Molecular Biomarkers For Predicting Tumor Recurrence And Prognosis In Hepatocellular Carcinoma After Liver Transplantation

Hepatocellular carcinoma (HCC) is one of the most prevalent cancer worldwide. China alone accounts for55%of the world's cases, because of the high prevalence of chronic hepatitis B virus infection and liver cirrhosis. Currently, liver transplantation (LT) is the only potentially curative therapeutic modality that can treat both cancer and liver dysfunction simultaneously, and offers a reasonable survival benefit for selected patients with HCC and end-stage liver disease. However, the long-term survival of patients following surgery remains unsatisfactory due to the high frequency of recurrence and metastasis. Therefore, a better understanding of the molecular mechanisms underlying HCC recurrence, identifying more accurate predictive biomarkers and establishing a novel model that can assess tumor recurrence and prognosis is highly desirable, which may offer an improved diagnostic and prognostic capability and the development of effective therapeutic strategies. Part Ⅰ Global transcriptome profiling identifies MACC1associated with tumor aggresiveness and poor clinical outcome in hepatocellular carcinomaAims:Metastasis-associated in colon cancer-1(MACC1) acts as a promoter of metastasis in several tumors, however, its cellular function and molecular mechanism in hepatocellular carcinoma (HCC) remain unclear. We aim to evaluate the biological effects and prognostic significance of MACC1and its downstream genes in HCC.Methods:Global transcriptome profiling based on AgilentG3Human GE8*60K gene chip analysis identifies HCC related transcripts. We examined the expression of MACC1by quantitative PCR, western blot, and immunochemical analyses. The effects of MACC1overexpression or depletion in HCC cells were determined in viability, apoptosis, migration and invasion assay and observed its tumorigenesis in nude mice. Changes in gene expression induced by MACC1were identified by microarray and immunoblot analyses.Results:Transcriptomic analysis of8HCC tissues identifies MACC1as an upregulated gene in poorly differentiated tumors, compared with the well or moderately differentiated tumors. MACC1was overexpressed in HCC cell lines and tumor tissues, and was positively correlated with their metastatic potential. MACC1re-expression promoted cell viability, migration and invasion; and upregulated the expression of Met, FAK, and phospho-FAK. While down-regulation of MACC1induced the opposite effects, and it also increased the response of HCC cells to apoptotic stimuli and sensitized doxorubicin and etoposide chemotherapy. Microarray analysis revealed that MACC1modulated the expression of multiple genes involved in tumor metastasis. In160HCC patients who underwent liver transplantation (LT) therapy, those with MACC1high or FAKhigh in HCCs showed a significantly shorter overall survival and higher cumulative recurrence rates after liver transplantation (LT), compared with MACC1low or FAK1ow group. Furthermore, in patients exceeding the Milan criteria or the UCSF criteria, those with a high expression level of MACC1revealed a significantly higher cumulative recurrence rates and shorter overall survival. Multivariate analysis indicated that MACC1alone or combination of MACC1/FAK was an independent prognostic factor for overall survival and cumulative recurrence.Conclusions:MACC1is a critical oncogene in controlling HCC metastasis. MACC1may regulate tumor cell viability, apoptosis, invasion and tumorigenesis through HGF/Met and FAK pathway. MACC1or combination of MACC1/FAK could serve as a novel biomarker in predicting the prognosis of HCC after LT. Part Ⅱ Predictive value of long noncoding RNA for tumor recurrence and prognosis in hepatocellular carcinoma following liver transplantationAims:We aim to screen and identify novel long noncoding RNA (lncRNA) related to hepatocellular carcinoma (HCC), and evaluate the biological effects and its underlying molecular mechanisms of lncRNAs in HCC. Based on the above findings, we will establish a model based on lncRNA that can assess the risk of tumor recurrence and prognosis after liver transplantation (LT), and evaluate its predictive power.Methods:One hundred and sixty consecutive liver transplant recipients with HCC were selected. Their perioperative laboratory examination results, treatment protocol and the status of HCC recurrence and survival were the primary parameters used to assess their risk of post-transplant HCC recurrence and overall survival. LncRNA were screened by quantitative PCR in10HCC cell lines and76paired HCC tissues. The effects of HOTAIR depletion in HCC cells were determined in viability, apoptosis, and invasion assays. A model that may be generally used to evaluate the risk of post-transplant HCC recurrence and overall survival was established by Cox's proportional hazard model.Results:We identified seven lncRNAs (HOTAIR, MALAT1, ncRAN(L), ncRAN(S), lncRNA-HEIH, TUC338and ANCR) that were overexpressed in HCC tissues. siRNA suppression of HOTAIR in a liver cancer cell line reduced cell viability and cell invasion, sensitized Tumor necrosis factor-α induced apoptosis, and increased the chemotherapeutic sensitivity of cancer cells to cisplatin and doxorubicin. We found that the cumulative recurrence-free survival and overall survival in HCC patients with a high expression level of HOTAIR, MALAT1, ncRAN(L), ncRAN(S), lncRNA-HEIH, TUC338or ANCR was significantly lower than those with low expression. Thus we established a model based on lncRNA ("lncRNA signature") that can assess the risk of tumor recurrence and prognosis after LT. Risk scores=(0.727×HOTAIR expression level)+(0.603×MALAT1expression level)+(0.666×ncRAN(L) expression level)+(0.455×ncRAN(S) expression level)+(0.662×lncRNA-HEIH expression level)+(0.699×TUC338expression level)+(0.605×ANCR expression level), HCC patients receiving LT were segregated into high/low risk lncRNA signature groups based on Receiver Operating Characteristics (ROC) analysis (cut-off point2.5). Patients with high risk lncRNA signature had significantly worse prognosis than those with low risk lncRNA signature. The1-year,3-year, and5-year cumulative recurrence-free survival rates and overall survival rates of the HCC patients with high risk lncRNA signature were much lower than those with low risk lncRNA signature. Cox multivariate analysis revealed that lncRNA signature was an independent prognostic factor for predicting tumor recurrence and overall survival of patients with HCC after LT. When the patients were stratified according to the Milan, UCSF or Hangzhou criteria, in patients who matched these criteria, those with low risk lncRNA signature revealed a significantly higher overall survival rates and cumulative recurrence-free survival rates, the5-year cumulative recurrence-free survival rates were89.8%,88.6%,83.8%respectively, and the5-year overall survival rates were82.1%,80.7%,79.8%respectively. The ROC analysis revealed that the area under the curve of lncRNA signature was larger than single lncRNA, thus had better predictive power. Conclusions:The high expression level of seven lncRNAs (HOTAIR, MALAT1, ncRAN(L), ncRAN(S), lncRNA-HEIH, TUC338and ANCR) were identified as the major risk factors associated with HCC recurrence and prognosis after LT. A novel model incorporating these factors could effectively evaluate the risk of post-transplant HCC recurrence and the overall survival of the patients.