Molecular Mechanism Of CD147-Annexin A2 Interaction In Regulating Cancer Cell Movement

Invasion and metastasis are the main characteristics of tumor. The majority of deaths associated with tumor are due to the metastasis of the original tumor cells[1]. Cell movement plays an important role in tumor invasion and metastasis. The study of tumor cell movement is the hot spot in tumor biology researches. CD147 and Annexin A2 are found to be involved in rearrangement of the actin cytoskeleton in tumor cells. Our previous work has found that CD147 regulates Annexin A2-activated RhoA signaling in hepatocellular carcinoma(HCC) cells, but the detailed molecular mechanism is far from clear. This study is composed of the following three parts.Part Ⅰ CD147 interacts directly with Annexin A2.Using co-immunoprecipitation and mass spectrometry, Annexin A2 was identified as a potential binding partner for CD147. Colocalization analysis based on Pearson’s correlation coefficient(PCC) showed that there was a high degree of colocalization between CD147 and Annexin A2 in lung cancer and HCC cells. Fluorescence resonance energy transfer assay(FRET) demonstrated that there is a direct interaction between CD147 and Annexin A2 in living cells. We purified the extracellular portion of CD147 and Annexin A2 and characterized the kinetic binding parameters using purified proteins and surface plasmon resonance(SPR). We also truncated CD147 and demonstrated that the extracellular portion of CD147 interacts directly with Annexin A2 using His Pull-Down, FRET and SPR.Part Ⅱ CD147 regulates tyrosine phosphorylation of Annexin A2.We first determined the effects of Src and CD147 on tyrosine phosphorylation of Annexin A2. We found that Src could phosphorylate Annexin A2 and CD147 negatively regulated tyrosine phosphorylation of Annexin A2, which was consistent with the previous results. When Src and CD147 were simultaneously overexpressed, we found that CD147 could inhibit Annexin A2 phosphorylation by Src. In vitro kinase assay showed that the extracellular portion of CD147 and I domain could inhibit Annexin A2 phosphorylation by Src, however, C2 domain showed no effect on Annexin A2 phosphorylation. We determined the interaction between these constructs using FRET and found that only I domain could interact directly with the N-terminal domain of Annexin A2. These results demonstrate that CD147 inhibits Annexin A2 phosphorylation by Src via direct interaction between its I domain and the N-terminal domain of Annexin A2.Part Ⅲ CD147 regulates tumor cell movement via p-Annexin A2.We determined the effects of CD147 and Annexin A2 on tumor cell movement and found that CD147 might regulate tumor cell movement via regulating Annexin A2 phosphorylation. We evaluated the expression of DOCK family GEFs in cells transfected with Annexin A2 siRNA and found that DOCK3 was significantly down-regulated. Knockdown and specific rescue approaches revealed that DOCK3 expression was regulated by Annexin A2 phosphorylation, indicating that p-Annexin A2 might regulate tumor cell movement via DOCK3. DOCK3 was identified as a GEF for Rac1 and WAVE2 expression was suppressed by DOCK3. Furthermore, downregulation of DOCK3 resulted in increased lamellipodia formation and DOCK3 inhibited WAVE2 expression via suppressing β-catenin signaling. Further studies confirmed that CD147 regulated lamellipodia formation and tumor cell movement via p-Annexin A2/DOCK3/β-catenin/WAVE2 signaling axis in CD147 knockout HCC and colon cancer cells as well as in CD147 knockdown lung cancer cells. Cytoskeletal rearrangement and cell motility achieve dire significance during tumor metastasis. We investigated whether CD147 influenced tumor metastasis in vivo. We established an intrasplenic injection model for metastatic HCC cells and found that CD147 could promote HCC metastasis in nude mice.