Validation of Therapeutic Target Genes in Human Liver Cancer

人类肝癌治疗靶基因的验证

• 批准号: 8937992
• 负责人: Snorri Thorgeirsson
• 金额: $ 31.18万
• 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/8937992
• 关键词: Address; Affect; Apoptosis; BCL2 gene; Biliary; Biological Assay; CDKN2A gene; Cell Cycle Progression; Cell Cycle Regulation; Cell Line; Cells; Cholangiocarcinoma; Chromatin; Classification; Clinical Management; Common Hepatic Duct; Data; Diagnosis; Disease; Dose; Down-Regulation; Drug Formulations; Drug Targeting; Duct (organ) structure; Encapsulated; Epigenetic Process; Epithelium; Excision; Extrahepatic; FGFR2 gene; Fatty acid glycerol esters; Frequencies; GLUT4 gene; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genomics; Glycolysis; Growth; Hepatic; Heterogeneity; Hilar; Histone Deacetylase; Human; Inbred ICR Mice; Incidence; Injection of therapeutic agent; Interlobular Bile Duct; Intrahepatic Cholangiocarcinoma; Ligands; Lipids; Liver; Location; Luciferases; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Medicine; Modality; Modeling; Modification; Molecular; Monitor; Mus; Neoplasm Metastasis; Neoplasm Transplantation; Operative Surgical Procedures; PPAR gamma; Pathway interactions; Patients; Pattern; Peripheral; Pharmaceutical Preparations; Pharmacologic Substance; Prevalence; Primary carcinoma of the liver cells; Protein Isoforms; Receptor Activation; Refractory Disease; Relative (related person); SCID Beige Mouse; Safety; Sampling; Signal Transduction; Small Interfering RNA; Staging; Subgroup; Survival Analysis; Survival Rate; Systemic Therapy; TP53 gene; Therapeutic; Therapeutic Effect; Tissues; Tumor Burden; Ubiquitination; Unresectable; Up-Regulation; Validation; Xenograft Model; Xenograft procedure; base; bioluminescence imaging; cancer type; cell growth; cohort; combinatorial; exome sequencing; gamma secretase; hazard; hepatocellular carcinoma cell line; histone deacetylase 2; histone modification; human FRAP1 protein; inhibitor/antagonist; intrahepatic; lipid biosynthesis; lipid metabolism; mouse model; nanoparticle; new therapeutic target; notch protein; novel; novel therapeutics; outcome forecast; pre-clinical; prognostic; receptor; response; safety testing; therapeutic development; therapeutic target; tumor; tumor growth; tumor progression

