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Expression and Prognosis Analysis of Asparagine Synthetase and Its Functional Role in Hepatocellular Carcinoma

Author: ZhangBo
Tutor: YangJiaHe
School: Second Military Medical University
Course: Surgery
Keywords: Hepatocellular carcinoma (HCC) asparagine synthetase (ASNS) L-asparaginase prognosis
CLC: R735.7
Type: PhD thesis
Year: 2013
Downloads: 21
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Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignant neoplasms and the third leading cause of death from cancer worldwide. Despite many kinds of treatments, such as surgical resection, local ablation, transplantation, transcatheter arterial chemoembolization (TACE) and so on, the prognosis still remains dismal. Across all countries,5-year overall survival of HCC is only3-5%. The poor prognosis of patients with HCC is largely due to the high frequency of recurrence and metastasis after surgical resection, in addition to resistance to systemic chemotherapy. Therefore, the limited therapeutic options and poor prognosis have triggered the search for molecular markers related to clinical outcomes, which will provide new targets for intervention of progression of HCC.The asparagine synthetase (ASNS) gene, encoding the enzyme that catalyzes the biosynthesis of asparagine from aspartate in an ATP-dependent reaction for which glutamine is the nitrogen source. The transcription of ASNS gene is highly regulated by the nutritional status of the cell. Early studies showed that elevated expression of ASNS was correlated with drug resistance of leukemic cells to L-asparaginase, which is a universally used component of childhood acute lymphoblastic leukemia (ALL) and some forms of acute myeloblastic leukemia (AML) treatments. Similarly, ASNS was considered as a causal, predictive biomarker for L-asparaginase activity in ovarian cancer cell. Furthermore, it has also been showed that enhanced expression of ASNS protects pancreatic cancer cells from apoptosis induced by glucose deprivation and cisplatin. Recent studies suggested that ASNS was up-regulated in castration-resistant prostate cancer (CRPC) and depletion of asparagine using ASNS inhibitors might be a novel strategy for targeting CRPC cells. However, the expression and the functional roles of ASNS in solid tumours still remain uncertain, especially in HCC. The significance of ASNS expression level in the prognostic evaluation of HCC patients who undergo hepatectomy has not been reported yet. By proteomics approach, we identified ASNS higher expressed in HCC tumour tissues compared with normal liver specimens. Hence, it is meaningful to investigate the clinical significance and biological function of ASNS in the development of HCC.This study aimed to investigate the ASNS expression pattern and determine its contribution to HCC progression and clinical prognostic values. In addition, the functional role of ASNS in HCC development and its therapeutic potential were also addressed. The results indicated that expression of ASNS was significantly correlated with clinical characteristics, predicting HCC patients’outcome after surgical resection and chemotherapy target for HCC.In the present study, we investigated ASNS is overexpressed in HCC tumour specimens.By proteomics approach, we identified that ASNS protein level was much higher in HCC tumour tissues than normal liver specimens. Real-time RT-PCR assay was performed for transcripts of ASNS from frozen paired samples derived from58patients with HCC and18normal live tissues. The expression of ASNS mRNA varied greatly between tumour and their adjacent tissues, with76%(44of58) tumour tissues expression higher than normal like tissues. The upregulation of ASNS was further confirmed by Immunoblotting and immuneohistochemistry assay.ASNS protein was stained in liver tissues of the DEN carcinogenesis rat by immuneohistochemistry to observe the dynamic expression of ASNS in the process from hepatitis, cirrhosis to HCC. ASNS expression was significantly higher in DEN treated liver than control group after injecting DEN for8weeks, and up to highest expression in12-14weeks, which period is advanced cirrhosis and early HCC stage. While the ASNS expression was moderately decreased in16-20weeks, which period is advanced HCC stage. In accordance with the findings in animal model experiments, in clinical samples, high expression of ASNS was associated with smaller tumour size and early tumour stage and low ASNS expression was correlated with larger tumour size and advanced tumour stage.The relationship between ASNS expression levels in tumour tissues and the clinicopathological characteristics of269patients was examed in the TMA analysis. Pearson χ2test indicated that expression of ASNS was significantly correlated with serum AFP level (P=0.040), tumour size (P=0.002), microscopic vascular invasion (P=0.003), tumour encapsulation (P=0.007), TNM stage (P=0.039) and BCLC stage (P=0.039). Other clinical characteristics were not closely related to the expression of ASNS, including age, gender, tumour number, intrahepatic metastasis, distant metastasis, portal venous invasion, cirrhosis, and hepatitis background. Kaplan-Meier survival curves with comparisons of high ASNS expression versus low expression in269HCC patients are shown. ASNS expression levels were negatively correlated with1-year,3-year and5-year survival rates (52.6%,29.2%and25.3% for high ASNS expression versus37.4%,20.0%and13.9%for low expression; P=0.002,0.005and0.002). Additionally, the1-year,3-year and5-year disease-free survival rates in high ASNS expression