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: 8349389
• 负责人: John Jessup
• 金额: $ 9.12万
• 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/8349389
• 关键词: 3-Dimensional; Adherence; Adherent Culture; Affect; Alleles; Amino Acids; Anoikis; Apoptosis; Apoptotic; Biological Assay; Caspase; Cell Fraction; Cell Line; Cell physiology; Cells; Clinical; Codon Nucleotides; Collaborations; Colon Carcinoma; Colorectal Cancer; Complementary DNA; Cytoplasm; DNA Restriction Enzymes; Data; Department of Defense; Dideoxy Chain Termination DNA Sequencing; Digestion; Embryo; Embryonic Development; Family member; Funding; Gel; Gene Expression; Genes; Glioblastoma; Grant; Growth; Human; Immunohistochemistry; In Vitro; Individual; Infection; Laboratories; Large Intestine Carcinoma; Liver; Malignant - descriptor; Malignant Epithelial Cell; Measures; Mediating; Metastatic Neoplasm to the Liver; Mitochondria; Modeling; Mus; Neoplasm Metastasis; Pathway interactions; Patients; Peptides; Population; Pre-Clinical Model; Preparation; Primary carcinoma of the liver cells; Proteins; Reporter; Reporting; Resected; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Serum-Free Culture Media; Side; Specimen; Stem cells; Stomach; Suspension Culture; Suspension substance; Suspensions; Testing; Transcript; Transcription Coactivator; Transfection; Translating; Tumorigenicity; United States National Institutes of Health; Work; annexin A5; base; cancer stem cell; caspase-3; caspase-8; caspase-9; cellular transduction; chemotherapy; embryonic stem cell; in vivo; monolayer; neoplastic cell; overexpression; pluripotency; pre-clinical; promoter; research study; self-renewal; small hairpin RNA; stemness; stressor; therapy development; transcription factor; tumor; two-dimensional

Nanog Regulates Other Stem Cell Transciption Factors Nanog controls the expression of Oct4 and SOX2 during early embryogenesis. We have found that inhibition of NanogP8 with allele specific shRNA Np8-1 inhibits expression of Oct4 and SOX2. Thus, targeting one factor may inhibit the others so that single agent targeting may effect multiple downstream pathways. In addition, we have shown that Nanog and NanogP8 affect stemness by reducing spherogenicity, tumorigenicity and metastatic potential in colorectal carcinoma cells (CRC). We have now found that allele-specific inhibition of NanogP8 reduces the size of the side population, while overexpression of Nanog or NanogP8 increases the side population. Thus, Nanog and NanogP8 are instrumental in modulating the stemness of CRC. Nanog and NanogP8 are expressed in clinical liver CRC metastases Ten de-identified samples of colorectal carcinoma resected from patients treated at the NIH Clinical Center were analyzed for expression of Nanog and NanogP8 transcripts and protein. RT-PCR was followed by cutting the cDNA with the restriction endonuclease AlwNI that specifically cuts NanogP8 but not Nanog. Seven liver metastases contained a Nanog-related transcript as did 3 adjacent liver specimens. Six of the 7 specimens contained NanogP8 with and one only Nanog. One of the adjacent liver specimens contained NanogP8 while the 2 others only expressed Nanog transcripts. All results were confirmed by Sanger sequencing. Immunohistochemistry (IHC) was also performed and confirmed that Nanog protein was present in the cytoplasm of the tumor cells that contained detectable Nanog or NanogP8 transcripts but not in CRC that lacked such transcripts. In addition, IHC of sample 9 demonstrated the presence of Nanog protein in a sample where only NanogP8 transcripts were identified. Thus, based on this small sample that needs confirmation and expansion approximately 70% of clinical CRC express a Nanog family member and 60% express NanogP8. NanogP8 Can be Translated into Protein In collaboration with the Veenstra laboratory at NCI-Frederick, we attempted to use tandem MS/MS to identify the NanogP8 protein in CRC cell lines. Extracts of CRC cell lines were immunoprecipitated, isolated by SDS-PAGE, subjected to in gel tryptic digestion and then MS/MS. Four Nanog-related peptides (KTWFQNQRM, KYLSLQQMQELSNILNLSYKQ, KKEDKVPVKK, and KGKQPTSAENSVAKK) were identified in extracts from Clone A overexpressing NanogP8. Interestingly, the last peptide is unique to NanogP8, which includes the shift from Lys (K) in Nanog to Asn (N) in NanogP8 at codon 82. This amino acid change was also reported by Ambady (2) and is consistent with our gene sequencing data. Nanog/NanogP8 proteins were not identified in extracts of CRC cell lines without overexpression by transfection presumably because CRC cell lines express low levels of endogenous proteins. NanogP8 Promoter