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: 8553034
• 负责人: John Jessup
• 金额: $ 8.01万
• 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/8553034
• 关键词: 3T3 Cells; Acidity; Address; Adherence; Adherent Culture; Affect; Alleles; Amino Acids; Anoikis; Apoptosis; Apoptotic; Appearance; Belgium; Binding; Binding Sites; Biological Assay; Caliber; Camptothecin; Carcinoma; Caspase Inhibitor; Cell Death; Cell Line; Cell membrane; Cell physiology; Cells; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 15; Clinical; Clinical Management; Coculture Techniques; Code; Collaborations; Colon Carcinoma; Colorectal Cancer; DNA Binding; DNA Damage; Data; Dose; Embryo; Fluorescence; Focal Adhesions; Future; Gene Expression; Genes; Glioblastoma; Goals; Grant; Green Fluorescent Proteins; Growth; Human; Immune response; Immunoglobulins; In Vitro; Individual; Induction of Apoptosis; Injection of therapeutic agent; Journals; Kinetics; Large Intestine Carcinoma; Lentivirus Vector; Link; Llama; Malignant - descriptor; Malignant Epithelial Cell; Manuscripts; Measures; Mediating; Modeling; Mus; NOD/SCID mouse; Natural Immunity; Neoplasm Metastasis; Nodule; Nucleotides; Oncogenes; PTK2 gene; Pathway Analysis; Pathway interactions; Phosphatidylserines; Plasmids; Preclinical Testing; Primary carcinoma of the liver cells; Production; Prognostic Factor; Promoter Regions; Proteins; Proto-Oncogene Proteins c-akt; RNA; Ramp; Regulation; Relative (related person); Reporter; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Scaffolding Protein; Series; Serum-Free Culture Media; Site; Specificity; Stimulus; Stress; Subfamily lentivirinae; Suspension Culture; Suspension substance; Suspensions; Testing; Topoisomerase-I Inhibitor; Topotecan; Transcript; Transcription Coactivator; Viral; Virus; Xenograft Model; authority; cancer stem cell; caspase-3; caspase-8; caspase-9; chemotherapy; cytotoxicity; embryonic stem cell; gene therapy; human DNA; in vivo; inhibitor/antagonist; interstitial; killings; leukemia; malignant stomach neoplasm; monolayer; neoplastic cell; overexpression; particle; pluripotency; pre-clinical; pressure; prevent; professor; promoter; protein expression; research study; self-renewal; small hairpin RNA; stemness; subcutaneous; therapy development; transcription factor; tumor; tumor growth; vector; vector control; 3T3 Cells; Acidity; Address; Adherence; Adherent Culture; Affect; Alleles; Amino Acids; Anoikis; Apoptosis; Apoptotic; Appearance; Belgium; Binding; Binding Sites; Biological Assay; Caliber; Camptothecin; Carcinoma; Caspase Inhibitor; Cell Death; Cell Line; Cell membrane; Cell physiology; Cells; Chromosomes, Human, Pair 12; Chromosomes, Human, Pair 15; Clinical; Clinical Management; Coculture Techniques; Code; Collaborations; Colon Carcinoma; Colorectal Cancer; DNA Binding; DNA Damage; Data; Dose; Embryo; Fluorescence; Focal Adhesions; Future; Gene Expression; Genes; Glioblastoma; Goals; Grant; Green Fluorescent Proteins; Growth; Human; Immune response; Immunoglobulins; In Vitro; Individual; Induction of Apoptosis; Injection of therapeutic agent; Journals; Kinetics; Large Intestine Carcinoma; Lentivirus Vector; Link; Llama; Malignant - descriptor; Malignant Epithelial Cell; Manuscripts; Measures; Mediating; Modeling; Mus; NOD/SCID mouse; Natural Immunity; Neoplasm Metastasis; Nodule; Nucleotides; Oncogenes; PTK2 gene; Pathway Analysis; Pathway interactions; Phosphatidylserines; Plasmids; Preclinical Testing; Primary carcinoma of the liver cells; Production; Prognostic Factor; Promoter Regions; Proteins; Proto-Oncogene Proteins c-akt; RNA; Ramp; Regulation; Relative (related