Gene of the month: Axl.

Matthew Brown1, James R. M. Black1, Rohini Sharma1, Justin Stebbing1, David J. Pinato1

1.  Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, IRDB Building, Du Cane Road, W12 0HS, London (UK).

Competing interests: None to disclose.

Word Count: 3,834 Tables: 0 Figures: 1 References: 89

Running Title: Gene of the month: Axl.

Keywords: Axl, oncogene, cancer, therapy.

*To whom correspondence should be addressed:

Dr David J. Pinato, MD MRes MRCP PhD

NIHR Academic Clinical Lecturer in Medical Oncology

Imperial College London Hammersmith Campus, Du Cane Road, W12 0HS, London (UK)

Tel: +44 020 83833720 E-mail:

ABSTRACT.

The interaction between Axl receptor tyrosine kinase and its main ligand Gas6 has been implicated in the progression of a wide number of malignancies. More recently, overexpression of Axl has emerged as a key molecular determinant underlying the development of acquired resistance to targeted anticancer agents. The activation of Axl is overexpression-dependent and controls a number of hallmarks of cancer progression including proliferation, migration, resistance to apoptosis and survival through a complex network of intracellular second messengers. Axl has been noted to influence clinically meaningful endpoints including metastatic recurrence and survival in the vast majority of tumour types. With Axl inhibitors having gained momentum as novel anticancer therapies, we provide an overview of the biologic and clinical relevance of this molecular pathway, outlining the main directions of research.

INTRODUCTION.

Axl, previously known as UFO, is a receptor tyrosine kinase (RTK) that forms part of the TAM family of RTKs together with Tyro3 and Mer. Evidence suggesting the oncogenic potential of Axl has been ever-present from the point of its initial isolation from chronic myelogenous leukaemia (CML).[1] Further work in to the functionality of this gene has shown its mechanistic involvement in determining a wide variety of cancerous hallmarks including: proliferation, survival, evasion from apoptosis, enhanced angiogenesis, invasiveness and, more recently, resistance to targeted anticancer therapies.[2,3] Furthermore, Axl has been shown to influence the clinical behaviour of a number of cancer histotypes, holding prognostic significance in breast, lung, ovarian, renal, gastrointestinal cancers as well as many other solid and hematopoietic malignancies.

STRUCTURE.

The Axl locus is located on chromosome 19 at position q13.2 and extends over 44 kb.[1,4] The promoter region of the gene is rich in GC repeats, which are important for the epigenetic control of Axl expression through methylation of guanine nucleotides.[5] The full length receptor is encoded within 20 exons, however, two splice variants are observed differing by the exclusion of the 27 bp exon 10.[4] Exon 10, although encoding part of the second fibronectin type III repeat, would appear to have no functional relevance to the protein as both variants hold transforming capabilities and are present within neoplastic cells.[1,6,7] Once translated, the protein alone constitutes 104 kDa and when fully post-translationally modified, at six N-linked glycosylation sites in the extracellular domain, a protein of 140 kDa is produced.[1]

Axl is a trans-membrane RTK, consisting of an extracellular ligand-binding domain and a cytoplasmic kinase domain. The extracellular portion consists of two N-terminal immunoglobulin-like domains followed by two fibronectin type III repeats giving it the appearance of a cell adhesion molecule.[1,8–10] C-terminal to the short single pass transmembrane domain, that follows the extracellular portion, is the kinase domain of Axl. An interesting feature of the kinase domain of Axl is a conserved KWIAIE sequence as opposed to the (K/T)W(T/M)APE motif usually characterising other RTKs.[1]

AXL LIGANDS.

