Gas6 Levels Research Articles

Preoperative Serum Axl/Gas6 Allow Stratification of Oncological Outcome in Patients Undergoing Liver Resection for Hepatocellular Carcinoma

Introduction: Liver resection is routinely performed in patients suffering from primary liver malignancies. Still, this group of neoplastic entities is known to have an unfavourable prognosis. Thus, preoperative risk assessment is vital for clinical decision making. Recently, Axl and its ligand Gas6 were found to be associated to unfavourable tumor characteristics in hepatocellular carcinoma (HCC). Further, a negative correlation with overall survival (OS) and disease-free survival (DFS) were observed, which suggests its potential use as a prognostic marker in HCC patients. Method: Serum was collected in 27 patients prior to liver resection for HCC. Levels of Axl/Gas6 were measured via ELISA and evaluated as markers for OS and DFS. Results: Indeed, Axl and Gas6 were elevated in patients with an OS of less than one year and showed a high predictive potential for death within this period as assessed via receiver operating characteristics (p=0.033,AUC=0.809, respectively). Using the Youden index, optimal cut-offs were identified at 45.00 ng/mL for Axl and at 50.00 ng/mL for Gas6. Interestingly, patients above the cut-off for Axl showed significantly reduced OS in Kaplan-Meier analysis (p=0.045), and a tendency towards earlier tumor recurrence (p=0.067). Similarly, patients with increased Gas6 levels tended to display reduced OS (p=0.072), while there was no association to DFS. Conclusion: The present data underlines a potential benefit of incorporation of Axl/Gas6 in clinical routine. Especially combination with other biological markers could further increase validity. Accordingly, clinical decision making and treatment evaluation could be adapted in concordance with these markers.

The AXL/Gas-6 pathway as critical negative regulator of inflammation to orchestrate in human liver regeneration

Presenter: Patrick Starlinger MD | Mayo Clinic, Rochester Background: AXL and its corresponding ligand GAS-6 are critically involved in hepatic immunomodulation. Accumulating evidence further suggests their importance in regenerative processes. Essential might be their pleiotropic inhibitory effect on innate inflammatory responses, mediated by shifting macrophage phenotype from a pro-inflammatory M1 to an anti-inflammatory, pro-regenerative M2 polarization. Given experimental evidence, we aimed to assess the relevance of the AXL-pathway in human liver regeneration and, consequently, its association with clinical outcome after hepatic resection. Methods: Preoperative sAXL, GAS-6 concentrations and postoperative clinical outcome were analyzed in patients undergoing partial hepatectomy. Perioperative dynamics of interleukin (IL)-6 and neutrophil density were assessed as markers of inflammation, and sCD163, reflecting a shift in macrophage polarization. Results: Preoperatively elevated sAXL and GAS-6 levels significantly predicted postoperative liver dysfunction (area under the curve = 0.721 and 0.722; P < 0.005) and worse clinical outcome. These patients were found to be unable to respond with an immediate burst upon induction of liver regeneration. Abolished AXL pathway response interfered with sufficient formation/generation of regeneratory macrophages, as shown by a reduced increase of sCD163, an indicator of M2-polarization. Concomitantly, a distinct association of neutrophil infiltration (δ - GAS-6 and δ - Neutrophils: R = - 0.941 (P = 0.002)) and IL-6 release with an absent increase of TAM-signaling indicated pronounced postoperative inflammation. This was further supported by increased intrahepatic secondary necrosis as reflected by M65. Conclusion: We provided evidence that sAXL and GAS-6 represent not only potent and easily accessible preoperative biomarkers for the postoperative outcome, but that AXL/GAS-6 signaling is of critical relevance for human liver regeneration. Chronic exhaustion of the sAXL/GAS-6 system results in a lack of their immediate release upon induction of liver regeneration, resulting in an overwhelming immune activation, presumably due to absent M2 macrophage polarization.

Proof of Concept: Protein S As an Immune Modulatory Agent to Control Pancreatic Cancer

