Cancer stem cells and human liver cancer

癌症干细胞与人类肝癌

• 批准号: 8552944
• 负责人: Snorri Thorgeirsson
• 金额: $ 63.6万
• 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/8552944
• 关键词: ABCG2 gene; Address; Adult; Antibodies; Biological Assay; Blood Vessels; Bone Marrow Stem Cell; Brain; Breast; CD44 gene; Cancer Biology; Cancer Model; Cancer Patient; Cancer cell line; Cell Density; Cell Line; Cell Lineage; Cell Separation; Cell model; Cells; Classification; Clinical; DNA; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; Data; Diagnosis; Employee Strikes; Epidermal Growth Factor Receptor; Epigenetic Process; Epithelial; Epithelial Cells; Evaluation; Event; Exhibits; Exposure to; Frequencies; Gene Expression; Gene Expression Profile; Gene Targeting; Generations; Genes; Genetic; Genomics; Glioblastoma; Goals; HRAS gene; Hepatic; Hepatocyte; Heterogeneity; Human; Hypoxia; In Vitro; Incidence; Individual; Injection of therapeutic agent; Liver; Liver neoplasms; Lung; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of liver; Membrane Transport Proteins; Mesenchymal; Methylcellulose; MicroRNAs; Minor; Modeling; Mutation; NOD/SCID mouse; Neoplasm Metastasis; Oncogenes; Oncogenic; Organ; Outcome; Pathway interactions; Penetrance; Pharmaceutical Preparations; Play; Population; Proliferating; Property; Prostate; Proteins; Proteomics; Pump; Radio; Regulation; Reporting; Research Personnel; Resistance; Rest; Role; Serum; Serum-Free Culture Media; Side; Signal Pathway; Simian virus 40; Site; Solid; Staging; Stem cells; Subgroup; TACSTD2 gene; Target Populations; Testing; Therapeutic; Thyroid Gland; Tissues; Translations; Transplantation; Tumor-Derived; Tumorigenicity; United States; Western Europe; Zebularine; adult stem cell; beta catenin; c-myc Genes; cancer cell; cancer stem cell; cell transformation; cell type; clinically relevant; density; established cell line; genome sequencing; hepatoma cell; in vitro Assay; in vivo; inhibitor/antagonist; interest; mouse model; new therapeutic target; novel; outcome forecast; overexpression; pluripotency; prospective; research study; response; self renewing cell; self-renewal; small hairpin RNA; stem; stem cell biology; stem cell population; stemness; theories; tool; trait; transcriptomics; transdifferentiation; tumor; tumor initiation; tumorigenesis; vasculogenesis

Different mechanisms could explain the origin and heterogeneity of CSC such as (i) differentiation arrest (stem cells), (ii) dedifferentiation (mature cells) and (iii) transdifferentiation (bone marrow stem cells). It is conceivable that all 3 mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSC and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Our most recent results in this project include: (1)Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase-1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal, superior tumor-initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. Conclusion: Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs; (2)Cancer heterogeneity is dynamically regulated by genetic, epigenetic and cellular microenvironmental factors. These factors also play important roles in transcriptional reprogramming and cell fate changes in cancer cells. DNA methylation constitute an important part of the epigenetic mechanism modulating cancer and stem cells. We have studied the interaction between epigenetic alterations and local microenvironment in regulation of