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[This corrects the article DOI: 10.3389/fmed.2021.787150.].

Tobacco smoking is a well-known risk factor for both fibrogenesis and fibrotic progression; however, the mechanisms behind these processes remain enigmatic. RTKs (receptor tyrosine kinases) have recently been reported to drive profibrotic phenotypes in fibroblasts during pulmonary fibrosis (PF). Using a phospho-RTK array screen, we identified the RTK AXL as a top upregulated RTK in response to smoke. Both expression and signaling activity of AXL were indeed elevated in lung fibroblasts exposed to tobacco smoke, whereas no significant change to the levels of a canonical AXL ligand, Gas6 (growth arrest-specific 6), was seen upon smoke treatment. Notably, we found that smoke-exposed human lung fibroblasts exhibited highly proliferative and invasive activities and were capable of inducing fibrotic lung lesions in mice. Conversely, genetic suppression of AXL in smoke-exposed fibroblasts cells led to suppression of AXL downstream pathways and aggressive phenotypes. We further demonstrated that AXL interacted with MARCKS (myristoylated alanine-rich C kinase substrate) and cooperated with MARCKS in regulating downstream signaling activity and fibroblast invasiveness. Pharmacological inhibition of AXL with AXL-specific inhibitor R428 showed selectivity for smoke-exposed fibroblasts. In all, our data suggest that AXL is a potential marker for smoke-associated PF and that targeting of the AXL pathway is a potential therapeutic strategy in treating tobacco smoking-related PF.

Background Lupus nephritis (LN) is the most common and serious complication of systemic lupus erythematosus (SLE). LN pathogenesis is not fully understood. Axl receptor tyrosine kinase is upregulated and contributes to the pathogenic progress in LN. We have reported that Axl disruption attenuates nephritis development in mice. Methods In this study, we analyzed the gene expression profiles with RNA-seq using renal cortical samples from nephritic mice. Axl-KO mice were bred onto a B6.lpr spontaneous lupus background, and renal disease development was followed and compared to the Axl-sufficient B6.lpr mice. Finally, anti-glomerular basement membrane (anti-GBM) Ab-induced nephritic mice were treated with Axl small molecule inhibitor, R428, at different stages of nephritis development. Blood urine nitrogen levels and renal pathologies were evaluated. Results Transcriptome analysis revealed that renal Axl activation contributed to cell proliferation, survival, and motility through regulation of the Akt, c-Jun, and actin pathways. Spontaneous lupus-prone B6.lpr mice with Axl deficiency showed significantly reduced kidney damages and decreased T cell infiltration compared to the renal damage and T cell infiltration in Axl-sufficient B6.lpr mice. The improved kidney function was independent of autoAb production. Moreover, R428 significantly reduced anti-GBM glomerulonephritis at different stages of GN development compared to the untreated nephritic control mice. R428 administration reduced inflammatory cytokine (IL-6) production, T cell infiltration, and nephritis disease activity. Conclusions Results from this study emphasize the important role of Axl signaling in LN and highlight Axl as an attractive target in LN.

Lymphangiogenesis has gained considerable interest due to its established role in cancer progression and dissemination of metastatic cells through lymph nodes. Deciphering the molecular mechanisms that govern lymphangiogenesis within lymph nodes holds promise for revealing novel targetable molecules and pathways to inhibit metastasis. In this study, we revealed a previously unrecognized role of AXL, a tyrosine kinase receptor, in the lymphatic vessel formation. We first validated the expression of AXL in lymphatic endothelial cells (LECs), followed by functional studies using RNA interference and pharmacological inhibition with R428/Bemcentinib. These approaches provided compelling evidence that AXL promotes LEC migration in both 2D and 3D culture systems. Our findings demonstrated that AXL activation was induced by VEGF-C (Vascular Endothelial Growth Factor C) and further amplified downstream signaling via the AKT pathway. In vivo, the role of AXL in lymphatic vessel sprouting was demonstrated using R428 in a model of VEGF-C-induced lymphangiogenesis in lymph nodes. Interestingly, we discovered that AXL was predominantly expressed in MARCO + LECs. Strikingly, under metastatic conditions, there was a notable increase in the density and penetration extent of these AXL-expressing LECs into the lymph node parenchyma. Collectively, our findings pinpoint AXL as a potent enhancer of lymphangiogenesis operating through the VEGF-C/AKT pathway. Furthermore, the identification of AXL expression within a distinct LEC subpopulation, particularly in the context of metastasis, underscores the intricate interplay between AXL signaling and lymphatic dynamics within the lymph node microenvironment.

