The role of cancer stem cells in liver cancer heterogeneity and subtypes

在一项比较慢性肝病和肝细胞癌特征的基因表达阵列研究中，我们发现了一种分子特征，可以区分这些患者发展为晚期疾病的风险。上皮细胞粘附分子被确定为该标记的先导基因，该基因的沉默导致肝癌细胞生长受到抑制。我们的研究结果支持这样一种观点，即在单个肿瘤形成之前可以获得大量的前致癌基因，并提示肝癌存在另一种机制，即在肿瘤开始时可以同时发生多个肿瘤启动事件。这些癌前基因在癌前组织中的鉴定及其激活或失活的机制可能会改善未来对高危人群的诊断，并可能指导新的化学预防策略。我们还使用全局分子谱分析EpCAM阳性和阴性肝细胞癌之间的基因表达差异。我们发现EpCAM可以显著地将肝细胞癌分化为两种类似肝谱系的亚型。特别是，epcam阳性的肝细胞癌表现出肝干/祖细胞的明显特征，包括wnt- β连环蛋白通路的激活。epcam阴性的肝细胞癌呈现成熟肝细胞的特征。我们还发现，通过对甲胎蛋白状态的额外评估，肝细胞癌可以进一步分为四种不同的亚型。这四种亚型与肝癌的预后相关，EpCAM和甲胎蛋白双阳性的细胞预后最差。此外，这些亚型类似于肝脏谱系的某些阶段，EpCAM/甲胎蛋白阳性细胞显示出具有肝干/祖细胞特征的独特分子特征。此外，这些细胞表现为预后不良的肝细胞癌亚型，能够在体外和体内模型中引发高度侵袭性肝细胞癌。提示EpCAM和甲胎蛋白是肝细胞癌的有效诊断指标，可作为判断预后的便捷分类系统。此外，EpCAM和甲胎蛋白可能作为下游分子维持HCC的干性，并作为HCC起始细胞的良好标记物。我们还探讨了EpCAM在具有干细胞/祖细胞特征的肝细胞癌亚型中升高的机制。我们发现wnt- β -catenin通路的激活调节EpCAM的表达。我们证明EpCAM是wnt- β -连环蛋白信号的生物传感器，并通过直接的Tcf结合元件相互作用在该途径中转录上调。我们的数据表明，EpCAM表达和wnt- β -连环蛋白信号传导的融合可以维持肝癌细胞的生长。通过阻断EpCAM阳性肝癌细胞的EpCAM/wnt- β -catenin信号通路，可以实现对肝癌细胞生长的抑制。基于这些发现，我们提出EpCAM/wnt- β -catenin信号传导在维持肝细胞癌干细胞生长中起作用，并且基于EpCAM表达的肝细胞癌分类可能在临床环境中对肝细胞癌患者进行分层，这些患者可能受益于β -catenin/EpCAM辅助治疗。MicroRNAs是内源性小的非编码rna，其调节基因表达与肿瘤发生的功能联系。一种全局microRNA微阵列方法被用于探索某些microRNA是否与肝细胞癌干细胞相关。我们发现保守的microRNA-181家族成员在肝癌干细胞中表达上调。抑制microRNA-181导致肝细胞癌干细胞数量和肿瘤启动活性的减少，而添加microRNA-181导致该细胞类型的富集。在进一步的研究中，我们发现microRNA-181可以直接靶向肝脏分化的转录调节因子和wnt- β -catenin信号传导抑制剂。此外，我们最近发现Wnt/ β -连环蛋白信号在HCC中转录激活microRNA-181s。这些结果表明，microRNA-181家族成员、Wnt/ β -catenin信号传导和肝癌干细胞之间存在一种新的调控联系，并暗示分子靶向microRNA-181或Wnt/ β -catenin信号传导可能会根除肝细胞癌。我们最近评估了肝内胆管癌（ICC）中与HCC干性和预后相关的分子特征。利用Affymetrix mRNA和Nanostring microRNA微阵列技术，亚洲ICC病例可以分为两个主要亚组，其中一个亚组与先前鉴定的具有干细胞基因表达特征的HCC具有相同的基因表达特征。对ICC特异性mRNA和microRNA表达谱的综合分析显示，在具有干细胞基因表达特征的ICC中，miR-200c信号传导与上皮-间充质转化（EMT）的共同信号通路被优先激活。miR-200c的失活导致EMT的诱导，而miR-200c的激活导致EMT的减少，包括细胞迁移和侵袭的减少。我们还发现，已知的肝干/祖细胞标志物NCAM1是miR-200c的直接靶点。我们的研究结果表明ICC和HCC具有共同的茎样分子特征和不良预后。我们的研究表明，EMT的特定成分可能被开发为侵袭性干细胞样ICC的关键生物标志物和临床相关治疗靶点。

In a gene expression array study comparing signatures of chronic liver diseases with hepatocellular carcinoma, we revealed a molecular signature that separates these patients for their risk of developing advanced disease. Epithelial cell adhesion molecule was identified as the lead gene in this signature and silencing of this gene resulted in growth suppression of hepatocellular carcinoma cells. Our results support the notion that large numbers of procarcinogenic genes can be acquired before a solitary tumor is formed, and suggest that an alternative mechanism exists for hepatocellular carcinoma, whereby multiple tumor-initiating events can simultaneously occur at the beginning of tumor initiation. The identification of these procarcinogenic genes in premalignant tissues and the mechanisms for their activation or inactivation may improve the future diagnosis of high-risk populations and may guide new strategies for chemoprevention. We have also used global molecular profiling to analyze the gene expression differences among EpCAM positive or negative hepatocellular carcinoma. We found that EpCAM could significantly differentiate hepatocellular carcinoma into two subtypes that resembled liver lineages. In particular, EpCAM-positive hepatocellular carcinoma displayed distinct features of hepatic stem/progenitor cells including an activation of the wnt-beta catenin pathway. Meanwhile, EpCAM-negative hepatocellular carcinoma displayed features of mature hepatocytes. We also found that hepatocellular carcinomas could be further stratified into four distinct subtypes with the additional assessment of alpha-fetoprotein status. These four subtypes were associated with prognostic outcome of hepatocellular carcinoma and cells double positive for EpCAM and alpha-fetoprotein had the worst prognosis. Furthermore, these subtypes resembled certain stages of liver lineages and EpCAM/alpha-fetoprotein-positive cells displayed a distinct molecular signature with features of hepatic stem/progenitor