Targeting Gas6/TAM in cancer cells and tumor microenvironment

Gas6/TAM have a significant role in the survival of cancer cells. For instance, in the osteosarcoma cell lines MG63 and U2OS, Axl activation by recombinant human Gas6 can protect tumour cells from apoptosis caused by serum starvation [21], and knockdown of Axl inhibits osteosarcoma cell proliferation and induces apoptosis. Moreover, when Mer expression in the AML cell lines Nomo-1 and Kasumi-1 was decreased by two independent shRNA constructs, the rate of myeloblast apoptosis increased two- to threefold in response to serum starvation [42]. In this study, researchers also transplanted NOD-SCID-gamma mice with Nomo-1 myeloblasts with reduced levels of Mer, and the results showed significantly prolonged survival of NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts compared to that of those transplanted with control cell lines. The underlying mechanisms of Gas6/TAM involvement in cancer cell survival should also be studied in the future with regard to genes mutated or upstream regulators. Gas6 and Axl interactions activate the PI3K-protein kinase B (Akt) pathway, which promotes cellular survival [29, 32]. Activation of Akt downregulates Bad, a proapoptotic mediator, and increases the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) by an NF-κB-dependent mechanism [30]. Gas6 binds to Mer and causes activation of Grb2, which promotes survival through Ras and MEK1 and increased expression of ERK1/2 [43]. Gas6/Mer interaction also activates P38 mitogen-activated protein kinase (MAPK) to promote survival [39], whereas Gas6/Tyro3 stimulates the Akt survival pathway, which leads to nuclear translocation of NF-κB and upregulation of NF-κB target genes (Fig. 1) [6].

Gas6 is a mitogen that is upregulated during the G0–G1 phase of the cell cycle [16]. Research concerning the role of Gas6 in different cancers has indicated that Gas6/TAM induces cancer cell proliferation. For instance, Gas6 promotes proliferation of prostate carcinoma cell lines expressing Axl by inducing phosphorylation of Akt and MAPK [28]. Knockdown of Axl reduces the proliferation of osteosarcoma cells, possibly via downregulation of the Akt pathway [21]. In animal experiments, knockdown of Axl in transfected NSCLC and thyroid cancer cells reduces tumour growth in nude mice [34, 37], and Mer knockdown reduces the growth of subcutaneous NSCLC xenografts in nude mice [22]. Another study revealed that the underlying mechanism of schwannoma cell proliferation after Gas6/Axl interaction involves Src, focal adhesion kinase (FAK) and NF-κB [6]. Additional mechanisms involved in the proliferation of cancer cells include the induction of ERK signalling due to Gas6 binding to Axl (Fig. 1) [24].

Migration is induced in several types of cancer, including prostate and breast cancers, in response to Gas6. For instance, Lee et al. studied the DU145 (prostate cancer) and A431 (skin cancer) cell lines and discovered that binding of Gas6 to Axl induces migration mainly by upregulating Slug expression [44]. Expression of Slug, a member of the Snail family, is enhanced in metastatic breast cancer, lung cancer, mesothelioma and melanoma [45] and is involved in metastatic prostate cancer cell invasion and migration [46, 47]. Interestingly, Yang, B et al. found that phosphorylated Axl localizes to active myosin filaments and phosphorylates tropomyosin at a tyrosine residue critical for adhesion formation, which may indicate the involvement of Axl in neoplastic migration [48]. However, this process is ligand-independent (Fig. 1).

As noted above, Gas6 plays a significant role in the development of numerous cancer types [8, 68, 69]. Moreover, Gas6 is involved in resistance to cancer therapy [70, 71]. In a study in which plasma DNA was sequenced to analyse acquired resistance to cancer therapy, Gas6 was found to contribute to resistance to breast cancer therapy [70]. An increase in the abundance of a splicing isoform of Gas6 was observed after further treatment of one patient with lapatinib in combination with capecitabine [72]. In the same study, activation of the Axl kinase pathway was found to cause resistance to tyrosine kinase inhibitors in NSCLC as well as resistance to lapatinib in estrogen receptor-positive (ER-positive), human epidermal growth factor receptor-2 (HER 2)-positive breast cancer cell lines. All of these findings suggest that targeting Gas6 may be an effective approach to treating tumours. Therefore, targeting Gas6 may effectively aid existing cancer treatments. Although there is no existing research on Gas6-specific inhibitors, a recent study exploited a novel way of inhibiting Gas6/Axl signalling, which may inspire the development of better cancer therapies [73]. The authors engineered an Axl ‘decoy receptor’ that binds to Gas6 with high affinity to inhibit its function, offering an alternative approach to drug discovery efforts that directly target Gas6. Four mutations within the high-affinity Axl variant cause structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change in Gas6. When reformatted as an Fc fusion, the engineered decoy receptor binds to Gas6 with femtomolar affinity, constituting an 80-fold improvement compared with the binding of the wild-type Axl receptor and allowing effective sequestration of Gas6 and specific abrogation of Axl signalling. Moreover, this increased Gas6 binding affinity is critical and correlates with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo. The results suggest a novel method for inhibiting Gas6/Axl signalling [73].

