Expression Of Receptor Tyrosine Kinase Axl Research Articles

Axl Expression in Renal Mesangial Cells Is Regulated by Sp1, Ap1, MZF1, and Ep300, and the IL-6/miR-34a Pathway.

Axl receptor tyrosine kinase expression in the kidney contributes to a variety of inflammatory renal disease by promoting glomerular proliferation. Axl expression in the kidney is negligible in healthy individuals but upregulated under inflammatory conditions. Little is known about Axl transcriptional regulation. We analyzed the 4.4 kb mouse Axl promoter region and found that many transcription factor (TF)-binding sites and regulatory elements are located within a 600 bp fragment proximal to the translation start site. Among four TFs (Sp1, Ap1, MZF1, and Ep300) identified, Sp1 was the most potent TF that promotes Axl expression. Luciferase assays confirmed the siRNA results and revealed additional mechanisms that regulate Axl expression, including sequences encoding a 5′-UTR mini-intron and potential G-quadruplex forming regions. Deletion of the Axl 5′-UTR mini-intron resulted in a 3.2-fold increases in luciferase activity over the full-length UTR (4.4 kb Axl construct). The addition of TMPyP4, a G-quadruplex stabilizer, resulted in a significantly decreased luciferase activity. Further analysis of the mouse Axl 3′-UTR revealed a miRNA-34a binding site, which inversely regulates Axl expression. The inhibitory role of miRNA-34a in Axl expression was demonstrated in mesangial cells using miRNA-34a mimicry and in primary kidney cells with IL-6 stimulated STAT3 activation. Taken together, Axl expression in mouse kidney is synergistically regulated by multiple factors, including TFs and secondary structures, such as mini-intron and G-quadruplex. A unique IL6/STAT3/miRNA-34a pathway was revealed to be critical in inflammatory renal Axl expression.

PO-246 AXL receptor tyrosine kinase expression as a prognostic marker and therapeutic target in neuroendocrine tumours

IntroductionNeuroendocrine tumours (NETs) present a clinical challenge due to their late presentation, limited treatment options and a lack of accurate biomarkers to guide their management. Increasing evidence has confirmed the oncogenic potential of the receptor tyrosine kinase Axl, which is implicated in several hallmarks of cancer progression. This study aimed to evaluate the prevalence, prognostic role and therapeutic potential of Axl expression and its ligand Gas6 in NETs.Material and methodsTissue microarray blocks were constructed from a consecutive cohort of 54 patients with surgically resected NETs. The prevalence of the expression of Axl and Gas6 as well as markers of hypoxia were identified and correlated with clinicopathologic features and overall survival. To test the hypothesis of whether Axl is involved in the metastatic progression of NETs, immunostaining for Axl and Gas6 was evaluated in paired primary and metastatic tumour specimens in a group of post mortem cases (n=7) with adequately preserved tissues.Results and discussionsIn the 54 consecutive patients, n=46 (85%) presented with a pancreatic primary, n=24 (44%) were well-differentiated tumours and 35% were metastatic. Axl/Gas6 overexpression was identified in n=28 (52%) of the resection specimens.In isogeneic primary/metastatic NETs, Axl was overexpressed in 29% of primary tumour specimens and 61% of metastatic deposits and interestingly, Gas6 was also expressed in 29% of primaries and 61% of metastases. Axl and Gas6 expression did not correlate with VEGF-A or CaIX, nor was it predictive of overall survival in univariate analyses. However, Axl and Gas6 overexpression correlated significantly with decreased HiF1α expression levels.ConclusionOverexpression of Axl and Gas6 was found in >50% of NETs and correlated with decreased HiF1α expression. However, it did not influence patient’s overall survival.

Increased Mer and Axl receptor tyrosine kinase expression on glomeruli in lupus nephritis.

Mer and Axl receptor tyrosine kinases (MerTK and AxlTK) play important roles in the clearance of apoptotic cells and the inhibition of inflammatory responses. Previous studies demonstrated that they might participate in glomerular injury in mice model. This study aimed to elucidate the expression of MerTK and AxlTK on glomeruli and analyze their clinical significance in lupus nephritis (LN) patients. Twenty-nine LN and 10 primary nephrotic syndrome (NS) patients were recruited. The expression of MerTK and AxlTK on glomeruli was measured by immunohistochemistry. Correlations between the levels of MerTK and AxlTK and clinical data were investigated. Statistical differences in each group were calculated by one-way analysis of variance, t test, or Mann-Whitney U test. Correlations were evaluated with Pearson's or Spearman's correlation tests. Both MerTK and AxlTK were expressed mainly on mesangial cells. LN patients demonstrated more expression of MerTK and AxlTK than primary NS patients (1.19±1.01×10-2 vs 0.21±0.29×10-2, 7.25±2.69×10-2 vs 3.10±1.22×10-2, p<0.01). In LN patients, MerTK expression correlated with AxlTK (r=0.529, p<0.01). LN patients with class IV expressed more MerTK and AxlTK (1.50±1.03×10-2 and 7.56±2.93×10-2). The expression of MerTK and AxlTK varied according to the deposition of immunoglobulin and complements on glomeruli. Both MerTK and AxlTK expressions were increased on glomeruli and varied according to pathological classifications. Thus, we assumed that both two subsets might participate in the pathogenesis of LN.