person); Reporter; Research; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Scaffolding Protein; Series; Serum-Free Culture Media; Site; Specificity; Stimulus; Stress; Subfamily lentivirinae; Suspension Culture; Suspension substance; Suspensions; Testing; Topoisomerase-I Inhibitor; Topotecan; Transcript; Transcription Coactivator; Viral; Virus; Xenograft Model; authority; cancer stem cell; caspase-3; caspase-8; caspase-9; chemotherapy; cytotoxicity; embryonic stem cell; gene therapy; human DNA; in vivo; inhibitor/antagonist; interstitial; killings; leukemia; malignant stomach neoplasm; monolayer; neoplastic cell; overexpression; particle; pluripotency; pre-clinical; pressure; prevent; professor; promoter; protein expression; research study; self-renewal; small hairpin RNA; stemness; subcutaneous; therapy development; transcription factor; tumor; tumor growth; vector; vector control

NANOGP8 Function in Stemness: A major question in our research is why the retrogene NANOGP8 located on Chromosome 15 is activated in human carcinomas and leukemias. Its coding region differs from NANOG?s located on chromosome 12 by 5 nucleotides that cause only 2 amino acid changes in a protein of 305 amino acids. It has not been clear that NANOGP8 could rescue stemness if NANOG is lost. This year we have found that when shRNA inhibits the expression of both NANOG and NANOGP8 and as a consequence the ability of human colorectal carcinoma cells (CRC) to form spheroids, the re-expression of NANOGP8 rescues the ability of CRC lines to form spheroids whereas re-expression of NANOG rescues only one of the cell lines. Since the capacity of cells to form spheroids is a major in vitro measure of stemness, the demonstration that NANOGP8 rescues stemness in these carcinomas suggests that NANOGP8 is able to replace NANOG as a core transcription factor. These data are in a manuscript undergoing final review at the journal Oncogene. Mechanism of Apoptosis: A goal for this year under aim 2 was to elucidate whether allele-specific shRNA inhibition of NANOG or NANOGP8 induces apoptosis. Lentiviral vector delivered shRNA to either NANOG causes the appearance of phosphatidylserine, a marker for apoptosis, in monolayer culture in 3 human colorectal carcinoma cells. However, since NANOG or NANOGP8 expression is low in monolayer culture, the amount of apoptosis is low in monolayer cultures. When human colorectal carcinoma cells that normally grow attached to a substrate in vitro are placed in suspension culture without an ability to attach to a substrate, they die from a form of apoptosis termed anoikis that is driven mainly by the activation of Caspase 8 and the extrinsic pathway of apoptosis. Since the relative expression of both NANOGs increases in suspension culture, we investigated whether inhibition of either NANOG caused apoptosis and if so what pathway for apoptosis. We found that inhibition of NANOGP8 and to a lesser extent NANOG induces apoptosis through the intrinsic pathway by activation of Caspases 9 and 3 to reduce tumor growth. Confirmation of this observation was provided by stimulating growth of CRC cells in suspension culture by blocking the effects of allele-specific shRNAs by treatment with inhibitors of Caspases 3 and 9 as well as the overexpression of NANOGP8 and NANOG. These data suggest that the induction of apoptosis may be a useful component for therapy. Synergy with Chemotherapy: The interaction between inhibition of the NANOGs and a model of chemotherapy has been addressed. Since camptothecins are important agents for the clinical management of CRC, we have assessed whether the effects of Topotocan, a model camptothecin, might be synergistic with lentiviral vector delivered