Embryonic Transcription Factor Function in Human Colorectal Cancer Stem Cells

shRNA对NANOG的表达抑制亲本NANOG的表达，显著降低CRC克隆A和CX-1细胞的球形性。在克隆A和CX-1细胞中重新表达NANOG或NANOGP8蛋白，NANOGP8的重新表达增加了球状性和侧群体的大小。NANOG的过表达增加了克隆A细胞的球形性，但对CX-1细胞没有影响。此外，我们证明了NANOGP8是可翻译的。这是以前没有做过的。数据收录在Zhang等人的稿件中。癌基因，2012年10月22日。doi: 10.1038 / onc.2012.461。在以5:1 ~ 16:1的MOI启动悬浮培养后，在3天内加入慢病毒将shRNA传递给NANOG （shNG-1）或NANOGP8 (shNp8-1)，可抑制克隆A、CX-1和LS 174T人CRC细胞的三维（3d）生长，抑制率近50%。这种3-D生长的抑制作用进一步扩展到在电镀效率试验中复制存活细胞时再生潜力的15-30%的下降。转导效率为45%。这表明慢病毒（LV） shNG-1和/或shNp8-1可能诱导细胞凋亡。我们之前已经发表了悬浮培养诱导caspase 8依赖的外源性途径凋亡，而不激活caspase 9和CX-1克隆细胞的内在途径。当我们通过细胞渗透荧光凋亡实验评估LV shNG-1、shNp8-1或对照LV shNEG对caspase 3,8,9活性的诱导作用时，悬浮培养中shNp8-1诱导caspase 9的激活程度大于shNG-1。当CRC在单层培养时，LV递送的shNG-1或shNp8-1通常不会引起caspases的显著激活。有趣的是，有时LV shNEG控制载体在单层培养中确实激活了半胱天蛋白酶——这可能反映了单层培养中Toll受体的表达，导致单层培养中CRC细胞内先天免疫机制的激活。综上所述，这些结果表明shRNA对NANOGs，特别是NANOGP8，激活了细胞凋亡的内在途径，并表明LV递送的shNp8-1和/或shNG-1可能与化疗或靶向治疗协同作用。这一点目前正在写成一份手稿。我们已经评估了LV shRNA对NANOG和/或NANOGP8是否与细胞凋亡间接途径的特定方面具有协同作用。由于该通路涉及BH3结构域蛋白的参与，并受抗凋亡Bcl-2家族成员和Mcl-1的控制，因此我们研究了LV shRNA对Mcl-1和Bcl-2表达的影响。LV shNp8-1抑制MCL-1的表达和AKT的激活，LV shNp8-1也抑制MCL-1，但抑制程度较轻。所有LV制剂对Bcl-2及其家族成员均无影响。LV shNp8-1在3种CRC细胞系克隆A、CX-1、LS 174T中抑制MCL-1。我们测试了LV shRNA是否与细胞毒性化疗（以Topotecan，喜树碱为模型）和抗bcl -2治疗（细胞渗透拟肽ABT-737）具有协同作用。尽管这两种处理都增加了单层培养CRC细胞中NANOG和NANOGP8基因的表达，但LV shNp8-1或LV shNG-1的加入与Topotecan或ABT-737具有协同作用。在本实验中，LV shNEG不增加细胞凋亡。在药物或抗bcl -2药物治疗前、同时或治疗后添加LV shRNA的序列也不影响协同作用。这些结果表明，shRNA抑制NANOGP8或NANOG可能抑制Mcl-1，与抗bcl -2治疗协同作用。手稿正在准备中。临床前研究将很快开始，特别是抗bcl -2疗法即将进入临床。通过NANOGP8基因改变调控的CX-1细胞RNA-seq数据分析，NEDD9是一个关键基因。NEDD9是一种连接粘附斑块中的c-src和FAK并激活AKT的支架蛋白。因此，NEDD9可能是一种抗凋亡分子，因为它有助于FAK的组成性激活。RT-PCR证实，在CRC细胞系中，NANOG的shRNA可抑制NEDD9的表达，而NANOG的过表达可增加NEDD9的表达。此外，ChIP实验证实NANOG结合了NEDD9的启动子。用siRNA修饰NEDD9的初步结果正在进行中，以确定NEDD9表达的抑制是否会影响AKT的激活和/或Mcl-1的表达。此外，我们还评估了NANOG和NEDD9作为预后因素在一系列近400例原发性结肠癌中的作用，这些结肠癌是由癌症诊断项目（Cancer Diagnosis Program， DCTD， NCI）生产的组织微阵列。在与博士合作。Scott Lawrence, Stephen Hewitt， Daekwan Seo和Robert Kinders采用定量免疫荧光（qIFA）和免疫组织化学（IHC）方法评估原发性结肠癌的NANOG和NEDD9蛋白。将原发性结肠癌分为训练集和测试集，分别进行Kaplan-Meier和Cox多元回归分析。数据表明，NANOG和NEDD9在包含肿瘤分期、局部侵袭、分级、年龄和部位的模型中是一个重要的预后因素。这些结果被编入一篇手稿。作为一种研究工具，我们选择评估一种可能从crc基因组中去除NANOG的TALEN。有趣的是，为我们设计TALEN的Cellectis公司实际上针对的是外显子1上的相同序列，这与我们在TRC财团中发现的最活跃的商业shRNA中发现的序列相同。我们的TAELN的问题是，该序列不仅存在NANOG，还存在NANOGP8以及NANOGP4和NANOGP7的罕见转录本。我们分离转染TALEN后可能失去NANOG的克隆的策略是分离增殖速度比其他克隆慢的克隆，因为增殖抑制是NANOG抑制的早期迹象。不幸的是，这确实拖慢了我们的评估。我们发现，我们可以减少75%的NANOG蛋白表达，亲本NANOG减少至少50%，NANOGP8完整。然而，发生的一件事是，即使NANOG水平在蛋白质或转录水平上没有改变，克隆在4周后普遍增加了增殖。目前正在对适应机制进行调查。我们很难将结直肠癌作为异种移植物在皮下进行转导。虽然肿瘤内初步注射LV shRNA抑制肿瘤生长，但这是继发于先天免疫的，因为LV shNEG也有活性。我们最终证实了CX-1细胞的体内转导，但它仅限于在一个小的异种移植物中零星的10-15个转导细胞岛。因此，使用慢病毒递送的shRNA可能不可持续。因此，在接下来的一年里，我们将研究在肿瘤内使用条件复制腺病毒（CRAd）。这种CRAd也被称为溶瘤病毒，因为CRAd可以溶解肿瘤细胞，但它们也可以允许shRNA作为插曲表达。由于NANOGP8报告基因在这些CRC细胞系中非常活跃，我们将开始创建一个NANOGP8驱动的CRAd，它也可以传递shRNA。

NANOGP8 Function in Stemness shRNA to NANOG inhibited the expression of parental NANOG and decreased spherogenicity significantly by single CRC Clone A and CX-1 cells. Re-expression of either NANOG or NANOGP8 protein was achieved and re-expression of NANOGP8 increased both spherogenicity and the size of the side population in both Clone A and CX-1 cells. Over-expression of NANOG increased spherogenicity in Clone A cells but not CX-1 cells. In addition, we demonstrated that NANOGP8 is translated. This had not been previously done. The data are included in the manuscript by Zhang et al. Oncogene. 