Axl activates autocrine transforming growth factor-ß signaling in hepatocellular carcinoma.

UNLABELLED: In hepatocellular carcinoma (HCC), intrahepatic metastasis frequently correlates with epithelial to mesenchymal transition (EMT) of malignant hepatocytes. Several mechanisms have been identified to be essentially involved in hepatocellular EMT, among them transforming growth factor (TGF)-ß signaling. Here we show the up-regulation and activation of the receptor tyrosine kinase Axl in EMT-transformed hepatoma cells. Knockdown of Axl expression resulted in abrogation of invasive and transendothelial migratory abilities of mesenchymal HCC cells in vitro and Axl overexpression-induced metastatic colonization of epithelial hepatoma cells in vivo. Importantly, Axl knockdown severely impaired resistance to TGF-ß-mediated growth inhibition. Analysis of the Axl interactome revealed binding of Axl to 14-3-3ζ, which is essentially required for Axl-mediated cell invasion, transendothelial migration, and resistance against TGF-ß. Axl/14-3-3ζ signaling caused phosphorylation of Smad3 linker region (Smad3L) at Ser213, resulting in the up-regulation of tumor-progressive TGF-ß target genes such as PAI1, MMP9, and Snail as well as augmented TGF-ß1 secretion in mesenchymal HCC cells. Accordingly, high Axl expression in HCC patient samples correlated with elevated vessel invasion of HCC cells, higher risk of tumor recurrence after liver transplantation, strong phosphorylation of Smad3L, and lower survival. In addition, elevated expression of both Axl and 14-3-3ζ showed strongly reduced survival of HCC patients. CONCLUSION: Our data suggest that Axl/14-3-3ζ signaling is central for TGF-ß-mediated HCC progression and a promising target for HCC therapy.

E. coli endotoxin modulates the expression of Sirtuin proteins in PBMC in humans.

BACKGROUND: Sirtuin (SIRT) proteins are class I histone deacetylases displaying gene regulatory functions in inflammatory, cancer, and metabolic diseases. These SIRT actions involve the nuclear factor κ B and its inhibitor I κ B pathway. However, the regulation of SIRT in vivo is still unclear. MATERIAL AND METHODS: In a human endotoxemia model, 20 healthy male subjects received an intravenous bolus of 2 ng/kg body weight Escherichia coli endotoxin (LPS). SIRT expression was investigated in peripheral blood mononuclear cells (PBMC) with qPCR and Western blot before and 3 hours, 6 hours, and 24 hours after LPS challenge. Additionally, SIRT regulation was studied in vitro in cultivated PBMC after incubation with 20 ng/mL LPS. RESULTS: A downregulation by >40% of SIRT1 mRNA was detectable 3 hours after LPS and of SIRT3 mRNA 6 hours after LPS. SIRT3, IκBα, and IκB-ß protein expressions were decreased 3 and 6 hours after LPS. SIRT2 mRNA or protein expression did not change following LPS. These findings were consistent in vitro and associated with augmented phosphorylation of IκB-ß. DISCUSSION: In this E. coli endotoxemia model, SIRT1 and SIRT3 mRNA expressions in PBMC in humans were reduced after LPS challenge. This suggests that SIRT may represent an inflammatory target protein in vivo.

VPAC1 receptor expression in peripheral blood mononuclear cells in a human endotoxemia model.

BACKGROUND: Vasoactive intestinal peptide (VIP) exerts immune-modulatory actions mainly via VPAC1 receptor stimulation. VPAC1 may be a treatment target of inflammatory diseases, but little is known about the receptor expression profile in immune-competent cells in vivo. MATERIAL AND METHODS: 20 male healthy subjects received a single intravenous bolus of 2 ng/kg body weight Escherichia coli endotoxin (LPS). Receptor status was evaluated in peripherial blood cells before and 3, 6 and 24 h after LPS by FACS analysis and q-PCR. VIP plasma concentrations were measured by ELISA. RESULTS: Granulocytes accounted for 51% of leukocytes at baseline and 58 ± 37% were positive for VPAC1. The granulocyte population increased 2.6 fold after LPS, and a transient down-regulation of VPAC1 to 28 ± 23% was noted at 3 h (p < 0.001), which returned to baseline at 24 hours. Baseline VPAC1 expression was low in lymphocytes (6.3 ± 3.2%) and monocytes (11 ± 9.6%). In these cells, LPS up-regulated VPAC1 at 6 h (13.2 ± 4.9%, p < 0.001) and 24 h (31.6 ± 20.5%, p = 0.001), respectively. Consistent changes were noted for the VIP-receptors VPAC2 and PAC1. VPAC1, VPAC2 and PAC1 mRNA levels were unchanged in peripheral blood mononuclear cells (PBMC). VIP plasma concentration increased from 0.5 ± 0.3 ng/ml to 0.7 ± 0.4 ng/ml at 6 h after LPS (p < 0.05) and returned to baseline within 24 h. CONCLUSION: The time profile of VPAC receptor expression differs in granulocytes, monocytes and lymphocytes after LPS challenge in humans. Changes in circulating VIP concentrations may reflect innate immune responses.

