Axl Protein Expression Research Articles

Axl receptor tyrosine kinase is a novel target of apigenin for the inhibition of cell proliferation.

The Axl receptor tyrosine kinase (RTK), along with Tyro 3 and Mer, belongs to the TAM subfamily that promotes survival, stimulates proliferation and/or inhibits apoptosis. In various types of human cancer, including breast, lung and prostate cancer, Axl expression is increased and correlates with an advanced clinical stage. In this study, we examined whether apigenin has an effect on Axl expression, which in turn can affect cell proliferation. The treatment of the non‑small cell lung cancer (NSCLC) cells, A549 and H460, with apigenin decreased Axl mRNA and protein expression in a dose‑dependent manner. Axl promoter activity was also inhibited by apigenin, indicating that apigenin suppressed Axl expression at the transcriptional level. Upon treatment with apigenin, the viability of both the A549 and H460 cells was gradually decreased and the anti-proliferative effects were further confirmed by the dose‑dependent decrease in the clonogenic ability of the apigenin‑treated cells. Subsequently, we found that the viability and clonogenic ability of the cells treated with apigenin was less or more affected by transfection of the cells with a Axl-expressing plasmid or Axl targeting siRNA, compared to transfection with the empty vector or control siRNA, respectively. In addition, apigenin increased the expression of p21, a cyclin-dependent kinase inhibitor, but reduced the expression of X-linked inhibitor of apoptosis protein (XIAP). These cell cycle arrest and pro-apoptotic effects of apigenin were also attenuated or augmented by the up- or downregulation of Axl expression, respectively, which suggests that Axl is a novel target of apigenin through which it exerts its inhibitory effects on cell proliferation. Taken together, our data indicate that apigenin downregulates Axl expression, which subsequently results in the inhibition of NSCLC cell proliferation through the increase and decrease of p21 and XIAP expression, respectively.

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...

MP28-04 AXL IS A NOVEL PROGNOSTIC MARKER IN UPPER URINARY TRACT UROTHELIAL CARCINOM

INTRODUCTION AND OBJECTIVES: There are few molecular markers which are known to predict a poor prognosis in upper tract urothelial carcinomas (UTUC). Axl, which is in the TAM family of receptor tyrosine kinases, has been shown to be associated with tumor progression and cancer metastasis in some types of cancer, although the association with UTUC is still not clear. We therefore investigated the biological significance of Axl on UTUC outcome. METHODS: The protein expression of Axl by immunohistochemistry and its correlation with clinicopathologic features were investigated in surgical specimens obtained from 167 patients who had been surgically treated for UTUC at our institution. The mean follow-up period was 81.8 (1-291) months. The immunoreactivity for Axl was assessed independently by a single uropathologist. RESULTS: Axl labeling was strong in 85 of 167 (55%) cases and was weak in 82 of 167 (45%) cases. Overall strong positivity of Axl expression was statistically significantly associated with high grade tumors (p1⁄40.004), positive lymphovascular invasion (LVI) (p1⁄40.001), and occurrence of postoperative metastasis (p<0.001). In univariate analysis, patients with high pathological stage (p<0.001), high grade tumor (p<0.001), LVI positive (p<0.001), or strong Axl immunostaining (p<0.001) had a significantly lower rate of disease-specific survival. In multivariate analysis, high pathological stage (p1⁄40.010), high grade tumor (p1⁄40.012), and strong Axl immunostaining (p1⁄40.047) were statistically significant factors which predicted disease-specific survival. Using the 3 statistically significant variables in multivariate Cox regression analysis, we developed a prognostic factor-based model for risk stratification (Figure 1). Based on the odds ratio (OR) of diseasespecific survival, patients were divided into 3 risk groups: low (OR 1.00-5.23, 87 patients), intermediate (OR 7.01-9.07, 35 patients), and high (OR 18.2, 45 patients). The 3-year disease-specific survival rate was 90.3% in the low-risk group, 72.4% in the intermediate-risk group, and 35.7% in the high-risk group. The differences across groups were significant (low-risk vs. intermediate risk: p1⁄4 0.033, intermediate risk vs. high-risk: p<0.001, low-risk vs. high-risk: p<0.001). CONCLUSIONS: Axl protein expression is highly associated with tumor progression and metastasis of UTUC and might be a strong prognostic marker in UTUC patients treated by radical nephroureterectomy.

Differential expression of CRKL and AXL genes in lung adenocarcinoma subtypes according to the epidermal growth factor receptor and anaplastic lymphoma kinase gene status.

Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related mortality. Adenocarcinoma (AC) is the predominant histological type of NSCLC; however, AC consists of several subtypes. It has not yet been determined whether there is a correlation of CRKL and AXL expression with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) gene status in lung AC. We assayed exons 18 through 21 of the EGFR gene by direct sequencing; ALK rearrangement and the expression of CRKL and AXL were detected by immunostaining. A total of 212 cases of AC were included in this study, diagnosed using the novel classification system established by the International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society in 2011, including 69 acinar ACs, 17 lepidic predominant ACs (LPAs), 63 papillary, 14 mucinous, 17 micropapillary and 32 solid ACs. Of the 212 cases, 101 harbored EGFR mutations. The most common subtypes carrying delK745-S753 were papillary and acinar ACs. ALK rearrangement was found in 23 cases (11%) of lung ACs. Acinar and solid ACs were the most frequent subtypes with ALK aberrance, particularly in acinar ACs with cribriform structure (4/5 cases, 80%). The expression of CRKL was significantly different among the AC subtypes (P=0.01), with the highest and lowest expression levels of CRKL protein in papillary ACs and LPAs, respectively (P<0.05). AXL expression was also significantly different among the AC subtypes (P=0.002) and was correlated with lymph node infiltration in acinar ACs. ACs with EGFR mutations exhibited high levels of AXL protein expression compared to those without mutations (P<0.001). Acinar AC with cribriform structure is a distinct subtype that frequently harbors ALK rearrangement. The activation of AXL may be one of the factors contributing to the invasion of acinar and micropapillary ACs.

AXL is a key regulator of inherent and chemotherapy-induced invasion and predicts a poor clinical outcome in early-stage colon cancer.

Despite the use of 5-fluorouracil (5-FU)-based adjuvant treatments, a large proportion of patients with high-risk stage II/III colorectal cancer will relapse. Thus, novel therapeutic strategies are needed for early-stage colorectal cancer. Residual micrometastatic disease from the primary tumor is a major cause of patient relapse. To model colorectal cancer tumor cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) colorectal cancer cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins that underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumors from patients with early-stage colorectal cancer (N = 336) were examined for AXL expression. Invasive colorectal cancer cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability, and an attenuation of EGF receptor (EGFR)/HER2 autocrine signaling. RTK arrays showed significant increases in AXL levels in all invasive sublines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion, and targeting AXL using pharmacologic inhibition or RNA interference (RNAi) approaches suppressed basal and 5-FU-induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early-stage colorectal cancer tissues. We have identified AXL as a poor prognostic marker and important mediator of cell migration/invasiveness in colorectal cancer. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in colorectal cancer, in particular in the adjuvant disease in which EGFR/VEGF-targeted therapies have failed.

Axl gene knockdown inhibits the metastasis properties of hepatocellular carcinoma via PI3K/Akt-PAK1 signal pathway

The objective of this study is to clarify the possible role and mechanism of Axl in the tumorigenicity and metastasis process of hepatocellular carcinoma. The mRNA and protein expression levels of Axl in MHCC97-H and MHCC97-L cell lines were evaluated by real-time PCR and Western blot analysis. The key factor of phosphatidylinositol-3-kinase (PI3K)/Akt-p21-activated kinases-1 (PAK1) signaling pathway was studied after Axl expression was downregulated by shRNA. Finally, we analyzed the expression status of Axl protein expression in hepatocellular carcinoma tissues and its relationship with the prognosis of hepatocellular carcinoma. Axl was observed to be higher expressed in MHCC97-H cell lines compared to MHCC97-L cell lines. The downregulation of Axl in MHCC97-H cell lines resulted in the inhibition of the invasion ability of MHCC97-H cells both in vitro and in vivo. Interestingly, blocking PI3K/Akt signaling pathway by LY294002 or Akt siRNA could remarkably inhibit the PAK1 activation and cell invasion. Finally, the Axl protein expression was positively correlated with differentiation, lymph node metastasis, and clinical stage in patients with hepatocellular carcinoma patients (all P < 0.01). These findings suggest that Axl can also regulate the metastasis process of hepatocellular carcinoma and may serve as a new prognostic marker and therapeutic target for treating hepatocellular carcinoma metastasis.