2012 Oct 22. doi: 10.1038/onc.2012.461. [Epub ahead of print] Mechanism of Apoptosis Lentiviral delivered shRNA to NANOG (shNG-1) or NANOGP8 (shNp8-1) inhibits by nearly 50% the three-dimensional (3-D) growth of Clone A, CX-1 and LS 174T human CRC cells within 3 days when added a day after suspension cultures are initiated at a MOI of 5:1 to 16:1. This inhibition of 3-D growth extended to a further 15-30% decrease in regrowth potential when surviving cells were replated in a plating efficiency assay. Transduction efficiency was 45%. This suggested that lentiviral (LV) shNG-1 and/or shNp8-1 may induce apoptosis. We have previously published that suspension culture induces caspase 8-dependent extrinsic pathway apoptosis without activation of caspase 9 and the intrinsic pathway in Clone A and CX-1 cells. When we assessed the effects of LV shNG-1, shNp8-1 or the control LV shNEG on the induction of caspase 3, 8, 9 activity by a cell permeant fluorescent apoptosis assay, shNp8-1 induced activation of caspase 9 in suspension culture to a greater extent than shNG-1. LV delivered shNG-1 or shNp8-1 generally did not cause significant activation of the caspases when CRC were in monolayer culture. Interstingly, occasionally the LV shNEG contro vector did activate caspases in monolayer culture - this may reflect the expression of Toll receptors in monolayer cultures that lead to activation of innate immune mechanisms within CRC cells in monolayer culture. Taken together, the results suggest that shRNA to NANOGs, especially NANOGP8, activate the intrinsic pathway of apoptosis and suggest that LV delivered shNp8-1 and/or shNG-1 may be synergistic with chemo- or targeted therapy. This is currently being written up in a manuscript. We have assessed whether LV shRNA to NANOG and/or NANOGP8 is synergistic with specific aspects of the indirect pathway of apoptosis. Since this pathway involves the participation of BH3 domain proteins and is controlled by the anti-apoptotic Bcl-2 family members as well as Mcl-1 , the effect of LV shRNA on the expression of MCL-1 and Bcl-2 was performed. LV shNp8-1 inhibits the expression of MCL-1 as well as the activation of AKT with LV shNG-1 also inhibiting MCL-1 but to a lesser degree. None of the LV preparations have any effect on Bcl-2 or its family members. The LV shNp8-1 inhibits MCL-1 in the 3 CRC lines Clone A, CX-1, LS 174T. We have tested whether LV shRNA was synergistic with cytotoxic chemotherapy (using Topotecan, the camptothecin as a model) and anti-BCL-2 therapy (cell permeant peptidomimetic ABT-737). Even though both treatments increase NANOG and NANOGP8 gene expression of CRC cells in monolayer cultures the addition of LV shNp8-1 or LV shNG-1 is synergistic with either Topotecan or ABT-737. The LV shNEG does not increase apoptosis in this assay. Also the sequencing of the addition of LV shRNA's either before, simultaneously or after treatment with drug or anti-Bcl-2 agent does not affect the synergism. These results suggest that shRNA to inhibit NANOGP8 or NANOG may inhibit Mcl-1 that is synergistic with an anti-Bcl-2 therapy. A manuscript is under preparation. Preclinical studies will start soon, especially as the anti-BCL-2 therapy is entering the clinic. Regulation of NEDD9 Ingenuity Pathway Analysis of RNA-seq data from CX-1 cells modulated by alterations in NANOGP8 identified NEDD9 as a critical gene. NEDD9 is a scaffolding protein that connects