Embryonic Transcription Factor Function in Human Colorectal Cancer Stem Cells

人类结直肠癌干细胞中胚胎转录因子的功能

• 批准号: 7966200
• 负责人: John Jessup
• 金额: $ 5万
• 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/7966200
• 关键词: ABCA3 gene; ABCG2 gene; ALCAM gene; Activator Appliances; Adherent Culture; Affect; Anoikis; Antibodies; Apoptosis; Apoptotic; Binding; Biological; Biological Assay; Biology; CD44 gene; Camptothecin; Carcinoma; Cell Adhesion Molecules; Cell Cycle Inhibition; Cell Line; Cell Membrane Proteins; Cell Separation; Cell Size; Cells; Chemicals; Colon; Colon Carcinoma; Colorectal Cancer; Cultured Cells; DNA Sequence; Data; Dermis; Dyes; EGF gene; Embryo; Exclusion; Exposure to; Fibroblast Growth Factor 2; Flow Cytometry; Future; Gene Expression; Genes; Growth; HT29 Cells; Hour; Human; In Situ Nick-End Labeling; In Vitro; Individual; Injection of therapeutic agent; Large Intestine Carcinoma; Libraries; Malignant - descriptor; Measures; Methods; Mus; NOD/SCID mouse; Neoplasm Metastasis; Nude Mice; Patients; Pattern; Phase; Population; Relative (related person); Resistance; Role; Series; Serum-Free Culture Media; Side; Stomach; Suspension Culture; Suspension substance; Suspensions; Testing; Topotecan; Transcript; Transcription Coactivator; Transfection; Up-Regulation; Variant; Western Blotting; Work; abstracting; aldehyde dehydrogenase 1; base; c-myc Genes; cancer stem cell; chemotherapy; embryonic stem cell; in vivo; irinotecan; matrigel; open source; parent project; pluripotency; programs; promoter; research study; self-renewal; small hairpin RNA; small molecule libraries; stem cell technology; stressor; transcription factor; tumor; tumor growth; tumorigenic; vector control

i.) Abstract Nanog is one of several core transcription factors that maintain embryonic stem cells in a pluripotent, dedifferentiated state. Recent data indicate that expression of NANOG or its nearly identical retrogene NANOGP8 is increased in human gastric, hepatocellular and colon cancer. Our postulate is that NANOG/NANOGP8 is critical for the functions of putative CSC in colorectal carcinoma (CRC): self-renewal, pluripotency, spherogenicity and resistance to chemotherapy. Five human CRC lines were used in this study (HCC 2998, KM-12c, Clone A, Mip-101, CX-1). Anoikis (programmed cell death in suspension culture) was measured after 96 hours of suspension culture by TUNEL analysis of cells sorted for expression of ALDH1, CD44 or CD133 - alone or in combination-. No consistent pattern of resistance to anoikis was identified among the CRC lines. However, NANOG and/or Oct4 expression was increased in suspension culture and associated with resistance to anoikis. qRT-PCR analysis of spheroids of CX-1 and Clone A cultured in suspension in serum free medium indicated that NANOG transcript levels were increased by 43- and 12-fold respectively relative to monolayer cultures while the relative increase in transcript levels of Oct4, Sox2, Klf4, ABCG2, ABCA3, ALDH1A1, c-myc, CD44, CD133, CD166 and β-catenin was less than 5-fold except for Sox2 in Clone A which was 10-fold. CRC stably transfected with a GFP NANOG promoter were low expressors in monolayer culture, but expressed GFP as they transitioned to vertical growth and formed spheroids. Stable transfection with lentiviral NANOG shRNA decreased NANOG transcript levels and spherogenicity of CX-1 by 65% compared to vector controls in a single cell spherogenicity assay in serum-free medium. Finally, fewer NANOG shRNA transfected cells survived exposure to 0.3 M Topotecan for 48 than vector controls or parental cells. In summary, our data suggest that expression of NANOG/NANOGP8 occurs in human CRC and may be critical for such functions of putative CSC as spherogenicity and resistance to chemotherapy. ii.) Progress We developed an unbiased approach to isolating putative CSC by culturing cells in serum free medium supplemented with EGF and bFGF (Neurocult, Stem Cell