Cancer-Associated Fibroblasts Promote Angiogenesis of Hepatocellular Carcinoma by VEGF-Mediated EZH2/VASH1 Pathway

Abstract

Background: Hepatocellular carcinoma is a highly vascularized tumor, so it is critical to study its angiogenesis. Cancer-associated fibroblasts and enhancer of zeste homolog 2 play an important role in tumor angiogenesis and became significant hallmarks of cancer. But the relationship between enhancer of zeste homolog-2 and cancer-associated fibroblasts in response to angiogenesis and its precise mechanism remains unclear. Methods: The separation of cancer-associated fibroblasts was identified by immunofluorescence. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis was used to reveal the proliferation of human umbilical vein endothelial cells. Vascular endothelial growth factor level was quantified by enzyme-linked immunosorbent assay. The wound healing, transwell, and vascular tube formation assays were used to identify the capability of migration, invasion, and tube formation of human umbilical vein endothelial cells in vitro. The detection of tumor angiogenesis was also performed in vivo. Finally, the level of enhancer of zeste homolog-2 and vasohibin 1 were determined by quantitative real-time polymerase chain reaction and Western blotting. Results: In comparison to control and condition medium noncancerous fibroblasts groups, the condition medium cancer-associated fibroblasts could significantly promote the proliferation, migration, invasion, and angiogenesis of human umbilical vein endothelial cells. We found that cancer-associated fibroblasts promoted angiogenesis of human umbilical vein endothelial cells via vascular endothelial growth factor secretion in vitro and in vivo. The upregulation of enhancer of zeste homolog 2 by vascular endothelial growth factor inhibited the expression of vasohibin 1, thus promoting the proliferation and angiogenesis of human umbilical vein endothelial cells. Taken together, the cancer-associated fibroblasts of hepatocellular carcinoma regulate the enhancer of zeste homolog-2/vasohibin 1 pathway via vascular endothelial growth factor secretion, thereby promoting the proliferation and angiogenesis of human umbilical vein endothelial cells. Conclusion: Our study identified the relationship between cancer-associated fibroblasts and enhancer of zeste homolog-2 and confirmed the pivotal role of cancer-associated fibroblasts in angiogenesis of hepatocellular carcinoma. Cancer-associated fibroblasts promote angiogenesis of hepatocellular carcinoma by vascular endothelial growth factor–mediated enhancer of zeste homolog-2/vasohibin 1 pathway and may be a potentially useful therapeutic target for hepatocellular carcinoma.