Background: Pancreatic cancer (PC) remains one of the most difficult tumors to treat. PC is nearly always diagnosed late with 80% of PC patients having Stage 4 disease and an average life expectancy of a mere 4-6 months. It affects men and women equally. In spite of our understanding of KRAS as the driver mutation in the overwhelming majority of PC patients, drugs to target KRAS remain elusive so far. Chemotherapy and radiation remain the standard of care for PC patients, many of whom are unresectable at diagnosis. This bleak prognosis encourages scientists and drug developers to propose bold, creative treatment strategies to better manage PC. Protein S (PS) is a 75 KDa vitamin K-dependent potent natural anticoagulant. PC patients have much higher risk of thrombosis than the general population. In 2018, we have shown that HIF1α, commonly upregulated in hypoxic cancer tissues, suppresses PS expression. PS has coagulation-independent, receptor-mediated effects through the TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK). We recently published (in press) data that indicate that PS suppresses the growth of PC cells in vitro and that PROS1 (PS) gene expression is associated with better survival in PC. Survival analysis based on public domain RNASeq data shows that PROS1 mRNA expression in PC is significantly correlated with better overall survival (OS) up to 18 months (Figure 1). Additionally, very recent data indicate that PS receptor MERTK is a late costimulatory signal in CD8 T-cells, which is required for the establishment of immunological memory and anti-tumor immune responses in melanoma. Our proof of concept (POC) shown in the schematic is PS directly suppresses PC cell growth and promotes CD8-mediated tumor immunity against PC. Results &amp; Discussion: PS and its properties as a signaling molecule in the context of cancer progression and cellular aggressiveness have not been studied in-depth. The expression levels of the individual TAM receptors showed less than 1-fold variation among the cell lines tested; whereas, PS and GAS6 levels were significantly different. PS/GAS6 ratio directly correlates with the aggressiveness of PC cell lines. We found that the lower the ratio, the higher was the aggressiveness. We used three cell lines, MIA PaCa-2, PANC-1, and BXPC-3 with population doubling times of 40 hours, 52 hours and 72 hours, respectively (per information from ATCC). The basal levels of the three TAM receptors were relatively uniform among the three cell lines, i.e., less than 5% difference in expression level. Interestingly, although PANC-1 cells, with an intermediate degree of aggressiveness, had a PS/GAS6 ratio of 1, MIA-PaCa-2 cells had a significantly lower PS/GAS6 ratio indicating that more aggressive cell line has more GAS6. The least aggressive cell line BXPC-3 cells had a much higher PS/GAS6 ratio. PS overexpression reduced survival and proliferation of PANC-1 and MIA PaCa-2 cells. To further confirm that PS overexpression accelerated apoptosis of PC cells, we generated flow data using Annexin V/7AAD (7-Aminoactinomycin D) staining. Notably, PS gene knockdown by SiRNA or GAS6 overexpression resulted in increase in aggressiveness of those cell lines tested in our published data (In press). Conversely, GAS6 gene knockdown attenuated the aggressive phenotypes of these cell lines tested. Conclusion: We propose to test PS infusion in PC to downregulate HIF1α and modulate the tumor microenvironment (TME) to promote sustained tumor immunity. Our preliminary data and publicly available gene expression profiles suggest that PS may have therapeutic value in PC. Disclosures No relevant conflicts of interest to declare.

Integration of gene profile to explore the hub genes of lung adenocarcinoma: A quasi-experimental study.

Background:Lung cancer is a leading cause of morbidity diseases worldwide, but the key mechanisms of lung cancer remain elusive. This study aims to integrate of GSE 118370 and GSE 32863 profile and identify the key genes and pathway involved in human lung adenocarcinoma.Methods:R software (RStudio, Version info: R 3.2.3, Forrester, USA) were utilized to find the differentially expressed genes. All the differentially expressed genes were analyzed by gene ontology, kyoto encyclopedia of genes and genomes. Protein-protein interaction networks were constructed by STRING database and analyzed by Cytohubber and Module. The cancer genome atlas database was used to verification the expression of hub genes. Quantitative reverse transcription-PCR was used to verify the bio-information results.Results:Sixty-four lung adenocarcinoma and 64 adjacent normal tissues were used for integration analysis. Five hundred ninety-nine co-expression genes were locked. Biological processes mainly enriched in angiogenesis. Cellular component focused on extracellular exosome and molecular function aimed on protein disulfide isomerase activity. Cytohubber analysis showed that GNG11, FPR2, P4HB, PIK3R1, CDC20, ADCY4, TIMP1, IL6, CXC chemokine ligand (CXCL)12, and GAS6 acted as the hub genes during lung adenocarcinoma. Module analysis presented Chemokine signaling pathway was a key pathway. Quantitative reverse transcription-PCR showed that the expression level of GNG11, FPR2, PIK3R1, ADCY4, IL6, CXCL12, and GAS6 were significantly decreased and P4HB, CDC20 and TIMP1 were increased in human adenocarcinoma tissues (P < .05). The cancer genome atlas online analysis showed GNG11 was not associated with survival.Conclusions:This study firstly reported GNG11 acting as a hub gene in adenocarcinoma. GNG11 could be used as a biomarker for human adenocarcinoma. Chemokine signaling pathway might play important roles in lung adenocarcinoma.

Growth arrest-specific6 modulates adiponectin expression and insulin resistance in adipose tissue.

