Cancer stem cells and human liver cancer

癌症干细胞与人类肝癌

基本信息

批准号：
8937929
负责人：
Snorri Thorgeirsson
金额：
$ 103.93万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8937929
关键词：
ABCG2 gene Acceleration Address Adult Apoptosis Apoptotic Biliary Biological Assay Biology Bone Marrow Stem Cell Breast Cancer Biology Cancer Model Cell Adhesion Molecules Cell Density Cell Differentiation process Cell Line Cell Lineage Cell Separation Cells Characteristics Cholangiocarcinoma Commit DNA DNA Methylation Data Dose Epidermal Growth Factor Epidermal Growth Factor Receptor Epigenetic Process Epithelial Epithelial Cells Event Gatekeeping Gene Expression Gene Expression Profile Gene Silencing Gene Targeting Generations Genes Genetic Heterogeneity Genomics Glioblastoma Goals HRAS gene Hepatic Hepatoblastoma Hepatocarcinogenesis Hepatocyte Hepatocyte Growth Factor Heterogeneity Histologic Human Hypermethylation Hypoxia Immunocompromised Host Immunodeficient Mouse In Vitro Individual Keratin-19 Link Liver Liver Regeneration Liver neoplasms Malignant - descriptor Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of liver Mediating Membrane Transport Proteins Mesenchymal MicroRNAs Minor Mitogen-Activated Protein Kinases Modeling Molecular Morphogenesis Mus Mutation NOTCH1 gene Oncogenic Organ Pathway interactions Phenotype Population Primary carcinoma of the liver cells Proliferating Property Prostate Proteins Proteomics Proto-Oncogene Proteins c-akt Pump Radio Relative (related person)Reporting Repression Research Personnel Resistance Rest Role STAT protein STAT3 gene Side Signal Transduction Solid Solid Neoplasm Staging Stem cells Subgroup System TACSTD2 gene Testing Tetracyclines Therapeutic Thyroid Gland Tissues Transgenes Translations Transplantation Tumorigenicity Up-Regulation Vimentin Zebularine adult stem cell base c-myc Genes cancer cell cancer stem cell cancer therapy cell transformation cell type cellular transduction cholangiocyte density genetic manipulation genome sequencing hepatoma cell human HNF4A protein in vitro Assay in vivo inhibitor/antagonist insight interest loss of function monolayer neoplastic cell new therapeutic target overexpression progenitor prognostic programs receptor research study response restoration self renewing cell self-renewal small hairpin RNA stem stem cell biology stemness subcutaneous theories transcription factor transcriptomics transdifferentiation treatment duration tumor tumor growth

项目摘要

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) Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3-day treatment of established and primary human HCC-derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self-renewal potential. Differences in self-renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1-G5 were examined. Transient ZEB exposure produced differential cell density-dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes characterized by a stable overexpression of cancer stem cell-related and key epithelial-mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis- and differentiation-related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self-renewal, irrespective of cell density or ZEB exposure. Spheres formed by low-density cells treated with ZEB or shDNMT1 displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. In conclusion, these results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors; (2) Human primary liver cancer is classified into biologically distinct subgroups based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of primary liver cancer. We transduced mouse primary hepatic progenitor cells, lineage-committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic H-Ras and SV40LT. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling. Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and self-renewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells coexpressed hepatocyte (hepatocyte nuclear factor 4 alpha), progenitor/biliary (keratin 19, epithelial cell adhesion molecule, A6), and mesenchymal (vimentin) markers and showed dysregulation of genes that control the epithelial-mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage stage-dependent genetic changes to malignant transformation. Activation of c-Myc and its target genes was required to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of c-Myc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice. From these data we conclude that any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC by activating different cell type-specific pathways. Identification of common and cell of origin-specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer; (3) The relative contribution of hepatocyte growth factor (HGF)/MET and epidermal growth factor (EGF)/EGF receptor (EGFR), two key signal transduction systems in the normal and diseased liver, to fate decisions of adult hepatic progenitor cells (HPCs) has not been resolved. Here, we developed a robust culture system that permitted expansion and genetic manipulation of cells capable of multilineage differentiation in vitro and in vivo to examine the individual roles of HGF/MET and EGF/EGFR in HPC self-renewal and binary cell fate decision. By employing loss-of-function and rescue experiments in vitro, we showed that both receptors collaborate to increase the self-renewal of HPCs through activation of the extracellular signal-regulated kinase (ERK) pathway. MET was a strong inducer of hepatocyte differentiation by activating AKT and signal transducer and activator of transcription (STAT3). Conversely, EGFR selectively induced NOTCH1 to promote cholangiocyte specification and branching morphogenesis while concomitantly suppressing hepatocyte commitment. Furthermore, unlike the deleterious effects of MET deletion, the liver-specific conditional loss of Egfr facilitated rather than suppressed progenitor-mediated liver regeneration by switching progenitor cell differentiation toward hepatocyte lineage. These data provide new insight into the mechanisms regulating the stemness properties of adult HPCs and reveal a previously unrecognized link between EGFR and NOTCH1 in directing cholangiocyte differentiation. (4) Activation of c-MYC is an oncogenic hallmark of many cancers including liver cancer,and is associated with a variety of adverse prognostic characteristics. Despite a causative role during malignant transformation and progression in hepatocarcinogenesis, consequences of c- MYC activation for the biology of hepatic cancer stem cells (CSCs) are undefined. Here, distinct levels of c-MYC over-expression were established by using two dose-dependent tetracycline inducible systems in 4 hepatoma cell lines with different p53 mutational status. CSCs were evaluated using side-population approach as well as standard in vitro and in vivo assays. Functional repression of p53 was achieved by lentiviral shRNA transduction. The results show that c-MYC expression levels have a differential impact on liver CSC characteristics. At low levels, c-MYC activation led to increased proliferation and enhanced CSC properties including activation of reprogramming transcription factors and CSC marker expression (e.g. NANOG, OCT4 and EpCAM), expansion of side population and acceleration of tumor growth upon subcutaneous transplantation into immunocompromised mice. However, when exceeding a threshold level, c-MYC induced a pro-apoptotic program and loss of CSC potential both in vitro and in vivo. Mechanistically, c-MYC induced self-renewal capacity of liver cancer cells was exerted in a p53 dependent manner. Low c-MYC activation increased spheroid formation in p53-deficient tumor cells, whereas p53-dependent effects were blunted in the absence of MYC overexpression. Our results confirm the role of c-MYC as a master regulator during hepatocarcinogenesis and establish a new gatekeeper role for p53 in repressing c-MYC induced CSC phenotype in liver cancer cells.

