Hypoxia-inducible factor 1 alpha-activated angiopoietin-like protein 4 contributes to tumor metastasis via vascular cell adhesion molecule-1/integrin β1 signaling in human hepatocellular carcinoma

Abstract

Angiopoietin-like protein 4 (ANGPTL4) plays complex and often contradictory roles in vascular biology and tumor metastasis, but little is known about its function in hepatocellular carcinoma (HCC) metastasis. In the present study, we showed that hypoxia-inducible factor 1a (HIF-1a) directly up-regulates ANGPTL4, and its stableness positively correlates with ANGPTL4 expression in HCC tissue. Overexpression of ANGPTL4 significantly increased HCC cell transendothelial migration in vitro and intrahepatic and distal pulmonary metastasis in vivo, whereas silencing ANGPTL4 expression or treatment with a neutralizing antibody specific for ANGPTL4 protein resulted in a reduced transendothelial migration. We also found that serum ANGPTL4 is higher in HCC patients, compared to healthy control, and correlates with intrahepatic metastasis and histological grade. Further, secreted ANGPTL4 promotes transendothelial migration and metastasis of HCC cells in vitro and in vivo through the up-regulation of vascular cell adhesion molecule-1 (VCAM-1) of human umbilical vein endothelial cells and the activation of the VCAM-1/ integrin b1 axis. Conclusion: ANGPTL4 is a target gene of HIF-1a and acts as an important regulator in the metastasis of HCC. Serum ANGPTL4 correlates with tumor progression and metastasis and might be used to indicate prognosis in HCC patients. (HEPATOLOGY 2011;54:910-919) Abbreviations: 2ME2, 2-methoxyestradiol; AFP, alpha-fetoprotein; ANGPTL4, angiopoietin-like protein 4; ChIP, chromatin immunoprecipitation; CM, conditioned medium; DFO, deferoxamine mesylate; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HIF-1a, hypoxia-inducible factor 1a; HREs, hypoxia-responsive elements; HUVECs, human umbilical vein endothelial cells; IgG, immunoglobulin G; IL-1b, interleukin-1 beta; IjB-b, inhibitor of nuclear factor kappa B beta; kb, kilobase; LPS, lipopolysaccharide; NF-jB, nuclear factor kappa light-chain enhancer of activated B cells; shRNA, short-hairpin RNA; VCAM-1, vascular cell adhesion molecule-1.From the

Emerging Roles of Angiopoietin-like 4 in Human Cancer

“…Indeed, Zhang and colleagues identified a functional HIF-1a-binding site located 1.6 kb upstream of the human ANGPTL4 gene (28). This finding was further confirmed by another independent group using the hepatocellular carcinoma cell lines MHCC-97L and SMMC-7721 (27). The tumor microenvironment is constantly under chronic inflammatory pressure (29).…”

“…Furthermore, clinical studies have correlated ANGPTL4 expression with venous and lymphatic invasion in human gastric and CRCs, which further emphasizes the role of ANGPTL4 in tumor metastasis (47)(48)(49). ANGPTL4 was found to promote transendothelial migration and metastasis of hepatocellular carcinoma cells through the upregulation of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells and the activation of the VCAM-1/integrin b1-signaling axis (27). In this mechanism, the increased VCAM-1 expression on endothelial cells facilitates the attachment of the metastatic cancer cells in circulation and the subsequent trans-endothelial extravasation to establish a metastatic tumor.…”

Tunicamycin Potentiates Cisplatin Anticancer Efficacy through the DPAGT1/Akt/ABCG2 Pathway in Mouse Xenograft Models of Human Hepatocellular Carcinoma

“…Real-time PCR was subsequently carried out using SYBR Premix Ex Taq (TaKaRa Biotechnology) as described previously (26). The primers used to amplify the target genes are listed in Supplementary Table S2, and the expression levels were normalized against those of the internal reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).…”

“…The above cDNA sequences were cloned into the lentiviral expression vector pWPXL (Addgene) by replacement of the GFP fragment. Virus packaging and cell transfection were conducted as previously described (26).…”

“…The effective siRNA sequences are shown in Supplementary Table S2. RNA interference was conducted as described previously (26).…”