RNAi-Based Therapy of Hepatocellular Carcinoma

描述（由申请人提供）：本研究的总体目标是研究cyc E siRNA治疗肝细胞癌（HCC）的治疗潜力。我们的实验室最近发现细胞周期蛋白E (cyc E)，一个在70%的HCC中过表达的癌基因，在增殖和细胞存活中起着重要作用，可能成为HCC的一个有希望的治疗靶点。我们还发现，通过靶向cyc E编码区的siRNA，过表达的cyc E可被抑制高达90%。HCC中cyc E的缺失在培养细胞和裸鼠中诱导了细胞生长的显著抑制。因此，我们假设cyc E siRNA可能作为一种新的有效的治疗剂来治疗cyc E过表达的HCC。将实施四个具体目标来检验这一假设。(1)研究cyc E siRNA对小鼠皮下和原位肝癌模型的治疗作用。对于原位模型，将表达荧光素酶的HCC细胞系肝内注射到小鼠体内，使其在肝脏中产生肿瘤。一种改进的脂质体递送系统（DOTAP:Chol）将通过肿瘤内注射或全身静脉注射用于siRNA转移。通过体内成像系统监测肿瘤体积和转移情况，确定治疗效果。我们还将比较不同递送系统（非肝靶向与肝靶向递送系统）治疗的肿瘤抑制效果，以制定HCC的最佳临床前治疗策略。(2)体外和体内模型比较cyc E siRNA对HCC细胞和正常肝细胞或组织的影响。siRNA将被转染到永生的正常人类肝细胞或HCC细胞中，这些细胞之间的生长特性将被充分分析其差异。siRNA的体内毒性将通过对小鼠主要器官的酶和病理分析进行检测。(3)评价cyc E siRNA联合化疗药物的治疗效果。我们已经证明cyc E siRNA和阿霉素联合使用对抑制HCC细胞生长具有协同作用。为了测试cyc E过表达是否与化疗耐药有关，将产生具有不同cyc E表达水平的稳定细胞系，并检查它们对多种化疗药物的反应。cyc E siRNA和化学药物联合使用的协同效应将在动物中进一步测试。我们还将确定介导这些协同效应的机制，包括NF-kappaB、Akt和Bcl2生存途径的分析。(4)评价肿瘤特异性表达载体cyc E siRNA的体内外抗肿瘤作用。我们通过肝脏肿瘤特异性启动子（AFP）生成了表达cyc E siRNA的质粒。该载体的有效性和特异性将在小鼠肝癌细胞系和肝癌异种移植模型中进行系统评估。本研究产生的数据将为进一步了解HCC发展过程中涉及的分子事件提供有价值的信息，并导致基于cyc E sirna的有效治疗的发展，以降低HCC死亡率。

DESCRIPTION (provided by applicant): The overall goal of this study is to address the therapeutic potential of cyc E siRNA on the treatment of hepatocellular carcinoma (HCC). Our lab recently discovered that cyclin E (cyc E), an oncogene overexpressed in 70% of HCC, played a substantial role in proliferation and cell survival and could serve as a promising therapeutic target for HCC. We also found that overexpressed cyc E could be suppressed up to 90% by siRNA targeting the coding region of cyc E. Depletion of Cyc E in HCC induced significant inhibition of cell growth both in cultured cells and in nude mice. Therefore, we hypothesize that cyc E siRNA may serve as a novel and effective therapeutic agent to treat cyc E-overexpressing HCC. Four specific aims will be carried out to test this hypothesis. (1) To determine the therapeutic effects of cyc E siRNA using both subcutaneous and orthotopic HCC models in mice. For the orthotopic model, HCC cell lines expressing luciferase will be intrahepatically injected into mice to produce tumors in liver. An improved liposomal delivery system (DOTAP:Chol) will be used for siRNA transfer by intratumoral injection or systemic treatment through intravenous injection. The tumor volume and metastasis will be monitored by in vivo image system to determine the therapeutic efficacy. We will also compare the tumor suppression effects among treatments with different delivery systems (non-liver targeting versus liver-targeting delivery system) to develop an optimal preclinical therapeutic strategy for HCC. (2) To examine the effect of cyc E siRNA on HCC cells versus normal hepatocytes or tissues in in vitro and in vivo models. The siRNA will be transfected into immortalized normal human hepatocytes or HCC cells and the growth properties among those cells will be fully analyzed for their differences. The in vivo toxicity of siRNA will be examined by enzymatic and pathological analysis on major organs in mice after the treatments. (3) To evaluate the therapeutic efficacy of cyc E siRNA in combination with chemodrugs. We have demonstrated that combination of cyc E siRNA and doxorubicin exhibited a synergism on inhibition of HCC cell growth. To test if cyc E overexpression is involved in chemoresistance, stable cell lines with different cyc E expression levels will be generated and examined for their responses to multiple chemodrugs. The synergistic effects from the combinations involving cyc E siRNA and chemodrugs will be further tested in animals. We will also identify the mechanisms mediating these synergistic effects, including analysis of NF-kappaB, Akt and Bcl2 survival pathways. (4) To assess the in vitro and in vivo antitumor effect of cyc E siRNA from a tumor specific expression vector. We have generated a plasmid which expresses cyc E siRNA via a liver tumor specific promoter (AFP). The efficacy and specificity of this vector will be systematically assessed using both HCC cell lines and HCC xenograft models in mice. The data generated from this study will provide valuable information to further understanding the molecular events involved in the development of HCC as well as lead to the development of effective cyc E siRNA-based therapy to reduce HCC mortality.