| Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide, with predominant incidence in East Asia and Saharan Africa. New insights into the pathogenesis of this lethal disease are urgently needed. A powerful way to identify driver genes with causal roles in carcinogenesis is to detect the genomic regions that undergo frequent alterations in cancers. Although previous studies have identified common somatic copy number alterations (CNA) in HCC, these surveys primarily used lower resolution technologies and have thus provided a constrained view of the copy number alteration pattern. To overcome these limitations, we applied whole-genome single nucleotide polymorphisms array6.0to define a comprehensive genomic profile of a set of58paired HCC primary tumors and adjacent non-tumor liver tissues. Firstly, a total of1,241significantly somatic CNA were identified including963amplifications and278deletions. Furthermore, by integrating SNP array-based copy number data with gene expression profile, we identified a total of362deregulated genes in aberrant chromosomal regions that included known cancer-related genes such as DDEF1, SCRIB, and DLC1. Among the362deregulatd genes, we selected20candidate genes (13amplified and7deleted) and tested their effects on cancer cell proliferation in vitro and in vivo. One novel tumor suppressor (tripartite motif-containing35[TRIM35]) and two putative oncogenes (hairy/enhancer-of-split related with YRPW motif1[HEY1] and small nuclear ribonucleoprotein polypeptide E [SNRPE]) were discovered by various in vitro and in vivo experiments. Moreover, our results demonstrated that decreases of TRIM35expression are a frequent event in HCC and the expression levels of TRIM35were negatively correlated with tumor size, histological grade, and serum alpha-fetoprotein content.Additionally, a novel CNA in HCC (1q24.1-24.2amplicon) was selected for further study, which lead to the discovery of increased DNA dosage and mRNA expression level of the MPZL1(myelin protein zero-like1) gene. The expression level of MPZL1gene was observed to be positively correlated with tumor stages and intrahepatic metastasis of HCC. Furthermore, we found that MPZL1genes can increase the in vitro and in vivo metastatic potentials of HCC cells through ectopic expression or shRNA-mediated knockdown of the MPZL1gene. Moreover, the MPZL1gene can promote the metastatic abilities of HCC cells through phosphorylation and activation of Cortactin protein, which was mediated by Src kinase. Therefore, MPZL1is a novel amplified pro-metastaic gene in HCC. Taken together, those results denonstrated that integrating of copy number and expression profile data offers the potential for indentifying novel cancer genes in HCC pathogenesis.Part ICharacterization of the landscape of genomic copy number alterations in hepatocellular carcinomaAlthough previous studies have identified common CNA in HCC, they primarily used lower resolution technologies and have thus provided a constrained view of the copy number changes. To overcome these limitations, we applied whole-genome single nucleotide polymorphisms array6.0to define a comprehensive genomic profile of58paired HCC primary tumors, and a total of1,241significantly CNA were identified including963amplifications and278deletions. These regions were highly concordant with previous findings including the recurrent gains at8q,1q,7q,6p,20q,17q and the losses at8p,4p,16q, and17p. Otherwise, several novel CNA were also identified, including gains of1p,2q,3q,6q,8p,9p,10p,13q,18p,19p,22q and losses of3q,4p,8q,12p,14q, and18q. In summary, we have acquired a comprehensive catalog of copy number alterations in HCC genome, which provided a unique opportunity to identify CNA-related genes in HCC.Part II Integrative analysis of cancer-related genes associated with copy number alterations in hepatocellular carcinomaTo discover candidate cancer genes in regions of CNA, we performed an integrative analysis of CNA and gene expression data, which lead to the discovery of362differentially expressed genes in the aberrant regions, including292upregulated genes in the regions of copy number gain and70downregulated genes in the regions of copy number loss. To further define the biological processes in which these362DEGs are involved, we performed gene ontology enrichment analysis of those genes. Additionally, to determine the regulatory relationships of those genes and the key players in hepatocarcinogenesis, we performed a network analysis to generate an interaction network containing relevant biological information for the362genes. The results showed that a set of core signaling pathways and their components were altered in the development and progression of HCC, in addition, the interaction between different cellular processes and signaling pathways involved in some key genes was explored in HCC. Finally, by combining the results of interaction network analysis with functional implications obtained from PubMed data mining, a set of20potential genes in the recurrent aberrant regions (amplification≥12samples and deletion≤4samples, respectively) were chosen for further functional assessments, and the expression levels were validated in HCC by Q-PCR. Therefore, the integrative analysis combined with bioinformatics can be an effective method to select candidates from a lengthy list of genes, and thus be amiable to determine the role and function of those genes in human carcinogenesis.PartⅢ Identification and characterization of a novel tumor suppressor candidate TRIM35deleted in hepatocelluar carcinomaIn order to determine the roles of20genes derived from aforementioned studies, we next analyzed the biological functions and cellular mechanisms of those genes. By using of various in vitro and in vivo functional experiments, one novel candidate tumor suppressor (TRIM35) and two novel putative oncogens (HEY1and SNRPE) were identified in HCC. Furthermore, our results showed that TRIM35could inhibit HCC cell proliferation by arresting cell cycle in G2/M phrase and induce cell apoptosis. To further determine the clinicopathologic significance of TRIM35in HCC, we performed immunohistochemical (IHC) analysis of TRIM35in a tissue array that included an independent set of207paired HCC and adjacent noncancerous tissues as well as10normal liver and16cirrhosis tissues. The results showed that the expression levels of TRIM35were negatively correlated with tumor grade, tumor size and serum alpha-fetoprotein (AFP) levels. In summary, one novel candidate of tumor suppressor and two novel potential oncogenes were explored in HCC, which provided an opportunity to understand the mechanism of hapatocarcinogenesis. Part IVMPZL1is a frequently amplified gene with a pro-metastatic function in hepatocellular carcinomaIn this study, by integrating copy number and gene expression data combined with functional studies, we found that a novel amplicon1q24.1-24.2, which can lead to the increased DNA dosage and expression level of MPZL1gene. Notably, the expression levels of the MPZL1gene positively correlate with tumor stage and intrahepatic metastasis of HCC specimens. The MPZL1gene was further observed to promote the metastatic potentials of HCC cells. Moreover, we found that the possible mechanisms by which the MPZL1promotes HCC metastasis is via inducing the phosphorylation and activation of the Cortactin protein, a ubiquitous actin-binding protein that was originally identified as a substrate for Src kinase. Overexpression of Cortactin in human tumors has been proposed to result in increased cell migration and metastatic potential. Additionally, we also found that the Src kinase can mediate the phosphorylation of Cortactin induced by MPZL1. Taken together, these findings suggested that MPZL1is a novel pro-metastatic gene in a recurrent region of copy number amplifications in HCC, which might increase the metastatic potential of HCC cells by phosphorylation and activation of the Cortactin protein. |
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