There are two major ligands involved with activation of the TAM RTKs: Gas6 and Protein S. Both Protein S and Gas6 are members of the vitamin K-dependent protein family and carry a 44% sequence homology.[11–13] When originally characterised, Gas6 was described as containing four different regions, which were preserved within the Protein S structure.[11,12] Region A at their N-terminal end is highly rich in γ-carboxyglutamic acid residues (Gla domain). This is followed by a loop region, which in protein S contains a thrombin-sensitive cleavage site that is lacking from Gas6, reflecting the differential involvement of the two proteins in haemostasis. Region C contains 4 epidermal growth factor-like repeats, and region D, at the C-terminus, is a sex hormone binding globulin-like region, containing two tandem Laminin G-like domains.[11–13]

Two regions of this ligand appear to hold high significance for its functionality. Firstly, the laminin G-like domains are vital for the ability of Gas6 to bind Axl and these domains alone are sufficient to allow activation of RTK activity.[12] The second region which appears to bear importance to the localisation of these ligands is the Gla repeats. These γ-carboxyglutamic acid residues associate with 7-8 Ca2+ ions, which in turn mediate their ability to bind to negatively charged phospholipids and clotting factors.[13,14]

Some more recently identified and lesser studied ligands are Tubby, Tubby-like protein 1 (Tulp-1) and Galectin-3.[2] There is a recognised differential affinity between this selection of ligands and each member of the TAM family. In fact, Gas6 has a 3-10 fold higher affinity for Axl compared to Mer and Axl appears to not be activated by the presence of Protein S despite the sequence homology with Gas6.[2,13,15]

AXL ACTIVATION AND SIGNAL TRANSDUCTION.

The activation of Axl and its downstream signalling pathway relies on several different mechanisms. Ligand-dependent homodimerisation is the standard method of activation in physiological conditions; however, several ligand-independent mechanisms are possible and are more relevant in cancer. These include homodimersation upon overexpression of Axl, heterodimerisation with other TAM family RTKs: Axl and Tyro3 heterodimers have in fact been observed in the absence of ligand in gonadotropin-releasing hormone (GnRH) secreting neurons.[16,17] Heterodimerisation with non-TAM receptors can occur; interactions with fibromyalgia syndrome-like tyrosine kinase 3 (FLT-3) and epidermal growth factor receptor (EGFR) have been previously described in the literature.[7,18,19] Furthermore, Axl phosphorylation has also been reported to occur in the presence of reactive oxygen species in rat vascular smooth muscle cells.[20,21]

Activation of Axl by Gas6 has been described as a two-step process. Firstly, there is the formation of a high affinity 1:1 Gas6/Axl complex.[9] Lateral diffusion of these Gas6/Axl complexes allows formation of a 2:2 Gas6/Axl complex leading to activation of Axl via trans-auto-phosphorylation of several tyrosine residues in the intracellular domain of the protein.[9] So far three tyrosine residues, Y821, Y866 and Y779, in the C-terminal kinase domain have been identified as functionally relevant in the interaction of Axl with downstream signalling molecules.[22]

Generally, activation of Axl in cancer is caused by overexpression as opposed to an activating mutation. The methods by which overexpression occurs are not well understood and may vary in different cellular settings, however, several potential mechanisms for overexpression have been identified. Transcriptional control of the Axl gene occurs through Sp1/Sp3 and Myeloid zinc finger 1 (MZF1) transcription factors.[5,23] The ability of Sp1/Sp3 to bind the promoter region may be regulated via CpG methylation. This was shown in two colorectal cell lines, Colo206f and WiDr, both have moderate expression of Sp1 and Sp3 but exhibited very limited expression of Axl; demethylation of CG sites in these cell lines was found to increase Axl expression in dose-dependent manner.[5]

Moreover, Axl expression is also regulated by three microRNAs (miRNAs), specifically miR-34 and miR-199a/b,[23] through transcriptional repression via targeting of consensus sequences within the 3’-untranslated region of nascent Axl mRNA. MiRNA expression is epigenetically regulated by promoter methylation and unsurprisingly genomic hypermethylation verified across a panel of cell lines correlates inversely to Axl expression levels.[24]