Anexelekto (AXL), a member of the TYRO3-AXL-MER (TAM) family of receptor tyrosine kinases (RTK), is overexpressed in varieties of tumor tissues and promotes tumor development by regulating cell proliferation, migration and invasion. In this study, we investigated the role of AXL in regulating glycolysis in human ovarian cancer (OvCa) cells. We showed that the expression of AXL mRNA and protein was significantly higher in OvCa tissue than that in normal ovarian epithelial tissue. In human OvCa cell lines suppression of AXL significantly inhibited cell proliferation, and increased the sensitivity of OvCa cells to cisplatin, which also proved by nude mice tumor formation experiment. KEGG analysis showed that AXL was significantly enriched in the glycolysis pathways of cancer. Changes in AXL expression in OvCa cells affect tumor glycolysis. We demonstrated that the promotion effect of AXL on glycolysis was mediated by phosphorylating the M2 isoform of pyruvate kinase (PKM2) at Y105. AXL expression was significantly higher in cisplatin-resistant OvCa cells A2780/DDP compared with the parental A2780 cells. Inhibition of AXL decreased the level of glycolysis in A2780/DDP cells, and increased the cytotoxicity of cisplatin against A2780/DDP cells, suggesting that AXL-mediated glycolysis was associated with cisplatin resistance in OvCa. In conclusion, this study demonstrates for the first time that AXL is involved in the regulation of the Warburg effect. Our results not only highlight the clinical value of targeting AXL, but also provide theoretical basis for the combination of AXL inhibitor and cisplatin in the treatment of OvCa.

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.

Cholangiocarcinoma (CCA) is a type of liver cancer originating from bile duct epithelium which has an unfavorable prognosis. Therefore, novel prognostic markers and effective therapeutic regimens are required. Opioid-binding protein/cell adhesion molecule-like (OPCML) is a tumor-suppressor protein that suppresses CCA cell proliferation via AXL receptor tyrosine kinase/signal transducer and activator of transcription 3 (AXL/STAT3) inactivation. However, this association in clinical samples remains unknown. We aimed to determine OPCML and AXL expression and investigate their association with clinicopathological features in patients with CCA. In addition, we also addressed whether OPCML enhanced the sensitivity of CCA cells to AXL inhibitor R428 in vitro. The expression of OPCML and AXL was determined by immunohistochemistry in 90 CCA tissue samples. The study of CCA cell line sensitivity to R428 was performed by cell viability assay. The expression of OPCML was significantly lower while AXL expression was substantially higher in CCA than in adjacent normal tissue (p<0.001). Furthermore, high AXL expression was significantly associated with lymph node metastasis (p=0.035). Interestingly, patients with combined low OPCML/high AXL expression had significantly shorter overall survival (p=0.007). OPCML enhanced the effect of AXL inhibitor R428 in AXL-expressing CCA cell lines. Combined expression of OPCML and AXL shows potential value as a prognostic marker and OPCML as an agent enhancing the effect of R428 may contribute to better prognosis for patients with CCA.

