Integrin β3Promotes TGF-β1/H₂O₂/HOCl-mediated Induction Of Metastatic Phenotype Of Hepatocellular Carcinoma Cells By Enhancing TGF-β1Signaling

Objective:Previous studies demonstrated that TGF-β1is an important factor for the development of hepatocellular carcinoma (HCC), and that neutrophil-derived ROS (H2O2/HOCI) could promote the development and metastasis of tumor. Moreover, integrin P3has been found to regulate TGF-β1signaling and to be related to the metastatic phenotype of HCC cells. In this study we investigated whether H2O2and HOCl could cooperate with TGF-β1to induce metastatic phenotype of non-metastatic HCC cells, and whether β3is required for the induction.Methods:Invasive capacity was evaluated using matrigel invasion assay and actin polymerization analysis. Gelatin zymography was used to detect the active MMP-2and MMP-9released by tumor cells. Experimental metastasis model was established by tumor cell injection via tail vein. Tumor cell extravasation was evaluated by analyzing the density of the tumor cells invading into the lung tissue. The metastatic lesions were analyzed by Immunofluorescence and histology. The activation of signaling pathways was detected by Western blot. The expression of genes was detected using real-time RT-PCR and Western blot. The protein level on cell surface was evaluated by flow cytometry. The knock-down and overexpression of β3was performed by transfection with β3shRNA(h) lentiviral particles and eukaryotic expression plasmid of β3, respectively. Anti-α3and anti-αvβ3 antibodies were used to block α3and β3on cell surface. Apoptosis-resistance of adherent cells was analyzed by Annexin V/PI staining. Anoikis-resistance of tumor cells was evaluated by flow cytometry after incubation of tumor cells in poly-HEMA-coated6-well plate.Results:The stimulation with either H2O2/HOCI or TGF-β1could not induce the high invasive capability of HCC cells. However, H2O2/HOCI could cooperate with TGF-β1to augment the invasive migration of non-invasive HCC cells. After prolonged stimulation with TGF-β1/H2O2/HOCl, the invasive capability of HCC cells was gradually augmented, resulting in the induction of metastatic potential. The stimulation of non-metastatic HCC cells with TGF-β1/H2O2/HOCI could induce the metastatic potential which was sufficient for HCC cells to extravasate from circulation and form metastatic foci in secondary sites. Importantly, TGF-β1/H2O2/HOCI induced the metastatic potential by inducing the sustained activation of MAPK pathways. TGF-β1/H2O2/HOCI could activate p38MAPK-β3positive-feedback loop in HCC cells, thus up-regulating the expression of β3. β3in turn enhanced TGF-β1signaling by augmenting the activity of Src, resulting in the sustained and enhanced activation of p38MAPK and ERK pathways. The sustained activation of p38MAPK and ERK pathways could increase the expression of a3and SNAI2, promoting the invasive capability of HCC cells. The enhanced activation of p38MAPK and ERK pathways also antagonized the pro-apoptotic effect of Smad pathway, augmenting the anoikis-resistance of HCC cells. Inhibiting β3expression or the activation of Src could suppress the promoting effects of TGF-β1/H2O2/HOCI on invasive migration and anoikis-resistance of non-metastatic HCC cells.Conclusion:Our data show that H2O2/HOCI could cooperate with TGF-β1to promote the invasive migration and anoikis-resistance of non-metastatic HCC cells, thus inducing their metastatic potential. Integrin β3played an important role in this induction process. Inhibiting β3could suppress the TGF-β1/H2O2/HOCl-mediated induction of metastatic phenotype of HCC cells, suggesting that targeting β3might be a potential approach in preventing the metastasis of hepatocellular carcinoma.