ABL Regulation by AXL Promotes Cisplatin Resistance in Esophageal Cancer

Esophageal adenocarcinoma (EAC) is characterized by resistance to chemotherapy and poor outcome. Although cisplatin (CDDP) has been used as a first-line therapy in patients with EAC, resistance remains a major clinical problem. The AXL receptor tyrosine kinase, originally isolated as a transforming gene from leukemia, is overexpressed in several solid tumors. Herein, we assessed AXL protein expression in human EACs and examined its role in CDDP resistance in human EAC cells. AXL overexpression was detected in more than 50% of tumors examined. Elevating AXL in nonoverexpressing cells doubled the CDDP IC(50) and increased cell survival three-fold, while attenuating AXL in overexpressing cells reduced survival two-fold. The effects of AXL modulation on cell survival were associated with changes in cellular and molecular markers of apoptosis. Mechanistic investigations revealed that AXL blocked CDDP-induced activation of endogenous p73β (TP73), reducing its protein half-life, and inhibited CDDP-induced levels of p-c-ABL(Y412) and p-p73β(Y99). These changes were associated with a disruption of c-ABL/p73β protein interactions due to association with c-ABL in the cytoplasm, thereby blocking nuclear accumulation of c-ABL and phosphorylation of p73β in response to DNA damage. Together, our results establish that AXL promotes CDDP resistance in esophageal adenocarcinoma and argue that therapeutic targeting of AXL may sensitize these cancers to DNA-damaging drugs.

Glycoxidised LDL Induced the Upregulation of Axl Receptor Tyrosine Kinase and Its Ligand in Mouse Mesangial Cells

Aim/HypothesisLow-density lipoprotein (LDL) is subjected to glycoxidation in diabetes, and a novel signalling mechanism by which glycoxidised LDL functions in glomerular mesangial cells remains to be ascertained.MethodsWe performed gene expression analysis in mouse glomerular mesangial cells treated with LDL modified by glycation and oxidation (GO-LDL, 100 µg/ml) for 48 h by using DNA microarray analysis and quantitative real-time PCR. We examined the GO-LDL-specific changes in gene and protein expression in mesangial cells and glomeruli of type 2 diabetic Zucker diabetic fatty (ZDF) rats.ResultsBy microarray profiling, we noted that GO-LDL treatment increased Axl receptor tyrosine kinase (Axl) mRNA expression (∼2.5-fold, p<0.05) compared with normal LDL (N-LDL) treatment in mesangial cells. Treatment with GO-LDL also increased the protein levels of Axl and its ligand Gas6 as measured by Western blotting. These increases were inhibited by neutralising Axl receptor-specific antibody. Silencing Gas6 by siRNA inhibited GO-LDL-induced Axl expression in mesangial cells. Axl and Gas6 protein were also increased in cells cultured in high glucose (30 mM) or methylglyoxal (200 µM). Gas6 treatment increased the expression and secretion of TGF-β1 protein, a key regulator of extracellular matrix expression in the glomeruli of diabetic kidneys. Immunohistochemical analyses of glomeruli from 20-week-old ZDF rats exhibited increased Axl protein expression. Rottlerin, a selective PKC-δ inhibitor, completely blocked Gas6-induced TGF-β1 expression.Conclusions/InterpretationThese data suggest that LDL modified by glycoxidation may mediate Axl/Gas6 pathway activation, and this mechanism may play a significant role in the pathogenesis of diabetic nephropathy.

Abstract A202: A role of AXL in pancreatic ductal adenocarcinoma.

Abstract Pancreatic ductal adenocarinoma (PDAC) is the 4th leading cause of cancer-related death in the US. Due to a combination of late diagnosis with highly metastatic nature and therapeutic resistance, it is a near lethal malignancy. The identification of genes forming the molecular basis of these attributes is essential for the development of improved therapies. AXL is a receptor tyrosine kinase that is implicated in many common epithelial malignancies and has been shown to play a role in invasion, migration and innate immunity. In this study, we investigated the clinical relevance and oncogenic potential of AXL in PDAC progression to assess its potential as a therapeutic target. We performed IHC analysis on a total of 306 human primary PDAC samples and found that AXL is highly expressed in 50% of samples tested, suggestive of a role in PDAC. Subsequent screening of cell lines by immunoblot demonstrated expression and activation of AXL protein in 17 of 19 PDAC cell lines. Subsequently, we selected 4 cell lines with varying levels of AXL expression, and generated derivatives of these lines with constitutive knockdown of AXL using shRNA technology. Two shRNAs were selected based on their effect on AXL expression. We found significant impairment of proliferation, the ability to form colonies in soft agar as well as on migration and invasion in a dose-dependent manner. These results suggest that AXL may be a contributing factor to the metastatic phenotype of PDAC. Targeted inhibition of AXL expression in vitro led to 5-fold reduction in ERK and 2-fold reduction in AKT activation compared to control cells, demonstrating that these signaling pathways mediate the effects of AXL on tumor growth and invasion. In order to address the role of AXL on tumor initiation in vivo, shRNA derivatives of Capan-2 and PaTu-8988T cells were injected subcutaneously into nude mice. We found that knockdown of AXL resulted in significant reduction of tumor growth in both models. However, the most striking observation was in diminished invasion in AXL-knockdown tumors compared to control xenograft tumors. Histopathological analyzes revealed extensive invasion of tumor cells beyond the cutaneous muscle in control xenograft tumors, whereas AXL-knockdown xenograft tumors lacked such invasion but had formed a fibrous capsule which separated the tumor mass from the skin. Furthermore, these tumors exhibited reduced degradation of matrigel matrix used in injection of tumor cells, suggesting that inhibition of AXL may lead to impairment of matrix-degrading enzymes. To assess if targeting of AXL might have potential therapeutic implications, we tested the effect of AXL inhibition on maintenance and progression of existing tumors. We used the doxycycline inducible shRNA system to knockdown expression once tumors had reached 120 mm3 in size in Capan-2 and SW1990 xenograft tumors. Inducible inhibition of AXL greatly reduced tumor growth resulting in 36% and 60% reduction respectively, compared to control xenograft tumors, demonstrating the significance of continued AXL expression in tumor maintenance. Our data suggest that inhibition of AXL in patients could lead to reduced tumor growth and invasion warranting further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A202.

