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: 8157691
• 负责人: John Jessup
• 金额: $ 11.03万
• 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/8157691
• 关键词: Colorectal Cancer; Digestion; Embryo; Human; Pharmaceutical Preparations; Transcription Coactivator; cancer stem cell; prevent; transcription factor

A.) Aim 1 was completed in year 1 B.) Aim 2 Using our Single Cell Spherogenicity Assay (SCSA), we completed our analysis of spherogenicity in our human CRC lines and found that spherogenicity generally parallels metastatic potential: LS 174T greater than CX-1 = Mip-101 greater than Clone A = KM-12c and HCC 2998. Expression of the core embryonic genes Nanog, OCT4, and SOX2 was analyzed in spheroids and monolayer cultures by qRT-PCR, normalized by the expression of GAPDH and expression in spheroids compared to the expression in monolayer cultures of each cell lines as a relative expression and indicates that Nanog gene expression is consistently increased in spheroids compared to the expression in that lines monolayer culture while the expression of the other core embryonic genes OCT4 and SOX2 is more variable. OCT4 is a tightly regulated gene in embryonic stem cells and this does not seem to change in these CRC lines since multiple qRT-PCR experiments indicate that the level of OCT4 transcripts in spheroids only ranges between 5- and 9-fold increased over basal levels in monolayer cultures of each CRC line. However, the levels of Nanog gene transcripts ranges from 8-fold increase over monolayer in Clone A to 300-fold in KM-12c when normalized to GAPDH. SOX2 transcript levels also were quite different among the CRC with CX-1 and HCC 2998 expressing low levels by normalized qRT-PCR in monolayer and spheroid, Clone A had a 10-fold change in SOX2 expression and MIP-101 and LS 174T had a 50 - to 60-fold increase in SOX2 levels compared to monolayer cultures. These data suggest that the relative of Nanog, SOX2 and OCT4 may be critical determinants of the malignant properties of human CRC. With this knowledge about the variability of expression of the 3 embryonic genes, we then began to focus on Clone A, CX-1 and LS 174T for subsequent research. This was done by specific gene knock down with lentiviral vector mediated shRNA to each of the 3 genes. Stable transductants were created for proof of principle experiments in which expression of each target gene was inhibited by 60% or more by qRT-PCR. Then transductants and vector controls were assessed first in the SCSA in vitro and then in vivo in NOD/SCID mice. in the SCSA, shRNA to Nanog consistently reduced spherogenicity by 94% in Clone A, 70% in CX-1 and 52% in LS 174T. In contrast, shRNA to OCT4 or SOX2 actually increased spherogenicity by over 200% in CX-1 and 500% in Clone A. However, in LS174T that had elevated levels of SOX2 gene expression, shRNA to OCT4 or SOX2 reduced spherogenicity by 76% and 52% respectively. These novel findings are real since shRNA to OCT4 or SOX2 significantly shortened time to appearance of tumors after injection subcutaneously in NOD/SCID mice with CX-1 and Clone A (with shRNA to SOX2). However, shRNA to OCT4 or SOX2 prolonged the mean time to