The Clinical And Experimental Study Of Early Recurrence/metastasis-associated Protein Annexin A4 In Hepatocellular Carcinoma(HCC) After Radical Hepatectomy

Hepatocellular carcinoma(HCC) is one of the most common malignancies in China, which ranked asthe second leading cause of death from cancer. Currently, surgical resection remains to be the standard choice for the treatment of HCC, however,more than 70% of HCC patients develop metastasis and recurrence within 5 years after surgery, and most of the recurrence/metastasis usually occur in the early stage after hepatic resection. The high incidence of recurrence/metastasis has become a major obstacle to improve the clinical outcomes of HCC patients. In such a scenario, identifying novel molecular biomarkers that are suitable for the prediction of early recurrence/metastasis of HCC will aid in developing more effective therapeutic strategies and therefore greatly benefit the many HCC patients. Annexin A4 is a member of Annexins. It has been reported that Annexin A4 was aberrantly expressed in various cancers, including ovarian, colorectal, gastric, renal, pancreatic cancers etc, and confirmed to be closely associated with the development and malignant progression of these cancers. In our previous quantitative proteomics study, the results have showed that Annexin A4 might be a very promising prognostic biomarker for the early recurrence/metastasis of HCC. However, to the best of our knowledge, the research on clinical relevance of Annexin A4 expression with HCC is few and the related mechanism of Annexin A4 in HCC remains largely unknown. Therefore, the role of Annexin A4 in HCC requires further elucidation.Objective: 1. A large-cohort clinical study was conducted to examine the expression levels of Annexin A4 in HCC patients with different recurrent/metastatic time and systematically evaluated the prognostic value of Annexin A4 in HCC patients after curative resection. 2. To study the effects of Annexin A4 overexpression or knockdown on the biologicalbehaviors of HCC cells. 3. To investigate the underlying molecular mechanisms of Annexin A4 involving in the invasion and metastasis of HCC.Methods: 1. A total of 141 HCC specimens were randomly collected from HCC patients undergoing curative resection. The HCC patients enrolled in this study were divided into 3 groups according to the time of recurrence/metastasis after operation: the patients who had recurrence/metastasis within 1 year after operation; the patients whose recurrence/metastasis occurred between 1~2years after operation; the patients who had no recurrence/metastasis within 2 years of operation. By using Q-PCR, Western blot and immunohistochemistry, the expression of Annexin A4 was examined in HCC samples, and the clinical significance of expression of Annexin A4 was analyzed by SPSS19.0. 2. Q-PCR and Western blot was used to detected the expression of Annexin A4 in HCC cell lines with different metastatic potential. Stable Annexin A4 overexpressing or knockdown clones were established in HCC cell lines by using lipofectin transfection and sh RNA-mediated RNA interference. Afterwards, functional assays, such as CCK-8, flow cytometry and transwell assay, were performed to study the effects of Annexin A4 overexpression or silencing on tumor cell proliferation, cell cycle distribution, cell apoptosis, cell migration and invasion in vitro. Subcutaneous tumor model as well as orthotopic transplantation model were established in nude mice to investigate the effects of Annexin A4 expression on the growth and invasion of tumors in vivo. 3.Comparative proteomic method was applied to identify differentially expressed proteins in either Annexin A4-overexpressing Hep G2 cells or Annexin A4-silencing MHCC-97 H cells as compared to their corresponding control cells. Then, some of the differentially expressed proteins were further verified by Q-PCR and Western blot to explore the molecular mechanisms of Annexin A4-mediated HCC invasion and metastasis.Results: 1. Q-PCR and Western blot analysis showed that the Annexin A4 expression was significantly up-regulated in the recurrent/metastatic HCC tumor, especially in the HCC tumors with early recurrence/metastasis, and this result was in concordance with our previous proteomic analysis. Clinicopathological analysis revealed that Annexin A4 expression was significantly associated with Portal vein tumor thrombosis(P=0.03) and advanced BCLC stage(P=0.002), but did not correlated with other clinicopathologic characteristics such as age, gender, serum AFP level, liver cirrhosis, maximal tumor size, tumor encapsulation and tumor differentiation. Kaplan–Meier analysis indicated that patients with high expression of Annexin A4 were more prone to develop tumor recurrence/metastasis(Log-rank test, P<0.001) and had shorter overall survival time than those with low expression(Log-rank test, P<0.001). Cox's multivariate proportional hazards model validated that Annexin A4 expression was an independent risk factor for TTR(P=0.032) and OS time(P=0.009) in HCC after curative resection. 