Activity is increased during 3-D growth consistent with gene expression of NanogP8 We have previously shown that NanogP8 transcripts are increased in spheroids of the CRC cell lines Clone A and CX-1 cultured in serum-free medium (SFM). We tested the postulate that the promoter activity of the NanogP8 gene increases as CRC cells transition from a two-dimensional monolayer culture to a three-dimensional spheroid culture. A lentiviral NanogP8 promoter reporter was obtained from Jeter et al (3). Monolayer cultures of Clone A and CX-1 had low levels of GFP expression compared to expression in spheroids of Clone A and CX-1. Counting GFP positive cells demonstrated a 6- to 14-fold increase in GFP+ cells in spheroids compared to monolayers for Clone A and CX-1, respectively. This is similar to the increase in transcript number assessed by qRT-PCR during transition from 2-D monolayers to 3-D spheroids. Mechanism of Action and Inhibition of 3-D Growth By Transduction with Lentiviral shRNAs to Nanog or NanogP8 To be a successful therapy, inhibition of Nanog by shRNA must inhibit the growth of established tumors in vivo in preclinical models. We obtained a grant from the Department of Defense to support such preclinical work but unfortunately that has not yet started. However, we have been busy performing experiments in vitro with our allele-specific shRNAs and their effects on regression of established 3-D spheroids and cells in suspension to determine the multiplicity of infection (MOI) needed to cause tumor regression. Our previous experiments demonstrated that treatment of CRC in monolayer induced Annexin V expression as an indication of apoptosis. Now we started by taking LV with shRNA to NanogP8 (shNp8-1), Nanog (shNG-1) and a control shRNA (shNEG) and transducing CX-1 and Clone A cells that were then cultured for 24 hr in monolayer and then transitioned to SFM. We first demonstrated that LVshNp8-1 transduction significantly reduced the mass of cells after 72 hr of suspension culture. We then began to transduce CRC growing in suspension in SFM on ULLA plates. Since our LV preparations express GFP, we first determined the fraction of cells transduced in suspension and found that 40% to 75% of CRC cells were transduced stably. We then assessed whether transduction with LV shRNA to Nanog or NanogP8 inhibited growth compared to either untreated CRC cells or cells treated with the control shNEG. The cumulative results of more than 7 different experiments indicated that treatment of CRC in 3-D growth significantly inhibits short term growth by 50% by 72 hr. We then sought to determine how the LV shRNA effects apoptosis in 3-D culture. We previously demonstrated that anoikis apoptosis caused by suspension culture is dependent on activation of Caspase 8 (4). We measured Caspase 3, 8 and 9 activity in Clone A and CX-1 treated with LV shRNA after 4 days of suspension culture, 3 days after LV treatment. Activity of Caspase 3, the executioner caspase, was increased in both CX-1 and Clone A by shRNA to Nanog and NanogP8 beyond what occurs with just suspension culture alone. Activity of Caspase 8 or 9 was increased in Clone A or CX-1, respectively, by shRNA Np8-1. Interestingly, non-transduced cells cells adjacent to transduced cells had similar levels of caspase 9 activity as transduced cells did. Finally, at least in CX-1. Caspase 9 and the intrinsic pathway that uses mitochondrial amplification in apoptosis is important for apoptosis mediated by shNp8-1 and shNG-1. SUMMARY Our work suggests that 1) inhibition of Nanog or NanogP8 inhibits the other core embryonic TFs SOX2 and Oct4, 2) the NanogP8 transcript can be translated so that the retrogene is a functional gene, 3) LV shRNA to Nanog or NanogP8 transduce cells growing in 3-D, 4) an MOI of 8 is sufficient to inhibit CRC short term growth, and 5) inhibition of growth by LV shRNA is mediated by activation of caspases, especially caspase 9 and 3. In vivo experiments will go forward when the DOD IDEA grant is funded. Finally, it is not clear why some tumors produce only Nanog transcripts while others produce either both NanogP8 and Nanog or only NanogP8. However, by producing allele-specific inhibitory shRNA to both genes it is possible to target either or both and offers the possibility of personalizing therapy. While we have not yet determined whether the combination of both allele specific inhibitory shRNAs is better than either alone in the in vitro suspension culture model, we will do this soon. This is important especially since inhibition of either gene works through the intrinsic pathway of apoptosis that is distinct from the extrinsic pathway that is important for anoikis.

