The Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Hepatocarcinogenesis is a multi-stage process in which precursor lesions progress into early hepatocellular carcinomas (eHCC) by sequential accumulation of multiple genetic and epigenetic alterations. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules (DN) as well as eHCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, clear differences were detected between DN and eHCC which included 460 differentially expressed genes. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the dysplastic nodules. In addition, gene set enrichment analysis (GSEA) revealed a remarkable enrichment of MYC up-regulated gene set in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5 as well as with the higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from high- and low-grade dysplastic nodules. In conclusion, our study identified unique expression patterns associated with the transition of high-grade dysplastic nodules to early HCC and demonstrated that activation of the MYC transcription signature is critical for the malignant conversion of pre-neoplastic liver lesions. Samples from forty-nine nodular liver lesions including 24 regenerative (cirrhotic) nodules (CN), 3 low-grade (LGDN), 12 high-grade dysplastic nodules (HGDN) and 10 early hepatocellular carcinomas (Early HCC) were used. The Human Operon V2 oligonucleotide library containing 22K features representing expressed sequences was printed to glass arrays in the Advanced Technology Center (National Cancer Institute, Gaithersburg, MD). The aRNA probes were fragmented and hybridized to the microarray slides following the standard procedures. All samples were hybridized against a common amplified reference RNA pooled from normal liver samples. Experimental duplicates were prepared following a reverse-flour design.

肝细胞癌发生（hepatocarcinogenesis）是一个多阶段进程，指前驱病变通过逐步累积多种遗传与表观遗传改变，进展为早期肝细胞癌（early hepatocellular carcinoma, eHCC）。为解析肝脏癌变早期阶段的分子事件，我们对肝硬化性（再生性）结节、发育异常结节（dysplastic nodules, DN）以及早期肝细胞癌开展了基因表达谱分析。尽管在再生性与发育异常阶段观察到显著异质性，但在发育异常结节与早期肝细胞癌之间检测到明确的表达差异，涉及460个差异表达基因。对该显著基因集的功能分析显示，MYC癌基因可作为发育异常结节恶性转化的潜在驱动基因。此外，基因集富集分析（gene set enrichment analysis, GSEA）结果表明，相较于发育异常病变，MYC上调基因集在早期肝细胞癌中呈现显著富集。MYC表达特征的存在与CSN5的表达上调以及8号染色体区域基因的整体转录速率升高显著相关。进一步基于MYC靶基因构建的分类器可有效区分早期肝细胞癌与高、低级别发育异常结节。综上，本研究明确了高级别发育异常结节向早期肝细胞癌转化相关的独特表达模式，并证实MYC转录特征的激活对于癌前肝脏病变的恶性转化至关重要。本研究共纳入49例结节性肝脏病变样本，包括24例再生性（肝硬化性）结节（CN）、3例低级别发育异常结节（low-grade dysplastic nodules, LGDN）、12例高级别发育异常结节（high-grade dysplastic nodules, HGDN）以及10例早期肝细胞癌（Early HCC）。实验采用人类Operon V2寡核苷酸文库，该文库包含22000个代表表达序列的探针位点，由美国国家癌症研究所（马里兰州盖瑟斯堡）先进技术中心打印至玻璃微阵列芯片。按照标准流程，将片段化的反义RNA（aRNA）探针与微阵列玻片进行杂交。所有样本均与从正常肝脏样本中混合制备的通用扩增参考RNA进行杂交。实验重复样本采用反向荧光标记设计完成制备。