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Potential conflict of interest: Nothing to report.
This work was supported, in part, by grants from the National Key Sci-Tech Special Project of China (grant 2008ZX10002-022), the National Key Program for Basic Research of China (973) (2009CB521803), the Program Of Shanghai Subject Chief Scientist (A) (09XD1403600), Shanghai Science and Technology Developing Program (07DJ14006), and Shanghai Natural Science Foundation (08ZR1418300).
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 1α (HIF-1α) 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 β1 axis. Conclusion: ANGPTL4 is a target gene of HIF-1α 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;)
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Hepatocellular carcinoma (HCC) is one of the most common malignancies and a leading cause of cancer-related mortality in the world.1 Tumor recurrence and metastasis are the main causes of death in patients with HCC. Accumulating evidence suggests that tumor metastasis is a complex process influenced by multiple procedures and multiple mediators in human cancers2, 3; however, the biology controlling HCC metastasis remains poorly understood.
Angiopoietin-like protein 4 (ANGPTL4) is a member of the angiopoietin family and encodes a secretory glycoprotein that is highly expressed in adipose tissue, liver, and placental tissue, as well as in ischemic tissues. ANGPTL4 is also known as pp1158, peroxisome proliferator-activated receptor-gamma–induced angiopoietin-related protein, fasting-induced adipose factor, or hepatic fibrinogen/angiopoietin-related protein. ANGPTL4 plays an important role in lipid metabolism,4-7 but little is known about its role in tumor metastasis. Previous studies have suggested that the functions of ANGPTL4 in regulating angiogenesis and metastasis are diverse and often contradictory in different tissue contexts. Similar to angiopoietins and other members of the angiopoietin-like family that have been shown to regulate angiogenesis,8-10 ANGPTL4 is a proangiogenic factor in arthritis, conventional renal cell carcinoma and breast cancer, and it induces endothelial cell sprouting11-14; however, data from several independent laboratories have demonstrated that ANGPTL4 is also a potent antiangiogenic factor.15-17 ANGPTL4 remarkably prevents the metastatic process through the inhibition of vascular permeability, tumor cell motility, and invasiveness in murine Lewis lung carcinoma (3LL) and melanoma (B16F0) cells.18 Of note, a more recent report showed that ANGPTL4 promotes tumor metastasis by disrupting the integrity of vascular endothelial cell layers and facilitating the passage of breast cancer cells.19 Another study of Kaposi's sarcoma further supports the hypothesis that ANGPTL4 can promote angiogenesis and vascular permeability.20 ANGPTL4 expression, in some tissues, correlates with metastasis-related clinicopathological characteristics in human gastric cancer, esophageal squamous cell carcinoma, and colorectal cancer,21-23 indicating that ANGPTL4 might play an important role in the metastasis of human cancer.
Hypoxia is commonly associated with the environment of solid tumors and promotes invasion, metastasis, and malignancy.24 High expression of hypoxia-inducible factor 1 alpha (HIF-1α) in HCC specimens (51.39%) is found and significantly correlates with venous invasion and lymph-node invasion.25, 26 Previous studies have shown that hypoxia up-regulates ANGPTL4 in human articular chondrocytes, endothelial cells, cardiomyocytes, and certain cancer cells.13, 20, 27-30 Moreover, ANGPTL4 mRNA is expressed in the perinecrotic areas of some human tumors.13 However, it is not fully understood whether hypoxia up-regulates ANGPTL4 in HCC, and the mechanism that hypoxia regulates ANGPTL4 expression remains unclear.
In our previous study, we found that ANGPTL4 could stimulate angiogenesis in vitro and in vivo.31 Here, we evaluated, for the first time, the relationship between the expression of ANGPTL4 and HIF-1α in HCC, and demonstrated that ANGPTL4 levels positively correlated with HIF-1α expression. Moreover, ANGPTL4 is a target gene of HIF-1α. Our comprehensive investigation of the role of ANGPTL4 in HCC metastasis indicates that ANGPTL4, particularly the secreted form, is a positive regulator of HCC metastasis.
