Cancer stem cells and human liver cancer

癌症干细胞与人类肝癌

• 批准号: 7965969
• 负责人: Snorri Thorgeirsson
• 金额: $ 40.7万
• 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/7965969
• 关键词: ABCG2 gene; ATP-Binding Cassette Transporters; Address; Adult; Affect; Agar; Antibodies; Biological Assay; Bone Marrow Stem Cell; Breast; Cancer Biology; Cancer Model; Cell Line; Cell Separation; Cell Transplants; Cells; Characteristics; Copy Number Polymorphism; Development; Dyes; Epigenetic Process; Event; Frequencies; Future; Gene Expression Profile; Genes; Genomics; Glioblastoma; Goals; Hepatoblastoma; Heterogeneity; Human; Hypoxia; In Vitro; Liver; Liver neoplasms; Malignant Neoplasms; Malignant neoplasm of liver; Measures; Membrane Transport Proteins; Methylation; Microarray Analysis; Minor; Modeling; Molecular; Mus; NOD/SCID mouse; National Cancer Institute; Oncogenic; Organ; Patients; Play; Population; Procedures; Proliferating; Property; Prostate; Proteomics; Pump; Radio; Regulation; Reporting; Research Personnel; Resistance; Rest; Role; Side; Solid; Solid Neoplasm; Sorting - Cell Movement; Staining method; Stains; Stem cells; Testing; Therapeutic; Thyroid Gland; Tissues; Translations; Transplantation; Tumorigenicity; Work; Zebularine; adult stem cell; base; cancer cell; cancer stem cell; cell transformation; cohort; genome sequencing; in vitro Assay; in vivo; interest; novel; overexpression; pluripotency; prognostic; self renewing cell; self-renewal; stem; stem cell biology; stemness; theories; therapeutic target; tool; transcriptomics; transdifferentiation; tumor; tumorigenic

The existence of tumor-initiating cancer stem cells (CSC) has been shown in a variety of solid tumors (e.g. breast, prostate, glioblastoma, liver). However, these CSC have highly variable antigenic and functional properties even when derived from the same tumor. These observations highlight a cardinal problem in CSC biology, namely, the heterogeneity of the CSC. 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 three mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSCs and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Although a variety of different stem cell markers have been used to isolate and characterize CSC from HCC, none of them seemed to be specific for liver malignancies and none of the isolated fractions showed uniform properties. Therefore we opted to use a functional approach to isolate CSC based on their ability to efflux Hoechst dye via ABC-transporters. This population is referred to as side population (SP). We consider the functional approach as less biased since it identifies the stem cells by their unique property of chemo-resistance. Given that epigenetic regulation plays a crucial role both in stem cell and cancer development, we chose epigenic modulation as an additional tool to narrow down the heterogeneity of SP-fraction. The specific objectives of this study are: (i) to characterize liver cancer stem cells heterogeneity using in vitro and in vivo functional assays; (ii) to investigate the effect of epigenetic modulation on the cancer stem cells by treatment with the DMNT1-inhibtior Zebularine (ZEB). Human HCC cell lines representing the different cell origins of primary liver cancer were selected for analysis including Huh7 (HCC), WRL68 (hepatoblastoma) and KMCH (mixed HCC/ICC). FACS analysis after Hoechst 33342-staining showed that the size of SP-fractions varied from 0.8% to 1.2%. In vitro treatment with 100 M ZEB for 3 days caused a significant reduction of the SP fraction