Nanog调节其他干细胞转递因子Nanog控制着早期胚胎发生过程中OCT4和SOX2的表达。我们发现，用特异性SHRNA NP8-1抑制NanogP8抑制OCT4和SOX2的表达。因此，靶向一个因素可能会抑制其他因素，以便靶向单个药物可能会影响多个下游途径。此外，我们已经表明，Nanog和NanogP8通过降低结直肠癌细胞（CRC）的球体性，肿瘤性和转移潜力来影响茎。 现在，我们发现，等位基因特异性抑制NanogP8会减少侧群的大小，而Nanog或NanogP8的过表达增加了侧群。因此，Nanog和Nanogp8在调节CRC的茎上有助于。 Nanog和NanogP8在临床肝CRC转移术中表达了十种从NIH临床中心分析的患者切除的结直肠癌样本，以表达Nanog和NanogP8转录本和蛋白质的表达。 RT-PCR之后是用限制性核酸内切酶Alwni切割cDNA，该ccr专门切割纳米P8而不是纳米。七个肝转移含有与纳米相关的转录本，以及3个相邻的肝样品。 7个标本中有6个包含Nanogp8，其中一个只有Nanog。相邻的肝样品之一包含纳米P8，而另外两个则只表示纳米转录本。所有结果均通过Sanger测序确认。还进行了免疫组织化学（IHC），并证实纳米蛋白存在于肿瘤细胞的细胞质中，其中包含可检测的Nanog或Nanogp8转录本，但在缺乏此类转录本的CRC中不存在。此外，样本9的IHC证明了在仅鉴定出NanogP8转录本的样品中存在纳米蛋白。 因此，基于这个需要确认和扩展的小样本，约有70％的临床CRC表达纳米家族成员和60％的表达NanogP8。 Nanogp8可以与NCI-Frederick的Veenstra实验室合作转化为蛋白质，我们尝试使用串联MS/MS来鉴定CRC细胞系中的NanogP8蛋白。将CRC细胞系的提取物免疫沉淀，通过SDS-PAGE分离，在凝胶胰蛋白酶消化中进行，然后在MS/MS中进行。在克隆的提取物中鉴定了四个与Nanog相关的肽（KTWFQNQRM，KYLSLQQMQMQMQMQMQMQMQMQMQMQMQMQMQMQRM，KKEDKVPVKK和KGKQPTSAENSVAKK）。有趣的是，最后一个肽是NanogP8独有的，其中包括从密码子82处的Nanogp8中的Lys（K）转移到Nanogp8中的ASN（N）。Ambady（2）也报道了这种氨基酸的变化，并且与我们的基因测序数据一致。 Nanog/NanogP8蛋白在CRC细胞系的提取物中未通过转染过表达而鉴定出来，这可能是因为CRC细胞系表达了低水平的内源性蛋白质。 在3-D生长期间，NanoGP8启动子活性与NanoGP8的基因表达一致，我们先前已经表明，在无血清培养基（SFM）中培养的CRC细胞系A和CX-1的球体中，NanoGP8转录物增加。我们测试了一个假设，即随着CRC细胞从二维单层培养为三维球体培养物，NanoGP8基因的启动子活性增加。 慢病毒Nanogp8启动子记者从Jeter等人（3）获得。与克隆A和CX-1的球体中的表达相比，克隆A和CX-1的单层培养物的GFP表达水平较低。 与克隆A和CX-1的单层相比，计算GFP阳性细胞的球体中GFP+细胞的增加6至14倍。这类似于QRT-PCR在从2-D单层到3-D球体的过渡过程中评估的转录数增加。 