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-1α, hypoxia-inducible factor 1α; HREs, hypoxia-responsive elements; HUVECs, human umbilical vein endothelial cells; IgG, immunoglobulin G; IL-1β, interleukin-1 beta; IκB-β, inhibitor of nuclear factor kappa B beta; kb, kilobase; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa light-chain enhancer of activated B cells; shRNA, short-hairpin RNA; VCAM-1, vascular cell adhesion molecule-1.
Patients and Methods
Materials and methods are outlined in Supporting Materials and Methods.
ANGPTL4 Is Induced by Hypoxia in HCC Cells.
To explore the effect of hypoxia on ANGPTL4 expression, HCC cell lines were cultured under hypoxic conditions (2% O2) for 24 hours. We found that ANGPTL4 expression was significantly increased at the mRNA and protein levels in HCC cell lines under hypoxic conditions (Fig. 1A,B). Deferoxamine mesylate (DFO), a known HIF-1α activator, induced HIF-1α expression in a concentration-dependent manner and up-regulated ANGPTL4 in MHCC-97L and SMMC-7721 cells (Fig. 1C).
To further investigate the effect of hypoxia on the induction of ANGPTL4 expression in vivo, MHCC-97L and SMMC-7721 cells were xenografted subcutaneously into BALB/c (nu/nu) mice. The xenograft tumor masses were stripped gently once a week after tumor implantation and then subjected to immunofluorescent staining. We found that ANGPTL4 was significantly up-regulated with increasing tumor size and increasing degree of hypoxia in the tumor masses, and ANGPTL4 colocalized with HIF-1α in the hypoxic areas surrounding the necrotic fields (Fig. 1D). To better understand the correlation between HIF-1α and ANGPTL4 expression in HCC tissues, immunohistochemical staining was performed. High expressions of HIF-1α and ANGPTL4 were found in tumor specimens, and ANGPTL4 protein levels in HCC tissues positively correlated with HIF-1α expression (r = 0.631, P < 0.001) (Fig. 1E,F; Table 1), suggesting that ANGPTL4 might be up-regulated by the expression of HIF-1α in HCC.
Table 1. Correlation Between HIF-1α and ANGPTL4 Expression in HCC Tissues
HIF-1α Mediates Hypoxia-Induced ANGPTL4 Overexpression in HCC Cell Lines.
To further clarify the role of HIF-1α in regulating ANGPTL4 expression, we knocked down HIF-1α and HIF-2α (another alpha subunit of the HIF protein) using siRNA in MHCC-97L and SMMC-7721 cells. Under hypoxic conditions, silencing of HIF-1α significantly down-regulated ANGPTL4 (Fig. 2A; Supporting Fig. 1A), whereas silencing of HIF-2α did not affect ANGPTL4 expression (Fig. 2B; Supporting Fig. 1B). 2-methoxyestradiol (2ME2), a small molecule, has promising antitumor and antiangiogenic activity and can down-regulate HIF-1α in a number of tumors.32 We found that hypoxia-induced HIF-1α and ANGPTL4 protein levels decreased after treatment with 2ME2 under hypoxic conditions in MHCC-97L and SMMC-7721 cells (Fig. 2C; Supporting Fig. 1C).
To investigate whether the ANGPTL4 gene is a direct target of HIF-1α, chromatin immunoprecipitation (ChIP) assays were performed in MHCC-97L and SMMC-7721 cells. Bioinformatics analysis showed five potential hypoxia-responsive elements (HREs) within a 2.2-kb (kilobase) region upstream of the transcriptional start site of ANGPTL4 (Fig. 2D). ChIP assay analysis showed that the anti-HIF-1α antibody could precipitate HRE1 (−206 to −202), but not other HREs (Fig. 2E; Supporting Fig. 1D). This result indicates that hypoxia can, indeed, significantly up-regulate ANGPTL4 by promoting HIF-1α expression.
ANGPTL4 Can Promote Transendothelial Migration of HCC Cell Lines In Vitro and Increase the Metastatic Potential of HCC Cells In Vivo.
