Heparanase and MMP-9 synergistically induce endothelial-mesenchymal transition in portal vein microvessels to promote hepatocellular carcinoma metastasis

Abstract

Background

Both heparanase (HPSE) and matrix metalloproteinase-9 (MMP-9) can promote metastasis of hepatocellular carcinoma (HCC), but it is not clear whether they co-induce endothelial-mesenchymal transition (EndoMT) in portal vein endothelial cells (PVECs) to facilitate HCC metastasis. This study aimed to investigate the combined effect of HPSE and MMP-9 on EndoMT in PVECs and subsequent intrahepatic metastasis of HCC and to explore the underlying mechanism.

Methods

This study employed gene knockdown, gene overexpression and inhibitor strategies to manipulate HPSE/MMP-9 expression or activity within HCC cells, which were non-contact co-cultured with human umbilical vein endothelial cells (HUVECs). Quantitative real-time polymerase chain reaction and western blotting techniques were employed to assess the expression levels of endothelial and mesenchymal cell markers in the co-cultured HUVECs, while double immunofluorescent analysis was performed to determine the localization. Expression of syndecan 1 (SDC-1), transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α) and pSmad2/3 was determined simultaneously in HUVECs, with the first three proteins detected in the supernatant. Transendothelial migration assays were executed to detect the migration rate of HCC cells. A nude mouse model of liver cancer metastasis was established, and hematoxylin and eosin staining of the liver was performed to observe the liver metastasis and portal vein tumor thrombus (PVTT). Furthermore, immunohistochemical analyses of human HCC tissues around portal vein were performed to investigate the relationship between HPSE/MMP-9 and mesenchymal cell marker expression.

Results

Compared with single knockdown of HPSE/MMP-9 in HCCLM3 cells, simultaneous knockdown was more effective in reducing the expression of mesenchymal markers and increasing the expression of endothelial markers in co-cultured HUVECs. The levels of SDC-1, TGF-β1, TNF-α and pSmad2/3 showed a coordinated downregulation pattern. The dual knockdown strategy suppressed the release of EndoMT activators from stimulated HUVECs, thereby suppressing EndoMT progression and significantly reducing HCC cell transendothelial migration in vitro. The application of HPSE/MMP-9 inhibitors and the TGF-β1-specific inhibitor in HCCLM3 cells, along with rescue experiments using HepG2 cells, yielded similar results. Nude mouse experiments showed that double knockdown HCCLM3 cells led to the lowest liver metastasis and PVTT rates. Immunohistochemical analyses displayed a gradual enhancement in the expression of mesenchymal cell markers with the increase in the expression or co-expression of HPSE/MMP-9.

Conclusion

HPSE/MMP-9 may have a synergistic effect on inducing EndoMT in PVECs, ultimately promoting HCC metastasis through the SDC-1/TGF-β1 (TNF-α)/pSmad2/3 pathway. Our research expands the current understanding of the intricate mechanisms involved in the dissemination of HCC.