2. Q-PCR and Western blot analysis showed that Annexin A4 expression was significantly increased in the highly metastatic cell lines relative to the poorly metastatic HCC cell lines. Moreover, Annexin A4 was abundantly expressed in MHCC-97 H cells at the highest level among these HCC cell lines, whereas lowest in Hep G2 cells. Stable overexpression of Annexin A4 in Hep G2 cells and successful sh RNA-mediated knockdown of Annexin A4 in MHCC-97 H cells were confirmed by Q-PCR and Western blot. In vitro functional analysis indicated that up-regulation of Annexin A4 actually promoted migration and invasion of Hep G2 cells, whereas down-regulation of Annexin A4 dramatically inhibited migration and invasion of MHCC-97 H cells. However, there were no changes in cell proliferation, cell apoptosis and cell cycle distribution when Annexin A4 expression was up- or down-regulated in HCC cells. In additon, the resullts from in vivo experiments indicated that silencing of Annexin A4 in MHCC-97 H cells reduced the incidence of lung metastasis in orthotopic transplantation model, but did not inflence subcutaneous tumor growth. In this study, we did not observe the effects of Annexin A4 overexpression on tumor invasion and metastasis in vivo, which mainly due to the poorly tumorigenic and metastatic potential of Hep G2 cells(ATCC). 3.The protein expression files of HCC cells with Annexin A4 overexpression or knockdown and their corresponding control cells were obtained through a proteomic-based approach and a variety of proteins were identified to be associated with Annexin A4 expression. Among these proteins, N-cadherin and vimentin were found to be up-regulated 1.85 folds and 1.53 folds in Hep G2 cells with Annexin A4 overexpression while down-regulated 0.39 folds and 0.45 folds in MHCC-97 H cells with Annexin A4 knockdown. The alterations of N-cadherin and vimentin expression in HCC cells implied that Annexin A4 might be involved in regulating EMT process. During cell culture, we observed that Hep G2 cells ectopically overexpressing Annexin A4 exhibited a spindle-like fibroblastic morphology, whereas MHCC-97 H cells with Annexin A4 knockdown took on a cobblestone-like appearance of normal epithelium. Furthermore, EMT-related markers were detected using q PCR and Western blot. Results showed that Annexin A4 overexpression in Hep G2 cells resulted in decreased expression of epithelial marker(E-cadherin) and increased expression of mesenchymal marker(N-cadherin and vimentin). Moreover, the expression of Zeb1, known as an important EMT-promoting transcription factor, were also increased when Annexin A4 was overexpressed. By contrast, slencing of Annexin A4 caused enhanced E-cadherin expression and reduced N-cadherin expression, together with a concomitant reduction of Zeb1 in MHCC-97 H cells, which indicated the reverse of EMT process. However, modulation of Annexin A4 expression in HCC cells did not influenced the expression of other well-known EMT transcriptional factors, including Twist, Snail, Slug. Collectively, these results suggested that Annexin A4 may facilitate HCC cell migration and invasion via induction of EMT, thereby promoting HCC development and metastasis.Conclusions: 1. Annexin A4 is an independent risk factor predicting early recurrence/metastasis and poor survival of HCC patients. It can serve as a novel prognostic biomarker for HCC. 2. Annexin A4 overexpression and knockdown did not affect cell proliferation, cell apoptosis and cell cycle distribution of HCC cells, but can markedly promote the migation and invasion of HCC cells. It may play a pro-metastatic role in HCC progression.3. Annexin A4 may induce EMT through up-regulating Zeb1 expression and consequently accelerated migration and invasion of HCC cells. However, the underlying molecular mechanisms by which Annexin A4 regulate Zeb1 deserve futher in-depth exploration in future studies.