Technologies) under low attachment conditions. We have primarily used two human CRC lines for this work: Clone A, a poorly differentiated colon cell line that is tumorigenic in the sub cutis but weakly metastatic after intrasplenic injection into athymic nude mice, and CX-1, a variant of HT-29 that is moderately differentiated, highly tumorigneic and metastatic. Initial attempts to reproducibly develop a flow cytometry based method of isolating a side population by cell size and dye exclusion were unsuccessful because the transporter function of the CRC was too high. A single cell spherogenic assay was developed in which single cells are placed in individual wells of a 96 well microtiter plate by limiting dilution in serum-free medium and then the % of cell forming spheroids are assessed at 1 - 2 weeks. CX-1 was more aggressive than Clone A in this assay with 33% of single cells forming spheroids compared to less than 8% of Clone A cells. No difference in plating efficiency between the CRC lines but p< 0.0004 for spherogenicity between the 2 CRC lines. Spheroids formed by the cell lines Clone A and CX-1 increase the gene expression of the core pluripotent TFs, especially NANOG that is increased 12 - 43-fold relative to the expression in monolayer culture. Increases in gene expression of transporter, cell membrane proteins or other core pluripotent TFs were not to the same degree. Western blots have been performed that demonstrate commensurate changes in expression of transporter and cell adhesion proteins. However, the antibodies for NANOG do not work well in western blot and are not presented. As an alternative we created stable constructs in Clone A and CX-1 of a NANOG promoter-driven GFP to confirm upregulation of NANOG expression in spheroids (described in Project 2). Once we confirmed that gene expression of NANOG was upregulated in spheroids, we then began to develop a lentiviral shRNA approach to silencing NANOG. Using the open source shRNA we isolated stably transfected clones of Clone A and CX-1 that were had at least a 70% decrease in NANOG gene expression. When we compared these clones to vector controls and the parental cell lines in the single cell spherogenicity assay, inhibition of NANOG expression decreased spherogenicity in CX-1. Work is in progress with Clone A. The effect of NANOG expression on resistance to chemotherapy has been preliminarily studied. The agent that has been used here is Topotecan (TOPO), a camptothecin that is similar to irinotecan that is used in colorectal carcinomas in patients. Preliminary experiments indicate that inhibition of NANOG expression increases sensitivity to TOPO in CRC. A complicating issue is that inhibition of NANOG causes S phase arrest and future aspects of this project will determine how inhibition of cell cycle affects sensitivity to Topotecan. iii.) Future Directions For the immediate future we are planning to assess the effect of NANOG on both tumorigienicity in the sub cutis and on metastatic potential after intrasplenic injection in NOD/SCID mice. Cells will be injected in a dilution series and experiments will be repeated at least once. Malignant growth assays in vivo will be carried out without matrigel so as to establish the role of NANOG in unmodified tumor growth. In addition, lentiviral shRNA to Oct4 and SOX2 the other major core pluripotent TFs will be stably transfected into the CRC and their effect on CSC assessed and compared to that obtained by silencing NANOG. After the biology of NANOG and other core pluripotent TFs is established in these CRC lines, we will begin aim 3 by creating an assay that may be used to screen compounds that inhibit NANOG binding to its DNA sequences in promoters. If successful, we will propose to the NExT program that the assay be used to screen chemical libraries for inhibitory agents and then if an agent is found it will be tested for ability to inhibit CSC function both in vitro and in vivo.

