Overexpression and correlation of HIF-2α, VEGFA and EphA2 in residual hepatocellular carcinoma following high-intensity focused ultrasound treatment: Implications for tumor recurrence and progression

Abstract

Rapid growth of residual tumors can occur as a result of their recurrence and progression. The present study aimed to investigate the expression of hypoxia inducible factor-2 subunit α (HIF-2α), vascular endothelial growth factor A (VEGFA), erythropoietin-producing hepatocellular A2 (EphA2) and angiogenesis in residual hepatocellular carcinoma (HCC), following treatment with high-intensity focused ultrasound (HIFU) ablation, in order to investigate the association between protein expression and tumor recurrence and growth. Athymic BALB/c (nu/nu) mice were subcutaneously inoculated with the HCC cell line HepG2, in order to create xenograft tumors. Approximately 30 days post-inoculation, eight mice were treated with HIFU, whereas eight mice received no treatment and acted as the control group. Residual tumor tissues were obtained from the experimental groups after one month. Levels of HIF-2α, VEGFA, EphA2 and cluster of differentiation 31 (CD31) expression was measured by immunohistochemical staining. CD31-positive vascular endothelial cells were counted to calculate microvascular density (MVD), and western blot analysis was performed to determine levels of HIF-2α, VEGFA, and EphA2 protein. It was found that the expression levels of HIF-2α, VEGFA, EphA2, and MVD proteins in residual HCC tissues were significantly higher than in the control group tissues (P<0.05). Tumor MVD was strongly correlated with VEGFA (R=0.957, P<0.01) and EphA2 (R=0.993, P<0.01) protein expression levels. Furthermore, there was a significant positive correlation between HIF-2α and EphA2 expression (R=0.991, P<0.01). The correlation between VEGFA and EphA2 expression was also positive (R=0.985, P<0.01). These data suggest that overexpression of HIF-2α, VEGFA and EphA2 is related to angiogenesis in residual HCC following HIFU ablation, potentially via their association with key mediators of recurrence.

Keywords: angiogenesis; erythropoietin-producing hepatocellular A2; hypoxia inducible factor-2 subunit α; residual hepatocellular carcinoma; vascular endothelial growth factor A.

Figure 1.

Residual hepatic tumor tissue following high intensity focused ultrasound ablation. (A) The cut surface of the residual tumor tissue. (B) Residual tumor tissue with hematoxylin and eosin staining, revealing (c) necrotic cells, (d) normal hepatoma cells, and (e) inflammatory cells, Magnification, ×200.

Figure 2.

Immunohistochemical analysis of HIF-2α, VEGFA, EphA2 and CD31 expression. The staining indicates protein levels in tumor tissue of (A-D) the control group vs. residual hepatic tumor tissue of (E-H) the experimental group one month after HIFU treatment. Magnification, ×200. HIF-2α, hypoxia-inducible factor-2α; VEGFA, vascular endothelial growth factor A; EphA2, ephrin A2 receptor; CD31, cluster of differentiation 31; HIFU, high intensity focused ultrasound.

Figure 3.

Western blot analysis of HIF-2α, VEGFA, and EphA2 expression, β-actin was used as internal control. Relative protein expression of (A) HIF-2α, (B) VEGFA, and (C) EphA2 were quantified and normalized against β-actin. *t=7.32, P<0.01; **t=10.41, P<0.01; ^#t=16.67, P<0.01. HIF-2α, hypoxia-inducible factor-2α; VEGFA, vascular endothelial growth factor A; EphA2, ephrin A2 receptor.

Figure 4.

Increased HIF-2α, EphA2 and VEGFA protein expression in residual tumor tissue. Based on immunohistochemical detection, EphA2 expression was found to be significantly correlated with HIF-2α expression and with VEGFA expression in residual hepatic tumor tissue. *r=0.991, P<0.01; ^#r=0.985, P<0.01.