Embryonic Transcription Factor Function in Human Colorectal Cancer Stem Cells

目的3：开发抑制NANOG和NANOGP8在体内表达的治疗方法。慢病毒是体外将shRNA导入细胞的常用方法。我们的初步数据表明，慢病毒（LV）可能是一种有用的载体，可以传递shRNA来抑制已建立的肿瘤中的基因表达。然而，我们发现使用LV作为体内基因治疗似乎不适合这种非复制病毒。注射后8天内，病灶内治疗伴有LV 5次感染（MOI）的NOD/SCID小鼠3 - 6mm皮下CX-1结节可使肿瘤生长降低50%。这可能是最大临床剂量。8天后肿瘤生长迅速反弹。3天后处死小鼠时，shRNA组的肿瘤体积和重量与对照组相似。这些肿瘤中的NANOG总转录物水平与生长反应相似，在肿瘤注射后2天，与注射对照shRNA的肿瘤相比，NANOG水平较低，但在牺牲时，NANOG水平与对照相同。因此，LV shRNA对NANOGP8或NANOG（因为两者都存在于两种肿瘤中，抑制其中一种似乎会抑制另一种，因为两种启动子都含有NANOG结合位点）在转导细胞中控制肿瘤生长，但非转导细胞最终生长，导致单次注射后肿瘤生长进展。结果，我们创造了可复制的表达shRNA的溶瘤腺病毒。5型腺病毒（Ad5）是onyx15的主干，onyx15是一种条件复制腺病毒（CRAd），在失去p53功能的肿瘤细胞中复制。早期试验表明，病毒在头颈部鳞状癌患者中复制，但宿主免疫限制了全身给药CRAd的效用。Ad5也有肝毒性。我们用嵌合纤维创造了腺病毒，它结合了3型旋叶和5型纤维，创造了Ad5/3。这种嵌合体降低了肝性（和肝毒性）。这将病毒受体从柯萨奇腺病毒受体（CAR基因）转化为粘粒蛋白2 (DSG2)，这是一种在上皮细胞和内皮细胞中表达的半粒体中的蛋白质。我们的病毒还含有对照shNEG和等位基因特异性shNG-1和shNp8-1的h1驱动shrna以及绿色荧光蛋白（GFP）报告基因。这些病毒将在人类细胞中复制和裂解，而不是小鼠细胞，具有野生型E1a病毒启动子，并被指定为Ad5/3-E1aWT- shneg， Ad5/3-E1aWT- shnp8和Ad5/3-E1aWT（最后一个没有shRNA或GFP报告细胞）。此外，我们已经创建了一个具有5/3纤维的条件复制腺病毒（CRAd5/3），其中NANOGP8启动子的前1kb驱动E1a的一部分。该CRAD 5/3-E1aNp8-shNp8也含有shNp8-1和GFP报告基因，可在支持NANOGP8表达的细胞中复制，但不能在NANOGP8阴性的人类细胞中复制。2014年1月中旬，我们首次在克隆A和CX-1中检测了Ad5/3- e1awt和Ad5/3- e1awt - shnp8携带野生型启动子的Ad5/3’s，每个CRC细胞的病毒颗粒的不同稀释度。正如预期的那样，增加病毒的MOI会增加感染率，但即使在MOI只有35 -10%的CRC被感染，到第7天80%的细胞表达GFP。在我们的LV实验中，我们在3天内实现了CRC细胞存活率的最大降低，抑制率为50%。相比之下，暴露于Ad5/3-E1aWT或Ad5/3-E1aWT- shnp8的3-D球形和聚集体CRC细胞在10天内的细胞生长下降至未处理对照组的15%。CRAd Ad5/3-E1aNp8-shNp8活性稍低，但在悬浮培养10 d后达到50%的抑制生长。数据表明，野生型和CRAd病毒在裂解人CRC细胞时都以球状体的形式在细胞间传播，可能是通过连接细胞的桥粒。此外，与不支持Ad5/3复制的小鼠3T3细胞共培养抑制腺病毒的裂解活性，但仅当基质：CRC细胞比例为1:1时，这比大多数人CRC转移的发生率高。此外，数据表明，与不含shNp8的野生型病毒相比，含有NANOGP8 shRNA的Ad5/3更具活性。肿瘤内治疗研究正在进行中，以评估Ad5/3和CRAD在体内的作用。因此，我们开发了一种体外3-D肿瘤生长模型，这可能是预测体内治疗效果的有益模型，以及几种新的病毒制剂，其体内活性将很快进行评估。目标4。评估抑制NANOG或NANOGP8是否会增加对癌症治疗的反应。我们已经提交了一篇论文，证明抑制NANOG/NANOGP8基因表达可以抑制MCL-1的表达以及AKT的表达和激活。这种效应增强了BH3肽在NOD/SCID小鼠全身治疗模型中的体外和体内的功效，并抑制了CSC支持克隆再生的能力。目前正在临床癌症研究中心（Mattoo AR, Jingyu Zhang J, Luis A. Espinoza LA, Jessup JM）进行研究。抑制NANOG/NANOGP8导致结直肠癌细胞MCL-1下调，从而增强BH3模拟物的治疗效果。)这导致了一项后续研究，在该研究中，CSC抑制剂selinomycin被用来代替shRNA。该药物还增强了BH3抑制剂在4种人类结直肠癌细胞系中的3种中的作用。selinomycin不影响的细胞系是它不抑制MCL-1的细胞系。shNp8对MCL-1均有抑制作用，并增加ABT-737和ABT-199的活性。我们现在正在使用纳米链基因表达阵列来评估shRNA如何改变基因表达。去年我们报道了nanog的lvshrna增强拓扑替康对结直肠癌的活性。由于分子机制尚不清楚，这一研究尚未发表，我们主要关注nanog和BH3死亡蛋白之间的相互作用。最后，我们发现LV shNp8-1对NANOGP8的抑制激活了Caspase 9和ULLA板中无血清培养基悬浮培养引起的凋亡应激的内在凋亡途径。这一途径是一篇正在审查的论文的主题（Mattoo AR, Zhang J, Espinoza LA, Korokhov N, Jessup JM）。抑制NANOGP8或NANOG激活细胞凋亡的内在途径。正在审查中)，也可能有助于增加可能受益于caspase依赖性细胞死亡的药物的活性。纳米链基因表达系统的初步结果表明，抑制NANOG或NANOGP8会导致5 - 10个重要转录因子的改变，从而导致基因表达的二次修饰。初步研究表明，在一些CRC细胞系中，shNp8-1和较小程度的shNG-1激活自噬，当自噬水平相当高时，这也会导致细胞凋亡。综上所述，我们的数据支持NANOGP8和NANOG是CRC中干细胞功能的主要调节因子，抑制它们的表达可能会导致干细胞的显著减少，并可能增加对精准药物的反应。

Aim 3 To develop therapies that inhibit NANOG and NANOGP8 expression in vivo Lentivirus is commonly used for the introduction of shRNA into cells in vitro. Our preliminary data suggested that lentivirus (LV) might be a useful vector to deliver shRNA to inhibit gene expression in established tumors. However, we found that the use of LV as an in vivo gene therapy appears not to be practical for this nonreplicating virus. Intralesional treatment of 3 - 6 mm subcutaneous nodules of CX-1 in NOD/SCID mice with a 5 multiplicity of Infection (MOI) of LV decreased tumor growth by 50% for up to 8 days after injection. This is likely to be the maximum