in all three cell lines. In contrast, the colony-forming ability of SP cells as well as colony size was increased as measured by a quantitative soft-agar-based assay. ZEB treatment did not affect the viability of FACS sorted SP cells, although it reduced viability of the non-SP cells. To further assess tumorigenic potential of SP and non-SP cells, we transplanted 102-104 FACS sorted SP and non-SP cells into NOD/SCID mice. Consistent in all three HCC cell lines, an overall increase in tumor frequency was observed for SP cells regardless of ZEB treatment during the first 8 wk after transplantation. Limited dilution analysis (LDA) revealed a remarkable enrichment of tumor initiating cells within the SP fraction from all cell lines as compared to non-SP cells (3.1-fold, P=0.009 and 4.3-fold, P=0.001, at 6 and 8 wk, respectively). However at 10 wk, the differences became less pronounced although the tumor frequency in the group of mice receiving 100 SP cells remained higher (7/12 vs. 3/12 in non-SP). ZEB treatment significantly increased the number of tumor initiated cells in SP fraction. As few as 100 cells produced tumors in ZEB-treated SP cells from all cell lines (Figure 3, as shown for Huh7), while 1000 treated non-SP cells gave rise to few or no tumors (Huh7) within 20 weeks of observation. LDA analysis performed at 10 wk revealed a 15-fold increase in the tumor-initiating capacity of SP cells compared to non-SP (P=5.45x10x6). Treated SP cells showed a significant increase in tumorigenic potential as compared to their untreated counterpart (7-fold, P=0.001). Thus, ZEB-treatment significantly enhanced the tumorigenicity of the SP fraction while slightly reducing the tumor forming ability of non-SP fraction. To address the molecular mechanisms underlying these differences, we performed qRT-PCR analysis of selected genes. CSC-associated genes, such as ABCG2, CD133, GPC3 and c-KIT, as well as pluripotency related genes (OCT4, NANOG) were selectively overexpressed in the SP cells. Consistent with our in vivo and in vitro results, ZEB treatment significantly amplified the differences in the expression levels of CSC and stemness associated genes between SP and non-SP cells. These results suggest that (i) SP fraction is highly enriched for cells possessing tumor initiating ability and CSC properties; (ii) ZEB treatment can significantly reduce the heterogeneity of SP cells thus increasing the frequency of CSC; and (iii) the combination of an isolation procedure based on stem-like functional characteristics with epigenetic modulation provides an important tool for investigating cancer stem cells biology. Future plans include (i) to define antigenic characteristics of the cancer stem cells before and after ZEB treatment; (ii) to define specific gene expression signatures using microarray analysis; (iii) to integrate gene expression signatures with copy number variation (CNV) analysis obtained from the same groups; (iv) to characterize the effect of epigenetic modulation on the cancer stem cells by methylation specific PYRO-sequencing; (v) obtain primary human liver tumors to validate the ongoing work in cell lines; (vi) to test the usefulness of these integrative specific genomic signatures for prognostic prediction, both retrospectively and prospectively, in cohorts of HCC patients available to us at the National Cancer Institute (USA); and (vii) to employ the integrative genomic signatures for identification of novel and specific molecular therapeutic targets for the HCC derived CSC.