Background and Objectives: TAM receptors—Tyro3, Axl, and Mer—and their ligand Growth Arrest-Specific 6 (Gas6) represent a pleiotropic system implicated in fibrosis. Increased Gas6 and Axl expression have previously been observed in lung samples and fibroblast cultures from Idiopathic Pulmonary Fibrosis (IPF) patients. The study explored the contribution of Gas6/TAM system in fibrosis development and the impact of its pharmacological inhibition in fibroblasts. Materials and Methods: IPF fibroblasts (IPF FBs) and control human pulmonary fibroblasts (HPFs) were treated with R428 (Axl-specific inhibitor), LDC1267 (TAM inhibitor), or Nintedanib (an IPF-approved drug) to evaluate the influence of these drugs on cell proliferation, migration, and the expression of pro-inflammatory and pro-fibrotic genes. Fibroblast-to-myofibroblast differentiation was induced by TGF-β. The impact of IPF FBs and HPF on macrophage polarization was investigated through a co-culture of fibroblasts with monocyte-derived macrophages, with the further gene expression analysis of markers of the M1 (pro-inflammatory) or M2 (pro-fibrotic) polarization forms. Results: Cell proliferation was monitored in fibroblasts treated with TGF-β, the drugs, and their combination. In the presence of LDC1267 and Nintedanib, minor differences in cell confluence were detected between IPF FBs and HPFs; R428 (1 μM) seemed to have a higher inhibitory impact on IPF FBs. Regarding cell migration, the fibroblasts treated with LDC1267 exhibited slower wound closure. R428 treatment led to a relative wound closure of 76% in HPFs but only 56% in IPF FBs (60 h). R428 (1 μM) significantly reduced the expression of the pro-fibrotic markers ACTA2, COL1A1, and FN1 in HPFs and IPF FBs compared to TGF-β treatment. HPFs and IPF FBs co-cultured with monocyte-derived macrophages demonstrated a significantly increased expression of MRC1 while the expression of FN1, TNFα, and CXCL10 was moderately increased. Conclusions: These findings suggest that R428 and LDC1267 modulate the proliferation, migration, and gene expression of activated fibroblasts via TAM signaling. Fibroblast-mediated effects on macrophage polarization underscore the relevance of intercellular crosstalk in fibrotic disease.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer with high risk of relapse and metastasis. TNBC is a heterogeneous disease comprising different molecular subtypes including those with mesenchymal features. The tyrosine kinase AXL is expressed in mesenchymal cells and plays a role in drug resistance, migration and metastasis. We confirm that AXL is more expressed in mesenchymal TNBC cells compared to luminal breast cancer cells, and that its invalidation impairs cell migration while having no or little effect on cell viability. Here, we found that AXL controls directed migration. We observed that AXL displays a polarized localization at the Golgi apparatus and the leading edge of migratory mesenchymal TNBC cells. AXL co-localizes with F-actin at the front of the cells. In migratory polarized cells, the specific AXL inhibitor R428 displaces AXL and F-actin from the leading edge to a lateral area localized between the front and the rear of the cells where both are enriched in protrusions. In addition, R428 treatment disrupts the polarized localization of the Golgi apparatus towards the leading edge in migratory cells. Immunohistochemical analysis of aggressive chemo-resistant TNBC samples obtained before treatment reveals inter- and intra-tumor heterogeneity of the percentage of AXL expressing tumor cells, and a preference of these cells to be in contact with the stroma. Taken together, our study demonstrates that AXL controls directed cell migration most likely by regulating cell polarity.

Proliferative diabetic retinopathy (PDR), characterized mainly with abnormal epiretinal angiogenesis forming fibrovascular membranes (FVMs), threatens vision of people with diabetes; FVMs consist of extracellular matrix and a variety of cell types including vascular endothelial cells. Axl, one of receptor tyrosine kinases, can be activated indirectly by vascular endothelial growth factor-A (VEGF-A) via an intracellular route for promoting angiogenesis. In this study, we revealed that growth arrest-specific protein 6 (Gas6), a specific ligand of Axl, was elevated in vitreous from patients with PDR and that Axl was activated in FVMs from patients with PDR. In addition, we demonstrated that in cultured human retinal microvascular endothelial cells (HRECs), Axl inhibition via suppression of Axl expression with Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein 9 or through inactivation with its specific inhibitor R428 blocked PDR vitreous-induced Akt activation and proliferation of HRECs. Furthermore, PDR vitreous-heightened migration and tube formation of HRECs were also blunted by restraining Axl. These results indicate that in the pathogenesis of PDR, Axl can be activated by Gas6 binding directly and by VEGF-A via an intracellular route indirectly, suggesting that Axl plays a pivotal role in the development of PDR and that Axl inhibition shows a bright promise for PDR therapy.