Aims/IntroductionObesity is characterized by disturbed adipocytokine expression and insulin resistance in adipocytes. Growth arrest‐specific 6 (GAS6) is a gene encoding the Gas6 protein, which is expressed in fibroblasts, and its related signaling might be associated with adipose tissue inflammation, glucose intolerance and insulin resistance. The aim of this study was to investigate the associations among Gas6, adipocytokines and insulin resistance in adipocytes.Materials and MethodsMature Simpson Golabi Behmel Syndrome adipocytes were treated with high levels of insulin to mimic insulin resistance, and were examined for the expressions of Gas6, cytokines and adipocytokines from preadipocytes in differentiation. In an animal study, high‐fat diet‐induced obese mice were used to verify the Gas6 expression in vitro.ResultsDuring the differentiation of adipocytes, the expression of Gas6 gradually decreased, and was obviously downregulated with adipocyte inflammation and insulin resistance. Gas6 levels were found to be in proportion to the expression of adiponectin, which has been regarded as closely relevant to improved insulin sensitivity after metformin treatment. Similar results were also confirmed in the animal study.ConclusionsOur results suggest that Gas6 might modulate the expression of adiponectin, and might therefore be associated with insulin resistance in adipose tissues.

Serum Gas6 and Axl as non-invasive biomarkers of advanced histological stage in primary biliary cholangitis.

Advanced histological stage is an important factor in individual risk stratification in patients with primary biliary cholangitis (PBC). Non-invasive biomarkers for advanced histological stage are needed. We assessed the utility of Gas6 and Axl as biomarkers for advanced histological stage in patients with PBC. A total of 113 biopsy-proven PBC patients and 20 healthy controls were included in this study. Serum Axl and Gas6 were measured using enzyme-linked immunosorbent assay. The Gas6/albumin ratio and Axl/albumin ratio were also evaluated as biomarkers of histological stage. Serum Axl (42.6ng/mL vs. 30.6ng/mL, P < 0.001) and Gas6 (21.1ng/mL vs. 18.8ng/mL, P = 0.007) levels in PBC patients were significantly higher than those in healthy controls. The Axl/albumin ratio was 10.4, and the Gas6/albumin ratio was 7.6 in patients with PBC. Gas6 and Axl were significantly correlated with aspartate aminotransferase, bilirubin, albumin, and platelets. Gas6 and Axl levels in patients with an advanced Scheuer stage and an advanced Nakanuma stage were significantly higher than those in other patients. The area under the receiver operating characteristic curve (AUROC) of Axl, Gas6, Axl/albumin, and Gas6/albumin for diagnosing Scheuer stage 4 was 0.733, 0.837, 0.845, and 0.893, respectively. The AUROC of Axl, Gas6, Axl/albumin, and Gas6/albumin for diagnosing Nakanuma stage 4 was 0.794, 0.834, 0.869, and 0.898, respectively. High levels of Gas6 and Axl were associated with advanced histological stage in PBC patients. Furthermore, the Gas6/albumin ratio and the Axl/albumin ratio showed a high AUROC for diagnosing advanced histological stage.

Selective Increment of Synovial Soluble TYRO3 Correlates with Disease Severity and Joint Inflammation in Patients with Rheumatoid Arthritis.

Objective To investigate the role of TAM receptors in rheumatoid arthritis (RA) by determining synovial tissue TAM receptor expression, synovial fluid levels of soluble TAM receptors, and the relationship between soluble TAM receptors, joint inflammation and disease activity. Methods TAM receptor expression was determined by immunohistochemistry on the synovium from RA and osteoarthritis (OA) patients. Soluble (s) Tyro3, sAxl, sMer, and their ligand Gas6 were measured by ELISA in the synovial fluid of RA (n = 28) and OA (n = 12) patients and cytokine levels by multiplex immunoassay in RA samples. Correlation analyses were performed among sTAM receptors with local cytokine levels; systemic disease parameters like erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and anticyclic citrullinated peptide antibodies (ACPA); and disease activity scores (DAS28-ESR) in RA patients. Results TAM receptors were expressed on different locations in the synovial tissue (lining, sublining, and blood vessels), and a similar expression pattern was observed in RA and OA patients. Synovial fluid sTyro3 and sMer were significantly enhanced in RA compared to OA patients, whereas no significant differences in sAxl and Gas6 levels were found. In RA samples, sTyro3 levels, but not sMer, correlated positively with proinflammatory local cytokines and the systemic factor erythrocyte sedimentation rate. Moreover, stratification analysis showed high sTyro3 levels positively correlated with higher DAS28-ESR and in RF and ACPA double positive RA patients. Conclusion sTyro3 in the synovial fluid of RA patients correlates with local inflammatory molecules and systemic disease activity. These findings suggest that the reduced negative control of cell activation by TAM receptors due to their shedding in the synovial fluid, mainly sTyro3, favoring joint inflammation in RA patients.

Testosterone ameliorates vascular aging via the Gas6/Axl signaling pathway.

