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

癌症干细胞在肝癌异质性和亚型中的作用

• 批准号: 10262173
• 负责人: Xin Wei Wang
• 金额: $ 34.58万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262173
• 关键词: 3-Dimensional; Adjuvant Therapy; Age; Alginates; Animal Model; Apical; Asians; Biliary; Binding; Biological Assay; Biological Markers; Biological Models; Biosensor; Bone Marrow; Cancer Etiology; Cell Adhesion Molecules; Cell Culture Techniques; Cell Lineage; Cells; Cessation of life; Characteristics; Chromatin; Classification; Clinical; Cultured Cells; Disease; Elements; Environment; Epithelial; Epithelial Cells; Epithelium; FDA approved; Family member; Flow Cytometry; Gene Chips; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Goals; Growth; Hep3B; Hepatic; Hepatocyte; Human; In Vitro; Intrahepatic Cholangiocarcinoma; Lead; Libraries; Link; Liver; Maintenance; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of liver; Mesenchymal; Messenger RNA; MicroRNAs; Modeling; Molecular; Molecular Profiling; Molecular Target; Morphogenesis; NCAM1 gene; Natural Products; Nature; Oncoproteins; Organoids; Outcome; Pathology; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pimozide; Primary carcinoma of the liver cells; Property; Proteins; Recurrence; Research; Resistance; Risk; Role; Signal Pathway; Signal Transduction; Small RNA; Specimen; Structure; Subgroup; System; Testing; Time; Tumorigenicity; advanced disease; alpha-Fetoproteins; anti-cancer; base; beta catenin; cancer heterogeneity; cancer stem cell; cancer subtypes; cancer therapy; cell growth; cell motility; cell type; chemotherapeutic agent; chronic liver disease; clinically relevant; deep sequencing; diagnostic biomarker; disease diagnosis; genomic profiles; hepatocellular carcinoma cell line; high throughput screening; in vivo; in vivo Model; inhibitor/antagonist; miRNA expression profiling; molecular drug target; nano-string; novel; oncogene addiction; outcome forecast; pre-clinical research; prognostic; scaffold; screening; stem; stem cell population; stem cells; stem-like cell; stemness; therapeutic target; trait; transcriptome; tumor; tumorigenic; two-dimensional

In a gene expression array study comparing signatures of chronic liver diseases with hepatocellular carcinoma (HCC), we found a molecular signature that separates patients for their risk of developing advanced disease. Epithelial cell adhesion molecule (EpCAM) was identified as the lead gene and its silencing resulted in growth suppression of HCC cells. EpCAM could significantly differentiate HCC into two subtypes. HCC could be further stratified into four distinct subtypes with the additional assessment of alpha-fetoprotein (AFP) status. These four subtypes were associated with HCC prognostic outcome and cells double positive for EpCAM and AFP had the worst prognosis. Furthermore, these subtypes resembled certain stages of liver lineages and EpCAM/AFP-positive cells displayed a distinct molecular signature with features of hepatic stem/progenitor cells. Moreover, these cells, characterizing a poor prognostic HCC subtype, were capable of initiating highly invasive HCC in in-vitro and in-vivo models. Thus, EpCAM and AFP are useful diagnostic markers for HCC which can be used as a convenient classification system for prognosis. Furthermore, EpCAM and AFP may act as downstream molecules to maintain HCC stemness and as markers for HCC initiating cells. We have also recently explored whether integrative genomic profiling of a well-defined HCC subset of extreme EpCAM+ AFP+ could uncover survival-related driver genes in HCC. We found that YY1-associated protein 1 (YY1AP1) is a critical oncoprotein specifically activated in EpCAM+AFP+ HCC. YY1AP1 silencing eliminates oncogene addiction by altering the chromatin landscape while YY1AP1 expression promotes HCC proliferation and is required for the maintenance of stem cell features. Thus YY1AP1 may serve as a key molecular target for EpCAM+ AFP+ HCC subtype. We also investigated the mechanism by which EpCAM is elevated in HCC subtypes with stem/progenitor cell features. We found that the activation of wnt-beta-catenin pathway regulates EpCAM expression. In fact, EpCAM is a biosensor for wnt-beta-catenin signaling and is transcriptionally up-regulated by this pathway through direct Tcf binding element interactions. Inhibition of HCC cell growth could be achieved through