The most recent reeasearch includes (1) Intrahepatic cholangiocarcinoma (iCCA) is a treatment-refractory disease with heterogeneous underlying pathobiology, no approved therapy and rising incidence. This emphasizes the urgency for the development of therapeutic options for select patient subsets. In a pursuit for novel treatment options, we analyzed 15 iCCA patients with paired tumor and surrounding liver samples using whole-exome sequencing at a depth of 250X. In the prevalence screen of 144 cases, which involved the analysis of 48 cancer-related genes, we confirmed the deregulation of multiple potential causal pathways i.e., FGFR2, PI3K/mTOR, IDH1, TP53, CDKN2A and Notch1 in iCCA. Next, we examined the consequence of targeting Notch by blocking the receptor activation via the gamma-secretase (GS) complex both in a panel of 13 well-characterized CCA cell lines and in murine xenografts. A comprehensive analysis of the notch-signaling network revealed four distinct prognostic patient groups. Notch1 expression is a strong independent prognostic predictor of survival (?2=17.2, P 0.0007) with a 17% 5-year survival rate. Immunostaining confirmed a preferential Notch1 expression and receptor activation in patients with poor prognosis. Multiple notch-related genes, which include the receptor ligands, target-genes and key regulators of biliary differentiation, were significantly deregulated in tumors compared to matched liver samples. Also, we found a significant deregulation of all components of the GS complex. The effect of targeting Notch1 activation was evaluated using two GS inhibitors (R04929097 and YO-01027), which ranged from 0% to 100% sensitivity, and enabled the classification of cells based on drug-response. Importantly, the effect of blocking the GS activity on tumor growth was assessed in two xenograft models, which only demonstrated a beneficial drug-response on reduced tumor burden in GS inhibitor-sensitive iCCA. Our study revealed multiple putative drug targets, and highlights the effect of blocking the Notch receptor activation in iCCA. These data emphasize the usefulness of genomics-based medicine in selecting optimal therapy for defined subsets of patients. (2) Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL-2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPAR-gamma signaling and other regulators of glycolysis (ChREBP-alpha, GLUT4) and lipogenesis (SREBP1C, FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer. (3) We have previously demonstrated therapeutic effects of lipid nanoparticles (LNP) loaded with single siRNA targeting CSN5 or WEE1 against human HCC cell lines in an orthotropic mouse models.To test the safety and the efficacy of a combinatorial versus single siRNA therapy in the orthotopic mouse model and to identify molecular mechanism(s) involved in therapeutic responses by global transcriptome analyses. LNP formulations of chemically modified siRNAs targeting CSN5 and WEE1 were produced by Tekmira Pharmaceuticals. Safety was assessed in ICR mice after 9 injections. SCID-beige mice were used for intra-hepatic (Huh7-luciferase) tumor transplantation. Mice with established tumors were treated intravenously with 2 mg/kg of a single siRNA + 2 mg/kg ?gal siRNA or 2 mg/kg each of siCSN5:siWEE1 siRNA co-encapsulated in the same LNP. Tumors were assayed following 1 to 9 injected doses. Tumor progression in the Huh7 orthotopic model was monitored by bioluminescence imaging and metastases were evaluated at endpoint. Safety data show that combinatorial siRNA is well tolerated compared to single or control siRNA. We observed significant inhibition of tumor growth and metastases in mice treated with active siRNAs compared to LNP containing a non-targeting control siRNA. Significant targeted silencing was observed in tumors after single or repeat administration with no interference between the siRNAs for the CSN5:WEE1 combination. Potency was not lost with siCSN5:siWEE1 LNP, relative to the most efficacious single agent. Microarray analyses of the tumors demonstrate an extensive difference of gene expression between treatment groups. Interestingly, the microarray analyses of the surrounding liver show a minimal modification of gene expression in this non-tumor tissue. In conclusion, we have demonstrated that LNP-based combinatorial siRNA therapy is safe and effective in a human mouse model of HCC, with a significant decrease of tumor size associated with a massive downregulation of the targeted genes. Global gene expression of the surrounding liver is minimally affected by this therapy compared with what seen in the tumor.

