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，例如 (i) 分化停滞（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，所有 3 种机制都可能被致癌事件破坏，导致 CSC 的分类并解释其异质性。定义和表征这种异质性对于理解 CSC 生物学和有效的治疗转化至关重要。我们在该项目中的最新成果包括：（1）表观遗传机制通过维持干细胞的多能性和促进更成熟的衍生物的分化，在干细胞生物学中发挥着关键作用。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 分离和严格评估。为了检验这一假设，用 zebularine (ZEB)（一种有效的 DNA 甲基转移酶 1 抑制剂）处理原代人类癌细胞和肝癌细胞系，并使用侧群 (SP) 方法分离假定的 CSC。使用标准体外和体内测定法测试了 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。在多种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、连续移植中卓越的肿瘤启动能力和直接细胞追踪实验来判断，表观遗传调节增加了从人类癌细胞中分离出的 SP 部分中具有 CSC 特性的细胞的频率。综合转录组分析揭示了干性相关基因丰富的共同特征，尽管每个单独的 CSC 基因表达特征都表现出不同致癌途径（例如 EGFR、SRC 和 MYC）的激活。常见的 CSC 特征与恶性进展相关，恶性进展在低分化肿瘤中丰富，并且高度预测肝癌和其他癌症的预后。结论：表观遗传调控可能为CSC的前瞻性分离和深入分析提供工具。肝脏 CSC 基因特征是由独特的癌基因特异性和常见干性特征之间的有害相互作用定义的。这些数据应有助于识别针对 CSC 独特和共同特征的治疗工具； (2)癌症异质性受到遗传、表观遗传和细胞微环境因素的动态调节。这些因子还在癌细胞的转录重编程和细胞命运变化中发挥重要作用。 DNA甲基化是调节癌症和干细胞的表观遗传机制的重要组成部分。 我们使用 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和局部微环境在癌症干细胞 (CSC) 特性调节中的相互作用。七种人肝癌细胞系，包括从原发性 HCC 建立的两种早期传代细胞系，以高密度 (HD) 和低密度 (LD) 铺板，并暴露于 100 µM Zeb 3 天。此后，在无药物、无血清和无粘附的条件下，细胞被解离并以克隆密度接种数代（G1至G5代）。 检查了来自 G1 至 G5 球体的细胞在自我更新、基因表达、致瘤性和转移潜力方面的差异。 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：瞬时暴露于 Zeb 在 5/7 的测试 HCC 细胞系中产生了不同的细胞密度依赖性反应。在被评分为 Zeb 敏感的细胞系中，LD 培养物 (LDZ) 的 3 天药物预处理导致初级球体形成显着增加。在无药物和无血清培养基中的甲基纤维素球形成测定中，这种效应持续至少五次后续的球生成。与此形成鲜明对比的是，未经处理的 LD 细胞未能形成初级球体，而 HD 和 HD Zeb 处理 (HDZ) 细胞的球体形成潜力在前三代中迅速下降。通过 DNMT1 shRNA 的稳定慢病毒转导，Zeb 敏感细胞系 Huh7 中 DNMT1 的消耗同样增加了 LD 细胞的自我更新潜力，这提供了密度依赖性效应受到表观遗传调节的证据。相反，尽管 DNMT1 蛋白完全耗尽，Zeb 抗性 WRL68 和 HepG2 的表型反应独立于药物和铺板方案。LDZ 细胞中球体形成的增加与 CSC 相关标记物（CD133、CD44）的稳定过表达密切相关。 、EpCAM），以及参与 ESC 自我更新的关键基因（Bmi1、alapha-SUZ12）和上皮-间质转化 (EMT)（β-连环蛋白、ZEB1、VIM、SNAL1）。同样，当 LDZ、HD 和 HDZ 球体解离并皮下注射时。在NOD/SCID小鼠（100个细胞/注射部位）中，LDZ细胞产生肿瘤的速度更快，外显率更高，并且表现出更频繁的脑和肺转移。基因再激活和致瘤性均逐渐增加 G1 至 G4。来自 G1-G4 LDZ 细胞的肿瘤也越来越具有血管性。 G1-G4 LDZ 球体的全局转录组分析证实，常见的 LDZ 特征以与致癌信号通路相关的基因为特征，包括肿瘤起始、转移和血管生成，并且能够预测肝癌患者的临床结果。我们得出的结论是，DNA甲基化和细胞微环境联合调节诱导的肝癌细胞系的表观遗传重编程可以增强和稳定CSC特性；(3)将人类肝癌分类为生物学上不同的亚组表明其起源于不同的肝谱系细胞。为了阐明谱系阶段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)、成肝细胞 (HB) 和终末分化的成体肝细胞 (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 衍生的肿瘤表现出 c-Myc 靶基因的特异性富集。我们的结果表明，任何肝谱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。原发性人类肝癌（PLC）是全球第三大致命癌症，在美国和西欧的发病率不断上升。 PLC 可由肝上皮细胞谱系和成体肝祖细胞产生。鉴定对致癌转化敏感的细胞对于诊断和治疗都至关重要。利用PLC的嵌合小鼠模型，我们证明肝谱系内的任何细胞都可以成为恶性转化的目标，并表现出肿瘤发生的癌症干细胞模式。常见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演示提供了独特的治疗机会。