The role and mechanism of HIF-1α-mediated glypican-3 secretion in hypoxia-induced tumor progression in hepatocellular carcinoma

Objective: To explore the expression and secretion mechanism of glypican-3 (GPC3) in hepatocellular carcinoma (HCC) cells under hypoxic conditions, and its role in tumor progression.Methods: Huh7 cells with and without the knockdown of hypoxia-inducible factor 1-alpha (HIF-1 alpha) were cultured under 1% O-2 for varying durations to induce hypoxia. The expression levels of GPC3, HSP70, CD63, STX11 and SYT7 in the cytoplasm and exosomes of Huh7 cells were evaluated by western blotting and immunofluorescence. GPC3 protein expression was further measured in cells treated with GW4869 under hypoxic conditions. Huh7 cells and human umbilical vein endothelial cells (HUVECs) were cultured with the exosomes extracted from the control and GPC3-knockdown cells, the cell proliferation, migration, epithelial-mesenchymal transition (EMT), invasion, and in vitro angiogenesis were analyzed. Tumor xenografts were established to assess the role of GPC3-deficient exosomes in tumor growth.Results: Hypoxic culture conditions downregulated GPC3, STX11 and SYT7 protein levels in the Huh7 cells and upregulated GPC3 mRNA, and also increased GPC3 protein expression in the exosomes. HIF-1 alpha knockdown, as well as treatment with GW4869, upregulated GPC3 protein in the Huh7 cells grown under 1% O-2, but downregulated exosomal GPC3. Furthermore, exosomes derived from the GPC3-knockdown cells inhibited the proliferation and migration of Huh7 cells, decreased the expression of N-cadherin, vimentin, beta-catenin, c-Myc and cyclin D1, and increased that of E-cadherin. Likewise, the GPC3-deficient exosomes also suppressed the invasion and tube formation ability of the HUVECs compared to that of control cells. Consistent with the in vitro results, the GPC3-deficient exosomes also repressed tumor growth in vivo.Conclusion: Hypoxia promoted secretion of exosomal GPC3 through the activation of HIF-1 alpha. GPC3-deficient exosomes inhibited the proliferation, migration and EMT of HCC cells via the Wnt/beta-catenin signaling pathway, and suppressed the angiogenic potential of HUVECs. This provided a novel understanding of the role of exosomal GPC3 in HCC progression.