Expression And Role Of Metadherin In Hepatocellular Carcinoma, A Novel Therapeutic Target

Introduction：Hepatocellular carcinoma (HCC) is the fifth most common cancer and thethird most common cause of cancer mortality worldwide. Since the1990s, it hasbeen ranked the second leading cause of cancer deaths in China.Epidemiological studies have identified major risk factors for HCC, includinginfection with hepatitis B and C virus (HBV and HCV), exposure to aflatoxinB1, a high intake of alcohol, as well as metabolic diseases of liver. Among theserisk factors, growing evidence support the point that HBV infection as the mostcommon cause of HCC. According to a recent study,53%of HCC casesworldwide are associated with HBV infection. Because of endemic HBVinfection, HBV-related HCC has a higher prevalence in China than in Europeand America. Despite remarkable advances in diagnosis and therapeutictechniques, only a minority of patients with HCC are diagnosed at early stagesand are candidate for curative treatments, and the recurrence rate may be as high as50%at3years. In contrast, the remaining patients face extremely poorprognoses. The main cause of the extremely poor prognoses for patients withHCC is that there are not effective therapeutic target gene and biomarkers usedto diagnose or evaluate HCC because of unclear molecular mechanismsresponsible for HCC. In addition, the molecular mechanisms responsible for thepathogenesis of HBV-related HCC are completely different from those ofHCV-related HCC. Furthermore, conventional antiviral treatment, such aslamivudine, inhibits HBV replication but cannot reverse HBV-related changesin downstream signaling pathways, which are closely related to the occurrenceand development of HCC. The overexpression of oncoprotein metadherin(MTDH, also known as astrocyte elevated gene-1, AEG-1) has beenfrequently observed in a variety of primary human tumors. Furthermore,published studies on some cancers have revealed that MTDH is a novel anduseful prognostic marker for cancer progression, and its overexpression isassociated with an unfavorable prognosis. The data that have accumulated overthe last few years suggest that that overexpression of MTDH enhances severalhallmarks of cancers, including aberrant proliferation, resistance to apoptosisand increased adhesion and invasiveness through the activation of differentsignaling molecules including Ha-ras, NF-κB and Wnt/β-catenin, which overlapwith the signal pathways associated with HBx. However, the role of MTDH inHCC remains unclear. In the present study, we evaluated expression of MTDHin HCC and investigated its role in carcinogenesis of HCC.Methods：In the present study, by western blotting and qRT-PCR we detected MTDHexpression in normal liver, chronic hepatitis B and HBV-related HCC tissues.By immunohistochemistry method, MTDH protein expression was confirmed. Furthermore, We analyzed the correlations between MTDH expression level andclinicopathological factors and investigated its prognosis role in HBV-relatedHCC. After detecting expression of MTDH in3HCC cell lines, MTDH-shRNAand pCMV-MTDH were used to transfect different HCC cell lines and assessthe function of MTDH in tumor progression by MTT, flow cytometry, westernblotting, matrigel-coated transwell cell culture chambers, scrach test and nudemouse xenograft assays.Results：1． The data showed that MTDH expression levels were elevated in thehepatitis B tissues and especially in the HBV-related HCC tissues compared tonormal liver tissues. MTDH expression was mainly localized in the cytoplasmicregion, and nuclear MTDH staining was not detected in any sections. Nosignificant difference was found between hepatitis B patients with cirrhosis andthose without cirrhosis (P=0.139). Furthermore, a statistical analysis revealedthat MTDH expression significantly correlated with the American JointCommittee on Cancer (AJCC,7thedition) stage (P=0.020), T classification(P=0.007), N classification (P=0.044), vascular invasion (P=0.006), andhistological differentiation (P=0.020) in the HBV-related HCC patients. For theM classification (P=0.083), all of the6patients with M1exhibited high MTDHexpression. The log-rank test revealed that the MTDH expression levelcorrelated significantly with the survival time of patients with HCC (P=0.001).Furthermore, the median survival time of patients with high MTDH expressionlevel was30months (95%confidence interval,23.4-36.8months), while themedian survival time of those with low expression was49months (95%confidence interval,42.5-52.8months). The5-year cumulative survival rate was65.1%in the low MTDH expression group, whereas it was only19.4%in thehigh MTDH expression group. Multivariate survival analysis revealed that MTDH expression, AJCC stage, T classification, and tumor differentiation areindependent prognostic factors.2． MTDH was upregulated in HepG2、MHCC97-H、Huh7cells, andhighest in MHCC97-H, lowest in HepG2. Using MTDH-shRNA, we establishedstable MHCC97-H cell clone in which MTDH expression was inhibited at bothtranscriptional and translational levels. The data showed that the specificdownregulation of MTDH inhibits potential of proliferation, promotes apoptosisand induced cell arrest in the G0/G1phase of cell cycle in HCC cells. MTDHalso affected the expression of downstream molecules that are implicated inoncogenesis of HCC, including caspase-3, murine double minute-2, signaltransducer and activator of transcription3, forkhead transcription factors-3a andextracellular signal-regulated kinase1/2. On the contrary, overexpression ofMTDH resulted in the opposite effect.3． Establishment of subcutaneous xenograft in flanks of nude micerevealed that tumor growth and MTDH expression in tumor were significantlyinhibited by MTDH–shRNA compared to the control.Conclusion：Taken together, these results suggest that MTDH could be a potentialprognostic marker for overall survival and tumor progression and play key rolein oncogenesis of HCC. In addition, according to our findings, HBV may have arole as an initiator of MTDH activation during the initiation of HBV-relatedHCC. MTDH plays a key role in oncogenesis of HCC via regulating differentsignaling pathways or molecules. MTDH may be a effective molecular target ofRNAi for new anticancer agents to prevent HCC progression and metastasis.