Accumulating evidence suggests important roles for the Axl receptor tyrosine kinase in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development [21, 25]. For example, Axl is highly expressed in invasive breast cancer cells, and Axl knockdown blocks the invasive phenotype. Moreover, high Axl expression in primary breast tumours is a strong independent predictor of poor patient outcomes [74]. As mentioned in Graham et al.’s review, a wide range of small-molecule kinase inhibitors that target the Axl receptor have been described in several studies, including Foretinib, Cabozantinib, Merestinib, Bosutinib, Gilteritinib, Crizotinib, Amuvatinib, Sunitinib, MGCD265, ASLAN002, NPS-1034, LDC1267, SGI-7079, TP-0903, UNC2025, S49076 and BGB324 [75]. However, in most cases, Axl was not the intended primary target but a secondary target resulting from the similarities among the kinase domains of Axl and other receptor tyrosine kinases (RTKs), such as MET or Mer. Consequently, these inhibitors often show less potency for Axl than their main target. Intriguingly, BGB324, also known as R428, was found to be an Axl-selective inhibitor, and has advanced to clinical trials [74]. R428 inhibits Axl with low nanomolar activity and blocks Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations have revealed favourable effects after oral administration, with R428-treated tumours displaying a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In agreement with an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumour models. Furthermore, R428 administration reduced the metastatic burden and extended survival in MDA-MB-231 intracardiac and 4 T1 orthotopic mouse models of breast cancer metastasis. Additionally, R428 acted synergistically with cisplatin to enhance suppression of liver micrometastases [74]. Notably, in addition to breast cancer, R428 has been shown to inhibit Axl signalling in glioblastoma multiforme (GBM), AML and Ewing sarcoma, indicating the effectiveness of R428 for targeting Axl [76‐78]. Indeed, R428 is now in clinical development [77], and several ongoing controlled trials involving R428 at various clinical centres aimed at identifying its maximum tolerated dose are registered at ClinicalTrails.​gov (Identifier: NCT02922777, NCT02488408, NCT02424617 and NCT02872259). These studies include trials of R428 in NSCLC, AML and metastatic melanoma, and results are expected soon. In addition to specific inhibitors, shRNA knockdown of Axl and inhibition of Axl with siRNA are also effective approaches to inhibiting Axl signalling [22, 79]. In fact, the first clinical trial using R428 for the treatment of acute myeloid leukaemia and non-small cell lung cancer is currently being conducted [80], and several other new inhibitors specific for TAM receptors are being tested in a preclinical stage [81, 82]. Stable shRNA knockdown of Axl significantly reduces tumour growth in a xenograft model of breast carcinoma [65], and inhibition of Axl with siRNA in human umbilical vein endothelial cells blocks endothelial tube formation in vitro, suggesting that inhibition of Axl may restrict the angiogenesis required for breast tumour cell growth [79]. Several monoclonal antibodies specifically targeting AXL have been reported, including 12A11 [83], Mab173 [84], YW327.6S2 [85] and, more recently, D9 and E8 [86]. All these results may provide avenues for potential therapeutic targeting of Axl.

Mer is often overexpressed or activated in various malignancies with oncogenic properties, and several studies on Mer-targeted therapies have been performed. For instance, treatment of melanoma cells with UNC1062, a novel Mer-selective small-molecule tyrosine kinase inhibitor, has been shown to reduce Mer-mediated downstream signalling activation, induce apoptosis in culture, reduce colony formation in soft agar, and inhibit the invasion of melanoma cells [39]. Moreover, an improved Mer-selective small-molecule tyrosine kinase inhibitor, UNC2025, has been shown to exert anti-tumour effects in GBM lines [87]. These findings establish Mer as a therapeutic target in melanoma and provide a rationale for the continued development of Mer-targeted therapies. shRNA targeting of Mer is used to reduce Mer-mediated downstream signalling, which decreases colony formation by up to 59% and diminishes tumour volume by 60% in a human melanoma murine xenograft model [39]. Furthermore, shRNA constructs have been used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1, and this reduction in Mer protein significantly increases the rate of myeloblast apoptosis by two- to threefold in response to serum starvation [42]. Mer590, a novel monoclonal antibody targeting Mer, has also shown effective results, rapidly and robustly reducing surface and total Mer levels in multiple cell lines. Mechanistically, Mer downregulation is mediated by receptor internalization and degradation, leading to inhibition of downstream signalling through Akt and ERK1/2. Functionally, these effects result in increased apoptosis, increased chemosensitivity to carboplatin, and decreased colony formation. In addition to carboplatin, Mer590 interacts cooperatively with shRNA-mediated Mer inhibition to augment apoptosis [88].

Studies in which Tyro3 is targeted are rare. However, Ekyalongo et al. did find that Tyro3 knockdown using siRNA induced the greatest suppression of proliferation in ER-positive/HER2-non-amplified (luminal-type) cells and, to a lesser extent, in ER-negative/HER2-amplified (HER2-type) cells [23]. Conversely, no inhibition of proliferation was observed in ER-negative/HER2-non-amplified (triple-negative type) cells [23]. Such proliferation suppression is correlated with G0-G1/S-phase arrest. These findings indicate the effectiveness of targeting Tyro3 in breast cancer and the feasibility of Tyro3 knockdown via siRNA.