clinical dose. After 8 days there was rapid rebound of tumor growth. When mice were sacrificed 3 days later, the tumors in the shRNA groups were similar in volume and weight to the controls. NANOG total transcript levels in these tumors paralleled the growth response in that NANOG levels were low 2 days after tumor injection compared to tumors injected with control shRNA but levels were the same as controls at the time of sacrifice. Thus, LV shRNA to NANOGP8 or NANOG (since both are present in both tumors and inhibiting one seems to inhibit the other because both promoters contain NANOG binding sites) controls tumor growth in transduced cells but non-transduced cells eventually grow, leading to progressive tumor growth after single injection. As a result, we created replicating oncolytic adenoviruses that express shRNA. Adenovirus type 5 (Ad5) was the backbone of ONYX 15 that was a conditionally replicating adenovirus (CRAd) that replicated in tumor cells that lost function of p53. Early trials demonstrated that the virus replicated in patients with head and neck squamous carcinoma but that host immunity limited the utility of systemically administered CRAd. Also Ad5 is hepatotoxic. We have created adenoviruses with a chimeric fiber that combines a type 3 knob with a type 5 fiber to create Ad5/3. Such chimeras have reduced hepatotropism (and hepatotoxicity). This converts the receptor for the virus from the coxsackie adenovirus receptor (CAR gene) to desmoglein 2 (DSG2), a protein in hemidesmosomes expressed in epithelia and endothelial cells. Our viruses also contain the H1-driven shRNAs of the control shNEG and the allele-specific shNG-1 and shNp8-1 along with a green fluorescent protein (GFP) reporter. These viruses will replicate in and lyse human but not mouse cells and have the Wild Type E1a viral promoter and are designated Ad5/3-E1aWT-shNEG, Ad5/3- E1aWT -shNp8 and Ad5/3- E1aWT (this last without shRNA or GFP reporter). In addition, we have created a conditionally replicating adenovirus with a 5/3 fiber (CRAd5/3) where the first 1 Kb of the NANOGP8 promoter drives a part of E1a. This CRAD 5/3-E1aNp8-shNp8 also contains shNp8-1 as well as the GFP reporter and replicates in cells that support NANOGP8 expression but not in NANOGP8 negative human cells. The Ad5/3's with wild type promoters were first tested in mid-January, 2014 with Ad5/3-E1aWT and Ad5/3-E1aWT-shNp8 in Clone A and CX-1 at various dilutions of viral particles per CRC cell. As expected, increasing the viral MOI increases the infection rate but even at MOIs of only 3 5 -10% of CRC are infected and by day 7 80% of cells express GFP. In our LV experiments we achieved a maximal decrease in CRC cell survival of 50% inhibition at 3 days. In contrast, exposure of CRC cells in 3-D spheroids and aggregates to Ad5/3-E1aWT or Ad5/3-E1aWT-shNp8 decreased cell growth in 10 days to 15% of that of the untreated controls. The CRAd Ad5/3-E1aNp8-shNp8 is a little less active but achieves a 50% inhibition of growth in 10 days of suspension culture. Data indicate that both the wild type and CRAd viruses spread from cell to cell in spheroids as they lyse the human CRC cells, possibly by traversing through the desmosomes that connect one cell to another. In addition, co-cultures with murine 3T3 cells that do not