用慢病毒shrnas转导向Nanog或Nanogp8的作用机理和抑制3-D生长的生长，成为一种成功的疗法，SHRNA对Nanog抑制Nanog必须抑制临床前模型中体内已建立的肿瘤的生长。我们从国防部获得了支持此类临床前工作的赠款，但不幸的是尚未开始。 但是，我们一直在忙于在体外对我们的等位基因特异性shRNA进行体外进行实验，及其对悬浮液中已建立的3-D球体和细胞的回归的影响，以确定引起肿瘤回归所需的感染的多重性（MOI）。 我们以前的实验表明，单层诱导的膜联蛋白V表达的CRC作为凋亡的指示。现在，我们首先将LV与SHRNA一起使用NanogP8（SHNP8-1），Nanog（SHNG-1）和对照shRNA（SHNEG），并转导CX-1和克隆A细胞，然后在单层中培养24小时，然后过渡到SFM。我们首先证明，LVSHNP8-1转导可显着减少悬浮培养物72小时后细胞的质量。 然后，我们开始在ulla板上的SFM悬架中转导CRC。 由于我们的LV制剂表达了GFP，因此我们首先确定了在悬浮液中转导的细胞的分数，并发现40％至75％的CRC细胞被稳定转导。然后，我们评估了与未处理的CRC细胞或用对照SHNEG处理的未处理的CRC细胞或细胞相比，用LV SHRNA对Nanog或NanogP8的转导抑制了生长。 超过7种不同的实验的累积结果表明，在3-D生长中的CRC处理可显着抑制短期生长50％，降低72小时。 然后，我们试图确定LV SHRNA如何影响3-D培养物中的凋亡。 我们先前证明，悬浮培养引起的凋亡凋亡取决于caspase 8的激活（4）。我们测量了LV处理后3天，在悬浮培养后4天后，在克隆A和CX-1中测量了caspase 3、8和9的活性。 caspase 3的活性在CX-1和shRNA中的CX-1和克隆A中都增加了，而Nanog和Nanogp8仅仅是仅悬浮培养物而发生的。 shRNA NP8-1分别在克隆A或CX-1中增加了胱天冬酶8或9的活性。有趣的是，与转导的细胞相邻的非转导细胞具有与转导细胞相似的caspase 9活性。最后，至少在CX-1中。 caspase 9和在凋亡中使用线粒体扩增的内在途径对于由SHNP8-1和SHNG-1介导的凋亡很重要。 总结我们的工作表明，1）抑制Nanog或NanogP8抑制其他核心胚胎TFS SOX2和OCT4，2）可以翻译NanogP8转录本，以使逆转录曲面是功能性基因，3）LV ShRNA成长为Nanog或Nanogp8的生长，在3-d中生长在3-d，4）cr cr crtic and cr cr crtic and cr inc crtic and crtic and crtic and 4） LV SHRNA对生长的抑制是通过激活caspase的激活介导的，尤其是caspase 9和3。当DOD Idea Grant资助资金时，体内实验将继续进行。最后，尚不清楚为什么某些肿瘤仅产生纳米转录本，而另一些肿瘤同时产生NanogP8和Nanog或仅生产NanogP8。但是，通过对两个基因产生等位基因特异性抑制性shRNA，可以靶向或两者兼而有之并提供个性化治疗的可能性。虽然我们尚未确定在体外悬浮培养模型中，这两个等位基因抑制性shRNA的组合是否比任何一个单独的shrnas都更好，但我们很快就会做到这一点。 这很重要，尤其是因为对任何两个基因的抑制作用都通过凋亡的固有途径起作用，而凋亡的内在途径与对Anoikis很重要的外在途径不同。