多种实体瘤（例如乳腺癌、前列腺癌、胶质母细胞瘤、肝脏）中均已显示出肿瘤起始癌症干细胞（CSC）的存在。然而，即使来自同一肿瘤，这些 CSC 也具有高度可变的抗原和功能特性。这些观察结果凸显了 CSC 生物学中的一个主要问题，即 CSC 的异质性。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，所有三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。尽管多种不同的干细胞标记物已被用于从 HCC 中分离和表征 CSC，但它们似乎都不是肝脏恶性肿瘤的特异性，而且分离的部分也没有表现出统一的特性。因此，我们选择使用功能性方法来分离 CSC，因为它们能够通过 ABC 转运蛋白流出 Hoechst 染料。该群体被称为侧群体（SP）。我们认为功能性方法的偏见较小，因为它通过干细胞独特的化学抗性特性来识别干细胞。鉴于表观遗传调控在干细胞和癌症发展中都起着至关重要的作用，我们选择表观遗传调控作为缩小 SP 分数异质性的额外工具。本研究的具体目标是：（i）利用体外和体内功能测定来表征肝癌干细胞的异质性； (ii) 研究通过 DMNT1 抑制剂 Zebularine (ZEB) 治疗后表观遗传调节对癌症干细胞的影响。选择代表原发性肝癌不同细胞起源的人 HCC 细胞系进行分析，包括 Huh7 (HCC)、WRL68 (肝母细胞瘤) 和 KMCH (混合 HCC/ICC)。 Hoechst 33342 染色后的 FACS 分析表明 SP 级分的大小在 0.8% 至 1.2% 之间变化。用 100 M ZEB 体外处理 3 天导致所有三种细胞系中 SP 分数显着减少。相反，通过基于软琼脂的定量分析测定，SP细胞的集落形成能力以及集落大小均有所增加。 ZEB 处理不影响 FACS 分选的 SP 细胞的活力，尽管它降低了非 SP 细胞的活力。为了进一步评估 SP 和非 SP 细胞的致瘤潜力，我们将 102-104 个 FACS 分选的 SP 和非 SP 细胞移植到 NOD/SCID 小鼠中。与所有三种 HCC 细胞系一致，在移植后的前 8 周内，无论 ZEB 治疗如何，SP 细胞的肿瘤频率总体增加。有限稀释分析 (LDA) 显示，与非 SP 细胞相比，所有细胞系的 SP 级分中的肿瘤起始细胞显着富集（3.1 倍，P=0.009 和 4.3 倍，P=0.001，在 6 和 8 时）周）。然而，在第 10 周时，尽管接受 100 个 SP 细胞的小鼠组中的肿瘤发生率仍然较高（7/12 对比非 SP 中的 3/12），但差异变得不那么明显。 ZEB 处理显着增加了 SP 部分中肿瘤起始细胞的数量。所有细胞系中经 ZEB 处理的 SP 细胞中只有 100 个细胞产生肿瘤（图 3，如 Huh7 所示），而 1000 个处理的非 SP 细胞在观察 20 周内产生很少或没有肿瘤 (Huh7)。 10周时进行的LDA分析显示，与非SP细胞相比，SP细胞的肿瘤启动能力增加了15倍（P=5.45x10x6）。 与未处理的SP细胞相比，处理过的SP细胞的致瘤潜力显着增加（7倍，P=0.001）。因此，ZEB处理显着增强了SP部分的致瘤性，同时略微降低了非SP部分的肿瘤形成能力。为了解决这些差异背后的分子机制，我们对选定的基因进行了 qRT-PCR 分析。 CSC相关基因，如ABCG2、CD133、GPC3和c-KIT，以及多能性相关基因（OCT4、NANOG）在SP细胞中选择性过表达。与我们的体内和体外结果一致，ZEB 处理显着放大了 SP 和非 SP 细胞之间 CSC 和干性相关基因表达水平的差异。这些结果表明 (i) SP 部分高度富集具有肿瘤启动能力和 CSC 特性的细胞； (ii) ZEB处理可以显着降低SP细胞的异质性，从而增加CSC的频率； (iii)基于干细胞样功能特征的分离程序与表观遗传调节的结合为研究癌症干细胞生物学提供了重要工具。未来的计划包括 (i) 确定 ZEB 治疗前后癌症干细胞的抗原特征； (ii) 使用微阵列分析来定义特定的基因表达特征； (iii) 将基因表达特征与从同一组获得的拷贝数变异（CNV）分析相整合； (iv) 通过甲基化特异性 PYRO 测序来表征表观遗传调节对癌症干细胞的影响； (v) 获得原发性人类肝脏肿瘤以验证细胞系中正在进行的工作； (vi) 在美国国家癌症研究所（美国）提供的 HCC 患者队列中，回顾性和前瞻性地测试这些综合特异性基因组特征对预后预测的有用性； (vii) 利用整合基因组特征来鉴定 HCC 衍生的 CSC 的新颖且特定的分子治疗靶点。