Low serum testosterone level is associated with aging-related vascular stiffness, but the underlying mechanism is unclear. The Growth arrest-specific protein 6 (Gas6) /Axl pathway has been proved to play important roles in cell senescence. In this study, we intend to explore whether Gas6/Axl is involved in the effect of testosterone on vascular aging amelioration. Vascular aging models of wild type and Axl-/- mice were established by natural aging. Mice of these two gene types were randomized into young group, aging group and testosterone undecanoate (TU) treatment group. Mice were treated with TU (37.9 mg/kg) in the TU group, which treated with solvent reagent served as control. The aging mice exhibited decreases in serum testosterone, Gas6 and Axl levels and an increase in cell senescence, manifested age-related vascular remodeling. Testosterone treatment induced testosterone and Gas6 levels in serum, and ameliorated cell senescence and vascular remodeling in aging mice. Furthermore, we uncover the underlying molecular mechanism and show that testosterone treatment restored the phosphorylation of Akt and FoxO1a. Axl knockout accelerated cell senescence and vascular remodeling, and resisted the anti-aging effect of testosterone. Testosterone might exert a protective effect on vascular aging by improving cell senescence and vascular remodeling through the Gas6/Axl pathway.

Differential Expression Pattern of Epithelial Mesenchymal Transition Gens; AXL, GAS6, Claudin-1, and Cofilin-1, in Different Stages of Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC), is the fatal form of gynecological cancer. Almost 70% of ovarian cancer patients are detected at an advanced stage (III-IV) with metastases. Epithelial-mesenchymal transition (EMT) is a critical process associated with metastasis. This study investigated the expression levels of AXL, GAS6, Claudin-1, and Cofilin-1, as genes involved in EMT in relation to clinicopathologic features in ovarian cancer patients. In this descriptive study, 78 ovarian epithelial cancer patients were enrolled. Samples were provided by the Iran National Tumor Bank, founded by the Cancer Institute of Tehran University of Medical Sciences in 2017. The expression levels of AXL, GAS6, Claudin-1, and Cofilin-1 genes were investigated in a fresh, frozen tumor sample and normal adjacent tissue by real-time PCR (RT-PCR). Findings showed a significant relationship between the overexpression of AXL and TNM staging (P=0.03). The expression level of GAS6 decreased in more advanced stages (P=0.01). There is a negative relationship between Cofilin-1 expression level and TNM staging (P=0.002). Claudin-1 expression level was higher in low stages compared with that in high stages (P=0.01). There was no relationship between gene expression levels of target genes with size and grade of the tumor. Given the importance of these genes in EMT, alteration in their expression pattern can contribute to the progression of the disease and distant metastasis of cancer cells. Additionally, knowing the alteration pattern of these genes expression can help to better understanding and prediction of the prognosis of EOC.

Increased plasma levels of Gas6 and its soluble tyrosine kinase receptors Mer and Axl are associated with immunological activity and severity of lupus nephritis.

Growth arrest-specific 6 (Gas6) and its receptors have been shown to play a crucial role in the homeostasis of the innate immune system by regulating apoptosis and inflammation. We aimed to verify whether an impairment of this system is associated with systemic lupus erythematosus (SLE) disease activity and with lupus nephritis (LN). Plasma Gas6 and the soluble cleaved form of the receptors MerTK (sMer) and Axl (sAxl) concentrations were measured in n=59 SLE patients (n=44 with nephritis, 75%) and analysed in relationship to clinical and laboratory data. Patients with LN were characterised by higher Gas6 (19.0 ng/mL [16.8-24.5] vs. 16.5 ng/mL [13.89-18.91]; p=0.03) and sAxl plasma levels than those without LN (31.36 ng/mL [25.1-41.4] vs. 20.2 ng/mL [15.6-30.7]; p=0.03); conversely sMer plasma concentrations were similar between groups. All the three biomarkers studied were directly correlated to creatinine and daily proteinuria, being inversely related to creatinine clearance. 39 patients had a proteinuria level of <0.5 mg/day, 14 between 0.5 and 3.5 mg/day and 5 had ≥3.5 g/day; Gas6, sAxl and sMer plasma concentrations significantly increased for increasing degree of proteinuria (test for trend p=0.0002; p=0.02; p=0.009, respectively).These correlations were confirmed in multiple linear regression analysis models accounting for gender, age, disease duration and concomitant treatment. Plasma Gas6, sAxl and sMer concentrations are associated with the severity of LN in patients affected by SLE. The excess cleavage of TAM receptors might contribute to LN pathogenesis.

Implications of the Receptor Tyrosine Kinase Axl in Gastric Cancer Progression.