cells. Moreover, these cells, characterizing a poor prognostic hepatocellular carcinoma subtype, were capable of initiating highly invasive hepatocellular carcinoma in in-vitro and in-vivo models. This work suggests that EpCAM and alpha-fetoprotein are useful diagnostic markers for hepatocellular carcinoma which can be used as a convenient classification system for prognosis. Furthermore, EpCAM and alpha-fetoprotein may act as downstream molecules to maintain HCC stemness and serve as good markers for HCC initiating cells. We also explored the mechanism by which EpCAM is elevated in hepatocellular carcinoma subtypes with stem/progenitor cell features. We found that the activation of wnt-beta-catenin pathway regulates EpCAM expression. We demonstrate that EpCAM is a biosensor for wnt-beta-catenin signaling and is transcriptionally up-regulated by this pathway through direct Tcf binding element interactions. Our data suggest that the convergence of EpCAM expression and wnt-beta-catenin signaling functions to maintain hepatocellular carcinoma cell growth. Inhibition of hepatocellular carcinoma cell growth could be achieved through blockade of EpCAM/wnt-beta-catenin signaling in EpCAM-positive hepatocellular carcinoma cells. With these findings, we propose that EpCAM/wnt-beta-catenin signaling functions to maintain hepatocellular carcinoma stem cell growth and that EpCAM expression-based classification of hepatocellular carcinoma could be useful in clinical settings to stratify hepatocellular carcinoma patients who may benefit from beta-catenin/EpCAM adjuvant therapies. MicroRNAs are endogenous small noncoding RNAs that regulate gene expression with functional links to tumorigenesis. A global microRNA microarray approach was used to explore whether certain microRNAs were associated with hepatocellular carcinoma stem cells. We found that the conserved microRNA-181 family members were up-regulated in hepatoceullar carcinoma stem cells. Inhibitionof microRNA-181 led to a reduction in number and tumor initiating activity of hepatocellular carcinoma stem cells while addition of microRNA-181 led to an enrichment of this cell type. In further studies, we showed that microRNA-181 could directly target transcriptional regulators of differentiation in the liver and an inhibitor of wnt-beta-catenin signaling. In addition, we have recently shown that Wnt/beta-catenin signaling transcriptionally activates microRNA-181s in HCC. These results suggest a novel regulatory link between microRNA-181 family members, Wnt/beta catenin signaling and liver cancer stem cells and implies that molecular targeting of microRNA-181 or Wnt/beta-catenin signaling may eradicate hepatocellular carcinoma. We have recently assessed molecular signatures related to HCC stemness and outcome in intrahepatic cholangiocarcinoma (ICC). Using Affymetrix mRNA and Nanostring microRNA microarrays, Asian ICC cases could be segregated into two main subgroups, one of which shared gene expression signatures with previously identified HCC with stem cell gene expression traits. Integrative analyses of the ICC-specific mRNA and microRNA expression profiles revealed that a common signaling pathway linking miR-200c signaling to epithelial-mesenchymal transition (EMT) was preferentially activated in ICC with stem cell gene expression traits. Inactivation of miR-200c resulted in an induction of EMT while activation of miR-200c led to a reduction of EMT including a reduction of cell migration and invasion. We also found that NCAM1, a known hepatic stem/progenitor cell marker, was a direct target of miR-200c. Our results indicate that ICC and HCC share common stem-like molecular characteristics and poor prognosis. Our work suggests that specific components of EMT may be exploited as critical biomarkers and clinically relevant therapeutic targets for an aggressive form of stem cell-like ICC.