c-src and FAK in adhesion plaques and activates AKT . Thus, NEDD9 may be an anti-apoptotic molecule since it contributes to constitutive activation of FAK. RT-PCR confirmed that NEDD9 expression was inhibited by shRNA to the NANOGs and increased by overexpression of either NANOG in the CRC lines tested. In addition, ChIP assays confirmed that NANOG binds the promoter of NEDD9. Preliminary results with siRNA to NEDD9 are underway to determine whether inhibition of NEDD9 expression affects AKT activation and/or Mcl-1 expression. In addition, we have assessed the role of NANOG and NEDD9 as prognostic factors in a series of nearly 400 primary colon carcinomas in a tissue microarray produced by the Cancer Diagnosis Program, DCTD, NCI. In collaboration with Drs. Scott Lawrence, Stephen Hewitt, Daekwan Seo and Robert Kinders both quantitative immunofluorescence (qIFA) and immunohistochemistry (IHC) were performed to assess NANOG and NEDD9 protein in the primary colon carcinomas. The primary colon carcinomas was divided into a training and test set and analyzed by Kaplan-Meier and then Cox multivariate regression analysis. The data suggest that NANOG and NEDD9 together are a significant prognostic factor in models that contain tumor stage, local invasion, grade, age, and site. These results are bing put into a manuscript. TALEN Evaluation We were chosen to evaluate a TALEN that might remoce NANOG from the genomes of CRCs as a research tool. Interestingly, the company Cellectis who designed the TALEN for our use actually targeted the same sequence in exon 1 as we had found in our most active commercially available shRNA from the TRC consortium. The problem for our TAELN is that the sequence is present not only NANOG but also NANOGP8 as well as rare transcripts of NANOGP4 and NANOGP7. Our strategy for isolating the clones that may have had a loss of NANOG after transfection with the TALEN was to isolate clones that are proliferating more slowly than other clones since inhibition of proliferation is the early sign of NANOG inhibition. Unfortunately, this has really slowed down our evaluation. We have found that we can reduce NANOG protein expression by 75% percent with reduction of parental NANOG by at least 50% and intact NANOGP8. However, one thing that has happened is that the clones have generally increased proliferation after 4 weeks even though NANOG levels have not changed at either the protein or transcript level. Investigation is currently ongoing into adaptive mechanisms. Transduction in Vivo We have had difficulty in transducing CRC growing as xenografts in the sub cutis. Although preliminary intratumoral injection of LV shRNA inhibited tumor growth, it was secondary to innate immunity since the LV shNEG was also active. We have finally demonstrated transduction in vivo in CX-1 cells but it is limited to sporadic islands of 10-15 transduced cells within a small xenograft. As a result, the use of lentiviral delivered shRNA may not be sustainable. As a result, during the coming year we will investigate the use of conditionally replicating adenovirus (CRAd) within tumors. Such CRAds are also known as oncolytic virus because the CRAd may lyse tumor cells but they also may allow the expression of shRNA as an episome. Since a NANOGP8 reporter is quite active within these CRC lines, we will begin to create a NANOGP8-driven CRAd that may also deliver a shRNA.