Hypoxia plays an important role in promoting metastasis and tumor progression,24, 33 and ANGPTL4 facilitates the transendothelial passage of breast cancer cells by disrupting the integrity of vascular endothelial cell layers.19 In addition, we found that ANGPTL4 expression was significantly increased in metastatic HCC cell lines MHCC-97, MHCC-97L, MHCC-97H, and MHCC-LM3 (Supporting Fig. 2). To better understand the function of ANGPTL4 in HCC metastasis, we first established two stable ANGPTL4 overexpressing cell lines, SMMC-7721-lenti-ANGPTL4 and Huh7-lenti-ANGPTL4, using a lentivirus vector (Supporting Fig. 3A-C). Transendothelial migration assays were preformed, and the results showed that SMMC-7721-lenti-ANGPTL4 and Huh7-lenti-ANGPTL4 cells traversed more efficiently through the endothelial cell monolayer into the lower chamber of the transwell, when compared with the empty vector-infected cells (P < 0.001) (Fig. 3A,B).
To further clarify the role of ANGPTL4 in HCC metastasis in vivo, SMMC-7721-lenti-ANGPTL4 and SMMC-7721-lenti-control cells were orthotopically inoculated in the left hepatic lobe of mice with a microsyringe. Histological examination of lung and liver tissues indicated that ANGPTL4-overexpressing tumor-bearing mice had significantly higher numbers of pulmonary and intrahepatic metastatic nodules than those bearing vector control tumors (P < 0.05) (Fig. 3C).
We also verified the effects of endogenous ANGPTL4 on the transendothelial migration of HCC cells using short-hairpin (sh)RNA knockdown. As shown in Figure 3D, knockdown of ANGPTL4 markedly inhibited the migration of MHCC-97L cells (P = 0.001). Interestingly, treatment with a neutralizing antibody specific for ANGPTL4 full-length protein could partially block the transendothelial migration of MHCC-97L cells (P < 0.001) (Fig. 3E). To determine the potential role of HIF-1α-induced ANGPTL4 expression in HCC metastasis, a transendothelial migration assay was performed under hypoxic conditions. Results showed that treatment with the ANGPTL4-neutralizing antibody could partially inhibit the transendothelial migration of SMMC-7721 cells induced by hypoxia (P < 0.001) (Fig. 3F). These results indicate that ANGPTL4 is a positive metastatic regulator of HCC.
Secreted ANGPTL4 Promotes HCC Metastasis.
Bioinformatics analysis indicates that ANGPTL4 has a secretory signal peptide sequence and encodes a secretory glycoprotein. We detected and compared serum ANGPTL4 protein levels in 90 healthy individuals, 118 chronic hepatitis B patients, and 463 HCC patients. Serum ANGPTL4 levels were significantly higher in chronic hepatitis B patients (86.0 ± 81.8 ng/mL) than in normal controls (55.6 ± 30.9 ng/mL) (P = 0.002), but lower than in HCC patients (115.0 ± 117.0 ng/mL) (P = 0.010) (Fig. 4A). Further analysis showed that ANGPTL4 levels were significantly higher in HCC patients with intrahepatic metastasis (145.8 ± 112.0 ng/mL) than in those without intrahepatic metastasis (106.5 ± 66.9 ng/mL) (P < 0.001) (Fig. 4B). In addition, ANGPTL4 levels were higher in HCC patients with macrovascular invasion (188.7 ± 203.5 ng/mL) than in those without macrovascular invasion (116.3 ± 88.3 ng/mL) (Supporting Fig. 4A), whereas ANGPTL4 levels were not elevated in HCC patients with hepatitis B virus (HBV) infection, HCC patients with cirrhosis, or HCC patients with extrahepatic metastasis (Supporting Fig. 4B-D). To determine the relationship between serum ANGPTL4 levels and clinicopathologic parameters, a cut-off value of 93.5 ng/mL was determined with a sensitivity of 44.7% and specificity of 87.4% by receiver operating characteristic analysis (Supporting Fig. 4E). With respect to clinicopathologic features, we found that high levels of serum ANGPTL4 positively correlated with intrahepatic metastasis, histological grade, and liver cirrhosis present in HCC patients (Table 2), indicating that secreted ANGPTL4 plays an important role in HCC progression.
Table 2. Correlation Between Secreted ANGPTL4 Levels in HCC Patients and Their Clinicopathologic Characteristics
ANGPTL4 Negative (<93.5 ng/mL) n (%)
ANGPTL4 Positive (≥93.5 ng/mL) n (%)
P value represents the probability from a chi-square test for serum ANGPTL4 levels between variable subgroups.
P < 0.05.
