Embryonic Transcription Factor Function in Human Colorectal Cancer Stem Cells

a .)目标1于第一年完成。目的2使用我们的单细胞球形实验（SCSA），我们完成了我们的人类CRC细胞系的球形性分析，发现球形性通常与转移潜力相似：LS 174T大于CX-1 = Mip-101，大于克隆A = KM-12c和HCC 2998。采用qRT-PCR方法分析胚核基因Nanog、OCT4和SOX2在球形和单层培养中的表达；将GAPDH的表达归一化，并将各细胞系在球体中的表达与在单层培养中的表达相比，作为相对表达，表明Nanog基因在球体中的表达比在单层培养中的表达持续增加，而其他核心胚胎基因OCT4和SOX2的表达变化更大。OCT4在胚胎干细胞中是一个受到严格调控的基因，在这些CRC细胞系中似乎没有变化，因为多个qRT-PCR实验表明，在每个CRC细胞系的单层培养中，球体中OCT4转录本的水平仅比基础水平增加了5到9倍。然而，当归一化到GAPDH时，在KM-12c中，Nanog基因转录物的水平从单层的8倍增加到300倍。标准化qRT-PCR结果显示，CX-1和HCC 2998在单层和球形细胞中表达水平较低的结直肠癌中SOX2的表达水平也有很大差异，克隆A的SOX2表达变化了10倍，MIP-101和LS 174T的SOX2表达水平比单层培养的SOX2水平增加了50至60倍。这些数据提示Nanog、SOX2和OCT4的亲缘关系可能是人类结直肠癌恶性特性的关键决定因素。了解了3个胚胎基因的表达变异性后，我们开始对克隆A、CX-1和LS 174T进行后续研究。这是通过慢病毒载体介导的shRNA对3个基因中的每一个进行特异性基因敲除来完成的。稳定的转导剂被创建用于原理实验的证明，其中每个靶基因的表达被qRT-PCR抑制了60%或更多。然后首先在体外和NOD/SCID小鼠体内对SCSA进行了转导剂和载体对照的评估。在SCSA中，shRNA对Nanog的影响在克隆A中持续降低了94%的球形性，在CX-1中降低了70%，在LS 174T中降低了52%。相比之下，shRNA对OCT4或SOX2的处理实际上使CX-1的球状性提高了200%以上，在克隆a中提高了500%。然而，在SOX2基因表达水平升高的LS174T中，shRNA对OCT4或SOX2的处理分别使球状性降低了76%和52%。这些新发现是真实的，因为在CX-1和克隆A （shRNA to SOX2）的NOD/SCID小鼠皮下注射后，shRNA to OCT4或SOX2显著缩短了肿瘤出现的时间。然而，与父母对照相比，对OCT4或SOX2的shRNA使LS 174T患者的平均肿瘤出现时间延长了近50%。在所有3种CRC肿瘤中，shRNA to Nanog通过延长出现时间或减少肿瘤发生率显著降低致瘤性。此外，在实验转移试验中，shRNA完全阻止脾内注射CX-1后肝脏集落的形成。因此，shRNA对Nanog具有一致的抑制CRC恶性特性的作用，而shRNA对OCT4或SOX2则没有。此外，对转导剂的分析表明，shRNA对Nanog的OCT4和SOX2基因的表达抑制率为50%，但对OCT4或SOX2的抑制不影响Nanog基因的表达。最后，我们开发了一种筛选试验来检测Nanog家族成员中哪些在人类CRC中被激活。为了确定在人类结直肠癌中表达的基因产物，我们试图在NanogP8中确定一个位点，该位点位于可能适合核酸内切酶酶切的5个单核苷酸变化之一附近。对数据库的搜索显示，唯一可预测切割NanogP8 cDNA而不是Nanog的特异性商业上可用的内切酶是AlwNI在ATG下游143 nt 3处切割的5'…C、C、C、C、C、C……3 ' 3 '……G, c, c, c， G, c……5 '序列。设计引物扩增Nanog和NanogP8，然后对克隆A和CX-1细胞在基础条件下生长的总RNA进行RT-PCR，分别在含血清培养基的单层和ULLA板和无血清培养基的球形中分离总RNA。当NanogP8被AlwNI切割时，该实验在凝胶上产生两个裂解产物，而当NanogP8只存在时，没有裂解产物。CRC的单层培养细胞既表达Nanog，也表达NanogP8，很少表达NanogP7，但球状细胞主要表达NanogP8。通过对CX-1和克隆A的20 - 30个独立cdna克隆的直接测序证实了这一点，结果表明在球形培养中只有NanogP8表达，而10 -12.5%的单层cdna含有NanogP7或NanogP7转录本。有了这个，我们开始专注于只针对NanogP8而不是Nanog的治疗。这对临床研究很重要，因为Nanog和其他核心胚胎基因的功能发生在植入前和早期，在妇女知道自己怀孕之前。b .)目的3 -开发抑制NANOG体内表达的治疗方法。我们最初计划开发一种检测方法来开发针对Nanog的药物。然而，Nanog在癌细胞中的作用机制的不确定性，以及现在认识到活性基因是NanogP8，使我们重新考虑了我们最初的攻击计划。如下所述，即使在干细胞富集的细胞中，Nanog或NanogP8基因转录物的数量也相当低。因此，用慢病毒载体递送shRNA进行基因治疗可以充分抑制Nanog的恶性潜能。我们已经设计了等位基因特异性shRNA，可以抑制NanogP8而不是Nanog，并且正在决定NIH是否会支持这项专利申请。对NanogP8的特异性shRNA抑制克隆A、CX-1甚至攻击性的LS 174T的球形性。我们未来的计划是确定慢病毒载体是否抑制在无血清培养基中在ULLA条件下产生的球体的生长，然后如果确实如此（作为抑制实体瘤肿块的模型），我们将测试该shRNA抑制NOD/SCID小鼠中已建立的CX-1和LS 174T肝脏菌落的能力。我们还开发了所有三种CRC的荧光素酶表达构建体，这些构建体将用于促进体内测试以及对我们正在开发的其他几种shrna进行高通量筛选。一些手稿正在准备中，一旦专利申请的状态明确，将在明年提交。

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.