Hepatitis B Virus Small Envelope Protein Promotes Hepatocellular Carcinoma Angiogenesis via Endoplasmic Reticulum Stress Signaling To Upregulate the Expression of Vascular Endothelial Growth Factor A

Abstract

Hepatocellular carcinoma (HCC) is a hypervascular tumor, and accumulating evidence has indicated that stimulation of angiogenesis by hepatitis B virus (HBV) may contribute to HCC malignancy. The small protein of hepatitis B virus surface antigen (HBsAg), SHBs, is the most abundant HBV protein and has a close clinical association with HCC; however, whether SHBs contributes to HCC angiogenesis remains unknown. This study reports that the 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 counts 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, the overexpression of SHBs increased vascular endothelial growth factor A (VEGFA) expression at both the mRNA and protein levels. Higher VEGFA expression levels were also observed in xenograft tumors transplanted with SHBs-expressing HCC cells and in HBsAg-positive HCC tumor tissues than in their negative controls. As expected, in the culture supernatants, the secretion of VEGFA was also significantly enhanced from HCC cells expressing SHBs, which promoted HUVEC migration and vessel formation. Furthermore, all three unfolded protein response (UPR) sensors, inositol-requiring enzyme 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK), and activating transcription factor 6 (ATF6), associated with ER stress were found to be activated in 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 for 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 the overexpression of angiogenic factors like vascular endothelial growth factor A (VEGFA). However, a detailed mechanism of HBV-induced angiogenesis remains to be established. In this study, we demonstrate for the first time that the most abundant HBV protein, i.e., small surface antigen (SHBs), can enhance the angiogenic capacity of HCC cells by the 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 proangiogenic role in HBV-associated HCC and may represent a potential target for antiangiogenic therapy in HCC.

Keywords: HBV small surface proteins; angiogenesis; hepatocellular carcinoma; unfolded protein response; vascular endothelial growth factor A.

FIG 1

SHBs promotes angiogenesis of HCC. (A) In vitro growth curves of Huh7-Flag, Huh7-SHBs, HepG2-Flag, and HepG2-SHBs cells as determined by a CCK-8 assay. OD450, optical density at 450 nm. (B) Relative growth rates of Huh7-Flag versus Huh7-SHBs tumors and HepG2-Flag versus HepG2-SHBs tumors after subcutaneous inoculation of 2 × 10⁶ cells into nude mice. (C) Gross appearance of the dissected tumors xenografted from the indicated cells at the end of the experiment. (D) Tumor weight at the end of the study. (E) Density of microvessels as assessed by CD31 immunochemistry in the indicated xenograft tumors. (F) Density of microvessels as assessed by CD31 immunochemistry in HBsAg-negative and -positive HCC specimens. Bars, 50 μm. Original magnification, ×200. Values are means ± standard deviations (SD) from 3 to 5 independent experiments. *, P < 0.05.

FIG 2

The supernatant of SHBs-expressing cells promotes migration and tube formation of HUVECs. (A) Capillary tube formation in HUVECs cultured in conditioned medium (CM) derived from Huh7-Flag, Huh7-SHBs, HepG2-Flag, and HepG2-SHBs cells. (B) Migrated HUVECs after incubation in CM derived from Huh7-Flag, Huh7-SHBs, HepG2-Flag, and HepG2-SHBs cells in the transwell migration assay. (C) Relative motility of HUVECs cultured with CM derived from the indicated cells as assessed by a wound-healing assay. (D) Expression of SHBs and HBx in pRep-HBV-, pRep-HBV-SHBs(−)-, or pRep-HBV-HBx(−)-transfected Huh7 or HepG2 cells was assessed by Western blotting. (E) Capillary tube formation in HUVECs cultured in CM derived from HCC cells transfected with pRep-HBV, pRep-HBV-SHBs(−), pRep-HBV-HBx(−), or the empty control vector pRep-Sal. (F) Migrated HUVECs after incubation in CM derived from HCC cells transfected with pRep-HBV, pRep-HBV-SHBs(−), pRep-HBV-HBx(−), or the empty control vector pRep-Sal in the transwell migration assay. Values are means ± SD from 3 to 5 independent experiments. *, P < 0.05.