Protein kinase C α is involved in the regulation of AXL receptor tyrosine kinase expression in triple-negative breast cancer cells.

AXL receptor tyrosine kinase is overexpressed in triple-negative breast cancer (TNBC), and has a function in cancer progression and metastases. However, the mechanism underlying AXL gene regulation in TNBC remains unknown. In this study, the involvement of protein kinaseCα (PKCα) in the expression of AXL was investigated in human TNBC cells. The microarray data from other studies showed that PKCα is significantly correlated with AXL expression in TNBC cell lines. Tissue array analysis also confirmed their correlation in TNBC. The PKCα inhibitor Go6976 was used to treat MDA‑MB‑231 and Hs578T TNBC cells, which resulted in decreased expression of AXL and epithelia-mesenchymal transition-related gene vimentin, and decreased cell proliferation. An MZF‑1 acidic domain fragment (MZF-1 peptide), which was designed to downregulate PKCα expression, was transfected into the cells and resulted in inhibition of AXL expression. This effect was reversed by co‑treatment with the constitutive form of PKCα. Moreover, the downregulation of PKCα was also confirmed by treatment with TAT‑fused MZF‑1 peptide. Thus, the current study proposes that AXL may be correlated with PKCα‑dependent TNBC cells, and could be modulated by MZF‑1 peptides.

Axl Receptor Axis: A New Therapeutic Target in Lung Cancer

Abstract The Axl receptor tyrosine kinase belongs to the TAM (Tyro3, Axl, Mer) family of proteins and is upregulated in multiple types of cancers, including non-small cell lung cancer. In normal tissues, TAM receptor tyrosine kinases contribute to immune regulation. In cancer, Axl expression is associated with poor outcomes and is controlled through various transcriptional and epigenetic mechanisms. Preclinical studies have shown that Axl is involved in cancer cell migration, growth, survival and resistance to chemotherapy through activation of multiple downstream signaling pathways, such as Ras/MAPK, PI3K and Rac1. Axl is also expressed on endothelial cells and plays a role in angiogenesis. In preclinical models, Axl inhibition decreases cancer growth and impedes lung cancer metastases. Small molecule Axl inhibitors have been developed and are currently being used as monotherapy or in combination with cytotoxic chemotherapy or anti-EGFR therapy in early clinical trials. Here, we review Axl structure, functions, regulation, and preclinical and clinical studies in lung cancer.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Axl receptor tyrosine kinase expression in breast cancer

AimsTriple-negative breast cancer comprises a clinically aggressive group of invasive carcinomas. We examined a published gene expression screen of a panel of breast cancer cell lines to identify a potential...

Abstract 4324: Axl receptor tyrosine kinase expression regulates tumor cell invasion and migration in colorectal cancer

Abstract Background Colorectal cancer (CRC) is the third most common cancer in the UK with around 80% of patients undergoing surgery followed by adjuvant chemotherapy treatment. Among patients with clinicopathologically defined poor stage II and stage III CRC, there are subsets of patients who will not benefit from adjuvant 5-FU or 5-FU/oxaliplatin treatment. In fact, there is preclinical evidence to suggest that some of these patients may actually do worse when treated with adjuvant chemotherapy. The aim of this study was to develop in vitro adjuvant colon cancer models with the ultimate goal to identify novel treatment strategies for early stage CRC. Method Progressively invasive populations from parental HCT116 were generated using invasion chambers. Analysis of migration and invasion was carried out using the XCELLigence system, protein activity/expression using receptor tyrosine kinase array (RTK) and Western blotting, colony forming ability was assessed by survival assays and drug sensitivity using MTT assay and Flow cytometry. Results Six sublines of HCT116 cells were initially generated (I1-6) which displayed an increasing invasive/migratory phenotype compared to the parental cell line. At the molecular level, we found increases in basal activity of a number of RTK in the invasive sublines, such as Axl (a phenomenon which is characterized with a more aggressive nature in cancers) and also in other kinases such as ERK1/2, Akt and STAT3. Decreased activation of the EGFR and HER2 receptors was also noted in the invasive cell lines. In addition, exposure to sub-lethal 5-FU(IC20) doses resulted in significant increases in migration in these cell line models. Silencing of Axl resulted in significant decreases in migration of the invasive subpopulations even in the presence of 5-FU. Colony forming assays highlighted an increased ability to form colonies in the invasive sub-populations which also displayed drug resistance to both 5-FU and Oxaliplatin. The generation of invasive cell lines was also carried out in a number of other cell lines which confirmed that an increase in Axl expression could increase the migratory phenotype regardless of the genetic background of the cell and also that the results obtained are not an artefact of the cell line we have used. Conclusion Using our in-house developed in vitro preclinical cell line model we have begun to characterise the changes which tumour cells appear to go through during the transition to an invasive phenotype. We have highlighted Axl as a potential target for inhibition in early stage colorectal cancer as increases in total Axl protein levels correlate well with increases in invasive capacity in our models and silencing of the protein expression by siRNA results in ablation of the invasive potential in our model and also in a panel of CRC cell lines. Combination of an Axl small molecule inhibitor could potentially be a novel treatment strategy for early stage CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4324. doi:1538-7445.AM2012-4324

Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer

Axl is a receptor that induces proliferation, migration and invasion in cancer. In this study, we show that specific microRNAs (miRNAs) target the 3'-UTR of Axl. Luciferase-reporter assays with wild-type and deleted miR-34 and miR-199a/b seed sequences of Axl 3'-UTR confirmed the specificity of targeting. An inverse correlation between Axl protein and miR-34a expression in a panel of non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and breast cancer (BRC) cell lines was observed, while miR-199a/b expression was completely suppressed. Pre-miR transfection inhibited in vitro migration and invasion and, in vivo, reduced the number of distant lung- or liver-metastases in a chorion-allantoic-membrane (CAM) assay. Moreover, methylation-specific PCR on bisulfite-converted DNA obtained from the cell lines showed that the miR-34a promoter methylation status was inversely correlated with its expression, and that miR-199a/b promoter regions were methylated in all cells tested. In a panel of NSCLC tissues (n=44), miR-34a and miR-199a/b were found to be downregulated and significantly co-expressed. A lower expression of all three miRs was significantly associated with squamous histotypes, and, in a preliminary series, NSCLC patients with miR-34a upregulation showed a positive association towards a longer survival. These results indicate that Axl receptor expression can be regulated by miR-34a and miR-199a/b, which are suppressed by promoter methylation in solid cancer cells.

Abstract 2086: Regulation of Axl receptor tyrosine kinase expression, invasion and metastasis by miR-34a and mir-199a/b

Abstract Axl is a receptor tyrosine kinase over-expressed in different human cancers which induces proliferation, migration and invasion. In this study, we show that specific microRNAs (miR) can target the 3’-UTR of Axl and can modulate its expression. By a bioinformatics approach, we found conserved target sites for miR-34 and miR-199 within the Axl-3’-UTR at 31-49 nt and 29-56 nt, respectively. Luciferase-reporter assays with wild-type and deleted miR-34 and −199 seed sequences of Axl-3′UTR confirmed the specificity of targeting. An inverse correlation between Axl protein and miR-34a expression in a panel of non-small cell lung cancer (NSCLC), colorectal (CRC) and breast cancer (BRC) cell lines was observed, while no miR-199a or 199b expression was detected. Transient transfection of antagonist-miR or pre-miR for miR-34a and 199a significantly induced and reduced Axl-protein levels, respectively. Pre-miR transfection was able to inhibit in vitro migration and invasion of H1299 and RKO cells and, in vivo, to reduce the number of distant lung- or liver-metastasis in a chicken-embryo-metastasis assay. Moreover, methylation specific PCR on bisulfite-converted DNA obtained from the cell lines showed that the miR-34a promoter methylation status was significantly correlated with Axl expression, and that miR-199a/b promoter regions were methylated in all the cell lines tested. These results suggest that Axl receptor, together with its role in cancer cell growth, invasion and metastasis formation, can be regulated by miR34a and 199a/199b, and that one of the reasons for its over-expression in tumors can be the methylation of DNA that encodes these specific miRs which target Axl expression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2086.

Axl receptor tyrosine kinase expression in human lung cancer cell lines correlates with cellular adhesion

Axl is a receptor tyrosine kinase (RTK) with oncogenic potential and transforming activity. Since Axl bears structural similarities to cell adhesion molecules such as neural cell adhesion molecule (NCAM) (FNIII domains), it is thought that Axl might play a role in adhesion. In this study, we have analysed the expression of the Axl protein and its ligand, Gas6, in human lung cancer cell lines of different histological origin. Axl expression occurred in approximately 60% of non-small cell lung cancer (NSCLC) cell lines, which grow adherently, and in normal bronchial epithelial cells (NHBE), but not in cell lines of small cell lung cancer origin (SCLC), which grow in suspension. A number of SCLC sublines, which could be selected spontaneously or after oncogene transfection for adherent growth, all expressed Axl protein. Overexpression of Axl per se, however, did not induce any change in the adhesion phenotype. All Axl-expressing cell lines demonstrated a membrane-bound 140 kD form, as well as a soluble 85 kD form, detectable in supernatant, of Axl-RTK. Expression of the Axl ligand Gas6 was detected in approximately 80% of all cell lines investigated. We conclude from these data that loss of Axl expression is a feature of SCLC tumour cells. Axl expression appears to be a consequence of cellular adhesion and possibly influences differentiation in human lung cancers.