appearance of tumors with LS 174T by nearly 50% compared to the parental controls. shRNA to Nanog significantly reduced tumorigenicity by prolonging time to appearance or reducing tumor takes on limiting dilution analysis in all 3 CRC tumors. In addition shRNA completely prevented liver colony formation after intrasplenic injection of CX-1 in an experimental metastasis assay. Thus, shRNA to Nanog has a consistent inhibition of malignant properties of CRC whereas shRNA to OCT4 or SOX2 do not. Also analysis of transductants demonstrates that shRNA to Nanog inhibits OCT4 and SOX2 gene expression by 50% but that inhibition of OCT4 or SOX2 does not affect Nanog gene expression. Finally, we have developed a screening assay to detect which of the Nanog family members is activated in human CRC. In order to identify which gene product is expressed in human CRC, we sought to identify a site in NanogP8 around one of the 5 single nucleotide changes that might be amenable to endonuclease digestion. A search of databases revealed that the only specific commercially available endonuclease that was predicted to cut NanogP8 cDNA but not Nanog was 143 nt 3 downstream to the ATG by AlwNI which cuts at a 5'... C A G N N N C T G ... 3' 3'... G T C N N N G A C ... 5' sequence. Primers were designed to amplify both Nanog and NanogP8 and then RT-PCR was performed on total RNA isolated from Clone A and CX-1 cells growing under basal conditions in monolayer with serum-containing medium and in spheroids in ULLA plates and serum-free medium. The assay produces two cleavage products on a gel when NanogP8 is cleaved by AlwNI but no cleavage products when only Nanog is present. Monolayer cultures of CRC express both Nanog and NanogP8 and rarely NanogP7 but the spheroids express predominantly NanogP8. This was confirmed by direct sequencing of 20 30 clones of individual, separate cDNAs from CX-1 and Clone A that demonstrated only NanogP8 was expressed in spheroid cultures whereas 10 -12.5% of monolayer cDNAs contained Nanog or NanogP7 transcripts. With this we began to focus on therapy that would specifically target only NanogP8 and not Nanog. This is important for clinical studies since the function of Nanog and the other core embryonic genes occur during pre- and early implantation before the woman can know she is pregnant. B.) Aim 3 - To develop therapies that inhibit NANOG expression in vivo. We originally had planned to develop an assay to develop drugs that would target Nanog. However, the uncertainty of the mechanism of action of Nanog in carcinoma cells and now the realization that the active gene is NanogP8 has caused us to rethink our original plan of attack. As described below, the amount of Nanog or NanogP8 gene transcripts even in cells enriched for stemness is quite low. As a result, gene therapy with lentiviral vector delivered shRNA may inhibit Nanog sufficiently inhibit malignant potential. We have designed allele-specific shRNA that knocks down NanogP8 and not Nanog and are in the process of determining whether NIH will support a patent application for this. the specific shRNA to NanogP8 inhibits spherogenicity of Clone A, CX-1 and even the aggressive