FIG 3

SHBs induces VEGFA expression. (A) Western blot analysis of VEGFA, VEGFB, VEGFC, and VEGFD protein expression in Huh7-Flag, Huh7-SHBs, HepG2-Flag, and HepG2-SHBs cells. (B) ELISA of VEGFA protein expression in the indicated cell supernatants. (C) qRT-PCR analysis of VEGFA mRNA expression in the indicated cells. Transcript levels were normalized to GAPDH and expressed relative to the respective control cells. (D) VEGFA expression in the indicated xenograft tumors by anti-VEGFA immunohistochemistry (IHC). (E) Immunochemical staining of HBsAg and VEGFA in HCC tissues (n = 20). A significant positive correlation was found between SHBs and VEGFA expression in HCC tissues. Values are means ± SD from 3 to 5 independent experiments. *, P < 0.05.

FIG 4

SHBs promotes angiogenesis via inducing VEGFA expression. (A) ELISA of VEGFA protein expression in the supernatants of Huh7-Flag, Huh7-SHBs, HepG2-Flag, and HepG2-SHBs cells with or without VEGFA knockdown. (B) Western blot analysis of the phosphorylation status of VEGFR activation-associated signaling molecules in HUVECs cultured with CM from the indicated cells. (C) Capillary tube formation in HUVECs cultured in CM derived from the indicated cells. (D) Migrated HUVECs after incubation in CM derived from the indicated cells. (E) CM harvested from Huh7-SHBs, HepG2-SHBs, or control cells was pretreated with 1.0 μg/mL neutralizing antibodies against VEGFA, and tube formation of HUVECs cultured in the pretreated CM was then examined. (F) Number of migrated HUVECs after incubation in the pretreated CM. Values are means ± SD from 3 to 5 independent experiments. *, P < 0.05.

FIG 5

SHBs induces VEGFA via ER stress. (A) Effect of the ER stress inhibitors 4-PBA and TUDCA on the expression of BIP and VEGFA in Huh7-Flag, Huh7-SHBs, HepG2-Flag, and HepG2-SHBs cells. Cells were treated with 5 mM 4-PBA or 2 mM TUDCA for 24 h and analyzed by Western blotting. DMSO, dimethyl sulfoxide. (B) Migrated HUVECs after incubation in CM derived from the indicated cells with or without 4-PBA or TUDCA treatment in the transwell migration assay. (C) Effect of SHBs mutants G145R and KD (five mutations, W36L, T47K, N52D, V184A, and F220L), known to aggravate ER retention, on VEGFA protein expression. (D) ELISA of VEGFA protein expression in the supernatants of HCC cells transfected with pSHBs, pG145R, pKD, or the empty control vector pcDNA3.1. (E) Capillary tube formation in HUVECs cultured in CM derived from HCC cells transfected with pSHBs, pG145R, pKD, or the empty control vector pcDNA3.1. (F) Migrated HUVECs after incubation in CM derived from HCC cells transfected with pSHBs, pG145R, pKD, or the empty control vector pcDNA3.1. Values are means ± SD from 3 to 5 independent experiments. *, P < 0.05.

FIG 6

SHBs induces VEGFA expression via the UPR pathway. (A to D) Effect of siRNA-mediated knockdown of ATF6 (A), IRE1α (B), or PERK (C and D) on the expression of VEGFA and the activation markers of the ATF6/IRE1α/PERK pathway in Huh7-Flag, Huh7-SHBs, HepG2-Flag, and HepG2-SHBs cells detected by Western blotting (A; B, top three panels; and C), semiquantitative RT-PCR (B, bottom two panels), or quantitative real-time PCR (D). (E) ELISA of VEGFA protein expression in the supernatants of HCC cells transfected with siRNAs for ATF6, IRE1α, or PERK. (F) Cells were cotransfected with plasmids containing the VEGFA promoter/intron and the Renilla control vector after siRNA-mediated knockdown of ATF6, IRE1α, or PERK, and luciferase activities were measured. (G) Capillary tube formation in HUVECs cultured in CM derived from Huh7-Flag and Huh7-SHBs cells or HepG2-Flag and HepG2-SHBs cells with or without knockdown of ATF6, IRE1α, or PERK. (H) Migrated HUVECs after incubation in CM derived from Huh7-Flag and Huh7-SHBs cells or HepG2-Flag and HepG2-SHBs cells with or without knockdown of ATF6, IRE1α, or PERK. Values are means ± SD from 3 to 5 independent experiments. *, P < 0.05.

FIG 7

Schematic models for the mechanism by which SHBs promotes HCC angiogenesis via ER stress signaling to upregulate VEGFA expression.