cancer stem cell (CSC) properties using Zebularine (Zeb), a DNA-methyltransferase1 (DNMT1) inhibitor, in combination with the modulation of cell density in culture. Seven human hepatoma cell lines, including two early passage cell lines established from primary HCC, were plated at high (HD) and low density (LD), and exposed to 100 microM of Zeb for 3 days. Thereafter, cells were dissociated and plated at clonal density over several passages (generation G1 to G5) in drug-free, serum-free, and adherent-free conditions. The differences in self-renewal, gene expression, tumorigenicity and metastatic potential of cells derived from G1 to G5 spheres were examined. shDNMT1-Huh7 cell line was generated to address the role of DNMT1 in long-term self-renewal. Results: The transient exposure to Zeb produced differential cell density-depended responses in 5/7 tested HCC cell lines. In cell lines which were scored as Zeb-sensitive, a 3-day drug pre-treatment of LD cultures (LDZ) caused a remarkable increase in primary sphere formation. This effect persisted through at least five subsequent sphere generations in methylcellulose sphere-forming assays in drug- and serum-free medium. In striking contrast, untreated LD cells failed to form primary spheres while the sphere forming potential of HD and HD Zeb treated (HDZ) cells rapidly decreased over the first three generations. The depletion of DNMT1 in Huh7, a Zeb-sensitive cell line, by a stable lentiviral transduction of DNMT1 shRNA similarly increased the self-renewal potential of LD cells, providing evidence that the density-dependent effect was epigenetically regulated. Conversely, the phenotypic response of Zeb-resistant WRL68 and HepG2 was independent of drug and plating schema despite a complete depletion of DNMT1 protein.The increase in sphere formation in LDZ cells strongly correlated with a stable overexpression of CSC-related markers (CD133, CD44, EpCAM), as well as key genes involved in ESC self-renewal (Bmi1, alapha-SUZ12) and epithelial-mesenchymal transition (EMT) (beta-catenin, ZEB1, VIM, SNAL1). Likewise, when LDZ, HD and HDZ spheres were dissociated and injected s.c. into NOD/SCID mice (100 cells/injection site), the LDZ cells generated tumors more rapidly and with higher penetrance, and showed more frequent brain and lung metastasis. Both gene reactivation and tumorigenicity progressively increased G1 to G4. Tumors derived from G1-G4 LDZ cells were also increasingly more vascular. Global transcriptome analysis of LDZ spheres at G1-G4 confirmed that a common LDZ signature was characterized by genes associated with oncogenic signaling pathways including tumor initiation, metastasis, and vasculogenesis and was able to predict clinical outcome of liver cancer patients. We conclude that epigenetic reprogramming of liver cancer cell lines induced by a combined modulation of DNA methylation and cellular microenviroment can enhance and stabilize CSC properties;(3)Classification of human liver cancer into biologically distinct subgroups suggests its origin from different hepatic lineage cells. To clarify the contribution of the lineage stage in liver oncogenesis, we transduced H-Ras/SV40 large T into hepatic progenitor cells (HCP), hepatoblasts (HB) and terminally differentiated adult hepatocytes (AH). Regardless of origin, the transformed cell types acquired common cancer stem cell traits both in vitro and in vivo. However, expression