Abbreviations: ANGPTL4, angiopoietin-like protein 4; HCC, hepatocellular carcinoma; AFP, alpha-fetoprotein; HBV, hepatitis B virus.
To evaluate the function of secreted ANGPTL4 in the metastasis of HCC cells, conditioned medium (CM) was collected from COS7-lenti-ANGPTL4 or COS7-lenti-control cells, which were established by transduction with a lentiviral vector expressing ANGPTL4 or an empty lentiviral vector (Supporting Fig. 5B). CM assays were performed, and histological analysis of lung and liver tissues showed that ANGPTL4-containing CM could significantly promote lung metastasis of MHCC-97L cells, when compared with CM from vector control cells (P < 0.05) (Fig. 4C,D).
Secreted ANGPTL4 Can Up-Regulate Vascular Cell Adhesion Molecule-1 in Human Umbilical Vein Endothelial Cells.
It has been reported that lipopolysaccharide (LPS) administration and a variety of cytokines, including tumor necrosis factor γ, interleukin-1 beta (IL-1β), and interferon up-regulate ANGPTL4,34 and some of these factors also stimulate the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells.35, 36 In this context, the question arises as to whether secreted ANGPTL4 regulates VCAM-1 expression in human umbilical vein endothelial cells (HUVECs). VCAM-1, which is expressed on the surface of endothelial cells, mediates cellular adhesion via integrin α4β1.37, 38 The integrin α4β1/VCAM-1 pathway was reported to mediate the transendothelial migration of lymphoma cells.39 By western blotting, we found that integrin α4 and integrin β1 could be detected in HCC cell lines (Supporting Fig. 5C). After HUVECs were exposed to CM for 24 hours, VCAM-1 expression increased at protein level in the ANGPTL4-containing CM treatment group (Fig. 5A). When intrinsic VCAM-1 expression was knocked down in HUVECs (Fig. 5B), the transendothelial migration of SMMC-7721-lenti-ANGPTL4 cells was also significantly reduced (P < 0.05) (Fig. 5C). Consistent with these results, silencing of integrin β1 expression in SMMC-7721-lenti-ANGPTL4 cells resulted in a remarkable decrease in transendothelial migration (P < 0.05) (Fig. 5D,E). In addition, the transendothelial migration of SMMC-7721 cells was increased after treatment with ANGPTL4-containing CM (P < 0.05), but simultaneous treatment with integrin β1 antibody or ANGPTL4 antibody could reduce the transendothelial migration of SMMC-7721 cells induced by ANGPTL4-containing CM (P < 0.05) (Supporting Fig. 6A,B).
VCAM-1 expression in endothelial cells is usually regulated by the transcription factor, nuclear factor kappa light-chain enhancer of activated B cells (NF-κB), in a cytokine-responsive manner.35, 36 We analyzed the expression of molecules in the NF-κB-signaling pathway, and found that phosphorylation levels of p65 increased and inhibitor of nuclear factor kappa B beta (IκB-β) expression decreased in HUVECs after ANGPTL4-containing CM treatment (Supporting Fig. 6C).
Therefore, we concluded that ANGPTL4 could promote HCC cell transendothelial migration by up-regulating VCAM-1 in HUVECs.
Metastasis and recurrence are the most important prognostic factors for HCC patients.40, 41 Therefore, it is extremely important to explore the molecular events governing the pathogenesis of cancer metastasis in HCC. Here, we show that overexpression of ectopic ANGPTL4 could significantly enhance the transendothelial migration of HCC cells in vitro and promote both intrahepatic metastasis and lung metastasis in vivo. Knockdown of ANGPTL4 or treatment with ANGPTL4-neutralizing antibody decreased the metastatic potential of HCC cells in vitro. These results strongly suggest that ANGPTL4 acts as an enhancer of HCC cell metastatic potential. The role of ANGPTL4 in HCC cells fits with the previously described roles of ANGPTL4 as an inhibitor of endothelial integrity that mediates lung metastasis seeding19 or an enhancer of cell migration during wound healing, which shares numerous characteristics during cell migration in metastasis.42, 43 In some cancers, however, several reports have indicated discordant roles for ANGPTL4 in tumor dissemination; ANGPTL4 has been shown to act as a potent antiangiogenic factor15-17 and even prevents metastasis through reduced vascular permeability.18 These diverse results suggest that ANGPTL4 may have a contextual, tissue-specific and/or tissue-associated activity. It is also possible that the function of ANGPTL4 depends on its cleavage and the context of its interaction with extracellular matrix proteins that regulate cell-matrix communication.44 More details are still needed to elucidate the mechanism of how ANGPTL4 affects metastasis in different tumors.