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) 逆转 DNA 高甲基化和相关基因沉默是一种新兴的癌症治疗方法。在这里，我们讨论了表观遗传改变和细胞背景对肝细胞癌细胞（HCC）功能和转录重编程的影响。我们的策略采用 DNMT1 抑制剂 Zebularine (ZEB) 对已建立的原代人 HCC 衍生细胞系以不同细胞密度单层生长进行 3 天处理，然后进行 3D 培养，以鉴定具有自我更新潜力的细胞。检查了 G1-G5 代球体自我更新、基因表达、致瘤性和转移潜力的差异。瞬时 ZEB 暴露产生不同的细胞密度依赖性反应。在低密度生长的细胞中，ZEB 导致自我更新和致瘤性显着增加，与持久的基因表达变化相关，其特征是癌症干细胞相关和关键上皮间质转化基因的稳定过度表达。 DNMT1 表达恢复后，这些影响仍然存在。相反，在高细胞密度下，ZEB 导致自我更新和致瘤性逐渐下降，以及凋亡和分化相关基因的上调。使用短发夹 RNA (shRNA) 介导的 DNMT1 沉默永久减少 DNMT1 蛋白，使 HCC 细胞对细胞密度和 ZEB 效应不敏感。同样，表达低 DNMT1 基础水平的 WRL68 和 HepG2 肝母细胞瘤细胞也具有较高的自我更新能力，无论细胞密度或 ZEB 暴露如何。用ZEB或shDNMT1处理的低密度细胞形成的球体表现出高度的分子相似性，并且这种相似性在连续几代中得以维持，证实了DNMT1耗尽在增强癌症干细胞特性中的重要作用。总之，这些结果表明 DNA 甲基化是决定肝癌和其他可能的实体瘤中癌症干细胞库的关键表观遗传调控机制。 (2) 人类原发性肝癌根据细胞起源分为生物学上不同的亚组。最近描述了肝癌干细胞（CSC）。我们研究了不同谱系的肝细胞成为肝脏 CSC 的能力以及原发性肝癌的表型和遗传异质性。我们用编码致癌 H-Ras 和 SV40LT 的转基因转导小鼠原代肝祖细胞、谱系定型肝细胞和分化的成体肝细胞。通过标准体外和体内测定以及转录组分析来测试转导细胞的 CSC 特性及其形成肿瘤的能力。无论来源如何，所有转导细胞都获得了 CSC/祖细胞、侧群标记和体外自我更新能力。基于基因组和组织学分析，他们还形成了从胆管癌到肝细胞癌的广泛肝脏肿瘤，这些肿瘤类似于人类肝脏肿瘤。肿瘤细胞共表达肝细胞（肝细胞核因子 4 α）、祖细胞/胆汁（角蛋白 19、上皮细胞粘附分子、A6）和间充质（波形蛋白）标记物，并显示控制上皮-间质转化的基因失调。基因表达分析可以区分不同细胞起源的肿瘤，表明谱系阶段依赖性遗传变化对恶性转化的贡献。 c-Myc 及其靶基因的激活是将成年肝细胞重新编程为 CSC 以及肿瘤发展所必需的。在转化的成年肝细胞中稳定敲低 c-Myc 可降低其体外 CSC 特性，并抑制免疫缺陷小鼠的肿瘤生长。根据这些数据，我们得出结论，小鼠肝谱系中的任何细胞类型都可以通过激活不同的细胞类型特异性途径进行致癌性重编程，形成 CSC。鉴定常见的和细胞来源特异性的表型和基因变化可以为肝癌提供新的治疗靶点； (3) 正常和患病肝脏中的两个关键信号转导系统肝细胞生长因子（HGF）/MET和表皮生长因子（EGF）/EGF受体（EGFR）对成体肝祖细胞命运决定的相对贡献（ HPC）尚未解决。在这里，我们开发了一个强大的培养系统，允许在体外和体内对能够进行多谱系分化的细胞进行扩增和遗传操作，以检查 HGF/MET 和 EGF/EGFR 在 HPC 自我更新和二元细胞命运决定中的个体作用。通过体外功能丧失和拯救实验，我们发现两种受体通过激活细胞外信号调节激酶 (ERK) 途径协同增加 HPC 的自我更新。 MET 通过激活 AKT 以及信号转导子和转录激活子 (STAT3) 来强烈诱导肝细胞分化。相反，EGFR 选择性诱导 NOTCH1 促进胆管细胞规范和分支形态发生，同时抑制肝细胞定向。此外，与 MET 缺失的有害影响不同，肝脏特异性条件性 Egfr 缺失通过将祖细胞分化转向肝细胞谱系，促进而不是抑制祖细胞介导的肝再生。这些数据为调节成人 HPC 干性特性的机制提供了新的见解，并揭示了 EGFR 和 NOTCH1 在指导胆管细胞分化方面以前未被认识到的联系。 (4)c-MYC的激活是包括肝癌在内的多种癌症的致癌标志,并与多种不良预后特征相关。尽管 c-MYC 激活在肝癌发生的恶性转化和进展过程中具有致病作用，但其对肝癌干细胞 (CSC) 生物学的影响尚不清楚。在这里，通过在具有不同 p53 突变状态的 4 个肝癌细胞系中使用两个剂量依赖性四环素诱导系统，建立了不同水平的 c-MYC 过表达。使用侧群方法以及标准体外和体内测定法对 CSC 进行评估。 p53 的功能性抑制是通过慢病毒 shRNA 转导实现的。结果表明，c-MYC 表达水平对肝脏 CSC 特征有不同的影响。在低水平下，c-MYC 激活导致增殖增加和 CSC 特性增强，包括激活重编程转录因子和 CSC 标志物表达（例如 NANOG、OCT4 和 EpCAM）、侧群扩张以及皮下移植到免疫功能低下小鼠后加速肿瘤生长。然而，当超过阈值水平时，c-MYC 会在体外和体内诱导促凋亡程序和 CSC 潜力丧失。从机制上讲，c-MYC 诱导肝癌细胞的自我更新能力是以 p53 依赖性方式发挥的。低c-MYC激活增加了p53缺陷肿瘤细胞中球状体的形成，而p53依赖性效应在没有MYC过表达的情况下减弱。我们的结果证实了 c-MYC 在肝癌发生过程中作为主调节因子的作用，并建立了 p53 在抑制肝癌细胞中 c-MYC 诱导的 CSC 表型中的新看门人角色。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Epigenetic regulation of cancer stem cells in liver cancer: current concepts and clinical implications.

