Validation of Therapeutic Target Genes in Human Liver Cancer

人类肝癌治疗靶基因的验证

基本信息

批准号：
8553020
负责人：
Snorri Thorgeirsson
金额：
$ 38.16万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8553020
关键词：
Accounting Address Adenosine Adverse effects Affect Apoptosis Apoptotic Azacitidine Base Pairing Biological Assay CD29 Antigen Cell Cycle Cell Cycle Arrest Cell Cycle Progression Cell Cycle Regulation Cell Death Cell Line Cell Proliferation Cells Cirrhosis Cullin 1 Cyclin D1 Cytidine DNA DNA Methylation DNA Repair DNA biosynthesis DNA methyltransferase inhibition Development Diphosphates Dose Down-Regulation Drug Formulations Enzymes Epigenetic Process Evaluation Gene Expression Gene Silencing Generations Genes Genomics Glioblastoma Global Change Goals Growth Guanidines Hepatocarcinogenesis Human Hydrogen Bonding Immune response Immunologic Surveillance In Vitro Induction of Apoptosis Lipids Liver Luciferases Maintenance Malignant - descriptor Malignant Epithelial Cell Malignant Neoplasms Malignant neoplasm of liver Malignant neoplasm of pancreas Mediator of activation protein Messenger RNA Metabolic Activation Methylation Microarray Analysis Microscopy Modality Modeling Molecular Molecular Abnormality Molecular Profiling Molecular Target Mus Mutagenesis Neoplasm Metastasis Nuclear Nucleic Acids Nucleosides Oncogenic Pathogenesis Pathway interactions Patients Pattern Pharmaceutical Preparations Phenotype Phosphorylation Phosphotransferases Play Primary carcinoma of the liver cells Proliferating Proteins Pyrimidinones RNA Recurrence Reporter Ribonucleotide Reductase Role SKP2 gene Signal Transduction Small Interfering RNA Staging Survival Analysis TSC2 gene Testing Tissues Toxic effect Tumor Suppressor Proteins Tumor-Derived Ubiquitination Uridine Kinase Validation Variant Western Blotting Xenograft Model Zebularine adduct analog base cell growth cyclin B1 cyclin-dependent kinase 6 cytotoxicity demethylation hazard histone modification in vivo inhibitor/antagonist insight leukemia novel therapeutics nucleoside analog particle promoter response standard of care subcutaneous therapeutic target transcriptomics tumor tumor growth tumor progression

项目摘要

The most recent reeasearch includes (1)Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of the fifth subunit of COP9 signalosome (CSN5) in early HCC as compared with dysplastic stage. In the present study, we explored the possibility of CSN5 being a potential therapeutic target for HCC. Our results show that CSN5 knockdown by small-interfering (si) RNA caused a strong induction of apoptosis and inhibition of cell-cycle progression in HCC cells in vitro. The down-regulation of CSN5 was sufficient to interfere with CSN function as evidenced by the accumulation of neddylated Cullin 1 and changes in the protein levels of CSN-controlled substrates SKP2, p53, p27 and nuclear factor-kappaB, albeit to a different degree depending on the HCC cell line, which could account for the CSN5 knockdown phenotype. The transcriptomic analysis of CSN5 knockdown signature showed that the anti-proliferative effect was driven by a common subset of molecular alterations including down-regulation of cyclin-dependent kinase 6 (CDK6) and integrin beta1 (ITGB1), which were functionally interconnected with key oncogenic regulators MYC and TGFbeta1 involved in the control of proliferation, apoptotic cell death and HCC progression. Consistent with microarray analysis, western blotting revealed that CSN5 depletion increased phosphorylation of Smad 2/3, key mediators of TGFbeta1 signaling, decreased the protein levels of ITGB1, CDK6 and cyclin D1 and caused reduced expression of anti-apoptotic Bcl-2, while elevating the levels of pro-apoptotic Bak. A chemically modified variant of CSN5 siRNA was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of the CSN5 3/8 variant by stable-nucleic-acid-lipid particles significantly suppressed the tumor growth in Huh7-luc+ orthotopic xenograft model. Taken together, these results indicate that CSN5 has a pivotal role in HCC pathogenesis and maybe an attractive molecular target for systemic HCC therapy.Our most recent progress in this project include; and (2) Previously studies have shown that protein level and kinase activity of Wee1 are significantly elevated in HCC compared with surrounding cirrhotic tissues, although the underlying mechanisms are still unknown. Under normal conditions, Wee1 kinase plays an important role in maintaining G2 arrest through the inhibitory phosphorylation of cdc2 on Tyr-15. In the present study, we explored the possibility of Wee1 being a potential therapeutic target for HCC. To inactivate Wee1, three Wee1-specific small interfering (si) RNAs (Wee1-1, Wee1-2 and Wee1-3) were tested for growth inhibition in HCC cell lines as determined by MTT assay, FACS analysis and microscopy. To obtain insights into molecular changes caused by Wee1 silencing, global changes in gene expression were examined by illumina microarray. For in vivo evaluation of Wee1 as a therapeutic target, we employed orthotopic xenograft model using luciferase-expressing HCC reporter cell lines Huh7- and HepG2-luc+ and stable-nucleic-acid-lipid-particle (SNALP) as an optimal carrier of siRNA into liver. Among the tested siRNA molecules, the Wee1-2siRNA was the most effective in inhibiting Huh7 and HepG2 cell growth (80% and 84%, respectively) which was paralleled by a similar decrease in the levels of target mRNA and protein. Wee1 knockdown by siRNA also caused a block in cell cycle progression and induced apoptosis of HCC cells. The comparison of gene expression profiles in HepG2 cells treated with either control siRNA or Wee1-2siRNA identified 506 differentially expressed genes (P < 0.05 by bootstrap t-test). Genes functionally involved in cell proliferation, such as cdk2, cyclin B1, and Akt1, were down-regulated while cell cycle inhibitor p21 and tumor suppressor TSC2 were up-regulated. Western blotting showed that Wee1 silencing significantly increased the expression of p53 and p21 and decreased cyclin D1 protein levels in Wee1-deficient HepG2 cells, which could contribute to cell cycle arrest and induction of apoptosis. Wee1 5/6, a modified variant of Wee1-2siRNA, was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of Wee1 5/6 variant by SNALP significantly suppressed both Huh7 and HepG2 tumor growth in orthotopic xenograft model. In addition, administration of Wee1 5/6 SNALP increased the survival of mice bearing HepG2-derived tumors bearing mice in a dose-dependent manner. Taken together, these results indicate that Wee1 maybe an attractive molecular target for systemic HCC therapy.