No association of XRCC1 polymorphisms Arg194Trp and Arg399Gln with colorectal cancer risk.

BACKGROUND: X-ray repair cross complementation group 1 (XRCC1) plays a key role in base excision repair. The purpose of this study was to examine the association of two genetic polymorphisms in XRCC1 (rs1799782 and rs25487) with risk of colorectal polyps and colorectal cancer (CRC). METHODS: In the ongoing colorectal cancer study of Austria (CORSA), a total of 3091 Caucasian participants was genotyped using 5'-nuclease TaqMan assays. Multiple logistic regression was applied to compare individuals of the control group against three different case groups namely CRC cases, high-risk and low-risk polyps. RESULTS: The two investigated SNPs in XRCC1 were not found to be associated with neither CRC risk nor polyp risk. Comparing the CRC cases versus the controls the OR was 0.60 (95%CI 0.27-1.31) for the heterozygous polymorphic genotype of SNP rs1799782 and 1.47 (95%CI 0.81-2.65) for the homozygous polymorphic genotype of SNP rs25487. Comparing the high-risk polyp group versus the controls the OR was 2.64 (95%CI 0.61-11.42) for the homozygous polymorphic genotype of SNP rs1799782 and 0.89 (95%CI 0.60-1.33) for SNP rs25487, respectively. In an haplotype analysis also no statistically significant association was found. CONCLUSION: Our finding that none of the two investigated SNPs of XRCC1 were significantly associated with risk of CRC or polyps is consistent with the results of a recently published meta-analysis.

MNS16A tandem repeats minisatellite of human telomerase gene: a risk factor for colorectal cancer.

Telomerase reactivation and expression of human telomerase gene [human telomerase reverse transcriptase (hTERT)] are hallmarks of unlimited proliferation potential of cancer cells. A polymorphic tandem repeats minisatellite of hTERT gene, termed MNS16A was reported to influence hTERT expression. To assess the role of MNS16A as potential biomarker for colorectal cancer (CRC), we investigated for the first time the association of MNS16A genotypes with risk of colorectal polyps and CRC. In the ongoing colorectal cancer study of Austria (CORSA), 3842 Caucasian participants were recruited within a large screening project in the province Burgenland including 90 CRC cases, 308 high-risk polyps, 1022 low-risk polyps and 1822 polyp free controls verified by colonoscopy. MNS16A genotypes were determined by polymerase chain reaction from genomic DNA. Associations of MNS16A genotypes with CRC risk were estimated by logistic regression analysis computing odds ratios (ORs) and 95% confidence intervals (CIs). We identified five different variable number of tandem repeats (VNTRs) of MNS16A including VNTR-364, a newly discovered rare variant. VNTR-274 allele was associated with a 2.7-fold significantly increased risk of CRC compared with the VNTR-302 wild-type (OR = 2.69; 95% CI = 1.11-6.50; P = 0.028). In our CORSA study, the medium length VNTR-274 was identified as risk factor for CRC. Although, this population-based study herewith reports the largest cohort size concerning MNS16A thus far, further large-scale studies in diverse populations are warranted to confirm hTERT MNS16A genotype as potential biomarker for assessment of CRC risk.

Association of IGF1 and IGFBP3 polymorphisms with colorectal polyps and colorectal cancer risk.

PURPOSE: Insulin-like growth factor 1 (IGF1) is a peptide growth factor that promotes cell proliferation and inhibits apoptosis. The bioavailability of IGF1 is regulated by the insulin-like growth factor binding protein 3 (IGFBP3). The purpose of this study was to examine the association of genetic variants in IGF1 (rs6214, rs6220, and rs35767) and IGFBP3 (rs2854744 and rs2854746) with risk of colorectal polyps and colorectal cancer. METHODS: In this ongoing colorectal cancer study of Austria (CORSA), a total of 3,360 Caucasian participants, consisting of 178 colorectal cancer patients, 328 patients with high risk polyps, 1,059 patients with low risk colorectal polyps, and 1,795 colonoscopy-negative controls, were recruited within a large colorectal screening project in the province Burgenland and from three hospitals in Vienna. Multiple logistic regression was applied to compare individuals of the control group against three different risk groups, namely, colorectal cancer group, high risk polyp group, and low risk polyp group. RESULTS: Carriers of the homozygous polymorphic genotype of the SNP rs6214 were associated with an increased colorectal risk (OR = 1.79, 95% CI 1.04-1.90) compared to the colonoscopy-negative controls; this was also found when combining colorectal cancer cases and high risk polyp group (OR = 1.39, 95% CI 1.01-1.90). CONCLUSION: Our results suggest that the SNP rs6214 of IGF1 could have an impact on developing colorectal cancer and colorectal polyps with villous elements.