Abstract

BackgroundAngiogenesis 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.MethodsExpression 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.ResultsURG4/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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1378-7) contains supplementary material, which is available to authorized users.

Highlights

  • Consistent with the Western blotting analysis, Quantitative realtime RT-PCR (qRT-PCR) demonstrated that Up-regulated gene-4 (URG4)/ upregulator of cell proliferation (URGCP) mRNA was markedly upregulated in all seven Hepatocellular carcinoma (HCC) cell lines compared to the normal liver epithelial cell lines (Figure 1B)

  • These data suggest that URG4/URGCP is upregulated in HCC cells

  • Inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling activity inhibits the ability of URG4/URGCP to enhance the angiogenic capacity of HCC cells We further explored whether URG4/URGCP increased the angiogenic capacity of HCC cells by activating NFκB signaling

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Summary

Introduction

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. Blocking tumor angiogenesis could prevent the formation of tumor blood vessels and inhibit or slow the growth and spread of tumor cells [6,7,8]. 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 [14,15,16]. VEGF regulates the sprouting and proliferation of endothelial cells and can stimulate tumor angiogenesis [17]. 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

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