analyses distinguished tumors from different lineage stages demonstrating that distinct genetic changes occur during malignant transformation. Notably, AH-derived tumors showed specific enrichment of c-Myc target genes. Our results demonstrate that any hepatic lineage cell can be a target population for transformation via activation of diverse cell-specific pathways. Primary human liver cancer (PLC) is the third most lethal cancer worldwide with incidence rising in United States and Western Europe. PLC can arise from liver epithelial cell lineages and adult hepatic progenitor cells. Identification of cells that are susceptible to oncogenic transformation is critical for both diagnosis and treatment. Employing a mosaic mouse model of PLC we demonstrated that any cell within the hepatic lineage can be a target of malignant transformation and display a cancer stem cell mode of tumorigenesis. The identification of common and cell of origin specific phenotypic and genetic changes should provide novel therapeutic targets for the treatment of PLC. In particular, our demonstration of the general role of c-myc in PLC oncogenesis offers unique therapeutic opportunities.

不同的机制可以解释CSC的起源和异质性，如(i)分化阻滞（干细胞），（ii）去分化（成熟细胞）和（iii）转分化（骨髓干细胞）。可以想象，这三种机制都可能被致癌事件破坏，导致CSC的分类，并解释了它们的异质性。定义和表征这种异质性对于理解CSC生物学和有效的治疗翻译至关重要。我们的最新研究成果包括：(1)表观遗传机制在干细胞生物学中起着至关重要的作用，通过维持干细胞的多能性和促进更成熟的衍生物的分化。如果类似的机制与癌症干细胞（CSC）模型相关，那么表观遗传调节可能会丰富CSC群体，从而促进CSC的分离和严格的评估。为了验证这一假设，用一种有效的DNA甲基转移酶-1抑制剂zebularine （ZEB）处理原发性人类癌细胞和肝癌细胞系，并使用侧群体（SP）方法分离推定的CSCs。采用标准的体外和体内测定法检测zeb处理和未处理亚群的CSC特性。对分离的CSC进行全转录组分析以生成CSC签名。在不同的原发性人类癌症中评估了CSC特征的临床相关性。通过自我更新、连续移植中优越的肿瘤启动能力和直接细胞跟踪实验来判断，从人类癌细胞中分离的SP部分中，表观遗传调节增加了具有CSC特性的细胞频率。整合转录组分析揭示了富含干细胞相关基因的共同特征，尽管每个个体CSC基因表达特征显示了不同致癌途径的激活（例如，EGFR， SRC和MYC）。常见的CSC特征与恶性进展相关，在低分化肿瘤中丰富，并且高度预测肝脏和其他癌症的预后。结论：表观遗传调控可为CSC的前瞻性分离和深入分析提供工具。肝CSC基因特征是由独特的癌基因特异性和共同的干性特征的有害相互作用定义的。这些数据应该有助于确定针对CSCs独特和共同特征的治疗工具；(2)肿瘤异质性受遗传、表观遗传和细胞微环境因素的动态调控。这些因子在癌细胞的转录重编程和细胞命运变化中也起着重要作用。DNA甲基化是肿瘤和干细胞调控表观遗传机制的重要组成部分。我们利用dna甲基转移酶1 （DNMT1）抑制剂Zebularine （Zeb）结合培养细胞密度的调节，研究了表观遗传改变与局部微环境在调节癌症干细胞（CSC）特性中的相互作用。7个人肝癌细胞系，包括2个由原发性肝癌建立的早期传代细胞系，以高密度（HD）和低密度（LD）镀，暴露于100微米的Zeb中3天。然后，在无药物、无血清和无贴壁条件下，分离细胞并以克隆密度（G1至G5代）进行几代（G1至G5代）。研究了G1和G5球源性细胞在自我更新、基因表达、致瘤性和转移潜能方面的差异。shDNMT1-Huh7细胞系的产生是为了解决DNMT1在长期自我更新中的作用。结果：短暂暴露于Zeb在5/7的HCC细胞系中产生不同的细胞密度依赖性反应。在被标记为zeb敏感的细胞系中，LD培养物3天的药物预处理（LDZ）导致初级球体形成显著增加。在药物和无血清培养基中甲基纤维素成球试验中，这种效应持续了至少5代。与此形成鲜明对比的是，未经处理的LD细胞无法形成原代球体，而HD和HD Zeb处理（HDZ）细胞的球体形成潜力在前三代迅速下降。在zeb敏感细胞系Huh7中，通过稳定的慢病毒转导DNMT1 shRNA， DNMT1的耗竭同样增加了LD细胞的自我更新潜力，这提供了密度依赖效应受表观遗传调控的证据。相反，尽管DNMT1蛋白完全缺失，但耐zeb的WRL68和HepG2的表型反应不依赖于药物和电镀模式。LDZ细胞中球体形成的增加与csc相关标志物（CD133、CD44、EpCAM）以及参与ESC自我更新的关键基因（Bmi1、α - suz12）和上皮-间质转化（EMT）的关键基因（β -catenin、ZEB1、VIM、SNAL1）的稳定过表达密切相关。同样，当LDZ、HD和HDZ球体解离并注射sc到NOD/SCID小鼠体内（100个细胞/注射部位）时，LDZ细胞产生肿瘤的速度更快，外显率更高，并表现出更频繁的脑和肺转移。G1至G4期，基因再激活和致瘤性均逐渐增加。来自G1-G4 LDZ细胞的肿瘤也越来越多地呈血管化。G1-G4 LDZ球的全球转录组分析证实，一个共同的LDZ特征与肿瘤起始、转移和血管生成等致癌信号通路相关的基因有关，能够预测肝癌患者的临床结局。我们认为，DNA甲基化和细胞微环境的联合调控诱导肝癌细胞系的表观遗传重编程可以增强和稳定CSC的特性；(3)将人肝癌划分为生物学上不同的亚群，表明其起源于不同的肝细胞系。为了明确谱系分期在肝脏肿瘤发生中的作用，我们将H-Ras/SV40大T转导到肝祖细胞（HCP）、肝母细胞（HB）和终末分化成肝细胞（AH）中。无论来源如何，转化的细胞类型在体外和体内都获得了常见的癌症干细胞特征。然而，表达分析区分了不同谱系阶段的肿瘤，表明在恶性转化过程中发生了不同的遗传变化。值得注意的是，ah源性肿瘤显示出c-Myc靶基因的特异性富集。我们的研究结果表明，任何肝系细胞都可以通过激活不同的细胞特异性途径成为转化的目标群体。原发性人肝癌（PLC）是世界上第三大致命癌症，在美国和西欧的发病率正在上升。PLC可起源于肝上皮细胞系和成人肝祖细胞。鉴定易发生致癌转化的细胞对诊断和治疗都至关重要。利用PLC的马赛克小鼠模型，我们证明了肝脏谱系中的任何细胞都可以成为恶性转化的目标，并显示出肿瘤发生的癌症干细胞模式。共同和细胞起源特异性表型和遗传变化的鉴定应该为PLC的治疗提供新的治疗靶点。特别是，我们对c-myc在PLC肿瘤发生中的一般作用的论证提供了独特的治疗机会。