We demonstrated that ANGPTL4 expression significantly correlated with HIF-1α expression in HCC tissues. ANGPTL4 also colocalized with HIF-1α in hypoxic areas surrounding the necrotic fields in xenograft tumors. These data are consistent with the increased ANGPTL4 mRNA expression found in several other types of cancer, including renal cell carcinoma and glioblastomas, both of which are associated with dysregulation of HIF-1.13, 30 Our data showed that HIF-1α directly mediates hypoxia-induced ANGPTL4 expression through binding to HRE1 at −206 to −202 base pairs upstream of the transcriptional start site of ANGPTL4. HIF-1α induces the expression of many genes that lead to tumor proliferation, angiogenesis, metastasis, and progression.45 Our results showed that serum ANGPTL4 protein levels were elevated in chronic hepatitis B patients, and it was reported that ANGPTL4 could be induced by inflammation,11, 34 so we compared the roles of hypoxia and inflammation in the induction of ANGPTL4 in MHCC-97L cells. Results showed that ANGPTL4 protein levels were increased by inflammatory factors (such as IL-1β and IL-6) or hypoxia alone, whereas the role of hypoxia was more remarkable. Combined treatments with hypoxia and inflammatory factors additively increased ANGPTL4 levels (Supporting Fig. 7A,B). Although ANGPTL4 expression has been reported to correlate with venous invasion and lymphovascular invasion,21-23 our results did not identify a correlation between ANGPTL4 expression and metastatic features in primary HCC tissues (Supporting Table 1). HCC metastases are mainly intrahepatic in the clinic, and the liver tissue microenvironment plays an important role in intrahepatic metastasis.46 Furthermore, ANGPTL4 is a secreted glycoprotein. In this context, the role of secreted ANGPTL4 was investigated.
Our results showed that ANGPTL4 increased in the sera of HCC patients and correlated with the presence of macrovascular invasion, intrahepatic metastasis, and histological grade. CM containing ANGPTL4 enhanced lung metastasis of HCC cells in nude mice. In addition, we found that secreted ANGPTL4 up-regulated VCAM-1 in HUVECs and affected the transendothelial migration of HCC cells through the VCAM-1/integrin β1 axis. All of these results suggest that secreted ANGPTL4 might affect the tumor cell/endothelial cell interaction through the regulation of gene expression in endothelial cells and result in an increase in the transendothelial migration and even metastasis of HCC cells. ANGPTL4 also induces vessel permeability through a mechanism dependent on Rho and ROCK in Kaposi's sarcoma,20 which is consistent with the role of ANGPTL4 in breast cancer.19 Furthermore, ANGPTL4 expression is regulated by transforming growth factor beta19 and hypoxia, which are the main factors in the solid tumor microenvironment.45, 47 All of these studies indicate that ANGPTL4 might be a regulator of the tumor microenvironment. Although we showed ANGPTL4 is secreted and expressed consistently in HCC cells in both in vitro and in vivo experiments (Supporting Figs. 2B, 3A,C, and 5A,B), we found that serum ANGPTL4 levels are different from ANGPTL4 expression in tumor tissues in HCC patients. It is completely possible that serum ANGPTL4 is not exclusively secreted by tumor cells, because ANGPTL4 is also highly expressed in adipose tissue and ischemic tissues. This situation also occurs with serum alpha-fetoprotein (AFP) and tissue AFP in HCC.48
In conclusion, our results show that hypoxia-induced ANGPTL4 can significantly promote HCC cell invasion and metastasis in vitro and in vivo through the up-regulation of VCAM-1 in HUVECs; in addition, the ANGPTL4 gene is directly regulated by HIF-1α. These data suggest that the HIF-1α target gene, ANGPTL4, might be a potential target for cancer gene therapy, because HIF-1α has long been considered as an exceptional target for cancer therapy.49