Xiaoling Yu

A randomized controlled trial of Licartin for preventing hepatoma recurrence after liver transplantation.

Orthotopic liver transplantation (OLT) is the only curative therapy of HCC with underlying cirrhosis, but due to HCC metastasis and recurrence, its benefit is limited to a small population who meet the strict selection criteria. We previously reported that Licartin ([131I]mAb HAb18G/CD147) was safe and effective in treating HCC patients, and its antigen, HAb18G/CD147, was closely related to HCC invasion and metastasis. Here, we reported a randomized controlled trial to assess the post‐OLT antirecurrence efficacy of Licartin in advanced HCC patients. We randomized 60 post‐OLT patients with HCC, who were at tumor stage 3/4 and outside the Milan criteria before OLT, into 2 groups. Three weeks after OLT, the treatment group received 15.4 MBq/kg of Licartin, while the control group received placebo intravenously for 3 times with an interval of 28 days. At 1‐year follow‐up, the recurrence rate significantly decreased by 30.4% (P = 0.0174) and the survival rate increased by 20.6% (P = 0.0289) in the treatment group, compared with those in the control group. For the control group versus the treatment group, the hazard ratio for recurrence was 3.60 (95% confidence interval [CI], 1.50‐8.60) and that for death was 3.87 (95% CI, 1.23–12.21). Licartin treatment also resulted in an earlier decreased AFP level and a longer time of normal AFP level than placebo (P = 0.0016). No Licartin‐related toxic effects were observed. Conclusion: Licartin is a promising drug for preventing post‐OLT tumor recurrence in advanced HCC patients excluded by the currently strict criteria for OLT. HAb18G/CD147 can be a good drug target. (HEPATOLOGY 2007;45:269–276.)

Crystal structure of HAb18g/CD147 - Implications for immunoglobulin superfamily homophilic adhesion

CD147, a member of the immunoglobulin superfamily (IgSF), plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. Importantly, our previous studies have demonstrated that HAb18G/CD147 is a novel hepatocellular carcinoma (HCC)-associated antigen, and HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several matrix metalloproteinases, facilitating invasion and metastasis of HCC cells. In addition, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers. However, the structural basis underlying the multifunctional character of CD147 remains unresolved. We report here the crystal structure of the extracellular portion of HAb18G/CD147 at 2.8Å resolution. The structure comprises an N-terminal IgC2 domain and a C-terminal IgI domain, which are connected by a 5-residue flexible linker. This unique C2-I domain organization is distinct from those of other IgSF members. Four homophilic dimers exist in the crystal and adopt C2-C2 and C2-I dimerization rather than V-V dimerization commonly found in other IgSF members. This type of homophilic association thus presents a novel model for homophilic interaction between C2 domains of IgSF members. Moreover, the crystal structure of HAb18G/CD147 provides a good structural explanation for the established multifunction of CD147 mediated by homo/hetero-oligomerizations and should represent a general architecture of other CD147 family members.

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)

Function of HAb18G/CD147 in Invasion of Host Cells by Severe Acute Respiratory Syndrome Coronavirus

Abstract To identify the function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome (SARS) coronavirus (CoV), we analyzed the protein-protein interaction among HAb18G/CD147, cyclophilin A (CyPA), and SARS-CoV structural proteins by coimmunoprecipitation and surface plasmon resonance analysis. Although none of the SARS-CoV proteins was found to be directly bound to HAb18G/CD147, the nucleocapsid (N) protein of SARS-CoV was bound to CyPA, which interacted with HAb18G/CD147. Further research showed that HAb18G/CD147, a transmembrane molecule, was highly expressed on 293 cells and that CyPA was integrated with SARS-CoV. HAb18G/CD147–antagonistic peptide (AP)–9, an AP of HAb18G/CD147, had a high rate of binding to 293 cells and an inhibitory effect on SARS-CoV. These results show that HAb18G/CD147, mediated by CyPA bound to SARS-CoV N protein, plays a functional role in facilitating invasion of host cells by SARS-CoV. Our findings provide some evidence for the cytologic mechanism of invasion by SARS-CoV and provide a molecular basis for screening anti-SARS drugs

Extracellular Membrane-proximal Domain of HAb18G/CD147 Binds to Metal Ion-dependent Adhesion Site (MIDAS) Motif of Integrin β1 to Modulate Malignant Properties of Hepatoma Cells

Background: HAb18G/CD147 interacts with integrin β1 subunit. Results: Extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site (MIDAS) in the βA domain of the integrin β1 subunit. Conclusion: Interaction of HAb18G/CD147 with integrin β1 activates the downstream FAK signaling pathway, enhancing the malignant properties of hepatocellular carcinoma cells. Significance: This is first time binding sites of CD147 and integrin β1 are revealed. Several lines of evidence suggest that HAb18G/CD147 interacts with the integrin variants α3β1 and α6β1. However, the mechanism of the interaction remains largely unknown. In this study, mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, was used to study the CD147-integrin β1 subunit interaction. CD147 in human hepatocellular carcinoma (HCC) cells was interfered with by small hairpin RNA. Nude mouse xenograft model and metastatic model of HCC were used to detect the role of CD147 in carcinogenesis and metastasis. We found that the extracellular membrane-proximal domain of HAb18G/CD147 (I-type domain) binds at the metal ion-dependent adhesion site in the βA domain of the integrin β1 subunit, and Asp179 in the I-type domain of HAb18G/CD147 plays an important role in the interaction. The levels of the proteins that act downstream of integrin, including focal adhesion kinase (FAK) and phospho-FAK, were decreased, and the cytoskeletal structures of HCC cells were rearranged bearing the HAb18G/CD147 deletion. Simultaneously, the migration and invasion capacities, secretion of matrix metalloproteinases, colony formation rate in vitro, and tumor growth and metastatic potential in vivo were decreased. These results indicate that the interaction of HAb18G/CD147 extracellular I-type domain with the integrin β1 metal ion-dependent adhesion site motif activates the downstream FAK signaling pathway, subsequently enhancing the malignant properties of HCC cells.

