase Cbl-b has also been reported to counteract AXL in linking AXL with other signaling complexes. In particular, signaling by ubiquitination and subsequent degradation of the AXL–Nck2 interaction facilitates AXL-mediated modula- AXL (26, 27), enhancing the antitumor efficacy of NK cells tion of the integrin-linked kinase (ILK), a major component in the immune system. The schematic representation of the of signaling platforms at focal adhesions, thereby enabling Gas6/AXL signaling is illustrated in Fig. 2. AXL to regulate cytoskeleton dynamics (6). Furthermore, AXL has also been directly linked to control of contractility by its The EMT State Rewires the AXL Signaling ability to phosphorylate tropomyosin 2.1 (17). AXL signaling in cancers has also been implicated in processes such as Modality epithelial–mesenchymal transition (EMT; refs. 6, 18, 19), In a panel of 643 human cancer cell lines, elevated AXL wherein polarized epithelial cells lose their junctional integrity expression correlated positively with the mesenchymal pheno- to become motile, invasive, mesenchymal cells (20, 21). type, particularly in NSCLC and breast cancer. It was further The modulation of AXL required to regulate signaling has demonstrated that AXL inhibition was synergistic with antimi- not been extensively documented. The phosphatase C1-TEN, totic drugs in EMTed systems that presented with resistance to which is known to prevent signaling downstream of Akt tyrosine kinase inhibitors(TKI;ref.28). 3726 Cancer Res; 77(14) July 15, 2017 Cancer Research Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst June 30, 2017; DOI: 10.1158/0008-5472.CAN-17-0392 Targeting AXL in Cancer Gas6 AXL Integrins Calcium PLCγ signaling Shc ILK Nck2 C1-TEN PINCH-1 Grb2 Ras SRC SOCS-1 Raf1 Rac PI3K P38 Rac/ MEK1 MAPK Rho AKT Pro-inflammatory ERK1/2 MAPKAP Pak cytokine Kinase 2 production Caspase-3 S6 Bad NFκB Proliferation HSP25 JNK Actin reorganization/ Survival cell migration © 2017 American Association for Cancer Research Figure 2. Gas6/AXL signaling schema. Axl activation by ligand Gas6 in a 2:2 stoichiometry results in phosphorylation of the Axl kinase domain. Signal transduction through the Ras/Raf/MEK/ERK pathway as well as Src converges on proliferation and migration phenotype. Signal transmission through the PI3K kinase axis involving S6, AKT, or JNK results in cell survival and protection from apoptosis. Gas6/AXL signaling also interlinks with integrin-linked kinase to affect actin reorganization through integrins. It has also been shown to modulate calcium signaling through PCLg and proinflammatory cytokine production through SOCS-1. Given the relationship between drug resistance, changes in sig- in protracted downstream phosphorylated ERK (pERK) tem- naling, and emergence of an invasive phenotype is well appre- poral response, motility, and invasion. This addiction to the ciated, EMT state-mediated rewiring of the RTK signaling nodes Gas6/AXL signaling node sensitizes the mesenchymal system has identified AXL as a key player.