最新的研究包括（1）肝细胞癌（HCC）靶向治疗的开发仍然是一个重大挑战。我们最近发现，与发育不良阶段相比，早期 HCC 中 COP9 信号体第五亚基 (CSN5) 的表达升高。在本研究中，我们探讨了 CSN5 作为 HCC 潜在治疗靶点的可能性。我们的结果表明，小干扰 (si) RNA 敲低 CSN5 可在体外强烈诱导 HCC 细胞凋亡并抑制细胞周期进展。 CSN5 的下调足以干扰 CSN 功能，如 neddylated Cullin 1 的积累以及 CSN 控制底物 SKP2、p53、p27 和核因子 kappaB 的蛋白质水平的变化所证明的那样，尽管程度不同，但取决于在 HCC 细胞系上，这可以解释 CSN5 敲低表型。 CSN5 敲低特征的转录组学分析表明，抗增殖作用是由常见的分子改变子集驱动的，包括细胞周期蛋白依赖性激酶 6 (CDK6) 和整合素 beta1 (ITGB1) 的下调，这些改变在功能上与关键致癌因素相互关联。调节因子 MYC 和 TGFbeta1 参与控制增殖、凋亡细胞死亡和 HCC 进展。与微阵列分析一致，蛋白质印迹显示，CSN5 缺失增加了 Smad 2/3（TGFbeta1 信号转导的关键介质）的磷酸化，降低了 ITGB1、CDK6 和细胞周期蛋白 D1 的蛋白水平，并导致抗凋亡 Bcl-2 的表达减少，同时升高了促凋亡 Bak 的水平。然后根据生长抑制作用和最小程度地诱导不需要的免疫反应，选择 CSN5 siRNA 的化学修饰变体用于体内应用。通过稳定核酸脂质颗粒全身递送 CSN5 3/8 变体显着抑制 Huh7-luc+ 原位异种移植模型中的肿瘤生长。总而言之，这些结果表明 CSN5 在 HCC 发病机制中发挥着关键作用，并且可能是系统性 HCC 治疗的一个有吸引力的分子靶点。我们在该项目中的最新进展包括： (2)之前的研究表明，与周围肝硬化组织相比，HCC中Wee1的蛋白水平和激酶活性显着升高，但其潜在机制仍不清楚。正常情况下，Wee1 激酶通过抑制 Tyr-15 上 cdc2 的磷酸化，在维持 G2 停滞中发挥重要作用。在本研究中，我们探讨了 Wee1 作为 HCC 潜在治疗靶点的可能性。为了灭活 Wee1，测试了三种 Wee1 特异性小干扰 (si) RNA（Wee1-1、Wee1-2 和 Wee1-3）在 HCC 细胞系中的生长抑制作用，通过 MTT 测定、FACS 分析和显微镜检查确定。为了深入了解 Wee1 沉默引起的分子变化，通过 Illumina 微阵列检查了基因表达的整体变化。为了体内评估 Wee1 作为治疗靶点，我们采用原位异种移植模型，使用表达荧光素酶的 HCC 报告细胞系 Huh7- 和 HepG2-luc+ 以及稳定核酸脂质颗粒 (SNALP) 作为 siRNA 的最佳载体肝。在测试的 siRNA 分子中，Wee1-2siRNA 在抑制 Huh7 和 HepG2 细胞生长方面最有效（分别为 80% 和 84%），同时靶标 mRNA 和蛋白质水平也有类似的下降。 siRNA 敲除 Wee1 也会导致细胞周期进程受阻并诱导 HCC 细胞凋亡。对用对照 siRNA 或 Wee1-2siRNA 处理的 HepG2 细胞中的基因表达谱进行比较，鉴定出 506 个差异表达基因（通过 bootstrap t 检验，P < 0.05）。与细胞增殖功能相关的基因，如 cdk2、cyclin B1 和 Akt1 下调，而细胞周期抑制剂 p21 和肿瘤抑制因子 TSC2 上调。 Western blotting显示，在Wee1缺陷的HepG2细胞中，Wee1沉默显着增加了p53和p21的表达，并降低了cyclin D1蛋白水平，这可能有助于细胞周期停滞和诱导细胞凋亡。 Wee1 5/6 是 Wee1-2siRNA 的改良变体，基于生长抑制作用和最小程度诱导不需要的免疫反应，被选择用于体内应用。在原位异种移植模型中，通过 SNALP 全身递送 Wee1 5/6 变体显着抑制 Huh7 和 HepG2 肿瘤生长。此外，Wee1 5/6 SNALP 的施用以剂量依赖性方式增加了携带 HepG2 衍生肿瘤的小鼠的存活率。总而言之，这些结果表明 Wee1 可能是系统性 HCC 治疗的一个有吸引力的分子靶点。