Similarly to many other RTKs, Axl transmits a signal that is external to the cell through a series of multiple networks of protein interactions within the cytoplasm. Key to the signal-transducing properties of the receptor are a number phosphorylated tyrosine residues in its kinase domain which act as a multi-substrate docking site.[22] This allows Axl to influence a variety of different downstream pathways and processes, as illustrated in Figure 1. Two of the substrates that can bind to phosphorylated Axl are p85α and p85β, two of the regulatory subunits of PI3K.[22] These proteins bind at either tyrosine 821 or tyrosine 779, an interaction that forms a major axis of Axl signalling by providing it with an influence over the PI3K/AKT pathway, as can be seen in figure 1 the PI3K/AKT pathway plays a role in multiple aspect of Axl’s oncogenic potential.

A second, equally important pathway affected by Axl signalling is the Ras/ERK pathway, which is activated through the binding of GRB2 to tyrosine 821 of the Axl kinase domain.[22] Tyrosine residue 821 is also functionally crucial to enable the interaction between Axl and Src, Lck and PLCγ, although PLCγ can also bind through tyrosine 866.[22]

A number of emerging downstream targets are being characterised for their functional interaction with Axl activation. For example, in migrating GnRH cells it has been shown that Axl is involved with activation of the Rho family GTPase Rac.[25] In a yeast two-hybrid study several downstream targets were observed to relate to Axl activation, including SOCS-1, Nck2, C1-TEN and RanBMP.[26] Axl is also capable of lateral activation of Met, a paralog of Axl that is implicated in the promotion of metastatic potential, through its direct interaction with Src.[27]

FUNCTIONS.

Axl has a role in many aspects of cellular biology across multiple cell types, including phagocytosis, cell migration, platelet aggregation and inflammation.[28] The expression of phosphatidylserine on the surface of apoptotic cells allows binding of the Gla residues of Gas6 and Protein S, thereby activating Axl and recruiting macrophages and dendritic cells expressing TAM receptors on their cell surfaces.[7,29] Inhibition of the pro-inflammatory response is another key role played by Axl within a normal cellular environment. Axl expression is activated by type 1 interferons which are produced downstream of Toll-Like Receptor signalling.[30] Once active Axl then causes upregulation of Suppressor of Cytokine Signalling 1 and 3 (SOCS1 and SOCS3) leading to attenuation of the inflammatory signal.[10] Due to its role in several major cellular signalling pathways, aberrant activation of Axl in the context of cancer is capable of influencing a number of features underlying the malignant phenotype.

Proliferation and Survival.

There is consolidated evidence to suggest that signalling through Axl is sufficient for the promotion of survival in response to multiple pro-apoptotic stimuli including inorganic phosphate, tumour necrosis factor and adenovirus type 5 E1A protein.[31–33] Evasion from apoptosis relies upon the activation of several downstream effectors following the activation of the PI3K/Akt pathway. Firstly, activation of S6K was shown to support cell survival, as does the phosphorylation and consequent inactivation of Bad, a pro-apoptotic Bcl-2 family protein.[34,35] Furthermore, Akt driven phosphorylation of inhibitor of nuclear factor κ-B kinase subunit α (IKK-α), instigates phosphorylation and degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα).[36,37] Degradation of IκBα removes inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) allowing its translocation to the nucleus and subsequent induction of further anti-apoptotic proteins, namely Bcl-2 and Bcl-xL.[36,38] Pre-conditioning with Gas6 has been shown to suppress the activation of Caspase 3 in HUVEC cells, confirming the anti-apoptotic properties of Gas6/Axl signalling.[38] Another potential pro-survival pathway activated by PI3K is the c-Jun N-terminal Kinase (JNK) pathway, involving a cascade initiated by PI3K activation of Rac and Rho, Rho-family GTPases. This proceeds through PAK, p21-activated kinase, which has been known to lead to activation of JNK.[7,35]

A proliferative effect is observed in some cell lines as a result of Axl signalling, although it is not as commonly seen as the pro-survival effect. The role of PI3K in proliferative signalling appears to be variable, with it being required in some studies but dispensable in others,[34,39] however, the Ras/ERK pathway has been more heavily implicated as a vital factor in governing the mitogenic capabilities of Axl. Activation of several components of this pathway including Ras, Raf-1, MEK-1 and ERK occurs through the Axl/Grb2 interaction and concurrent activation of all these components is necessary to elicit a mitogenic response.[35,39]

Invasion and Metastasis.