support replicationof Ad5/3 inhibited the lytic activity of the adenoviruses but only when stromal:CRC cell ratio was 1:1 which is higher than occurs in most human CRC metastases. In addition, data demonstrate that the Ad5/3 with shRNA to NANOGP8 was more active than the wild type virus without shNp8. Intratumoral treatment studies are just underway to assess the effect of Ad5/3's and the CRAD in vivo. Thus, we have developed an in vitro 3-D tumor growth model that may be a beneficial model for predicting treatment effects in vivo as well as several new viral agents whose activity in vivo will be assessed shortly. Aim 4. Assess whether inhibition of NANOG or NANOGP8 increases responses to cancer therapies. We have submitted a manuscript that demonstrates that inhibition of the NANOG/NANOGP8 gene expression inhibits MCL-1 expression as well as the expression of AKT and its activation. This effect potentiates the efficacy of BH3 peptides both in vitro and in vivo in a systemic treatment model in NOD/SCID mice as well as inhibiting the ability of CSC to support clonogenic regrowth. This is currently under review at Clinical Cancer Research (Mattoo AR, Jingyu Zhang J, Luis A. Espinoza LA, Jessup JM. Inhibition of NANOG/NANOGP8 results in MCL-1 down regulation in colorectal cancer cells to enhance the therapeutic efficacy of BH3 mimetics.). This has led to a follow-up study in which the CSC inhibitor selinomycin is used in place of shRNA to the NANOGs. The drug also potentiates the effect of the BH3 inhibitors in 3 of 4 human CRC cell lines. The 1 cell line that selinomycin does not affect is the one in which it does not inhibit MCL-1. Treatment with shNp8 did inhibit MCL-1 in all 4 lines and increased the activity of ABT-737 and ABT-199. We are now using Nanostring gene expression arrays to assess how shRNA alters gene expression. Last year we reported that the LVshRNAs to the NANOGs potentiate the activity of Topotecan on CRC. This has not been published because the molecular mechanism is still unclear and we focused on the interaction between the NANOGs and BH3 death proteins. Finally, we have found that inhibition of NANOGP8 by LV shNp8-1 activates Caspase 9 and the intrinsic pathway of apoptosis during apoptotic stress caused during culture in suspension in serum-free medium in ULLA plates. This pathway is the subject of a manuscript under review (Mattoo AR, Zhang J, Espinoza LA, Korokhov N, Jessup JM. Inhibition of NANOGP8 or NANOG Activates the Intrinsic Pathway of Apoptosis. Under review) and may also help increase the activity of drugs that may benefit from caspase-dependent cell death. Preliminary results with the Nanostring gene expression system suggests that inhibition of NANOG or NANOGP8 leads to the alteration of 5 - 10 important transcription factors that then lead to secondary modifications in gene expression. Preliminary work suggests that in some CRC lines shNp8-1 and to a lesser extent shNG-1 activate autophagy and that this also leads to apoptosis when the level of autophagy is quite high. In summary, our data support the thesis that NANOGP8 and NANOG are master regulators of stem cell function in CRC and that inhibition of their expression may cause a pronounced decrease in stemness and possibly an increase response to agents of precision medicine.