Institute of Biology, Leiden University, The Netherlands.
J Pathol. 2011 Sep;225(1):19-28. doi: 10.1002/path.2936. Epub 2011 Jul 8.
Although the underlying molecular mechanism of hepatocellular carcinoma remains unclear, signalling pathways essential in cell survival and growth are altered, including the Raf-MEK-MAPK pathway. This pathway can be activated by hepatitis B or C virus infections and the ectopic expression of the Raf-1 oncogene is frequently seen in hepatocellular carcinomas. In addition, the Raf-MEK-MAPK pathway was also shown to be deregulated in zebrafish liver tumours. Based on the genetic conservation between zebrafish and human liver tumours, the zebrafish was used as an animal model to better understand the molecular basis of hepatocellular carcinoma. Here we establish an inducible oncogenic zebrafish cell model, in which oncogenic human Raf-1(ΔRaf1) can be post-transcriptionally activated in zebrafish liver cells by administration of 4-hydroxytamoxifen (4HT). The ΔRaf1 activation resulted in the hyperactivation of the zebrafish MEK-ERK cascade, promoted cell growth and proliferation, and inhibited apoptosis. The mitogenic transformation of the ZFL-ΔRaf1-ER cells was confirmed by in vivo allo-transplantation and in silico microarray analyses. Gene expression profiling of cells treated with 4HT and a MEK-inhibitor identified a Raf-MEK-dependent signature set. This transcriptome response was compared to zebrafish and human liver cancer transcriptomes. We identified, and validated by quantitative PCR, a set of genes transcriptionally regulated by hyperactive MAPK signalling in ZFL-ΔRaf1-ER cells, zebrafish liver tumours and human liver tumours, suggesting that the in vitro zebrafish liver cell model can be used for further study of the molecular basis of human hepatocellular carcinoma. The molecular targeting of the commonly regulated hepatocellular carcinoma genes using the ZFL-ΔRaf1-ER cell model can be applied for high-throughput preclinical target discovery.
虽然肝细胞癌的潜在分子机制尚不清楚，但细胞存活和生长所必需的信号通路发生了改变，包括 Raf-MEK-MAPK 通路。该通路可被乙型或丙型肝炎病毒感染激活，Raf-1 癌基因的异位表达在肝细胞癌中经常见到。此外，Raf-MEK-MAPK 通路在斑马鱼肝肿瘤中也显示出失调。基于斑马鱼和人类肝肿瘤之间的遗传保守性，将斑马鱼用作动物模型，以更好地理解肝细胞癌的分子基础。在这里，我们建立了一种可诱导的致癌性斑马鱼细胞模型，其中致癌性人 Raf-1(ΔRaf1)可通过给予 4-羟基他莫昔芬 (4HT) 在斑马鱼肝细胞中转录后激活。ΔRaf1 的激活导致斑马鱼 MEK-ERK 级联的过度激活，促进细胞生长和增殖，并抑制凋亡。ZFL-ΔRaf1-ER 细胞的有丝分裂转化通过体内同种异体移植和计算机芯片分析得到证实。用 4HT 和 MEK 抑制剂处理的细胞的基因表达谱分析确定了一个 Raf-MEK 依赖性的特征集。将这个转录组反应与斑马鱼和人类肝癌转录组进行比较。我们鉴定了一组受 ZFL-ΔRaf1-ER 细胞、斑马鱼肝肿瘤和人类肝肿瘤中高活性 MAPK 信号转录调节的基因，并通过定量 PCR 进行了验证，这表明体外斑马鱼肝细胞模型可用于进一步研究人类肝细胞癌的分子基础。使用 ZFL-ΔRaf1-ER 细胞模型对常见调节的肝癌基因进行分子靶向治疗可应用于高通量临床前靶标发现。
