Pu Zhao

Overexpression of HAb18G/CD147 promotes invasion and metastasis via α3β1 integrin mediated FAK-paxillin and FAK-PI3K-Ca2+ pathways

Abstract.Mechanism of HAb18G/CD147 underlying the metastasis process of human hepatoma cells has not been determined. In the present study, we found that integrin α3β1 colocalizes with HAb18G/CD147 in human 7721 hepatoma cells. The enhancing effect of HAb18G/CD147 on adhesion, invasion capacities and matrix metalloproteinases (MMPs) secretion was decreased by integrin α3β1 antibodies (p<0.01). The expressions of integrin downstream molecules including focal adhesion kinase (FAK), phospho-FAK (p-FAK), paxillin, and phospho-paxillin (p-paxillin) were increased in human hepatoma cells overexpressing HAb18G/CD147. Deletion of HAb18G/CD147 reduces the quantity of focal adhesions and rearranges cytoskeleton. Wortmannin and LY294002, specific phosphatidylinositol kinase (PI3K) inhibitors, reversed the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reducing cell adhesion, invasion and MMPs secretion potential (p<0.01). Together, these results suggest that HAb18G/CD147 enhances the invasion and metastatic potentials of human hepatoma cells via integrin α3β1-mediated FAK-paxillin and FAKPI3K-Ca2+ signal pathways.

HAb18G/CD147 promotes cell motility by regulating annexin II‐activated RhoA and Rac1 signaling pathways in hepatocellular carcinoma cells

Tumor cells can move as individual cells in two interconvertible modes: mesenchymal mode and amoeboid mode. Cytoskeleton rearrangement plays an important role in the interconversion. Previously, we reported that HAb18G/CD147 and annexin II are interacting proteins involved in cytoskeleton rearrangement, yet the role of their interaction is unclear. In this study we found that the depletion of HAb18G/CD147 produced a rounded morphology, which is associated with amoeboid movement, whereas the depletion of annexin II resulted in an elongated morphology, which is associated with mesenchymal movement. The extracellular portion of HAb18G/CD147 can interact with a phosphorylation‐inactive mutant of annexin II and inhibit its phosphorylation. HAb18G/CD147 inhibits Rho signaling pathways and amoeboid movement by inhibiting annexin II phosphorylation, promotes membrane localization of WAVE2 and Rac1 activation by way of the integrin‐FAK‐PI3K/PIP3 signaling pathway, and promotes the formation of lamellipodia and mesenchymal movement. Conclusion: These results suggest that the interaction of HAb18G/CD147 with annexin II is involved in the interconversion between mesenchymal and amoeboid movement of hepatocellular carcinoma cells. (HEPATOLOGY 2011)

Annexin II promotes invasion and migration of human hepatocellular carcinoma cells in vitro via its interaction with HAb18G/CD147

HAb18G/CD147, a member of the immunoglobulin family enriched on the surface of tumor cells, is reported to be correlated with invasion, metastasis, growth, and survival of malignant cells. Here, we found that annexin II, a 36‐kDa Ca2+‐ and phospholipid‐binding protein and in vivo substrate for tyrosine kinase and PKC, is a new interaction protein of HAb18G/CD147 in human hepatocellular carcinoma (HCC) cells. In the present study, we explored the unclear role of annxin II in HCC invasion and migration and the interaction effects between HAb18G/CD147 and annexin II. Our data show that downregulation of annexin II in HCC cells significantly decreased the secretion of MMP, migration ability, and invasive potential, and affected the cytoskeleton rearrangement of tumor cells. The MMP‐2 level and invasive potential of HCC cells were regulated by both annexin II and HAb18G/CD147. Also, interaction effects exist between the two molecules in tumor progression, including MMP‐2 production, migration, and invasion. These results suggest that annexin II promotes the invasion and migration of HCC cells in vitro, and annexin II and HAb18G/CD147 interact with each other in the same signal transduction pathway working as a functional complex in tumor progression. (Cancer Sci 2009)

The interaction of HAb18G/CD147 with integrin α6β1 and its implications for the invasion potential of human hepatoma cells

BackgroundHAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin α3β1. However, it has never been investigated whether α3β1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells.MethodsHuman SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin α6β1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin α6β1. Invasion potential was evaluated with an invasion assay and gelatin zymography.ResultsWe found that integrin α6β1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs) were partially blocked by integrin α6β1 antibodies (P < 0.01). Wortmannin, a specific phosphatidylinositol kinase (PI3K) inhibitor that reverses the effect of HAb18G/CD147 on the regulation of intracellular Ca2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P < 0.05). Importantly, no additive effect between Wortmannin and α6β1 antibodies was observed, indicating that α6β1 and PI3K transmit the signal in an upstream-downstream relationship.ConclusionThese results suggest that α6β1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

βig-h3 Interacts with α3β1 Integrin to Promote Adhesion and Migration of Human Hepatoma Cells

βig-h3 is a TGF-induced extracellular matrix (ECM) protein. Our previous evidence suggests that β ig-h3 may promote adhesion and invasion potential of human hepatoma cells, but the mechanism underlying this process is still unknown. The present study identifies a pivotal role for molecules of the β ig-h3 signal transduction pathway. We demonstrated that β ig-h3 co-immunoprecipitated with α 3β 1 integrin in human 7721 hepatoma cells. The addition of α 3β 1 integrin antibodies inhibited the elevated adhesion and migration in β ig-h3-over-expressing 7721 cells, but not in β ig-h3 siRNA transfected 7721 cells. The expression and activity of integrin downstream molecules FAK and paxillin show a positive correlation with β ig-h3 expression in different human hepatoma cells. Levels of focal adhesions and stress fibers were decreased in β ig-h3 siRNA transfected 7721 cells. We suggest that by interaction with α 3β 1 integrin, β ig-h3 activates FAK-paxillin signaling linkage, leads to cytoskeleton reorganization, and thus enhances the metastatic potentials of human hepatoma cells.

Dimerization is essential for HAb18G/CD147 promoting tumor invasion via MAPK pathway

HAb18G/CD147 is a transmembrane glycoprotein of the immunoglobulin superfamily (IgSF) and is reported to be correlated with invasion and metastasis of many cancers. The crystal structure of HAb18G/CD147 ectodomain has shown that it can form homodimers in crystal. However, the functional significance of HAb18G/CD147 dimerization remains unclear. In the present study, guided by the crystal structure, we performed extensive mutational and functional studies to identify residues critical for dimerization and molecular function of HAb18G/CD147. Fourteen mutants were purified and evaluated for their ability to form dimers in solution and in living cells. Subsequent functional validation revealed that K63E and S193A mutants, which disrupted CD147 dimerization both in solution and in living cells, showed clearly dominant-negative effects on MAPK activation, MMP2 induction and invasiveness in tumor cells. Taken together, the present study provides mutational and functional evidences demonstrating for the first time the functional importance of CD147 dimerization and its direct correlation with invasion and metastasis of tumor cells.

Morphological changes and molecular expressions of hepatocellular carcinoma cells in three-dimensional culture model

Metastatic processes of hepatocellular carcinoma (HCC) are highly associated with the breakdown of extracellular matrix (ECM). However, the regular two-dimensional (2D) culture system, in which only little ECM is involved, fails to provide a well-defined microenvironment for HCC functional research. HAb18G/CD147, a HCC-associated antigen, plays important roles in HCC progression, migration and invasion. In this study, we investigated whether HAb18G/CD147 enhanced the HCC migration and invasion in three-dimensional (3D) culture model through affecting the key molecules and enzymes involved in the metastatic processes, such as focal adhesion kinase (FAK), matrix metalloproteinases (MMPs) and cytoskeleton proteins. We found that, compared with those in 2D cell culture model, the expression of HAb18G/CD147 was significantly increased in 3D cell culture model, together with a high production of MMPs (P<0.01), an enhanced expression and activation of FAK (P<0.01) and a changed distribution of F-actin. In addition, the expressions of paxillin and E-cadherin, which enhance the adhesion and migration potentials, were also significantly increased in 3D cell culture model (P<0.01). All the results suggest that the enhanced expressions of HAb18G/CD147, MMPs, paxillin and FAK changed the distributions of cytoskeleton in the 3D reconstituted basement membrane (BM) and increased the adhesion and invasion potentials of HCC cells.