LS 174T. Our plan for the future is to determine whether lentiviral vector inhibits growth of spheroids that have been created in serum-free medium under ULLA conditions and then if it does (as a model of inhibition of solid tumor masses) we will test the ability of this shRNA to inhibit established liver colonies of CX-1 and LS 174T in NOD/SCID mice. We have also developed luciferase-expressing constructs of all three CRC that will be used to facilitate both the in vivo testing as well as to make a high throughput screen for several other shRNAs that we have in the pipeline. Several manuscripts are in preparation and will be submitted in the coming year once the status of the patent application is clear.

A.）AIM 1在第1年完成。）AIM 2使用我们的单细胞球源性测定（SCSA），我们完成了人类CRC系中球源性的分析，发现球源性通常与cx-1 = MIP-101大于CLS-101大于Clone a = KM-12C和HCC 299的cock 2998。通过QRT-PCR在球体和单层培养物中分析SOX2，与小细胞系的单层培养物中的表达相比，球体中的表达和球体中的表达归一化，并表明NANOG基因在该线培养物中的表达中的表达中的表达始终增加，而单层培养物的表达则更多地增加了nolayer corec的表达。 OCT4是胚胎干细胞中严格调控的基因，在这些CRC系中，这似乎并没有改变，因为多个QRT-PCR实验表明，球体中的OCT4转录物的水平仅在每种CRC系的单层培养物中基底水平上的5至9倍范围内仅增加了5至9倍。然而，当标准化为GAPDH时，Nanog基因转录物的水平从克隆A的单层比单层的8倍到300倍。 CRC中的Sox2转录水平在CX-1和HCC 2998中也有很大差异，单层和球体中的标准化QRT-PCR表达较低的水平，克隆A在Sox2水平上的SOX2表达变化10倍，SOX2表达变化10倍，而LS 174T的变化为50-至60倍。这些数据表明，Nanog，Sox2和Oct4的亲戚可能是人类CRC恶性特性的关键决定因素。凭借有关3个胚胎基因表达的可变性的知识，我们开始专注于克隆A，CX-1和LS 174T进行后续研究。这是通过特异性基因用慢病毒载体介导的shRNA对3个基因中的每个基因敲击而完成的。创建了稳定的转导剂，以证明原理实验，其中QRT-PCR抑制每个靶基因的表达60％或更多。然后，首先在SCSA中评估了转导剂和载体对照，然后在NOD/SCID小鼠的体内进行体内评估。在SCSA中，shRNA至nanog在克隆A中始终降低球体性的94％，CX-1中的70％和LS 174T的52％。相反，在CX-1中，SHRNA至OCT4或SOX2实际上增加了SHERNA，在CX-1中增加了200％以上。但是，在SOX2基因表达水平升高的LS174T中，SHRNA至OCT4或SOX2分别降低了球体源性，分别降低了76％和52％。这些新的发现是真实的，因为SHRNA至OCT4或SOX2显着缩短了在用CX-1和Clone A和Clone A（用SHRNA到SOX2）的NOD/SCID小鼠皮下注射后肿瘤出现的时间。然而，与父母对照组相比，SHRNA至OCT4或SOX2的平均延长时间延长了LS 174T肿瘤的出现近50％。 shRNA至nanog通过延长外观或减少肿瘤的延长时间可显着降低肿瘤性，这在所有3种CRC肿瘤中都进行了限制稀释分析。另外，shRNA在实验转移测定法中CX-1注射CX-1后完全阻止了肝落形成。因此，shRNA至Nanog具有CRC的恶性特性一致的抑制，而ShRNA对Oct4或Sox2则没有。对传染剂的分析也表明，shRNA至Nanog的shRNA抑制了OCT4和SOX2基因表达50％，但抑制OCT4或SOX2不会影响Nanog基因的表达。 最后，我们开发了一种筛选测定法，以检测人类CRC中的哪些Nanog家族成员被激活。为了识别人CRC中哪种基因产物的表达，我们试图在5个单核苷酸变化中的一种周围识别NanogP8中的位点，这可能是内核酸酶消化的。对数据库的搜索表明，预计将唯一可以切割Nanogp8 cDNA而不是Nanog的特定特定的市售内切核酸酶是Alwni的ATG下游143 NT 3 3'... c a g n n n n c t g ... c a g n n n n c t g ... 3'3'3'3'3'... g t c n n n n n n n n n n n g a c ... 5'序列。 设计了引物来扩增Nanog和NanogP8，然后对从克隆A和CX-1细胞分离的总RNA进行RT-PCR，在单层中生长的CX-1细胞，含血清的培养基，在ULLA平板和无血清培养基中的球体中进行。当NanogP8被Alwni裂解时，该测定法在凝胶上产生两种乳化产物，但仅存在Nanog时，没有切割产品。 CRC的单层培养物同时表达纳米和纳米P8和Nanogp7，但球体主要表达纳米型。 通过直接测序20 30个个体的克隆，与CX -1和克隆A进行直接测序，该克隆仅在球体培养物中表达了只有NanogP8的A，而单层cDNA中有10-12.5％的单层cDNA包含Nanog或Nanogp7转录本。 因此，我们开始专注于专门针对NanogP8而不是Nanog的治疗。这对于临床研究很重要，因为Nanog的功能和其他核心胚胎基因在妇女知道自己怀孕之前就发生了前和早期植入期间。 B.）目标3-开发抑制体内纳米表达的疗法。 我们最初计划开发一种用于开发靶向纳米的药物的测定法。然而，纳米在癌细胞中的作用机理的不确定性，现在意识到活性基因是nanogp8，使我们重新考虑了我们的最初攻击计划。如下所述，即使在富含茎的细胞中，Nanog或NanogP8基因转录物的量也很低。结果，用慢病毒载体进行的基因疗法递送SHRNA可能会抑制纳米足够抑制恶性潜力。我们设计了等位基因特异性的shRNA，它击倒Nanogp8而不是纳米，并且正在确定NIH是否会支持专利申请。对纳米P8的特异性shRNA抑制了克隆A，CX-1甚至侵略性LS 174T的球体。我们对未来的计划是确定慢病毒载体在ULLA条件下抑制在无血清培养基中创建的球体的生长，然后如果确实如此（作为抑制固体肿瘤块的模型），我们将测试该SHRNA在NOD/Scid/Scid小鼠中抑制CX-1和LS 174T的固定肝脏菌落的能力。 我们还开发了所有三种CRC的表达荧光素酶的构建体，这些构建体将用于促进体内测试，并为我们在管道中使用的其他几个SHRNA制作高吞吐量屏幕。一份专利申请的状态明确，将在准备中进行一些手稿，并将在来年提交。