Embryonic Transcription Factor Function in Human Colorectal Cancer Stem Cells

我)。Nanog是维持胚胎干细胞多能、去分化状态的几个核心转录因子之一。最近的数据表明，NANOG或其几乎相同的逆转录基因NANOGP8在人胃癌、肝细胞癌和结肠癌中的表达增加。我们的假设是，NANOG/NANOGP8对于结肠直肠癌（CRC）中假定的CSC的功能至关重要：自我更新、多能性、球状性和对化疗的抗性。本研究使用了5个人类结直肠癌系（HCC 2998、KM-12c、克隆A、Mip-101、CX-1）。悬浮培养96小时后，对单独或联合表达ALDH1、CD44或CD133的细胞进行TUNEL分析，测定悬浮培养中的细胞程序性死亡（Anoikis）。在结直肠癌品系中没有发现一致的耐药模式。然而，在悬浮培养中，NANOG和/或Oct4的表达增加，并与对anoikis的抗性有关。对CX-1和克隆A在无血清培养基中悬浮培养的球形细胞进行qRT-PCR分析发现，NANOG转录物水平较单层培养分别提高了43倍和12倍，Oct4、Sox2、Klf4、ABCG2、ABCA3、ALDH1A1、c-myc、CD44、CD133、CD166和&amp；#946转录物水平相对提高；-catenin在克隆A中除Sox2为10倍外均小于5倍。稳定转染GFP NANOG启动子的结直肠癌在单层培养中低表达，但在垂直生长并形成球体时表达GFP。在无血清培养基中进行单细胞球性实验，与载体对照相比，慢病毒NANOG shRNA稳定转染使CX-1的NANOG转录物水平和球性降低了65%。最后，与载体对照或亲本细胞相比，转染NANOG shRNA的细胞在暴露于0.3 M Topotecan中48天后存活的较少。总之，我们的数据表明NANOG/NANOGP8的表达在人类结直肠癌中存在，并且可能对推测的CSC的球形性和化疗耐药性等功能至关重要。二)。我们开发了一种无偏倚的方法，通过在无血清培养基中添加EGF和bFGF (Neurocult, Stem Cell Technologies)，在低附着条件下培养细胞来分离假定的CSC。我们主要使用了两种人类结直肠癌细胞系：克隆A，一种低分化的结肠细胞系，在皮下具有致瘤性，但在胸腺裸鼠脾内注射后转移性弱；CX-1， HT-29的一种变体，中等分化，高度致瘤性和转移性。由于CRC的转运蛋白功能太高，最初尝试开发一种基于流式细胞术的方法，通过细胞大小和染料排除来分离侧群，但没有成功。建立了单细胞成球试验，在无血清培养基中限制稀释，将单细胞置于96孔微滴板的单个孔中，然后在1 - 2周评估细胞形成球体的百分比。CX-1比克隆A更具侵袭性，33%的单细胞形成球体，而克隆A的单细胞形成球体的比例不到8%。两种CRC线之间的镀效率无差异，但两种CRC线之间的球度差异为0.0004。克隆A和CX-1细胞系形成的球体增加了核心多能tf的基因表达，特别是NANOG的表达量比单层培养增加了12 - 43倍。转运蛋白、细胞膜蛋白或其他核心多能性tf基因表达的增加程度不同。Western blot显示转运蛋白和细胞粘附蛋白的表达发生了相应的变化。然而，NANOG的抗体在western blot中效果不佳，因此没有出现。作为替代方案，我们在克隆A和CX-1中构建了NANOG启动子驱动的GFP的稳定构建体，以确认NANOG在球体中的表达上调（项目2中描述）。一旦我们确认NANOG的基因表达在球状体中上调，我们就开始开发一种慢病毒shRNA方法来沉默NANOG。利用开源shRNA，我们分离出稳定转染的克隆A和CX-1，它们的NANOG基因表达至少降低了70%。当我们将这些克隆与载体对照和亲本细胞系进行单细胞球形性试验时，抑制NANOG表达降低了CX-1的球形性。克隆a的研究正在进行中，NANOG表达对化疗耐药的影响已经初步研究。这里使用的药物是拓扑替康（TOPO），一种喜树碱，与伊立替康类似，用于结直肠癌患者。初步实验表明，抑制NANOG表达可增加结直肠癌对TOPO的敏感性。一个复杂的问题是，抑制NANOG会导致S期阻滞，该项目的未来方面将确定抑制细胞周期如何影响对拓扑替康的敏感性。iii)。在不久的将来，我们计划评估NANOG对NOD/SCID小鼠皮下致瘤性和脾内注射后转移潜力的影响。细胞将按稀释系列注射，实验将至少重复一次。为了确定NANOG在未经修饰的肿瘤生长中的作用，将在体内进行恶性生长试验。此外，将Oct4和SOX2（其他主要核心多能性tf）的慢病毒shRNA稳定转染到CRC中，并评估其对CSC的影响，并与沉默NANOG获得的效果进行比较。在这些CRC细胞系中建立NANOG和其他核心多能tf的生物学特性后，我们将通过创建一种可用于筛选抑制NANOG与其启动子DNA序列结合的化合物的检测来开始目标3。如果成功，我们将向NExT项目提议，将该试验用于筛选化学文库中的抑制剂，然后如果发现一种抑制剂，将在体外和体内测试其抑制CSC功能的能力。

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.