blockade of EpCAM/wnt-beta-catenin signaling in EpCAM-positive HCC cells. We propose that EpCAM/wnt-beta-catenin signaling functions to maintain HCC stem cell growth and that EpCAM expression-based classification of HCC could be useful in clinical settings to stratify HCC patients who may benefit from beta-catenin/EpCAM adjuvant therapies. We recently developed a high-throughput screening assay to identify inhibitors of EpCAM-dependent growth of HCC cells. EpCAM(+) and EpCAM(-) HCC cell lines were assessed for differential sensitivity to Wnt/beta-catenin pathway inhibitors. Libraries comprising 22 668 pure compounds and 107 741 crude or partially purified natural product extracts were tested, and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized by effects on EpCAM expression. Treatment of EpCAM(+) Hep3B cells resulted in loss of EpCAM expression by flow cytometry. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of EpCAM-dependent cell growth. Recently, we found that an FDA-approved psychiatric drug, pimozide (PMZ), has anti-cancer properties in HCC cell lines that express EpCAM. We demonstrate that PMZ effectively inhibits cell growth of HCC cells by disrupting the wnt/beta-catenin signaling pathway and reducing EpCAM expression. Thus, PMZ may be a useful molecular entity that could be repurposed as an anti-cancer therapy for treatment of HCC. A global microRNA microarray approach was used to explore whether certain microRNAs were associated with HCC stem cells. We found that microRNA-181 family members were up-regulated in HCC stem cells. Inhibition of microRNA-181 led to a reduction in number and tumor initiating activity of HCC stem cells while addition of microRNA-181 led to an enrichment of this cell type. We also 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 also recently explored whether specific microRNAs exist in hepatic cancer stem cells (CSCs) that are not expressed in normal hepatic stem cells by assessing the microRNA transcriptome of HCC specimens by small RNA deep sequencing. We found that miR-150, miR-155, and miR-223 were preferentially highly expressed in EpCAM+ HCC cells and their gene surrogates were associate with patient prognosis. Furthermore, suppressing miR-155 reduced EpCAM+ HCC cells, HCC tumorigenicity and shortened overall survival and time to recurrence of HCC patients. Thus, miR-155 might serve as a molecular target to eradicate the EpCAM+ CSC population in human HCCs. 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 miR-200c and epithelial-mesenchymal transition (EMT) signaling was preferentially activated in ICC with stem cell gene expression traits. Inactivation of miR-200c resulted in an induction of EMT while its activation reduced EMT and cell migration and invasion. NCAM1, a 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 and that specific EMT components may be critical biomarkers and clinically relevant therapeutic targets for an aggressive form of stem cell-like ICC. Pre-clinical research to delineate molecular mechanisms that drive cancer growth and progression is usually carried out in two-dimensional (2D) cell cultures. They are efficient and reliable but lack the appropriate cell-cell contact environment typically observed in vivo. Thus the 3D organotypic model provides an important alternative to both 2D culture and in vivo animal model systems. Recently, we demonstrated that HCC cells in porous alginate scaffolds can generate organoid-like spheroids that mimic numerous features of glandular epithelium in vivo, such as acinar morphogenesis and apical expression patterns of EpCAM. We show that the activation of Wnt/beta-catenin signaling, a pathway for maintaining HCC stemness, is required for EpCAM+ HCC spheroid formation and the maintenance of the acinous structure. We also demonstrated that EpCAM+ HCC cells cultured as spheroids are more sensitive to TGF-induced epithelial-mesenchymal transition with highly tumorigenic and metastatic potential in vivo compared to conventional 2D culture. HCC cells in EpCAM+ spheroids are more resistant to chemotherapeutic agents than 2D-cultured cells. The alginate scaffold-based organotypic culture system is a promising, reliable, and easy system that can be configured into a high-throughput fashion for the identification of critical signaling pathways and screening of molecular drug targets specific for HCC.