BackgroundGastric cancer (GC) is an aggressive malignancy with high lethality. Systematic chemotherapy is the main therapeutic strategy for advanced GC patients. The overexpression of Axl is associated with poor prognosis and regulates tumor growth and metastasis in many types of cancer. However, the role of Axl in GC progression remains elusive.Materials and MethodsWestern blot and quantitative real-time PCR assay (RT-PCR) assays were used to detect the expression of Gas6, Axl, ZEB1 and epithelial-mesenchymal transition (EMT)-related markers in GC cells. Cell proliferation was determined by EdU cell proliferation assay and CCK-8 assay. Transwell invasion assay was performed to explore the effect of Axl and ZEB1 on cell invasion. Tumor xenografts and lung metastasis models were conducted to examine the effect of Axl on the growth and lung metastasis of GC cells.ResultsIn our study, we found that high levels of Gas6 and Axl expression were associated with reduced overall survival (OS) in GC patients and the expression of Gas6 and Axl was upregulated in GC cell lines. Ectopic expression of Axl induced EMT and promoted GC cell invasion and proliferation. The knockdown of Axl inhibited EMT and suppressed the proliferation and invasion of GC cell. In vivo study showed that inhibition of Axl impaired tumor growth and lung metastasis of GC cells. Mechanistic investigations revealed that Axl promoted EMT, invasion, and proliferation via upregulating ZEB1 expression in GC cells.ConclusionOur results demonstrated that the Gas6/Axl/ZEB1 signaling pathway regulated EMT, invasion, and proliferation in GC cells and might represent a potential therapeutic target for GC treatment.

Qigesan inhibits esophageal cancer cell invasion and migration by inhibiting Gas6/Axl-induced epithelial-mesenchymal transition.

Qigesan (QGS) has been used to effectively treat esophageal cancer (EC) for decades in China, but the mechanism by which it suppresses EC metastasis remains unknown. In this study, we examined the effects of QGS on EC cell mobility. Using immunohistochemistry and immunofluorescence, expression of Gas6 and Axl, which promote tumor cell migration and invasion, was examined in carcinoma tissues and adjacent normal tissues from EC patients. Levels of Gas6, Axl, and the Gas6/Axl complex were also examined in ECA109 and TE13 EC cells treated with QGS. In addition, immunofluorescent staining and quantitative protein analysis were used to examine E-cadherin, N-cadherin, and Snail levels in ECA109 and TE13 EC cells after QSG administration, and cell mobility was assessed. The results demonstrated that levels of Gas6 and Axl expression are higher in EC tissues than in adjacent normal tissues. Moreover, QGS decreased Gas6/Axl levels, increased E-cadherin expression, decreased Snail and N-cadherin expression, and inhibited epithelial-mesenchymal transition (EMT) in EC cells. QGS thus suppresses EMT in EC by inhibiting Gas6/Axl binding.

Study on the relationship between growth arrest-specific protein 6 and acute myocardial infarction

Objective To explore the relationship between growth arrest-specific protein 6 (Gas6) and acute myocardial infarction (AMI). Methods Patients were included from Renmin Hospital of Wuhan University between January to June 2018. A total of 103 patients with angina pectoris aged 60.20±9.35 were included as angina pectoris group. A total of 102 patients with myocardial infarction aged 58.85±9.80 were included as AMI group. A total of 130 healthy individuals aged 63.14±10.40 were included as healthy control. Spearman analysis was performed to investigate the correlations between Gas6 and risk factors of (coronary heart disease, CHD). Logistic regression was performed to investigate the risk factor of myocardial infarction. ROC (receiver operating characteristic) curve was used to analyze the diagnostic performance of Gas6 to AMI. Results The levels of Gas6 in angina pectoris group [13.77 (10.57-17.03) ng/ ml, t=2.444, P=0.025] and AMI group[16.22 (12.70-20.09) ng/ml, t=4.965, P<0.001] was higher than control group [10.92 (8.90-14.92) ng/ml]. The levels of Gas6 in angina pectoris group was lower than AMI group (t=3.854, P<0.001). In the sensitivity analysis excluding hypertension and diabetes, the serum Gas6 level in AMI group (n=37) [15.05 (11.08-16.20) mg/L] was higher than that in control group [10.93 (8.91-14.93)mg/L, t=3.479, P=0.001] and angina group (n=42) [12.85 (9.10-16.20) mg/L, t=2.639, P=0.019]. CRP (C-reactive protein), WBC (white blood cell count), Glu (fasting glucose) and Cr (creatinine) were positively correlated with Gas6, r=0.194, 0.176, 0.180 and 0.120, P value=0.010, 0.012, 0.010 and 0.002, respectively. Logistic regression showed that Gas6 was a independent factor of myocardial infarction [OR and 95%CI were 1.080 (1.012-1.152), P=0.020]. AUC and 95%CI of ROC curve was 0.648 (0.572-0.723). Conclusion The levels of Gas6 may be positively associated with myocardial infarction risk. Key words: Acute myocardial infarction; Inflammation; Growth arrest-specific protein 6

Possible Association of Elevated Plasma Levels of Growth Arrest-Specific Protein 6 and the Soluble Form of Tyrosine Kinase Receptor Axl with Low Hepatitis C Viral Load in Patients with Type 2 Diabetes Mellitus.