The role of Axl in promoting the invasive potential of malignant cells is well documented by a number of studies showing a positive correlation between Axl expression and invasiveness.[40] Furthermore, disruption of Axl signalling through shRNA reduces cellular motility and invasion in experimental models.[41] Axl increases cellular motility and invasiveness through the promotion of the epithelial to mesenchymal transition (EMT), a process by which epithelial cells lose cell-cell contacts, polarity and switch to a more mobile mesenchymal phenotype. This process is often associated with expression of EMT-inducing transcription factors such as Twist, Snail and Slug. Whilst the precise molecular features underlying these phenotypic changes are not completely characterised, Axl appears to be involved in a form of positive feedback loop as several of the quintessential EMT transcription factors are both induced by and can themselves induce the expression of Axl.[41] Interestingly, vimentin, an intermediate filament that is present in mesenchymal cells, has also been shown to up-regulate expression of Axl.[42] Further work has shown that Axl may play a role in the initial induction of EMT as its expression in epithelial cells is capable of down-regulating E-cadherin and leading to the up-regulation of N-cadherin, Slug and Snail.[43] This suggests Axl may be involved with both the induction and maintenance of EMT signalling.

As well as leading to the upregulation of EMT transcription factors, activation of Axl has been implicated in influencing re-modelling of the actin cytoskeleton in GnRH neuronal cells. This re-modelling was found to be facilitated by a signalling cascade involving the Rho family protein Rac, p38 MAPK, MAPKAP kinase 2 and finally HSP25.[25] HSP25, and analogue of human heat shock protein 27, is capable of capping actin-filaments and is involved in cortical actin remodelling and membrane ruffling which are vital steps preceding cell migration.[25]

Angiogenesis.

In a normal cellular environment Axl is involved with repair of vascular injury. Expression of Gas6 along with the presence of reactive oxygen species (ROS) activates Axl in vascular smooth muscle cells which leads to increased resistance to apoptosis and migration of these cells.[44,45] In a neoplastic setting, Axl overexpression has been linked with increased angiogenesis,[46] this may be unsurprising especially as the tumour microenvironment is rich in ROS which may enhance activation of Axl. Consequently, multiple studies have shown Axl knockdown leads to reduced endothelial vessel formation, both in vitro and in vivo.[47,48]

Through profiling the changes in mRNA expression during Axl knockdown two potential downstream pathways have been identified. The first is DKK3, a member of the Dickkopf family usually involved with Wnt signalling, which was downregulated.[47] DKK3 has been shown to regulate endothelial tube formation and stable overexpression of this protein in the C57/BL6 melanoma model led to increased microvessel density.[49] The second pathway was identified through the upregulation of Ang-2, part of the angiopoietin pathway. Ang-2 acts to inhibit the interaction of Ang-1 and Tie2, which together promote endothelial cell survival.[47] Therefore activation of Axl leads to downregulation of Ang-2, freeing Ang-1 and Tie2 allowing their pro-angiogenic activity.

THE ROLE OF AXL IN CANCER PROGRESSION.

Considering the broad involvement of Axl in various aspects of cellular signalling and its implication in wide variety of hallmarks of cancer, it is unsurprising that the activation of Axl has been confirmed as a clinically meaningful trait across different solid and haematopoietic malignancies.[2] Furthermore, the degree to which Axl influences phenotypic events that can be lethal to the patient, such as progression to metastatic disease and resistance to treatment, makes Axl a prognostically appealing molecular marker in the clinical setting.

Lung carcinoma.