patients were lower than those in low ASNS expression patients, however, no significant difference was observed.Univariate analysis of recurrence-related and survival-related clinicopathological variables revealed that ASNS and serum AFP levels were predictors for OS. Gender, HBV, tumour size, intrahepatic metastasis, portal venous invasion, distant metastasis, microscopic vascular invasion, encapsulation, TNM stage and BCLC stage were statistically correlated with both recurrence and survival. These individual parameters were further subjected to multivariate Cox proportional hazards model, which demonstrated that ASNS expression level, together with HBV, intrahepatic metastasis, microscopic vascular invasion and BCLC stage was strongly associated with OS. ASNS was an independent prognostic indicator for the survival of HCC patients (HR0.744,95%CI0.565-0.979, P=0.035).We used lentivirus delivered shRNA targeting ASNS to infect HCC cells, and constructed stably transfected cells. Also, HCC cells were transient transfected with GV142/ASNS vector. We found that overexpression of ASNS in HCC cells suppressed cell proliferation by CCK-8assay. Furthermore, ASNS knockdown revealed increased proliferation and colony formation in ASNS knockdown cells compared with that of control cells. The Cell cycle distribution demonstrated ASNS knockdown could hasten cell cycle progression In addition, we found Cyclin Dl protein decreased in overexpressed ASNS cells, while increased in knockdown cells. Stably transfected HCC cells were inoculated into flanks of nude mice and the effect of ASNS on xenograft tumour growth was also observed. Compared with control cells, ASNS knockdown cells showed a significant increase in tumour size. These results indicated that ASNS knockdown promoted HCC growth both in vitro and in vivo.Transwell experiments showed that depletion of ASNS markedly increased cell migration and invasion capacity. Restoration of ASNS expression in SMMC7721cells repressed cell migration. For further examining the effect of ASNS knockdown, stably transfected MHCC97H cells were injected into tail vein of nude mice. Six weeks later, ASNS knockdown resulted in significantly increased number and sizes of pulmonary metastatic lesions. Furthermore, ASNS knockdown group had a shorter survival period in comparison with control group. These results demonstrate an enhanced metastatic potential in low ASNS expression HCC cells. HCC cells were treated with L-asparaginase and cell proliferation was detected by CCK-8. Compared with cells with high ASNS (MHCCLM3and MHCC97H), L-asparaginase exerted a much higher effect on cells with low ASNS expression (SMMC7721and PLC). Moreover, in the detection of stably transfected HCC cells, it showed decreased proliferation in ASNS knockdown cells in the presence of L-asparaginase compared with control. Futhermore, the effect of L-asparaginase was also observed in vivo. Nude mice bearing MHCCLM3tumour xenografts were treated with L-asparaginase every other day by intraperitoneal injection. ASNS knockdown group revealed a delayed tumour formation and a significant reduction in tumour size compared with that of control group. These results revealed an increased sensitivity of low ASNS expression cells to L-asparaginase.In this study, we presented the upregulation of ASNS at both mRNA and protein levels in HCC. The association of ASNS expression with clinicopathological features was further investigated in HCC patients by TMA, which showed a significant correlation of ASNS expression with many clinical features, indicating low expression of ASNS was associated with malignant clinicopathological characteristics. In DEN carcinogenesis rat, ASNS was high expressed from inflammation stage to advanced HCC stage and reached a peak at advanced cirrhosis and early HCC stage. Moreover, moderated decreased ASNS expression was observed in advanced cancer stage in comparison with early cancer stage, which was validated by human HCC clinical specimens. The Kaplan-Meier analysis showed that patients with HCC, who had high ASNS expression in general, had better prognosis than those with low expression. Multivariate analysis revealed that ASNS expression level was an independent and significant prognostic indicator affecting survival after surgical resection.In further confirming the role of ASNS in HCC, the result showed increased proliferation and colony formation ability in vitro and elevated tumourigenicity of xenografts in nude mice in low ASNS cells. In accordance with correlation analysis, these results imply that ASNS is a tumour growth suppressor in HCC. Overexpression of ASNS severely inhibited the migration capacity and ASNS knockdown significantly increased migration and invasion abilities in vitro. The result was further confirmed by pulmonary metastatic model in vivo.In later investigations, we also observed that ASNS overexpression increased apoptosis sensitivities of cells upon various stimuli. HCC cells with low ASNS expression were more sensitive to L-asparaginase than that exhibit high ASNS expression both in vitro and in vivo.In conclusion, this study explored the effects of ASNS in tumour progression and prognosis significant in HCC patients. ASNS expression level was an independent and significant prognostic indicator affecting survival after surgical resection. Low expression of ASNS might be an indicator for the patients treated with L-asparaginase.

Expression and Prognosis Analysis of Asparagine Synthetase and Its Functional Role in Hepatocellular Carcinoma

Abstract

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