shRNA. Topotecan is a Topoisomerase I inhibitor that induces DNA damage that in turn causes cytotoxicity through stimulation of both the extrinsic and intrinsic pathways of apoptosis. Topotecan induces cell death in a dose dependent fashion and with consistent kinetics that will generally achieve a > 90% kill in CRC lines sensitive to topotecan in 96 hr. Interestingly, relative transcript levels of both NANOGs increase at 72 hr after topotecan exposure. When lentiviral allele-specific shRNAs are added to CRC lines in monolayer culture along with topotecan, shRNA inhibition of NANOGP8 led to greater reduction in all 3 CRC lines than with Topotecan alone. Both the intrinsic and extrinsic pathways of apoptosis were stimulated. Further, the caspase inhibitors at the concentrations that blocked anoikis did not reverse the inhibition of growth caused by topotecan and shRNAs. This suggests that lentiviral shRNA to NANOG or NANOGP8 may be synergistic with topotecan treatment. Regulation of NEDD9: While it is possible to link inhibition of NANOGP8 or NANOG with the induction of apoptosis under conditions in which there is apoptotic stress, it is not clear what molecules link inhibition of either NANOG with the induction of apoptosis. To assess this, RNA-seq has been done in 2 CRC lines cultured in monolayer and suspension as well as with shRNA to NANOGP8 or with overexpression with NANOGP8. Ingenuity Pathway Analysis of the genes modulated by alterations in NANOGP8 identified NEDD9 as a critical gene. NEDD9 is a scaffolding protein for src and FAK in adhesion plaques that activates AKT and is an anti-apoptotic stimulus 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. Subsequent research is assessing whether this is the mechanism by which apoptosis is activated. We are currently assessing the role of NANOG and NEDD9 as prognostic factors in a series of nearly 400 primary colon carcinomas. Finally, the human DNA binding site for human NANOG has not been identified and we intend to define that site within this promoter. We have identified a minimal region of the promoter that responds to NANOG and in the coming year we will further narrow the binding site. These results are currently being put into a manuscript that will be submitted this year. Transduction in Vivo: About 6 months ago we started a DOD grant that will assess the potential of allele-specific lentiviral vector delivered shRNA as a gene therapy to inhibit growth of established tumor in preclinical xenograft models. Initial experiments indicated that intratumoral injection of lentiviral shRNA induced a modest nonspecific inhibition of tumor growth. Since the inhibition was as great to the negative control vector as the specific shRNAs, the lentiviral constructs induced an innate immune response. Repeated assessment of intratumoral injections with the lentiviral shRNA failed to reveal any significant transduction of tumor cells within the subcutaneous tumor nodules as measured by the presence of Green Fluorescent Protein (GFP) fluorescence. GFP is included in all of our lentiviral shRNA constructs as a reporter and is present in 45- 80% of cells within spheroids that are transduced in vitro. There was no GFP expression within tumor cells 3 ? 