Cancer stem cells and human liver cancer

癌症干细胞与人类肝癌

• 批准号: 8763313
• 负责人: Snorri Thorgeirsson
• 金额: $ 86.58万
• 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/8763313
• 关键词: ABCG2 gene; Address; Adult; Aftercare; Antineoplastic Agents; Apoptosis; BAY 54-9085; Biliary; Biological Assay; Bone Marrow Stem Cell; Breast; Cancer Biology; Cancer Model; Cell Adhesion; Cell Adhesion Molecules; Cell Cycle Arrest; Cell Density; Cell Line; Cell Lineage; Cell Proliferation; Cell Separation; Cell Survival; Cells; Cholangiocarcinoma; Commit; DNA; DNA Methylation; Data; Development; Disease; Disease Progression; Drug resistance; Epigenetic Process; Epithelial; Epithelial Cells; Event; Extracellular Signal Regulated Kinases; Family; Gene Expression; Gene Expression Profile; Gene Silencing; Gene Targeting; General Population; Generations; Genes; Genetic Heterogeneity; Genomics; Glioblastoma; Goals; Growth; Hepatic; Hepatoblastoma; Hepatocyte; Heterogeneity; Histologic; Homologous Gene; Human; Hypermethylation; Hypoxia; Immunodeficient Mouse; In Vitro; Investigation; Keratin-19; Label; Liver; Liver neoplasms; Malignant - descriptor; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of liver; Maps; Mediating; Membrane Transport Proteins; Mesenchymal; MicroRNAs; Minor; Modeling; Molecular; Mus; Mutation; Oncogenic; Organ; Pathway interactions; Patients; Population; Primary carcinoma of the liver cells; Proliferating; Property; Prostate; Protein Isoforms; Proteins; Proteomics; Proto-Oncogene Proteins c-akt; Pump; Radio; Recurrence; Reporting; Research Personnel; Resistance; Rest; Role; Side; Site; Solid; Solid Neoplasm; Staging; Stem cells; Subgroup; Testing; Therapeutic; Thymoma; Thyroid Gland; Tissues; Toxic effect; Transgenes; Translations; Transplantation; Tumorigenicity; Up-Regulation; Vimentin; Viral Oncogene; Zebularine; adult stem cell; aldehyde dehydrogenase 1; base; c-Myc Staining Method; cancer cell; cancer recurrence; cancer stem cell; cancer therapy; cell transformation; cell type; cellular transduction; density; experience; genome sequencing; hepatocellular carcinoma cell line; hepatocyte nuclear factor; improved; in vitro Assay; in vivo; inhibitor/antagonist; insight; interest; monolayer; neoplastic cell; new therapeutic target; novel; overexpression; progenitor; response; restoration; self renewing cell; self-renewal; small hairpin RNA; stem; stem cell biology; stem cell differentiation; theories; transcriptomics; transdifferentiation; treatment duration; tumor; tumor growth

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 HCC cells. Our strategy employed a 3 day treatment of established and primary human HCC derived cell lines grown as monolayer at various cell densities with the DNMT1 inhibitor Zebularine (ZEB) followed by a 3D culture to identify cells endowed with selfrenewal potential. Differences in self renewal, gene expression, tumorigenicity and metastatic potential of spheres at generations G1 to G5 were examined. Transient ZEB exposure produced differential cell densitydependent responses. In cells grown at low density, ZEB caused a remarkable increase in selfrenewal and tumorigenicity associated with longlasting 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 selfrenewal and tumorigenicty, and upregulation of apoptosis and differentiation related genes. A permanent reduction of DNMT1 protein using 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 shDNMT1A 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.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 HRas 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 selfrenewal 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 4alpha, 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 cMyc and its target genes wasrequired to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of cMyc 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 standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). Our objective was to test whether LRCC are resistant to sorafenib. We tested human HCC derived LRCC and non LRCC before and after treatment with sorafenib. LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless type MMTVintegration site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non uniform activation of specific isoforms of the sorafenib target proteins extracellular signal regulated kinases and v aktmurine thymoma viral oncogene homologue (AKT) in LRCC but not in non LRCC. A molecular pathway map for sorafenib treated LRCC is proposed. Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.

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环境对 HCC 细胞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 至 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，以及细胞凋亡和分化相关基因的上调。使用 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 shDNMT1A 处理的低密度细胞形成的球体显示出高分子相似性，这种相似性在连续几代中持续存在，证实了 DNMT1 耗竭在增强癌症干细胞特性中的重要作用。结论：这些结果将 DNA 甲基化确定为关键的表观遗传学决定肝癌和其他可能的实体瘤中癌症干细胞库的调节机制。(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 HRas 和 SV40LT 的转基因转导小鼠原代肝祖细胞、谱系定型成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 4α、祖细胞/胆汁（角蛋白 19、上皮细胞粘附分子、A6）和间质（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肿瘤不同的细胞起源，表明谱系阶段依赖性遗传变化对恶性转化的贡献，需要激活 cMyc 及其靶基因来重新编程成体。转化的成年肝细胞中 cMyc 的稳定敲低降低了其体外 CSC 特性，并抑制了免疫缺陷小鼠中肿瘤的生长。从这些数据中，我们得出结论，小鼠肝谱系中的任何细胞类型都可以发生致癌性重编程。通过激活不同细胞类型特异性途径识别常见和细胞起源特异性表型和遗传变化，可以为肝癌提供新的治疗靶点。(3)晚期的标准治疗。肝细胞癌 (HCC) 是索拉非尼，大多数患者在 6 个月内出现疾病进展。保留标签的癌细胞 (LRCC) 代表癌症干细胞 (CSC) 的一个新亚群。我们的目的是测试 LRCC 是否对索拉非尼具有耐药性。我们在索拉非尼治疗前后测试了人类 HCC 衍生的 LRCC 和非 LRCC。源自人 HCC 的 LRCC 对索拉非尼相对耐药。索拉非尼治疗后，HCC 细胞系中 LRCC 的比例增加，而一般癌细胞群的生长受到抑制。我们表明，与非 LRCC 相比，LRCC 表现出改善的活力和毒性特征，并减少了细胞凋亡。我们发现，用索拉非尼治疗后，LRCC 上调 CSC 标志物醛脱氢酶 1 家族、无翅型 MMTV 整合位点家族、细胞存活和增殖基因，并下调细胞凋亡、细胞周期停滞、细胞粘附和干细胞分化基因。这种现象伴随着 LRCC 中索拉非尼靶蛋白细胞外信号调节激酶和 v aktmurine 胸腺瘤病毒癌基因同源物 (AKT) 的特定亚型的非均匀激活，但非 LRCC 中则不然。提出了索拉非尼治疗 LRCC 的分子途径图。我们的结果表明，HCC 衍生的 LRCC 对索拉非尼相对耐药。由于 LRCC 可以产生仅需 10 个细胞的肿瘤，我们的数据表明这些细胞在疾病复发中具有潜在作用。对这一现象的进一步研究可能会为癌症生物学、癌症复发和耐药性提供新的见解，对基于 LRCC 的新型癌症疗法的开发具有重要意义。