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）肝内胆管癌（iCCA）是一种难治性疾病，其基础病理学具有异质性，尚无批准的治疗方法且发病率不断上升。这强调了为特定患者亚群开发治疗方案的紧迫性。为了寻求新的治疗方案，我们使用 250 倍深度的全外显子组测序分析了 15 名 iCCA 患者的配对肿瘤和周围肝脏样本。在 144 例病例的患病率筛查中，涉及 48 个癌症相关基因的分析，我们证实了 iCCA 中多个潜在因果通路的失调，即 FGFR2、PI3K/mTOR、IDH1、TP53、CDKN2A 和 Notch1。接下来，我们在一组 13 个特征良好的 CCA 细胞系和小鼠异种移植物中通过 γ-分泌酶 (GS) 复合物阻断受体激活，检查了靶向 Notch 的结果。对切​​迹信号网络的综合分析揭示了四个不同预后的患者组。 Notch1 表达是生存的强大独立预后预测因子 (?2=17.2, P < 0.0007)，5 年生存率为 17%。免疫染色证实预后不良的患者优先表达 Notch1 并激活受体。与匹配的肝脏样本相比，多个Notch相关基因，包括受体配体、靶基因和胆道分化的关键调节因子，在肿瘤中显着失调。此外，我们发现 GS 复合物的所有成分都显着放松管制。使用两种 GS 抑制剂（R04929097 和 YO-01027）评估靶向 Notch1 激活的效果，其灵敏度范围为 0% 至 100%，并能够根据药物反应对细胞进行分类。重要的是，在两个异种移植模型中评估了阻断 GS 活性对肿瘤生长的影响，这仅证明了对 GS 抑制剂敏感的 iCCA 中减少肿瘤负荷的有益药物反应。我们的研究揭示了多个假定的药物靶点，并强调了阻断 iCCA 中 Notch 受体激活的效果。这些数据强调了基于基因组学的医学在为特定的患者亚群选择最佳治疗方法方面的有用性。 (2) 组蛋白脱乙酰酶 2 (HDAC2) 是一种染色质修饰剂，参与细胞周期、细胞凋亡和分化的表观遗传调控，在人肝细胞癌 (HCC) 中普遍上调。在这项研究中，我们表明针对这种 HDAC 亚型的特异性靶向足以抑制 HCC 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 HCC 细胞生长。这些效应与控制细胞周期、细胞凋亡和脂质代谢的 HDAC 调节基因的失调有关，具体来说，是通过 p27 和乙酰化 p53 的上调以及 CDK6 和 BCL-2 的下调来实现的。我们发现 HCC 细胞中的 HDAC2 沉默也会强烈抑制 PPAR-gamma 信号传导以及糖酵解（ChREBP-α、GLUT4）和脂肪生成（SREBP1C、FAS）的其他调节因子，从而导致脂肪积累显着减少。值得注意的是，封装在脂质纳米颗粒中的 HDAC2 siRNA 的全身递送足以抑制小鼠异种移植模型中人类 HCC 的生长。我们的研究结果为 HDAC2 阻断作为肝癌的全身疗法提供了临床前概念验证。 (3) 我们之前已经在正交异性小鼠模型中证明了装载有针对 CSN5 或 WEE1 的单一 siRNA 的脂质纳米颗粒 (LNP) 对人 HCC 细胞系的治疗效果。原位小鼠模型并通过全局转录组分析来识别参与治疗反应的分子机制。针对 CSN5 和 WEE1 的化学修饰 siRNA 的 LNP 制剂由 Tekmira Pharmaceuticals 生产。注射 9 次后对 ICR 小鼠进行安全性评估。 SCID-米色小鼠用于肝内（Huh7-荧光素酶）肿瘤移植。用2mg/kg的单一siRNA+2mg/kg的μgal siRNA或共封装在同一LNP中的各2mg/kg的siCSN5:siWEE1 siRNA静脉内治疗已形成肿瘤的小鼠。注射1至9次后对肿瘤进行测定。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。安全数据表明，与单一或对照 siRNA 相比，组合 siRNA 具有良好的耐受性。与含有非靶向对照 siRNA 的 LNP 相比，我们观察到用活性 siRNA 治疗的小鼠的肿瘤生长和转移受到显着抑制。单次或重复给药后，在肿瘤中观察到显着的靶向沉默，CSN5:WEE1 组合的 siRNA 之间没有干扰。相对于最有效的单一药物，siCSN5:siWEE1 LNP 的效力并未丧失。肿瘤的微阵列分析表明治疗组之间的基因表达存在巨大差异。有趣的是，周围肝脏的微阵列分析显示该非肿瘤组织中基因表达的改变很小。总之，我们已经证明基于 LNP 的组合 siRNA 疗法在人类 HCC 小鼠模型中是安全有效的，肿瘤大小显着减小，与靶向基因的大量下调相关。与肿瘤中观察到的相比，周围肝脏的整体基因表达受这种疗法的影响最小。

项目成果

Genetic profiling of intrahepatic cholangiocarcinoma.

• 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.