5 days after intratumoral injection into small (3 mm diameter) nodules. We postulate that there may be 4 reasons why this may occur: 1) the amount of virus injected was too low for transduction, 2) interstitial pressure prevents binding of standard pseudotyped lentiviral particles to tumor cells, 3) intratumoral acidity decreases viral binding and fusion to tumor cell membranes so that transduction is blocked and 4) host stroma inhibits binding of vector to tumor cells. To assess postulate 1) we have developed a collaboration with Dr. J. Reiser at FDA/CBER who produces high quality/high titer lentivirus for selective targeting and use in clinical situations. To assess postulates 2 and 3 we have developed a collaboration with Dr. Breckpot in Belgium to develop a better and more selective binding agent that may overcome interstitial pressure and acidity. Associate Professor Karine Breckpot at the Brussels Vrje Universtat is a leading authority on innate immunity to lentivirus. She has developed a natural single chain immunoglobulin from llamas that recognizes CEA. She has given us this targeting plasmid that we are just beginning to incorporate into a lentiviral vector and with her and Dr. Reiser's guidance we are beginning to test viral binding and specificity in vitro before ramping up production for preclinical testing. To assess postulate 4) we will do co-cultures of mouse 3T3 cells with human CRC cells to assess whether host stroma also may participate in inhibition of lentiviral transduction.

Nanogp8在STEMNENS中的功能：我们研究的一个主要问题是为什么位于15号染色体上的Retrogene Nanogp8在人癌和白血病中被激活。 它的编码区域不同于位于12 x染色体上的Nanogs，在305个氨基酸的蛋白质中仅引起2种氨基酸的变化。 目前尚不清楚Nanogp8如果丢失了Nanog，可以挽救STEMNESS。 今年，我们发现，当ShRNA抑制Nanog和NanogP8的表达以及人类结直肠癌细胞（CRC）形成球体的能力时，NanogP8的重新表达挽救了CRC线的能力，而CRC线的能力仅形成球体，而Nanog仅挽救了Nanog的一部分细胞系。 由于细胞形成球体的能力是体外的主要体外量度，因此证明了NanogP8在这些癌中拯救了茎，这表明Nanogp8能够代替Nanog作为核心转录因子。 这些数据是在癌基因期刊上进行最终审查的手稿中。凋亡的机制：在目标2下，今年的目标是阐明等位基因特异性shRNA对纳米或纳米P8的抑制是否会诱导凋亡。 慢病毒载体将shRNA传递到纳米nog上，导致磷脂酰丝氨酸的出现，这是凋亡的标志物，在3个人结肠直肠癌细胞中的单层培养中。 但是，由于单层培养物中Nanog或NanogP8表达较低，因此单层培养物中凋亡的量很低。 当通常在体外附着在底物附着在底物的人结肠直肠癌细胞被置于悬浮培养物中而没有附着在底物上的能力时，它们死于所谓的厌氧症的形式，该形式称为厌氧菌，主要由caspase 8的激活和凋亡的外部途径驱动。 由于两种Nanogs的相对表达在悬浮培养中都增加，因此我们研究了抑制纳米的抑制是否引起了凋亡，如果是这样的凋亡途径。我们发现，通过激活胱天蛋白酶9和3的激活，抑制NanogP8并在较小程度上通过固有途径诱导凋亡，从而降低肿瘤的生长。 通过用caspase 3和9的抑制剂以及NanogP8和Nanog的过表达来阻止等位基因特异性shRNA的影响，通过刺激CRC细胞在悬浮培养中的生长来确认该观察结果。 这些数据表明，凋亡的诱导可能是治疗的有用组成部分。与化学疗法的协同作用：已经解决了纳米抑制与化学疗法模型之间的相互作用。 由于Camptothecins是CRC临床管理的重要药物，因此我们评估了topotocan模型camptothecin的影响是否可能与慢病毒载体递送的SHRNA具有协同作用。 拓扑替康是一种拓扑异构酶I抑制剂，诱导DNA损伤，进而通过刺激凋亡的外在和内在途径而引起细胞毒性。 拓扑替康以剂量依赖性的方式诱导细胞死亡，并具有一致的动力学，通常在96小时内对CRC线敏感的CRC线杀死> 90％。 有趣的是，两种纳米的相对转录水平在拓扑替康暴露后72小时增加。 当慢病毒等位基因特异性shRNA与单层培养中的CRC系一起添加到CRC系中时，SHRNA对NanoGP8的抑制会导致所有3种CRC系的降低都比单独使用托波特克can更大。 刺激了凋亡的内在和外在途径。 此外，在阻断厌氧菌的浓度下的caspase抑制剂并不能逆转拓扑替克和shRNA引起的生长的抑制作用。 这表明慢病毒shRNA至Nanog或Nanogp8可能与拓扑替康治疗协同作用。 NEDD9的调节：虽然可以在存在凋亡胁迫的条件下将纳米P8或Nanog的抑制作用与诱导凋亡的诱导联系，但尚不清楚哪种分子抑制对Nanog的抑制与诱导的凋亡。 为了评估这一点，RNA-Seq已在单层和悬浮液以及SHRNA培养的2个CRC系中进行，并用NanogP8进行过表达。 由NanoGP8改变调节的基因的独创性途径分析将NEDD9鉴定为关键基因。 NEDD9是SRC的脚手架蛋白，在粘附斑块中激活AKT，是一种抗凋亡刺激，因为它有助于FAK的本构激活。 RT-PCR证实NEDD9的表达被shRNA抑制了纳米人，并通过测试的CRC线中的任何一个Nanog的过表达增加。 此外，CHIP分析证实了Nanog结合NEDD9的启动子。 随后的研究是评估这是否是激活凋亡的机制。 我们目前正在评估Nanog和Nedd9作为预后因素的作用，这是一系列近400个原发性结肠癌。 最后，尚未确定人类纳米的人DNA结合位点，我们打算在该启动子中定义该位点。 我们已经确定了启动子的最低区域，该区域对Nanog做出了反应，在来年，我们将进一步缩小结合位点。 这些结果目前正在将今年提交的手稿中。体内转导：大约6个月前，我们成立了DOD赠款，该赠款将评估等位基因特异性慢病毒载体的潜力，将SHRNA作为一种基因疗法，用于抑制临床前异种移植模型中已建立的肿瘤的生长。 最初的实验表明，肺内shRNA的肿瘤内注射会诱导适度的非特异性抑制肿瘤生长。 由于抑制对阴性对照载体与特定的shRNA一样强，因此慢病毒构建体诱导了先天的免疫反应。 通过绿色荧光蛋白（GFP）荧光的存在测量，重复评估对慢病毒shRNA的肿瘤内注射症未能揭示在皮下肿瘤结节内的肿瘤细胞的任何显着转导。 GFP包括在我们所有的慢病毒shRNA构建体中作为记者，并且存在于45-80％的细胞中，这些细胞在体外转导的球体中。 肿瘤细胞3中没有GFP表达？肿瘤内注射到小（3毫米）结节后5天。 我们假设可能发生这种情况可能存在4个原因：1）注射病毒的量太低而无法进行转导，2）间质压力阻止标准的伪型肺内植物颗粒与肿瘤细胞的结合，3）肿瘤内酸度降低病毒和融合的肿瘤细胞对肿瘤细胞的结合和4）的构成构成脉动的构成。 为了评估假设1）我们已经与FDA/CBER的J. Reiser博士建立了合作，他在临床情况下生产了用于选择性靶向和使用的高质量/高滴度慢病毒。 为了评估假设2和3，我们已经与比利时的Breckpot博士建立了合作，以开发出一种更好，更具选择性的结合剂，该结合剂可能会克服间质压力和酸度。 布鲁塞尔VRJE Universtat的副教授Karine Breckpot是对慢病毒的先天免疫力的领先权力。她从骆驼开发了一种天然的单链免疫球蛋白，可以识别CEA。 她给了我们这个针对性的质粒，即我们刚刚开始纳入慢病毒载体，并在她和Reiser博士的指导下，我们开始在促进临床前测试之前在加大生产之前，开始在体外测试病毒结合和特异性。 要评估假设4）我们将与人CRC细胞共同培养小鼠3T​​3细胞，以评估宿主基质是否也可能参与慢病毒转导的抑制。