最新的研究包括：(1)肝内胆管细胞癌(ICCA)是一种治疗难治性疾病，具有异质性的病理生物学基础，目前尚无公认的治疗方法，发病率呈上升趋势。这强调了为选定的患者亚群开发治疗方案的紧迫性。为了寻求新的治疗方案，我们利用250倍深度的全外显子测序技术分析了15例iCCA患者的肿瘤和周围肝脏样本。在144例病例的患病率筛查中，包括48个癌症相关基因的分析，我们证实了多条潜在的致癌途径，即FGFR2、PI3K/mTOR、IDH1、TP53、CDKN2A和Notch1在iCCA中的表达下调。接下来，我们在13个特征良好的CCA细胞系和小鼠异种移植瘤中，研究了通过伽马分泌酶(GS)复合体阻断受体激活来靶向Notch的结果。对缺口信号网络的综合分析揭示了四个不同的预后患者组。NOTCH1的表达是一个强有力的独立预后预后因素(？2=17.2P 0.0007)，5年生存率为17%。免疫组织化学染色证实在预后不良的患者中Notch1优先表达和受体激活。与匹配的肝脏样本相比，多个缺口相关基因，包括受体配体、靶基因和胆管分化的关键调节因子，在肿瘤中显著解除调控。此外，我们发现GS复合体的所有组成部分都显著放松了监管。使用两种GS抑制剂(R04929097和YO-01027)评估靶向Notch1激活的效果，这两种抑制剂的敏感度从0%到100%不等，并实现了基于药物反应的细胞分类。重要的是，在两种异种移植模型中评估了阻断GS活性对肿瘤生长的影响，这两种模型仅显示了在GS抑制剂敏感的iCCA中减少肿瘤负担的有益药物反应。我们的研究揭示了多个可能的药物靶点，并强调了阻断ICA中Notch受体激活的作用。这些数据强调了以基因组学为基础的医学在为确定的患者亚群选择最佳治疗方案方面的有效性。(2)组蛋白脱乙酰基酶2(HDAC2)是一种染色质修饰物，参与细胞周期、细胞凋亡和分化的表观遗传调控，在肝细胞癌中普遍上调。在这项研究中，我们证明了这种HDAC异构体的特异性靶向足以抑制肝癌的进展。SiRNA介导的HDAC沉默通过阻断细胞周期进程和诱导细胞凋亡来抑制肝癌细胞的生长。这些效应与HDAC调节的控制细胞周期、细胞凋亡和脂质代谢的基因的解除调控有关，特别是通过上调p27和乙酰化的p53以及下调CDK6和bcl2的表达。我们发现，肝癌细胞中HDAC2的沉默也强烈地抑制了PPAR-Gamma信号和其他糖酵解(ChREBP-α，GLUT4)和脂肪生成(SREBP1C，Fas)的调节因子，导致脂肪积累显著减少。值得注意的是，系统地输送包裹在脂质纳米粒中的HDAC2 siRNA足以在小鼠异种移植模型中钝化人肝癌的生长。我们的发现为HDAC2阻断作为肝癌的系统治疗提供了临床前的概念验证。(3)我们已经在正交异性小鼠模型中展示了针对CSN5或WEE1的单一siRNA的脂质纳米粒对人肝癌细胞株的治疗作用。通过整体转录组分析，测试联合治疗与单一siRNA治疗小鼠模型的安全性和有效性，并确定参与治疗反应的分子机制(S)。针对CSN5和WEE1的化学修饰siRNA的LNP配方由Tekmira制药公司生产。在9次注射后对ICR小鼠进行安全性评估。用SCID-beige小鼠进行肝内(Huh7-荧光素酶)肿瘤移植。给荷瘤小鼠静脉注射单一siRNA 2 mg/kg+Gal siRNA 2 mg/kg或共包裹同一LNP的siCSN5：siWEE1 siRNA各2 mg/kg。在注射1~9次剂量后进行肿瘤检测。通过生物发光成像监测Huh7原位模型中的肿瘤进展，并在终点评估转移。安全性数据显示，与单一或对照siRNA相比，组合siRNA具有良好的耐受性。我们观察到，与含有非靶向对照siRNA的LNP相比，使用活性siRNAs治疗的小鼠肿瘤生长和转移受到显著抑制。在单次或重复给药后，肿瘤中观察到显著的靶向沉默，CSN5：Wee1组合的siRNAs之间没有干扰。与最有效的单一试剂相比，siCSN5：siWEE1 LNP的效力不会丧失。对肿瘤的微阵列分析表明，治疗组之间的基因表达存在广泛差异。有趣的是，对周围肝脏的微阵列分析显示，在这个非肿瘤组织中，基因表达的变化很小。总之，我们已经证明，在人-鼠肝癌模型中，基于LNP的组合siRNA治疗是安全和有效的，与大量下调靶基因相关的显著减小肿瘤大小有关。与肿瘤中看到的相比，这种疗法对周围肝脏的整体基因表达的影响最小。

项目成果

Genetic profiling of intrahepatic cholangiocarcinoma.

肝内胆管癌的基因分析。

• DOI: 10.1097/mog.0b013e3283523c7e
• 发表时间: 2012-05
• 期刊: Current opinion in gastroenterology
• 影响因子: 2.5
• 作者: Andersen JB;Thorgeirsson SS
• 通讯作者: Thorgeirsson SS

Definition of ubiquitination modulator COP1 as a novel therapeutic target in human hepatocellular carcinoma.

• DOI: 10.1158/0008-5472.can-10-0749
• 发表时间: 2010-11-01
• 期刊: Cancer research
• 影响因子: 11.2
• 作者: Lee YH;Andersen JB;Song HT;Judge AD;Seo D;Ishikawa T;Marquardt JU;Kitade M;Durkin ME;Raggi C;Woo HG;Conner EA;Avital I;Maclachlan I;Factor VM;Thorgeirsson SS
• 通讯作者: Thorgeirsson SS

Molecular targeting of CSN5 in human hepatocellular carcinoma: a mechanism of therapeutic response.

• DOI: 10.1038/onc.2011.126
• 发表时间: 2011-10-06
• 期刊: ONCOGENE
• 影响因子: 8
• 作者: Lee, Y-H;Judge, A. D.;Seo, D.;Kitade, M.;Gomez-Quiroz, L. E.;Ishikawa, T.;Andersen, J. B.;Kim, B-K;Marquardt, J. U.;Raggi, C.;Avital, I.;Conner, E. A.;MacLachlan, I.;Factor, V. M.;Thorgeirsson, S. S.
• 通讯作者: Thorgeirsson, S. S.