| Hepatocellular carcinoma (HCC) is one of the most prevalent cancers and the 5th leading cause of cancer-related deaths worldwide. Although radical hepatectomy is considered as one of the first line treatment for patients with early HCC, the surgical outcome is far from satisfactory due to the likelihood of intrahepatic and extrahepatic recurrence. In clinical practice, it is still difficult to predict the prognosis for patients with early HCC due to the similarity in clinicopathologic characteristics. In fact, early-stage HCC also tends to recur and the 5-year recurrence rate was up to 60%. Patients with early-stage HCC are recommended to receive hepatectomy. Meanwhile, the number of the early stage HCC patients who undergo hepatic resection has been gradually increasing as a result of progresses in the surveillance and early detection. Although great efforts have been made for investigating precise prognostic biomarker for early HCC, the optimal candidates with clinical applicability are still lacking and urgently required. Therefore, it is clinically meaningful that metastasis and prognosis related molecule and its functional mechanism were found. In this study, the relationship between expression of AAH and the prognosis of HCC patients undergoing curative resection was evaluated based on the former study that AAH was overexpressed in HCC tissues and could regulate the invasiveness and proliferation of HCC cells in vitro.Part 1 Expression and clinical meaning of AAH in hepatocellular carcinomaPurpose:The association between the overexpression of aspartyl-(asparaginyl)-β-hydroxylase (AAH) and the invasiveness of hepatocellular carcinoma (HCC) in vitro has been previously studied and reported. However, the prognostic value of AAH expression in HCC remains unclear. This study was to investigate the relationship between AAH expression and tumor recurrence and patients'survival.Methods:Overexpression of AAH was found by cDNA, and determined by RT-PCR, immunohistochemistry (IHC). The expression of AAH and prognosis of HCC patients undergoing hepatectomy, especially at BCLC 0/A stage patients, was evaluated in 233 consecutive patients recruited between January 2004 and June 2004 prospectively.Results:We identified AAH as the most overexpressed gene in HCC by cDNA microarray hybridization. A prospective observational study of 233 patients undergoing curative resections indicated that AAH expression was an independent factor affecting the recurrence (HR,3.161,95% CI,2.115-4.724, P<0.001) and survival (HR,2.712,95% CI,1.734-4.241, P< 0.001) by multivariate analysis. Patients with AAH overexpression had a poorer prognosis in contrast with those with AAH underexpression (P<0.001 for both recurrence and survival). In BCLC stage A patients with AAH over-or underexpression, the tumor recurrence and survival rates were also statistically different (45% and 85% vs.16% and 33% in 1-and 3-year cumulative recurrence rates; 73% and 37% vs.90% and 80% in 1-and 3-year survival rates? P<0.001 for both). Furthermore, in stage A patients with tumor=5cm in diameter, the time to recurrence (TTR) was 26.7±1.6 versus 51.9±2.8 months, and the one-and three-year survival rates were 97% and 52% versus 100% and 90% in AAH over-and underexpression patients (P<0.001 for both).Conclusion:Consequently, AAH overexpression in HCC was strongly correlated with worse surgical outcomes and this molecule likely provides a more precise prognostic predictor in early stage HCCs.Part 2 The relationship between Gas6/Ax1 pathway and AAH, and the biological function of Gas6/Ax1.Purpose:To investigate the mechanism of AAH regulating HCC proliferation and metastasisMethods:The expression of Gas6 and Ax1 in HCC tissues and cell lines SMMC-7721, HepG2, Hep3B, PLC, Hu7 and HCC adjacent nontumorous tissues, normal cell line L02 were evaluated. The active form of Axl expression was examined by western blot analysis when AAH was up-or down-regulated in SMMC-7721 cell line. In addition, the proliferation and invasiveness of the cell were also evaluated correspondingly. Finally, whether AAH participated in the interaction of Gas6 and Ax1 was confirmed by immunoprecipitation.Results:The expression of Ax1 in HCC tissues and cell lines were higher than that in nontumorous tissues and L02, but no difference was observed in Gas6 expression. And the active form of Axl expression was positively correlated with the expression of AAH. When Axl was up or down regulated, the proliferation and invasiveness of SMMC-7721 altered correspondingly. In addition, that AAH participated in the interaction of Gas6 and Ax1 was confirmed by immunoprecipitation. Conclusion:The mechanism of AAH regulating HCC proliferation and metastasis might be through the Gas6/Ax1 pathway, and Axl played an oncogene role in HCCPart 3 The relationship between Notchl and AAH, and the prognostic value of Notchl in early stage HCCPurpose:Multiple EFG-like domains were found in the ligands and receptors of Notch family. In this part, that whether Notchl could be regulated by AAH would be demonstrated. Considering the function of Notchl signaling pathway and its role in tumorigenesis, we inferred that abnormal expression of Notchl could be detected in the early process of HCC formation, and could predict the surgical outcomes of early stage HCC patients.Methods:The expression of Notchl-4 in HCC tissues and cell lines SMMC-7721, HepG2, Hep3B, PLC, and Hu7 was evaluated. The expression of Notchl was examined by western blot when AAH was up-of down-regulated in Hu7 cell line. The differential expression of Notchl was also examined by RT-PCR, western blot and immunohistochemistry. The correlation between expression level of Notchl and outcome after curative hepatectomy was investigated in training cohort of 246 patients with hepatocellular carcinoma recruited prospectively, and verified by the results of validation cohorts with 227 patients retrospectively. Patients with tumor =5cm in diameter from two cohorts which were recognized as training set (n=83), validation set (n=108), respectively, and patients at BCLC early (0/A) stage were further investigated.Results:Neither Notchl nor Notch2 could be found in HCC tissues nor cell lines, except that Notchl could only be found in Hu7 cell line. The expression of Notch3 and Notch4 were irregular. Expression of Notchl was positively correlated with the expression of AAH by western bot analysis. Compared with paired nontumorous tissue, tumor tissue had decreased expression of Notchl. In training set, patients with high Notchl expression had better prognosis than those with low expression (49.5±2.4 vs 21.0±5.5 months, P<.001 for time to recurrence; 1-,3-year,100%,87%vs 86%, 43%, P<.001 for survival). Multivariate analysis indicated that Notchl expression level was an independent prognostic factor for both recurrence and survival (hazard ratio,95% confidence interval,4.310,2.108-8.813; 6.093,2.494-14.881, respectively). The results were fully confirmed in the analyses of validation set. Expression level of Notch1 was also an independent prognostic factor,for BCLC 0/A stage patients from two cohorts.Conclusion:Although expression of Notchl correlated positively with that of AAH, the mechanism of AAH was not through Notchl pathway. The expression level of Notchl in tumor tissue was closely associated with time to recurrence and survival of patients with early-stage hepatocellular carcinoma undergoing hepatectomy.In conclusion, overexpression of AAH was found in HCC tissues and cell lines, and was associated with proliferation and metastasis of HCC. Overexpression of AAH in hepatocellular carcinoma is associated with worse surgical outcomes. Axl was regulated by AAH, and the mechanism of AAH might be through Gas6/Axl pathway. Although Notchl could be regulated by AAH, the function of AAH was not through the Notch pathway. Notchl might play a tumor suppressor role in HCC and its expression level was linked to time to recurrence and survival of patients with early stage HCC.
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