在一项比较慢性肝病和肝细胞癌（HCC）特征的基因表达阵列研究中，我们发现了一种分子特征，可以区分患者发展为晚期疾病的风险。上皮细胞粘附分子（Epithelial cell adhesion molecule, EpCAM）被确定为先导基因，其沉默导致HCC细胞生长受到抑制。EpCAM可将HCC区分为两种亚型。通过对甲胎蛋白（AFP）状态的额外评估，HCC可以进一步分为四个不同的亚型。这四种亚型与HCC预后相关，EpCAM和AFP双阳性细胞预后最差。此外，这些亚型与肝脏谱系的某些阶段相似，EpCAM/ afp阳性细胞表现出具有肝干/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HCC亚型，能够在体外和体内模型中引发高度侵袭性HCC。因此，EpCAM和AFP是一种有效的HCC诊断指标，可作为一种方便的预后分类系统。此外，EpCAM和AFP可能作为下游分子维持HCC的干性，并作为HCC起始细胞的标志物。我们最近还探讨了EpCAM+ AFP+的一个明确定义的HCC亚群的整合基因组分析是否可以揭示HCC中与生存相关的驱动基因。我们发现yy1相关蛋白1 （YY1AP1）是EpCAM+AFP+ HCC特异性激活的关键癌蛋白。YY1AP1沉默通过改变染色质景观消除癌基因成瘾，而YY1AP1表达促进HCC增殖，是维持干细胞特征所必需的。因此YY1AP1可能是EpCAM+ AFP+ HCC亚型的关键分子靶点。我们还研究了EpCAM在具有干细胞/祖细胞特征的HCC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。事实上，EpCAM是wnt- β -连环蛋白信号的生物传感器，并通过Tcf结合元件的直接相互作用在这一途径中转录上调。通过阻断EpCAM阳性HCC细胞中的EpCAM/wnt- β -catenin信号通路，可以实现对HCC细胞生长的抑制。我们建议EpCAM/wnt- β -catenin信号传导功能维持HCC干细胞生长，并且基于EpCAM表达的HCC分类可能在临床环境中对可能受益于β -catenin/EpCAM辅助治疗的HCC患者进行分层。我们最近开发了一种高通量筛选试验来鉴定肝癌细胞依赖epcam生长的抑制剂。评估EpCAM（+）和EpCAM(-) HCC细胞系对Wnt/ β -连环蛋白途径抑制剂的敏感性差异。共检测了包含22 668个纯化合物和107 741个粗或部分纯化的天然产物提取物的文库，鉴定出12个纯化合物和67个天然产物提取物供进一步研究。三种活性化合物和阳性对照进一步研究了对EpCAM表达的影响。流式细胞术显示，EpCAM(+) Hep3B细胞处理后EpCAM表达减少。具有多种可能分子靶标的化合物的鉴定表明，epcam依赖性细胞生长的调节可能存在多种机制。最近，我们发现fda批准的精神科药物吡莫胺（PMZ）在表达EpCAM的HCC细胞系中具有抗癌特性。我们证明PMZ通过破坏wnt/ β -连环蛋白信号通路和降低EpCAM表达有效地抑制HCC细胞的生长。因此，PMZ可能是一种有用的分子实体，可以作为肝癌治疗的抗癌药物。一种全局microRNA微阵列方法被用于探索某些microRNA是否与HCC干细胞相关。我们发现microRNA-181家族成员在HCC干细胞中上调。抑制microRNA-181导致HCC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。我们还发现，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。我们最近也通过小RNA深度测序评估肝癌标本的microRNA转录组，探索了在正常肝干细胞中不表达的肝癌干细胞（CSCs）中是否存在特异性microRNA。我们发现miR-150、miR-155和miR-223在EpCAM+ HCC细胞中优先高表达，其基因替代物与患者预后相关。此外，抑制miR-155可降低EpCAM+ HCC细胞，降低HCC的致瘤性，缩短HCC患者的总生存期和复发时间。因此，miR-155可能作为一种分子靶点来根除人类hcc中的EpCAM+ CSC群体。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，miR-200c和上皮-间充质转化（EMT）信号在具有干细胞基因表达特征的ICC中优先被激活。miR-200c的失活导致EMT的诱导，而其激活则减少了EMT和细胞的迁移和侵袭。NCAM1是一种肝干/祖细胞标志物，是miR-200c的直接靶点。我们的研究结果表明，ICC和HCC具有共同的干细胞样分子特征和不良预后，特异性EMT成分可能是侵袭性干细胞样ICC的关键生物标志物和临床相关治疗靶点。描述驱动癌症生长和进展的分子机制的临床前研究通常在二维（2D）细胞培养中进行。它们是有效和可靠的，但缺乏适当的细胞-细胞接触环境，通常在体内观察到。因此，3D器官型模型为2D培养和体内动物模型系统提供了重要的替代方案。最近，我们证明多孔藻酸盐支架中的HCC细胞可以产生类器官样球体，模仿体内腺上皮的许多特征，如腺泡形态发生和EpCAM的顶端表达模式。我们发现Wnt/ β -catenin信号的激活是维持HCC干细胞性的途径，是EpCAM+ HCC球形形成和维持腺泡结构所必需的。我们还证明，与传统的二维培养相比，EpCAM+ HCC细胞培养成球状体对tgf诱导的上皮-间质转化更敏感，具有高度的体内致瘤性和转移潜力。EpCAM+球体的HCC细胞比2d培养的细胞对化疗药物更耐药。海藻酸盐支架为基础的有机型培养系统是一种有前途的、可靠的、简单的系统，可以配置成一种高通量的方式，用于识别关键信号通路和筛选HCC特异性的分子药物靶点。