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.

Overexpression of receptor tyrosine kinase Axl promotes tumor cell invasion and survival in pancreatic ductal adenocarcinoma.

The receptor tyrosine kinase Axl has been reported to be overexpressed in a variety of human cancers. Although previous studies have identified the role of Axl in the transformation, proliferation, survival, and invasion in cancers, the expression and functions of Axl in pancreatic cancer have not been studied in detail. The expression of Axl protein in 12 pancreatic cancer cell lines and 54 patient samples of stage II pancreatic ductal adenocarcinoma (PDA) and their paired non-neoplastic pancreatic tissue samples were examined. Using univariate and multivariate analysis, Axl expression was correlated with survival and other clinicopathologic features. To examine Axl functions in PDA, the effects of Axl knockdown on the invasion ability and radiation-induced apoptosis in PDA cell lines were measured. Axl was overexpressed in 38 of 54 (70%) stage II PDA samples and 9 of 12 (75%) PDA cell lines. Axl overexpression was associated with higher frequencies of distant metastasis and poor overall and recurrence-free survivals (P = .03 and P = .04, respectively) independent of tumor size and stage or lymph node status in patients with stage II PDA. Knockdown of Axl expression in PDA cells abolished Gas6-mediated Akt activation, decreased invasion, and increased radiation-induced PARP cleavage and the percentage of apoptosis. This study showed that Gas6 and Axl are frequently overexpressed in PDA cells and are associated with a poor prognosis in patients with stage II PDA. Axl promotes the invasion and survival of PDA cells. Therefore, targeting the Axl signaling pathway may represent a new approach to the treatment of PDA.

Gas6-Axl Pathway

In vascular smooth muscle cells, Axl is a key receptor tyrosine kinase, because it is upregulated in injury, increases migration and neointima formation, and is activated by reactive oxygen species. Reaction of glutathione with cysteine residues (termed "glutathiolation") is an important posttranslational redox modification that may alter protein activity and protein-protein interactions. To investigate the mechanisms by which reactive oxygen species increase Axl-dependent vascular smooth muscle cell function we assayed for glutathiolated proteins that associated with Axl in a redox-dependent manner. We identified glutathiolated nonmuscle myosin heavy chain (MHC)-IIB as a novel Axl interacting protein. This interaction was specific in that other myosins did not interact with Axl. The endogenous ligand for Axl, Gas6, increased production of reactive oxygen species in vascular smooth muscle cells and also increased the association of Axl with MHC-IIB. Antioxidants ebselen and N-acetylcysteine decreased the association of Axl with MHC-IIB in response to both Gas6 and reactive oxygen species. Blocking the Axl-MHC-IIB interaction with the specific myosin II inhibitor blebbistatin decreased phosphorylation of Axl and activation of extracellular signal-regulated kinase 1/2 and Akt. Association of MHC-IIB with Axl was increased in balloon-injured rat carotid vessels. Finally, expression of MHC-IIB was upregulated in the neointima of the carotid artery after balloon injury similar to upregulation of Axl protein expression, as shown in our previous studies. These results demonstrate a novel interaction between Axl and MHC-IIB in response to reactive oxygen species. This interaction provides a direct link between Axl and molecular motors crucial for directed cell migration, which may mediate increased migration in vascular dysfunction.

Inhibition of Mer and Axl receptor tyrosine kinases in astrocytoma cells leads to increased apoptosis and improved chemosensitivity.

Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal-regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development.

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.

The Axl receptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target

Pancreatic cancer is a near uniformly lethal disease and a better understanding of the molecular basis of this malignancy may lead to improved therapeutics. The Axl receptor tyrosine kinase is implicated in cellular transformation and tumor progression, although its role in pancreatic cancer has not been previously documented. The immunohistochemical expression of Axl protein was assessed in a panel of 99 archival pancreatic cancers. Axl labeling was present in 54 of 99 (55%), and was absent in 45 of 99 (45%) cases, respectively. Axl expression in pancreatic cancer was significantly associated with lymph node metastases (P

Expression of Axl in Lung Adenocarcinoma and Correlation with Tumor Progression

We used the Transwell system to select highly invasive cell lines from minimally invasive parent cells, and we compared gene expression in paired cell lines with high and low invasive potentials. Axl was relatively overexpressed in the highly invasive cell lines when compared with their minimally invasive counterparts. However, there is only limited information about the role of Axl in cancer invasion. The biologic function of Axl in tumor invasion was investigated by overexpression of full-length Axl in minimally invasive cells and by siRNA knockdown of Axl expression in highly invasive cells. Overexpression of Axl in minimally invasive cells increased their invasiveness. siRNA reduced cell invasiveness as Axl was downregulated in highly invasive cells. We further investigated the protein expression of Axl by immunohistochemistry and its correlation with clinicopathologic features. Data from a study of 58 patient specimens showed that Axl immunoreactivity was statistically significant with respect to lymph node status (P < .0001) and the patient's clinical stage (P < .0001). Our results demonstrate that Axl protein kinase seems to play an important role in the invasion and progression of lung cancer.

Increased expression of Axl tyrosine kinase after vascular injury and regulation by G protein-coupled receptor agonists in rats.

Axl is a receptor tyrosine kinase originally identified as a transforming gene product in human myeloid leukemia cells. Cultured rat vascular smooth muscle cells also express Axl, where it has been proposed that Axl may play a role in cell proliferation. In the current study, we tested the hypotheses that Axl expression would parallel neointima formation in balloon-injured rat carotid, and that Axl expression would be regulated by growth factors present at sites of vascular injury. Ribonuclease protection assay showed dynamic increases in Axl mRNA in vessels, with peak expression 7 and 14 days after injury. Immunohistochemical analysis confirmed these results and demonstrated that Axl protein expression was localized primarily to cells of the neointima after injury. Northern blot analysis indicated increased mRNA expression for the secreted Axl ligand, Gas6, in injured carotids, with a time course paralleling that of Axl upregulation. Axl and Gas6 expression were temporally correlated with neointima formation, suggesting a role for Axl signaling in this process. Other studies, performed in cultured rat vascular smooth muscle cells, revealed positive regulation of Axl mRNA expression by thrombin or angiotensin II but not by basic fibroblast growth factor, platelet-derived growth factor-BB, or transforming growth factor-ss1. Western blot analysis confirmed these results, showing that Axl protein expression was specifically increased by thrombin or angiotensin II. Our results implicate Axl as a potential mediator of vascular smooth muscle migration and proliferation caused by vascular injury and G protein-coupled receptor agonists.

Recent Progress On the Role of Axl, A Receptor Tyrosine Kinase, in Malignant Transformation of Myeloid Leukemias

Receptor tyrosine kinases (RTK) play an important role in the signal transduction of normal and malignant cells. There are different families of RTKs which are mainly characterized by differences in the ligang-binding extracellular domains. Axl (or UFO/Ark) is the first member of a new class of RTK with two fibronectin type III domains and two immunoglobulin-like domains present at the extracellular domain. The axl-gene has been isolated by means of gene transfection studies using DNA of patients with chronic myelogeneous leukemia. For a previous and the present study, we used a sensitive reverse-transcriptase polymerase chain reaction assay to detect axl's mRNA in cells from normal and malignant hematopoietic tissue. Axl's mRNA expression was mainly detected in myelo-monocytic cells, whereas much weaker transcription was seen in lymphatic cells and in lymphatic leukemias. In normal bone marrow, axl was heavily transcribed in marrow stromal cells. Further, we analysed Axl protein expression using monoclonal antibody M50 in peripheral stem cell harvests; in most harvests, no co-expression of CD34 and Axl was detected. However, in one patient with AML in complete remission, Axl was co-expressed on 80% of the CD34-positive population.These data show that axl is preferentially expressed in monocytes and stromal cells. Furthermore, a fraction of CD34-positive progenitor cells may express Axl. The exact mechanism for transformation of myeloid progenitor cells through Axl, however, remains to be determined.