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.

最新的重新搜索包括（1）肝内胆管癌（ICCA）是一种治疗难治性疾病，具有异质性的病理生物学，没有批准的治疗和发病率上升。这强调了为精选患者子集开发治疗选择的紧迫性。为了追求新的治疗选择，我们使用全外活体测序在250倍的深度分析了15例配对肿瘤和周围肝样品的ICCA患者。在涉及48个与癌症相关基因的分析的144例病例的患病率筛查中，我们证实了多个潜在的因果途径的放松管制，即FGFR2，PI3K/MTOR，IDH1，TP53，CDKN2A，CDKN2A和Notch1在ICCA中。接下来，我们通过在13个特征良好的CCA细胞系和鼠类异种移植物中通过γ-分泌酶（GS）复合物阻断受体激活来检查靶向Notch的结果。对缺口信号网络的全面分析显示了四个不同的预后患者群体。 Notch1表达是一个强大的独立预后预测因子（？2 = 17.2，p 0.0007），生存率为17％。免疫染色证实了预后不良的患者的优先Notch1表达和受体激活。与匹配的肝脏样品相比，肿瘤中的多个与Notch相关的基因包括受体配体，靶基因和胆道分化的关键调节剂。另外，我们发现对GS复合物的所有组件的显着放松管制。使用两种GS抑制剂（R04929097和YO-01027）评估靶向Notch1激活的效果，范围从0％到100％敏感性，并使基于药物反应的细胞分类。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ICCA肿瘤负担减少的有益药物反应。我们的研究揭示了多个推定的药物靶标，并突出了阻断ICCA中缺口受体激活的效果。这些数据强调了基于基因组学的药物在为定义的患者子集选择最佳治疗方面的有用性。 （2）组蛋白脱乙酰基酶2（HDAC2）是一种染色质修饰剂，参与细胞周期，凋亡和分化的表观遗传调节，通常在人肝细胞癌（HCC）中上调。在这项研究中，我们表明该HDAC同工型的特定靶向足以抑制HCC的进展。 siRNA介导的HDAC沉默通过阻断细胞周期进展并诱导凋亡抑制HCC细胞的生长。这些作用与控制细胞周期，细胞凋亡和脂质代谢的HDAC调节基因的不受管制有关，特别是通过上调p27和乙酰化p53以及CDK6和BCL-2的下调。我们发现，HCC细胞中的HDAC2沉默也强烈抑制了PPAR-GAMMA信号传导和其他糖酵解的调节剂（Chrebp-Alpha，Glut4）和脂肪生成（SREBP1C，FAS），从而引发了明显的脂肪积累下降。值得注意的是，封装在脂质纳米颗粒中的HDAC2 siRNA的全身递送足以在鼠异种移植模型中钝化人类HCC的生长。我们的发现为HDAC2阻滞剂提供了临床前概念验证，作为肝癌的全身疗法。 （3）我们先前已经证明了脂质纳米颗粒（LNP）的治疗效应，该脂质靶向CSN5或WEE1在正骨小鼠模型中对人HCC细胞系的单个siRNA靶向对人类HCC细胞系的治疗作用。用于测试与整体小鼠模型（S）在整体上识别单siRNA治疗的安全性（S），以识别矫形小鼠模型（S）涉及分解形成（S）。分析。 Tekmira Pharmaceuticals生产了靶向CSN5和WEE1的化学改性siRNA的LNP制剂。 9次注射后，在ICR小鼠中评估了安全性。 SCID米型小鼠用于肝内（HUH7-荧光素酶）肿瘤移植。用已建立的肿瘤的小鼠用2 mg/kg的单个siRNA + 2 mg/kg？gal siRNA或2 mg/kg的小鼠进行SICSN5：SIWEE1 siRNA的每个共同封装在同一LNP中。 1至9剂剂量后，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，并在端点评估转移。安全数据表明，与单个或对照siRNA相比，组合siRNA的耐受性良好。我们观察到与含有非靶向对照siRNA的LNP相比，在用活性siRNA治疗的小鼠中对肿瘤生长和转移的显着抑制作用。单次或重复给药后，在肿瘤中观察到明显的靶向沉默，而CSN5：WEE1组合的siRNA之间没有干扰。相对于最有效的单剂，SICSN5：SIWEE1 LNP并没有损失效力。对肿瘤的微阵列分析表明，治疗组之间的基因表达存在很大差异。有趣的是，周围肝脏的微阵列分析显示，在这种非肿瘤组织中基因表达的修饰最少。总之，我们已经证明，基于LNP的组合siRNA治疗在HUCC的人小鼠模型中是安全有效的，并且与靶向基因的大量下调有关的肿瘤大小显着降低。与肿瘤中看到的疗法相比，周围肝脏的全球基因表达受到这种疗法的影响。

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.