This study aimed to investigate the plasma levels of Gas6 and soluble Axl (sAxl) in patients with chronic hepatitis C virus (HCV) infection with and without type 2 diabetes mellitus (T2DM). The study involved four groups; 50 patients with chronic HCV, 50 patients with T2DM, 50 patients with chronic HCV and T2DM, and 31 age- and sex-matched healthy controls. T2DM was diagnosed according to American Diabetes Association criteria, HCV antibodies were detected by enzyme-linked immunosorbent assays (ELISA) and confirmed by real-time-polymerase chain reaction. Plasma Gas6 and sAxl levels were assayed in all groups by ELISA. Significant low levels of GAS 6 in HCV/T2DM group versus HCV group were detected (7.92 ± 5.18 vs. 16.09 ± 7.36, respectively, p = 0.000), but higher than T2DM and control groups (p ≥ 0.05), although nonsignificant. HCV load was higher in the HCV group than the HCV/T2DM group (1,888,300 ± 5,595,070 vs. 1,417,900 ± 4,066,460 copies/mL, respectively, p = 0.632). Among HCV group, significant positive correlations were detected between Gas6 and sAxl levels with HCV viral load (r = 0.48, p = 0.000 and r = 0.43, p = 0.002, respectively), while among HCV/T2DM group, significant negative correlations were detected (r = -0.29, p = 0.04 and r = -0.34, p = 0.014, respectively). Significant negative correlations were detected between Gas6/sAxl levels and glycated hemoglobin (r = -0.36, p = 0.01 and r = -0.4, p = 0.003, respectively) in T2DM despite the positive correlations detected in HCV/T2DM (r = 0.27, p = 0.053 and r = 0.55, p = 0.000, respectively). In conclusion, Gas6/Axl system in combined HCV/T2DM diseases may affect the pathogenesis and can alter the biomarkers and complications of both diseases in a manner that differs from a solitary disease.

Inhibition of Heme Oxygenase-1 Attenuates Bortezomib Resistance in Multiple Myeloma Via Down-Regulating Gas6 Production

Background Multiple myeloma (MM), one of the most prevalent hematological malignancies, is characterized by the proliferation of abnormal plasma cells. The proteasome inhibitor bortezomib is a frontline drug in the treatment of MM; however, most patients will develop acquired resistance to bortezomib, which has been a critical challenge in clinic. Our previous studies have indicated that inhibition of heme oxygenase-1 (HO-1) can attenuate drug resistance to MM. Moveover, emerging evidence suggests that growth arrest-specific gene 6 (Gas6) may regulate tumor microenvironment and serve as a novel target for cancer therapy. However, whether HO-1 mediated-Gas6 production involved in the development of bortezomib resistance in MM remains unclear. Aims The aims to perform the present study were to: 1) assess the association between intracellular HO-1 and Gas6 expression in the bone marrow of CD138+ cells of patients with MM; 2) test the role of HO-1 and Gas6 in the development of bortezomib resistance in MM; 3) explore whether HO-1 mediate Gas6 production and the potential mechanism. Methods Patients with different stages of MM (n=18) and healthy donors (n=18) at Affiliated Hospital of Guizhou Medical University were included in this study. Baseline clinical characteristic data and bone marrow samples were obtained from study participants. CD138+ plasma cells were purified from bone marrow samples. The recombinant lentiviral vector of HO-1 was constructed and subsequent transfected into U266 and RPMI8226 cells. The expressions of mRNA and protein levels were determined by real-time PCR and western blot, respectively. Secreted Gas6 was determined by ELISA. Flow cytometry was used to assess cell apoptosis. Results In human study, the HO-1 mRNA and protein level in CD138+ cells of patients with MM showed a significant increase compared with those in healthy group, respectively (P&lt;0.01). With the development of MM, HO-1 mRNA and protein level were gradually increased from stage I to III. The similar findings were also observed in the expression of Gas6 mRNA and protein level (P&lt;0.01). Interestingly, spearman correlation analysis showed that higher HO-1 mRNA and protein level were associated with increased Gas6 mRNA and protein level, respectively (P&lt;0.05). To test whether HO-1 mediate Gas6 production, recombinant lentivirus-HO-1 vector and HO-1 siRNA were transfected into U266 and RPMI8226 cells to up/down-regulate HO-1 expression. Compared with control group, Gas6 mRNA and protein expression were significantly increased when U266 and RPMI8226 cells were up-regulated by recombinant lentivirus-HO-1 vector. Additionally, Gas6 mRNA and protein expression were also increased by the use of hemin (HO-1 inducer). In contrast, Gas6 expression presented a decrease in mRNA and protein level with the stimulation of HO-1 siRNA and HO-1 inhibitor ZnPPIX. Similar significantly trend was also observed in the culture medium. Moreover, the effect of HO-1 on bortezomib resistance in MM cells was markedly inhibited by the stimulation with Gas6 siRNA and Gas6 neutralizing antibody (the apoptosis rate in MM cells treated with PBS, Gas6 siRNA and Gas6 neutralizing antibody is 28%, 56% and 42%, respectively; P&lt;0.01). These results indicated that HO-1 up-regulated Gas6 production in the development of bortezomib resistance in MM. Furthermore, to explore the potential mechanism how HO-1 regulates Gas6 production, MAPK, an important pathway in drug resistance, was investigated. U266 or RPMI8226 cells transfected with recombinant lentiviral vector of HO-1 were stimulated with p38MAPK inhibitor SB203580, the MEK inhibitor PD0325901, the JNK inhibitor SP600125, the PI3K inhibitor LY294002, and the STAT3 inhibitor S3I-201, and the HO-1 level of cells and Gas6 levels of cells and culture medium were determined. Findings showed that HO-1 level did not significantly change, while Gas6 expression in cells and Gas6 level in culture medium showed markedly decrease by the treatment with MAPK pathway inhibitor compared with control group. These results indicated that HO-1 mediated Gas6 production via MAPK pathway. Conclusion These results demonstrate that inhibition of HO-1 attenuates bortezomib resistance in MM via down-regulating Gas6 production. Targeting these pathological interactions holds promise for improve MM treatment. Disclosures No relevant conflicts of interest to declare.

