Interaction Between HAb18G/CD147 And Annexin â…¡ Is Involved In Regulating HCC Cell Cytoskeleton Rearrangement And Cell Movement

Invasion and migration are two central processes of malignant tumors, which usually lead to tumor-associated death. Such migratory and invasive events are regulated by different proteolytic enzymes, including serine protease, cysteine protease and matrix metalloproteases(MMPs)[1]. These proteolytic enzymes degrade the extracellular matrix (ECM) and basement membrane surrounding blood vessels. During metastasis, cancer cells penetrate through the degraded basement membrane and ECM, become implanted in the underlying tissues and subsequently form secondary tumors[2].Among the proteases,MMPs is mainly regulated by tumor-stroma interactions via CD147, a highly glycosylated cell surface transmembrane protein belonging to the immunoglobulin superfamily[3]. CD147 was first identified as a factor shed from the surface of tumour cells responsible for stimulating MMP-1 production by fibroblasts. One of the important and most studied functions of CD147 is its role in induction of MMPs production via cell interactions– thus the derivation of its another name: extracellular matrix metalloproteinase inducer (EMMPRIN)[4].We previously developed an anti-HCC monoclonal antibody (mAb) HAb18 by using a cell suspension extracted from fresh human HCC tissues to immunize BALB/c mice and to prepare hybridoma[10-12]. Its antigen, HAb18G, was identified by screening the HCC cDNA expression library and was named as HAb18G/CD147 for being homologous to CD147[3, 13]. Studies have demonstrated that HAb18G/CD147 promotes the invasion and migration of HCC cells by stimulating both the fibroblast cells and tumor cells themselves to produce MMPs. Our previous study also showed that HAb18G/CD147 was involved in tumor metastasis and invasion as a signal transduction molecule by regulating Ca2+ inflow[9, 14]. All these findings show that HAb18G/CD147 may play important roles in HCC progression, including adhesion, migration, and enzymes degradation. But the mechanism has not been testified. It is reported that annexin II, a 36kDa Ca2+and phospholipids-binding protein, also plays important roles in tumor progression. We speculated that relationships may exist between CD147 and annexin II [15], but it has never been testified. In the present study, we first explored and proved the possibility of the above hypothesis. In the following experiments, we studied the role of annexin II in HCC invasion and migration. Then we further investigated the interaction between annexin II and HAb18G/CD147 in tumor invasion and migration. We also investigated the signaling transduction pathway that the two molecules may be involoved in, which affected the cytoskeleton rearrengement of HCC cells. The study has been divided into two parts.Part I: Annexin II promotes invasion and migration of human hepatocellular carcinoma cells in vitro via its interaction with HAb18G/CD147We carry out co-immunoprecipitation assay and immunofluorescent assay to investigate the interaction between annexin II and HAb18G/CD147. Annexin II was found co-immunoprecipitated with HAb18G/CD147 in HCC cell lysates, indicating that HAb18G/CD147 interact with annexin II in their native form in HCC cells. The results of immunofluorescent double staining showed co-localizations of annexin II with HAb18G/ CD147 in HCC cells. Knocking down the expression of annexin II in HCC cells by RNAi, we found that annexin II promoted adhension, migration and invasion potentials of HCC cells. Annexin II can also affect cytoskeleton rearrangement of HCC cells. The results also showed that annexin II and HAb18G/CD147 have interaction effects in HCC invasion and migration. They may work in the same signal transduction pathway in tumor invasion and migration.Part II: Mechanism of HAb18G/CD147 and annexin II in hepatocellular carcinoma cell cytoskeleton rearrangement and cell movementResults of Part I confirmed that annexin II can affect the cytoskeleton rearrangement. Our previous studies also indicated that HAb18G/CD147 is involved in the cytoskeleton rearrangement. Therefore, the present study focused on the mechanisms of HAb18G/CD147 and annexin II in HCC cell cytoskeleton rearrangement and cell movement. The results showed that downregulation the expression of HAb18G/CD147 and annexin II caused morphological changes of HCC cells, but the morphological changes caused by the two molecules were diametrically different. When the expression of HAb18G/CD147 was downregulated, HCC cells become rounded morphology. When the expression of annexin II was downregulated, HCC cells become elongated morphology. The former is similar with the amoeboid mode of movement and the later is similar with the mesenchymal mode of movement. Next, we detected the interaction site of annexin II and HAb18G/CD147 using MAPPIT system. We found that the extracellular portion of HAb18G/CD147 can interact with the annexin II prey. HAb18G/CD147 inhibited Rho signaling pathways and ameboid movement in HCC cells by inhibiting annexin II phosphorylation. HAb18G/CD147 can also promote the membrane localization of WAVE2 and Rac1 activation in HCC cell by integrin-FAK-PI3K/PIP3 signaling pathway, and then, promote the formation of lamellipodia and mesenchymal movement.All the above results suggest that annexin II promotes HCC cell invasion and migration, in vitro. Annexin II and HAb18G/CD147 have interaction effects in HCC invasion and migration. They may work as a functional complex in tumor invasion and migration in the same signal transduction pathway. HAb18G/CD147 regulates the interconversion between amoeboid mode of movement and mesenchymal mode of movement by regulating the activation of RhoA and Rac1 signaling pathways in HCC cells.