i。）抽象纳米是在多能，去分化状态下维持胚胎干细胞的几个核心转录因子之一。最近的数据表明，在人类胃，肝细胞癌和结肠癌中，纳米或其几乎相同的逆转录纳米P8的表达增加。我们的假设是，Nanog/NanogP8对于假定CSC在结直肠癌（CRC）中的功能至关重要：自我更新，多能性，球状性和对化学疗法的抗性。这项研究使用了五个人类CRC系（HCC 2998，KM-12C，克隆A，MIP-101，CX-1）。通过TUNEL分析分类用于表达AldH1，CD44或CD133的细胞，单独或组合 - - 悬浮培养物的悬浮培养物（悬浮培养物中的编程细胞死亡）（在悬浮培养物中）进行测量。在CRC线之间未发现对厌氧菌的抗性模式。但是，悬浮培养物中的纳米和/或OCT4表达增加，并且与对厌氧菌的抗性有关。 qRT-PCR analysis of spheroids of CX-1 and Clone A cultured in suspension in serum free medium indicated that NANOG transcript levels were increased by 43- and 12-fold respectively relative to monolayer cultures while the relative increase in transcript levels of Oct4, Sox2, Klf4, ABCG2, ABCA3, ALDH1A1, c-myc, CD44, CD133, CD166 and ＆＃946; - 蛋白蛋白小于5倍，除了克隆A中的Sox2，其10倍。在单层培养物中，用GFP Nanog启动子稳定转染的CRC是低表er的，但在过渡到垂直生长并形成球体时表示GFP。与在无血清培养基中单细胞球源性测定中相比，用慢病毒nanog shRNA稳定转染可降低纳米转录水平和CX-1的球源水平和65％。最后，与载体对照或亲本细胞相比，纳米shRNA转染的细胞在48中的暴露于0.3 m的拓扑替康幸存。总而言之，我们的数据表明，纳米/纳米P8的表达发生在人类CRC中，对于假定的CSC等功能，例如球源性和对化学疗法的抗性至关重要。 ii。）进步我们通过在低依恋条件下补充EGF和BFGF（神经性，干细胞技术）的无血清培养基中培养细胞，开发了一种无偏见的方法来分离推定的CSC。我们主要使用了两条人CRC系来进行这项工作：克隆A，一种分化差的结肠细胞系，在亚属cutis中是肿瘤性的，但在胞内注射到无静脉裸小鼠中后弱转移性，而CX-1（HT-29的变体）是中等差异化的，高度分化的，高度溶解的，较大的，转移性和转移性的。基于流式细胞术的最初尝试通过细胞大小和染料排除来隔离侧群的方法是不成功的，因为CRC的转运蛋白功能太高。 开发了单个细胞球源测定，其中通过限制无血清培养基中的稀释度，将单个细胞放置在96孔微量滴定板的单个孔中，然后在1-2周时评估了细胞形成球体的％。在该测定中，CX-1比克隆A更具侵略性，其中33％的单细胞形成球体，而不到8％的克隆A细胞。 CRC线之间的电镀效率没有差异，而是2个CRC线之间的球源性p <0.0004。细胞系克隆A和CX-1形成的球体增加了核心多能TF的基因表达，尤其是Nanog，相对于单层培养中的表达增加了12-43倍。转运蛋白，细胞膜蛋白或其他核心多能TF的基因表达的增加不相同。 已经进行了蛋白质印迹，证明了转运蛋白和细胞粘附蛋白表达的相应变化。但是，Nanog的抗体在蛋白质印迹中不能很好地工作，也没有提出。作为替代方案，我们在纳米启动子驱动的GFP的克隆A和CX-1中创建了稳定的构建体，以确认球体中纳米表达的上调（项目2中描述）。一旦我们确认纳米的基因表达在球体中上调，我们就开始开发一种慢病毒shRNA方法来沉降纳米。使用开源shRNA，我们分离了稳定的克隆A和CX-1的克隆克隆，纳米基因表达至少降低了70％。当我们将这些克隆与单细胞球体性测定中的载体对照和父母细胞系进行比较时，纳米表达的抑制降低了CX-1的球体性。克隆A。Nanog表达对化疗的耐药性的影响正在进行中。这里使用的药物是Toopotecan（Topo），这是一种类似于患者大肠癌中使用的虹膜蛋白酶。初步实验表明，抑制纳米表达会增加对CRC中TOPO的敏感性。一个复杂的问题是，抑制Nanog会导致S相倒置和该项目的未来方面，将决定细胞周期的抑制如何影响对拓平的敏感性。 iii。）未来的未来方向，我们计划评估Nanog对亚皮带下肿瘤金属的影响以及在NOD/SCID小鼠中注射泛胞经后的转移潜力。细胞将在稀释系列中注射，并将至少重复一次实验。在没有矩阵的情况下将进行体内恶性生长测定，以确定纳米在未修饰的肿瘤生长中的作用。此外，慢病毒shRNA到OCT4和SOX2还将稳定转染到CRC中，并将其对CSC评估并与沉默的Nanog获得的CSC进行稳定转染。在这些CRC系中建立了Nanog和其他核心多能TF的生物学之后，我们将通过创建一种可以用于筛选抑制纳米结合其在启动子中DNA序列的化合物的测定方法来开始AIM 3。如果成功，我们将向下一个程序提议，该测定法用于筛选抑制剂的化学文库，然后如果发现该剂的抑制作用，则将测试其在体外和体内抑制CSC功能的能力。