A Functional Role of GAS6/TAM in Nonalcoholic Steatohepatitis Progression Implicates AXL as Therapeutic Target

Background and AimsGAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development.MethodsGAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl-/-, Mertk-/- and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients.ResultsIn primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD- and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibrotic- and inflammatory-related genes in HFD-fed mice after bemcentinib administration. HFD-fed Mertk-/- mice exhibited enhanced NASH, while Axl-/- mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH.ConclusionAXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice.

Increased sMer, but not sAxl, sTyro3, and Gas6 relate with active disease in juvenile systemic lupus erythematosus.

Tyro3, Axl, and Mer (TAM) receptors and ligands mediate apoptotic bodies engulfment which alteration has been related with juvenile systemic lupus erythematosus (JSLE) pathogenesis. Thus, the aim was to determine their soluble levels. Serum sTyro3, sAxl, sMer, and Gas6 levels were measured using ELISA in 67 JSLE patients, 12 juvenile idiopathic arthritis (JIA) inflammatory and 20 healthy controls and related with SLEDAI-2K score, anti-dsDNA antibody, ESR, CRP, C3, C4 levels, and nephritis. JSLE patients with active disease (SLEDAI-2K> 4) had significantly increased sMer levels compared with healthy controls (median 8.4 vs. 6.0 ng/mL, p = 0.009) and inactive disease patients (5.2 ng/mL, p = 0.0003). sMer levels correlated with SLEDAI-2K (r = 0.44; p = 0.0004) and ESR (r = 0.24; p = 0.04), while sAxl correlated with SLEDAI-2K (r = 0.33; p = 0.008) and C4 levels (r = - 0.24; p = 0.04). JSLE patients taking glucocorticoid had increased sAxl and sMer levels. Moreover, sAxl correlated with sMer and sTyro3 levels. Patients with nephritis and those with focal or diffuse proliferative glomerulonephritis had these protein levels similar to healthy controls and patients without renal involvement. sTyro3 levels of JSLE patients taking glucocorticoid were decreased, and correlated with Gas6 and sAxl, while Gas6 levels correlated with age upon enrollment. JIA controls had protein levels similar to healthy controls and JSLE patients. This study reinforces that sMer is increased in active JSLE patients, yet sMer and sAxl correlates with disease activity parameters, and their alterations are disease-specific. However, further studies are needed to determine exact roles of sTyro3 and Gas6 in disease pathogenesis. Key Points • sMer and sAxl serum levels are related with active disease in JSLE patients • sMer correlated with SLEDAI-2K score in JSLE • sTyro3, sAxl, sMer and Gas6 levels did not related with nephritis in JSLE patients • sTyro3 and Gas6 exact roles in JSLE are not established and further studies are needed.