DOI：
10.1016/j.jhep.2010.05.003
发表时间：
2010-09
期刊：
Journal of hepatology
影响因子：
25.7
作者：
Marquardt JU;Factor VM;Thorgeirsson SS
通讯作者：
Thorgeirsson SS

Stem cells in hepatocarcinogenesis: evidence from genomic data.

DOI：
10.1055/s-0030-1247130
发表时间：
2010-02
期刊：
Seminars in liver disease
影响因子：
4.2
作者：
Marquardt JU;Thorgeirsson SS
通讯作者：
Thorgeirsson SS

Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types.

DOI：
10.1053/j.gastro.2013.03.013
发表时间：
2013-07
期刊：
Gastroenterology
影响因子：
29.4
作者：
Holczbauer Á;Factor VM;Andersen JB;Marquardt JU;Kleiner DE;Raggi C;Kitade M;Seo D;Akita H;Durkin ME;Thorgeirsson SS
通讯作者：
Thorgeirsson SS

Epigenetic reprogramming modulates malignant properties of human liver cancer.

DOI：
10.1002/hep.27026
发表时间：
2014-06
期刊：
HEPATOLOGY
影响因子：
13.5
作者：
Raggi, Chiara;Factor, Valentina M.;Seo, Daekwan;Holczbauer, Agnes;Gillen, Matthew C.;Marquardt, Jens U.;Andersen, Jesper B.;Durkin, Marian;Thorgeirsson, Snorri S.
通讯作者：
Thorgeirsson, Snorri S.

Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways.

DOI：
10.1002/hep.24454
发表时间：
2011-09-02
期刊：
HEPATOLOGY
影响因子：
13.5
作者：
Marquardt, Jens U.;Raggi, Chiara;Andersen, Jesper B.;Seo, Daekwan;Avital, Itzhak;Geller, David;Lee, Yun-Han;Kitade, Mitsuteru;Holczbauer, Agnes;Gillen, Matthew C.;Conner, Elizabeth A.;Factor, Valentina M.;Thorgeirsson, Snorri S.
通讯作者：
Thorgeirsson, Snorri S.