AMACR And Galectin-4 Serve As Multi-biomarker For The Early Recurrence/Metastasis Of HCC

Hepatocellular carcinoma(HCC) is one of the most common malignancies and also reported to be the third leading cause of cancer death in the world. While in China, it has become the second cause of death. The high deathrate of HCC is mainly due to the recurrence/metastasis after the curative liver resection. Intrahepatic recurrence and / or extrahepatic metastasis may ocurr in approximately 75 percent of patients who have had surgeical treatment. With the development of the imaging technique, the diagnosis of early recurrence/metastasis of HCC has been improved greatly, but the high prize causes its low utilization rate. Therefore, it is necessary to identify new biomarkers for predicting and distinguishing the early recurrence / metastasis, which will certainly give important guides for identifying patients with HCC with high risk of early recurrence / metastasis. In our previous quantitative proteomics study, the results have showed that AMACR and Galectin-4 might be the very promising prognostic biomarkers for the early recurrence / metastasis of HCC. However, we found no paper mentioning the clinical feather about these two proteins, and the mechanisms of AMACR and Galectin-4 involved in early recurrence / metastasis of HCC still remain unknown.Objective: 1. To evaluate AMACR and Galectin-4 proteins’ prognostic potential. 2. To investigate the capacity for AMACR and Galectin-4 serving as the multi-biomarker for the early recurrence / metastasis of HCC. 3. To illuminate the mechanisms of AMACR and Galectin-4 involvied in early recurrence / metastasis of HCC.Methods: 1. Samples from HCC early recurrence / metastasis group(R/M≤12Months), late recurrence / metastasis group(R/M12-24months) and no recurrence / metastasis group(R/M>24Months) respectively were examined for AMACR m RNA level by Q-PCR. Samples from patients(n=158) were used for the construction of tissue microarray. The expression level of AMACR was determined by immunohistochemical staining. A large-scale cohort clinical study between the expression of AMACR and some major clinical parameter has been performed to assess the prognostic potential of AMACR for the early recurrence / metastasis of HCC.2. Samples were divited into three groups: early recurrence / metastasis group, late recurrence / metastasis group and no recurrence / metastasis group. Galectin-4 m RNA level and protein level from every group was tested by Q-PCR and Western Blot respectively. Samples from patients(n=201) were used for the construction of tissue microarray. The expression level of galectin-4 was determined by immunohistochemical staining. A large-scale cohort clinical study between the expression of galectin-4 and some major clinical parameter has been performed to assess the prognostic potential of galectin-4 for the early recurrence / metastasis of HCC. 3. The circulation AMACR and galectin-4 in HCC patients’ serum has been detected by ELISA. A logistic regression analysis was used to draw a receiver operating characteristic(ROC) curve. The area under the curve(AUC) was calculated to compare the performance of different serum biomarkers as a diagnostic test. 4. We established AMACR and galectin-4 stable overexpressing and knocked-down HCCLM3 cell lines respectively. Transwell was used to examine the function of AMACR and galectin-4 in cancer cell migration and invasion. We evlaueated the impact of AMACR and galectin-4 to the tumor through nude mouse tumorigenicity assay. The different expressed proteins between the high galectin-4expressioncell line and low galectin-4cell line were analysed by proteomics.Results 1. Q-PCR analysis revealed that the AMACR expression was down-regulated in the early R/M(recurrence / metastasis) group comparing with the no R/M group(P=0.004). Some important clinical parameters such as: AFP(P=0.012), tumor numbers(P=0.005), dissemination to regional lymph nodes(P=0.042), tumor capsule(P=0.026), and Portal vein tumor thrombosis(P=0.049) are significantly correlated with the low expression of AMACR. The expression of AMACR was an independent factor for the survival of HCC patients(P=0.031). The median survival time was 17 months in the low expression group comparing with 45 months in the high expression group(log-rank, P<0.001).2. Q-PCR and WB showed that the galectin-4 expression was significantly decreased at both the m RNA level and the protein level in the early R/M(recurrent/metastatic) HCC tumor. Lower expression of galectin-4 was well associated with bigger tumor size(P=0.036), poor tumor differentiation(P=0.011), micro-vascular invasion(P=0.004), and more advance tumor-node metastasis(TNM) stage(P= 0.015). Cox’s multivariate proportional hazards model showed that galectin-4 expression was an independent poor prognostic factor for TTR(P=0.003) and OS time(P=0.002) in HCC after curative resection. Kaplan-Meier analysis indicated that patients with lower galectin-4 expression had higher R/M rate(log-rank, P<0.001) and shorter OS time(log-rank, P<0.001) than those with high galectin-4 expression(log-rank, P<0.001). 3. It was found that the serum levels of AMACR and galectin-4 were significantly higher in the early R/M(recurrence / metastasis) of HCC patients compared with those late R/M of HCC patients(P<0.05). The area under the receiver operating characteristic curve of AMACR and galectin-4 was 0.703 and 0.712, respectively. And AMACR in combination with galectin-4 improved the diagnostic accuracy in distinguishing early R/M from late R/M of HCC patients(AUC=0.799). 4. The ability of cancer cell migration and invasion could be significantly reduced through overexpression of galctin-4, but upregulated by knocking down of galectin-4 in vitro. Compared with high expression group, the low expression of galectin-4 could significantly accelerate the tumor grouth in vivo(P=0.0496). Proteomic analysis result: Between high expression of galectin-4 and low expression of galectin-4 cell lines, 127 proteins have different expressions. And most of them involved in translation, fatty acid or amino acids metabolism in HCC.Conclusions 1. AMACR and galectin-4 might be the potential prognostic markers for predicting early recurrence/metastasis of HCC after hepatectomy. The down regulation of AMACR or galectin-4 is significantly associated with poor prognosis of patients with HCC. 2. Combining detection of serum AMACR protein and galectin-4 protein improves the accuracy in diagnosis of early recurrent/metastasis of HCC. 3. Galectin-4 may impact the amino acid and fatty acid metabolism of hepatoma cell. Downregulating galectin-4 might enhance the ability of migration and invasion of hepatoma cell.