Tumor tissue and plasma levels of AXL and GAS6 before and after tyrosine kinase inhibitor treatment in EGFR‐mutated non–small cell lung cancer

BackgroundNon‐small cell lung cancer (NSCLC) positive for activating mutations of the epidermal growth factor receptor (EGFR) gene is initially sensitive to EGFR‐tyrosine kinase inhibitors (TKIs) but eventually develops resistance to these drugs. Upregulation of the receptor tyrosine kinase AXL in tumor tissue has been detected in about one‐fifth of NSCLC patients with acquired resistance to EGFR‐TKIs. However, the clinical relevance of the levels of AXL and its ligand GAS6 in plasma remains unknown.MethodsTumor tissue and plasma specimens were collected from 25 EGFR‐mutated NSCLC patients before EGFR‐TKI treatment or after treatment failure. The levels of AXL and of GAS6 mRNA in tumor tissue were evaluated by immunohistochemistry and chromogenic in situ hybridization, respectively. The plasma concentrations of AXL and GAS6 were measured with enzyme‐linked immunosorbent assays.ResultsAXL expression was detected in three of 12 (25%) and nine of 19 (47%) tumor specimens obtained before and after EGFR‐TKI treatment, respectively. All tumor specimens assayed were positive for GAS6 mRNA. The median values for the plasma AXL concentration before and after EGFR TKI treatment were 1 635 and 1 460 pg/mL, respectively, and those for the plasma GAS6 concentration were 4 615 and 6 390 pg./mL, respectively. There was no significant correlation between the plasma levels of AXL or GAS6 and the corresponding expression levels in tumor tissue.ConclusionPlasma concentrations of AXL and GAS6 do not reflect tumor expression levels, and their measurement is thus not a viable alternative to direct analysis of tumor tissue in EGFR‐mutated NSCLC.

A critical role of the Gas6-Mer axis in endothelial dysfunction contributing to TA-TMA associated with GVHD

AbstractEndothelial dysfunction in the early phases of hematopoietic stem cell transplantation (HSCT) contributes to a common pathology between transplant-associated thrombotic microangiopathy (TA-TMA) and graft-versus-host disease (GVHD), which are serious complications of HSCT. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family: Tyro3, Axl, and Mer. Serum Gas6 levels were significantly increased in HSCT patients with grade II to IV acute GVHD (aGVHD), and Gas6 and Mer expression levels were upregulated in aGVHD lesions of the large intestine and skin. The increased serum Gas6 levels were also correlated with elevated lactate dehydrogenase, d-dimer, and plasmin inhibitor complex values in HSCT patients with aGVHD. In human umbilical vein endothelial cells (ECs), exogenous Gas6 or the exposure of sera isolated from patients with grade III aGVHD to ECs induced the downregulation of thrombomodulin and the upregulation of PAI-1, as well as the upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, which were inhibited by UNC2250, a selective Mer tyrosine kinase inhibitor. In mouse HSCT models, we observed hepatic GVHD with hepatocellular apoptosis, necrosis, and fibrosis, as well as TA-TMA, which is characterized pathologically by thrombosis formation in the microvasculature of the liver and kidney. Of note, intravenous administration of UNC2250 markedly suppressed GVHD and TA-TMA in these mouse HSCT models. Our findings suggest that the Gas6-Mer axis is a promising target for TA-TMA after GVHD.

1978-P: Possible Regulation between Estrogen and Gas6 Gene in Adipogenesis

Background: The physiological withdrawal of estrogen during menopause brings about changes in fat metabolism. Previous cell-based studies showed estrogen had direct effects on adipocytes. Gas6 is the ligand for the Tyro-3, Axl and Mer (TAM) receptors, and Gas6/TAM signaling stimulates cellular responses. Recent studies indicate that Gas6 gene was involved in adipocyte development, and human Gas6 levels were associated with obesity. Our clinical data showed Gas6 levels were positively correlated with estradiol (E2) levels in the postmenopausal women. However, the regulation mechanism between E2 and Gas6 gene in adipocyte functions is still unknown. In this study, we try to investigate the possible role of Gas6 gene that involved in estrogen effect on adipocyte development. Materials and Methods: Human and mouse adipocyte cell lines, SGBS and 3T3-L1, were used as in vitro cell model and ovariectomy rat was used as the postmenopausal animal model. Results: Our data showed increased estrogen receptor α (ERα) and Gas6 expression after E2 treatment in both SGBS and 3T3-L1 cell lines. Meanwhile, E2 treatment can decrease adipogenesis-related genes expression. Furthermore, we used estrogen antagonism in adipocyte that blocked the ERα and then reduced Gas6 expression and reversed the expression of adipogenesis-related genes. The luciferase assay confirmed that Gas6 gene expression was activated after E2 treatment through the genomic pathway. In addition, estrogen may also regulate Gas6 expression through non-genomic AMPK pathway. Finally, our animal model data showed significantly decreased Gas6 expression in adipose tissue from ovariectomy rats compared to the sham operation group. Interesting, the Gas6 expression in adipose tissue was increased in ovariectomy rats after E2 supplement. Conclusion: Our study first elucidates the E2 effect on adipogenesis-related genes expression and adipocyte function and may partially through genomic and non-genomic pathways to activate Gas6 gene expression. Disclosure C. Lee: None.