Hepatocellular carcinoma (HCC) is a hypervascular tumor and accumulating evidence has indicated that stimulation of angiogenesis by HBV may contribute to HCC malignancy. The small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV viral protein and has a close clinical association with HCC, however, whether SHBs contributes to HCC angiogenesis remains unknown. This study reports that forced expression of SHBs in HCC cells promoted xenograft tumor growth and increased the microvessel density (MVD) within the tumors. Consistently, HBsAg was also positively correlated with MVD count in HCC patients’ specimens. The conditioned media from the SHBs-transfected HCC cells increased the capillary tube formation and migration of human umbilical vein endothelial cells (HUVECs). Intriguingly, overexpression of SHBs increased VEGFA expression at both mRNA and protein levels. A higher VEGFA expression level was also observed in the xenograft tumors transplanted with SHBs-expressing HCC cells and in HBsAg-positive HCC tumor tissues as compared to their negative controls. As expected, in the culture supernatants, the secretion of VEGFA was also significantly enhanced from HCC cells expressing SHBs, which promoted HUVECs migration and vessel formation. Furthermore, all the three unfolded protein response (UPR) sensors IRE1α, PERK and ATF6 associated with endoplasmic reticulum (ER) stress were found activated in the SHBs-expressing cells and correlated with VEGFA protein expression and secretion. Taken together, these results suggest an important role of SHBs in HCC angiogenesis and may highlight a potential target for preventive and therapeutic intervention of HBV-related HCC and its malignant progression.
IMPORTANCE
Chronic hepatitis B virus infection is one of the important risk factors for the development and progression of hepatocellular carcinoma (HCC). HCC is characteristic of hypervascularization even at early phases of the disease due to overexpression of angiogenic factors like vascular endothelial growth factor-A (VEGFA). However, a detailed mechanism in the HBV-induced angiogenesis remains to be established. In this study, we demonstrate for the first time that the most abundant HBV viral protein, i.e. small surface antigens (SHBs) can enhance the angiogenic capacity of HCC cells by upregulation of VEGFA expression both
in vitro
and
in vivo
. Mechanistically, SHBs induced endoplasmic reticulum (ER) stress which consequently activated unfolded protein response (UPR) signaling to increase VEGFA expression and secretion. This study suggests that SHBs plays an important pro-angiogenic role in HBV-associated HCC and may represent a potential target for anti-angiogenic therapy in the HCC.
Increased Oxidative Stress and RUNX3 Hypermethylation in Patients with Hepatitis B Virus-Associated Hepatocellular Carcinoma (HCC) and Induction of RUNX3 Hypermethylation by Reactive Oxygen Species in HCC Cells