Abstract
Background
Angiogenesis is essential for tumor growth. Hepatocellular carcinoma (HCC) is characterized by hypervascularity; high levels of angiogenesis are associated with poor prognosis and a highly invasive phenotype in HCC. Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is overexpressed in multiple tumor types and has been suggested to act as an oncogene. This study aimed to elucidate the effect of URG4/URGCP on the angiogenic capacity of HCC cells in vitro.
Methods
Expression of URG4/URGCP in HCC cell lines and normal liver epithelial cell lines was examined by Western blotting and quantitative real-time PCR. URG4/URGCP was stably overexpressed or transiently knocked down using a shRNA in two HCC cell lines. The human umbilical vein endothelial cell (HUVEC) tubule formation and Transwell migration assays and chicken chorioallantoic membrane (CAM) assay were used to examine the angiogenic capacity of conditioned media from URG4/URGCP-overexpressing and knockdown cells. A luciferase reporter assay was used to examine the transcriptional activity of nuclear factor kappa – light – chain - enhancer of activated B cells (NF-κB). NF-κB was inhibited by overexpressing degradation-resistant mutant inhibitor of κB (IκB)-α. Expression of vascular endothelial growth factor C (VEGFC), tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-8 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were examined by quantitative real-time PCR; VEGFC protein expression was analyzed using an ELISA.
Results
URG4/URGCP protein and mRNA expression were significantly upregulated in HCC cell lines. Overexpressing URG4/URGCP enhanced - while silencing URG4/URGCP decreased - the capacity of HCC cell conditioned media to induce HUVEC tubule formation and migration and neovascularization in the CAM assay. Furthermore, overexpressing URG4/URGCP increased - whereas knockdown of URG4/URGCP decreased - VEGFC expression, NF-κB transcriptional activity, the levels of phosphorylated (but not total) IκB kinase (IKK) and IκB-α, and expression of TNFα, IL-6, IL-8 and MYC in HCC cells. Additionally, inhibition of NF-κB activity in HCC cells abrogated URG4/URGCP-induced NF-κB activation and angiogenic capacity.
Conclusions
This study suggests that URG4/URGCP plays an important pro-angiogenic role in HCC via a mechanism linked to activation of the NF-κB pathway; URG4/URGCP may represent a potential target for anti-angiogenic therapy in HCC.
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Background
Angiogenesis, the formation of new blood vessels, occurs during numerous physiological and pathological processes [1]. Angiogenesis is required to maintain tumor growth and metastasis, and constitutes an important hallmark of tumor progression [2-5]. Tumor angiogenesis is the generation of a network of blood vessels that penetrates into the tumor to supply the nutrients and oxygen required to maintain and enable tumor growth and invasion. Consequently, blocking tumor angiogenesis could prevent the formation of tumor blood vessels and inhibit or slow the growth and spread of tumor cells [6-8]. Angiogenesis is widely regarded to be an effective therapeutic target and promising biomarker for the diagnosis of cancer; therefore, angiogenesis is an important field of research in biological and clinical oncology [9-13]. Tumor angiogenesis is a consequence of an imbalance between pro-angiogenic factors, such as the vascular endothelial growth factor (VEGF) family and IL-8/CXCL8, and inhibitors of angiogenesis, including endostatin, angiostatin and other related molecules [14-16]. VEGF regulates the sprouting and proliferation of endothelial cells and can stimulate tumor angiogenesis [17]. A number of currently-used anti-angiogenesis drugs function by inhibiting pro-angiogenic factors, for example the monoclonal antibody bevacizumab binds to VEGF and prevents it from binding to the VEGF receptors, and sunitinib and sorafenib are small molecules that attach to VEGF-R and inhibit the binding of VEGF [18,19]. However, the precise regulation and mechanisms of tumor angiogenesis are not yet fully explored and the identification of other novel specific, effective inhibitors of angiogenesis is urgently required to treat patients with cancer.
Hepatocellular carcinoma (HCC) accounts for 90% of all primary malignant liver cancers and is the fifth most common cancer and third most common cause of cancer-related mortality worldwide [20,21]. HCC has a much higher morbidity in Asia due to the high incidence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, especially in China where 55% of all cases of HCC worldwide occur [21]. HCC is characterized by hypervascularity indicative of angiogenesis, and tumor growth in HCC relies on the formation of new blood vessels [15]. VEGF has been reported to a play critical role in angiogenesis in HCC [22]. Targeting angiogenesis using pharmacologic strategies has recently been validated in several other solid tumor types [23]. Therefore, identification of an anti-angiogenic strategy for HCC may help to improve the treatment outcomes and extend survival for patients with HCC.
Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is located on chromosome 7p13 and was identified and initially characterized by Tufan et al. URG4/URGCP is upregulated in the presence of hepatitis B virus X antigen (HBxAg) and contributes to the development of HCC as it can promote hepatocellular growth and survival both in vitro and in vivo [24]. Previous studies demonstrated that URG4/URGCP is upregulated in human HCC and gastric cancer and URG4/URGCP could promote the proliferation and tumorigenicity of HCC and gastric cancer cells [41,42]. Activation of NF-κB signaling is negatively regulated by the IκBs, which bind and sequester NF-κB in the cytoplasm in an inactive state. IκBs are phosphorylated by IKKs, which leads to ubiquitin-mediated degradation of the IκBs and consequently enables the release and translocation of NF-κB to the nucleus [43-45]. Consistent with these well-studied processes, the present study demonstrated that overexpression of URG4/URGCP upregulated the level of p-IKK and p-IκBα and ultimately enhanced the activation of NF-κB. Additionally, when the cells overexpressing URG4/URGCP were transfected with the IκBα mutant, the capacity of CM from URG4/URGCP-overexpressing cells to enhance the angiogenic capacity of HCC cells was attenuated. These findings indicate that URG4/URGCP promotes the angiogenic capacity of HCC cells - at least in part - by activating the NF-κB/VEGFC signaling pathway.
Additionally, overexpression of URG4/URGCP upregulated a number of genes downstream of the NF-κB signaling pathway: TNF, IL-6, IL-8 and MYC. TNF-α is well-recognized to promote angiogenesis and drive remodeling of blood vessels in vivo [46-48]; interleukin-6 increases the expression of VEGF and can promote angiogenesis [49-51]; IL-8 has been shown to play an important role in tumor angiogenesis [52]; and Myc plays an essential role in vasculogenesis and angiogenesis during the development and progression of various types of cancer [53-55]. It would be interesting to explore whether TNF, IL-6, IL-8 or MYC play a role in angiogenesis and disease progression in HCC, and explore the correlation between the expression of these genes and VEGFC. The regulatory mechanism by which upregulation of URG4/URGCP modulates the NF-κB/VEGFC pathway and enhances the angiogenic capacity of HCC cells remains to be elucidated and should be investigated further.
Conclusion
In conclusion, this study demonstrates that URG4/URGCP is upregulated in HCC cell lines and enhances the angiogenic capacity of HCC cells via activation of the NF-κB signaling pathway. These results may provide new insight into the mechanisms that regulate angiogenesis in HCC; targeting URG4/URGCP may represent a promising therapeutic strategy for HCC.
Abbreviations
- HCC:
-
hepatocellular carcinoma
- URG4:
-
up-regulated gene-4
- URGCP:
-
upregulator of cell proliferation
- NF-κB:
-
nuclear factor kappa-light-chain-enhancer of activated B cells
- VEGF:
-
vascular endothelial growth factor
- PDGF:
-
platelet-derived growth factor
- FGFs:
-
fibroblast growth factors
- IκB:
-
inhibitor of kappa B
- IKK:
-
IκB kinase
- HUVEC:
-
human umbilical vein endothelial cells
- CAM:
-
chicken chorioallantoic membrane
- IL:
-
interleukin
- TNF-α:
-
tumor necrosis factor alpha
- HBV:
-
hepatitis B virus
- HCV:
-
hepatitis C virus
- HBxAg:
-
hepatitis B virus X antigen
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- ATCC:
-
American Type Culture Collection
- FBS:
-
fetal bovine serum
- qRT-PCR:
-
quantitive real-time RT-PCR
- SD:
-
standard deviation
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (grant numbers 30600156, 81071247), the Science and Technology Projects Foundation of Guangdong Province (grant numbers 2011B031800022, 2012B031800501) and Natural Science Foundation of Guangdong Province (grant numbers 2014A030313090, 2014A030313190).
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Authors’ contributions
JYY, SDL and HPL participated in the design of study. SZX, BZ, RXH, ZRZ, BHX, CHH and JSX performed experimental work. SZX, BZ, RXH, WCST and SQX performed the statistical analysis and helped to draft the manuscript. JYY, SDL and HPL provided administrative support and funded experiments. All authors read and approved the final manuscript.
Sizhong **ng, Bing Zhang and Ruixi Hua contributed equally to this work.
Additional files
Additional file 1: Figure S1.
Effect of URG4/URGCP on the angiogenic capacity of normal hepatic cell lines. A. Western blotting analysis of URG4/URGCP protein expression in Lo2 and THLE3 cells transduced with either pMSCV-URG4/URGCP or the control vector pMSCV; α-Tubulin was used as a loading control. B. Representative images (left) and quantification (right) of tube-like structures formed by HUVECs cultured on Matrigel-coated plates in the presence of CM from the indicated cells. C. Representative images (left) and quantification (right) of the number of migrated HUVEC cells in the Transwell migration assay after incubation in CM derived from the indicated cells. D. Representative images (left) and quantification (right) of neovessels formed in the CAM assay when stimulated by CM derived from the indicated cells. E. Quantitative real-time PCR analysis of VEGFC mRNA expression in the indicated cells; transcript levels were normalized to GAPDH and expressed relative to the respective vector control cells. F. ELISA of VEGFC protein expression in the indicated cell supernatants. Data is mean ± SD of three independent experiments; * P < 0.05.
Additional file 2: Figure S2.
Western blotting analysis of phosphorylated IκBα expression in the indicated cells; α-Tubulin was used as a loading control.
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**ng, S., Zhang, B., Hua, R. et al. URG4/URGCP enhances the angiogenic capacity of human hepatocellular carcinoma cells in vitro via activation of the NF-κB signaling pathway. BMC Cancer 15, 368 (2015). https://doi.org/10.1186/s12885-015-1378-7
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DOI: https://doi.org/10.1186/s12885-015-1378-7