Modulation of CD147-induced matrix metalloproteinase activity: role of CD147 N-glycosylation.

Degradation of the basement membrane by MMPs (matrix metalloproteinases) is one of the most critical steps in tumour progression. CD147 is a tumour-associated antigen that plays a key regulatory role for MMP activities. In the present study, mass spectrum analysis demonstrated that the purified native CD147 from human lung cancer tissue was N-glycosylated and contained a series of high-mannose and complex-type N-linked glycan structures. Moreover, native glycosylated CD147 existed exclusively as oligomers in solution and directly stimulated MMP production more efficiently than non-glycosylated prokaryotic CD147. The glycosylation site mutation results indicated that, among three N-glycan attachment sites, the N152Q mutants were retained in the endoplasmic reticulum and unfolded protein response signalling was activated. This improper intracellular accumulation impaired its MMP-inducing activity. Increased β1,6-branching of N-glycans as a result of overexpression of GnT-V (N-acetylglucosaminyltransferase V) plays an important role in tumour metastasis. In the present study, we identified CD147 as a target protein of GnT-V and found that overexpression of GnT-V resulted in an elevated level of CD147 at the plasma membrane and in cell-conditioned medium, thereby increasing the induction of MMPs. The present study reveals the important role of N-glycosylation of CD147 in its biological function and implied that targeting aberrant β1,6-branching of N-glycans on CD147 would be valuable for the development of novel therapeutic modalities against carcinoma.

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.

Increasing the culture efficiency of hybridoma cells by the use of integrated metabolic control of glucose and glutamine at low levels

The metabolism of HAb18 hybridoma cells was shifted to decrease metabolite accumulation and to improve culture efficiency by integrated metabolic control of glucose and glutamine at low levels. When glucose and glutamine levels were decreased to 0.5 and 0.3 mM respectively, lactate and ammonia production were reduced by 62.6 and 74% respectively, glucose‐to‐cell yield was increased from 0.23×109 to 0.66×109 cells·mmol−1, and glutamine‐to‐cell yield from 0.18×109 to 1.95×109 cells·mmol−1. Compared with high‐level glucose and glutamine fed‐batch cultures, low‐level glucose and glutamine led to higher cell density (1.0×107 versus 0.3×107 cells·ml−1), longer culture span (14 as opposed to 8 days) and higher antibody yield (250 as against 150 mg·l−1). These results indicate that hybridoma culture efficiency would be increased by the integrated control of glucose and glutamine at 0.5 and 0.3 mM respectively. In contrast with previously reported glucose‐and/or‐glutamine‐level‐controlled fed‐batch cultures, we demonstrated an efficient strategy of nutrient level selection and amino acid feeding. More importantly, our accurately and well‐distributed Equable Feeding Control System opens a new avenue for reducing metabolites to low levels by controlling nutrients at low levels.

New strategy for large-scale preparation of the extracellular domain of tumor-associated antigen HAb18G/CD147 (HAb18GED)

HAb18G/CD147, a cancer-associated biomarker, can promote tumor growth, invasion and metastasis. Here, we established a new strategy to express the extracellular domain of HAb18G/CD147 (HAb18GED) by using the FRT/Flp-In recombinase-based site-directed integration system. Two clones expressing HAb18GED were established, and 600 mg HAb18GED was obtained with more than 95% purity. Our results showed that purified HAb18GED exhibited strong activities to stimulate the secretion of pro-MMP2 and MMP-2 and promote the invasion of HCC cells. Overall, our study effectively overcame the difficulty of large-scale HAb18GED preparation and laid a solid foundation for the further studies on structure and functions of HAb18G/CD147.

CD147 reinforces [Ca 2+ ] i oscillations and promotes oncogenic progression in hepatocellular carcinoma

Oscillations in intracellular Ca2+ concentrations ([Ca2+]i) mediate various cellular function. Although it is known that [Ca2+]i oscillations are susceptible to dysregulation in tumors, the tumor-specific regulators of [Ca2+]i oscillations are poorly characterized. We discovered that CD147 promotes hepatocellular carcinoma (HCC) metastasis and proliferation by enhancing the amplitude and frequency of [Ca2+]i oscillations in HCC cells. CD147 activates two distinct signaling pathways to regulate [Ca2+]i oscillations. By activating FAK-Src-IP3R1 signaling pathway, CD147 promotes Ca2+ release from endoplasmic reticulum (ER) and enhances the amplitude of [Ca2+]i oscillations. Furthermore, CD147 accelerates ER Ca2+ refilling and enhances the frequency of [Ca2+]i oscillations through activating CaMKP-PAK1-PP2A-PLB-SERCA signaling pathway. Besides, CD147-promoted ER Ca2+ release and refilling are tightly regulated by changing [Ca2+]i. CD147 may activate IP3R1 channel under low [Ca2+]i conditions and CD147 may activate SERCA pump under high [Ca2+]i conditions. CD147 deletion suppresses HCC tumorigenesis and increases the survival rate of liver-specific CD147 knockout mice by regulating [Ca2+]i oscillations in vivo. Together, these results reveal that CD147 functions as a critical regulator of ER-dependent [Ca2+]i oscillations to promote oncogenic progression in HCC.