缺氧诱导信号通路对肝癌索拉非尼耐药机制的影响和对策

Liver cancer is the second most frequent cause of cancer death in men worldwide, and hepatocellular carcinoma (HCC) accounts for 70-85% of the total liver cancer burden. Traditional chemotherapeutic drugs have a very low response rate for HCC, and developing molecular targeting drugs has become the hot spot for combating HCC. Sorafenib is the unique first-line molecular targeting drug recommended for advanced HCC patients as it has been demonstrated that administration of sorafenib prolonged the survival of advanced HCC patients for 3-4 months. However, the effect of sorafenib in treating HCC is far from satisfaction due to drug resistance. The frequently observed hypoxic microenvironment inside solid tumors orchestrates a wide spectrum of molecular pathways that frustrate therapy, and is a major cause of tumor-resistance to radiotherapy and chemotherapy. Based on our previous research on tumoral hypoxia microenvironments, we hypothesize that there is a cross-link between sorafenib-resistance mechanisms and hypoxia-inducible signal pathways in HCC. We have previously demonstrated that blocking hypoxia-inducible pathways could enhance the efficacy of chemotherapy, transarterial embolization (TAE) and anti-angiogenic therapy in treat HCC. On the other hand, sorafenib enhanced hypoxia microenvironments by upregulating the key hypoxia-inducible molecules, including hypoxia-inducible factor (HIF) 1 and 2 and vascular endothelial growth factor (VEGF). Thus, these results indicate that the hypoxia-inducible signal pathway participates in the mechanisms of sorafenib-resistance in HCC. The present proposal aims to explore the novel mechanism underlining sorafenib-resistance by targeting hypoxia-inducible pathway, screening the key hypoxia-inducible molecules that take part in sorafenib-resistance, and finally utilizing our already established techniques to interfere these molecular targets in order to overcome sorafenib resistance to combat HCC. The specific aims include 1) screening significantly altered hypoxia-inducible molecules regulated by sorafenib in HCC cells; 2) screening significantly altered molecules in sorafenib-regulated molecular pathways under hypoxia in HCC cells; 3) investigating the effects of knocking-down HIF-1 and HIF-2 on sorafenib-regulated signal pathways under hypoxia in HCC cells; 4) clarifying the functions of the key molecules discovered from in vitro assays in the animal models of HCC tumors; 5) targeting hypoxia-inducible molecules to enhance the efficacy of sarofenib to treat HCC tumors established subcutaneously and orthotopically in animals. We hope this proposed project will underline at least in part the mechanisms accounting for sorafenib-resistance of HCC, and develop novel therapeutic strategies by targeting hypoxia-inducible pathways to combat HCC.

肝癌是发病率和死亡率最高的恶性肿瘤之一，传统化疗药物疗效低，分子靶向药物是肝癌研究的方向。代表性药物索拉非尼是目前唯一被证实可显著延长肝癌患者生存期的分子靶向药物，但因肝癌对其耐受导致疗效远不够理想。针对实体肿瘤独特的缺氧微环境，课题组提出肝癌对索拉非尼的耐药机制和缺氧信号通路存在分子交叉网络的观点，而找出其中的关键分子靶点将成为解决索拉非尼耐药的希望。课题组前期研究发现阻断缺氧信号通路，可以提高肝癌对化疗、TAE和抗新生血管生成治疗的疗效；而索拉非尼可以增加肝癌缺氧微环境，提高缺氧诱导因子的表达，提示缺氧信号通路参与了肝癌对索拉非尼的耐药机制。本项目拟在此基础上，进行肝癌对索拉非尼的耐药机制的新探索。通过体内外实验阐明缺氧信号通路与索拉非尼作用通路间的分子交叉网络，筛选影响肝癌对索拉非尼耐药的关键分子靶点，并通过干预这些分子靶点，以克服肝癌对索拉非尼的耐药，提高肝癌的治疗效果。

肝癌对索拉菲尼耐药是影响治疗效果的关键因素，肿瘤组织内的缺氧微环境诱导和激活多种信号通路和因子，导致对索拉非尼的耐药。课题组在国家自然基金项目（81272467）“缺氧诱导信号通路对肝癌索拉非尼耐药机制的影响和对策”的资助下，对影响肝癌索拉菲尼耐药的分子机制尤其是缺氧诱导信号通路进行研究，取得成果如下。 1）索拉菲尼可以抑制HIF-1，通过反馈机制使肝癌细胞缺氧诱导机制从HIF-1转换为依赖HIF-2的信号通路，激活TGF-/EGFR信号通路导致肝癌细胞的耐药。 敲除HIF-2下调可以提高索拉菲尼治疗肝癌的效果。2）2-甲氧基雌二醇（2ME2）显著降低HIF-1和HIF-2的表达, 抑制向细胞核的移位, 协同索拉非尼抑制肝癌细胞体外和体内的增殖和诱导细胞凋亡，抑制肿瘤血管生成。 3）采用浓度递增的方法建立了索拉菲尼耐药肝癌细胞株。耐药细胞株内AKT处于高度活化状态，自噬从保护机制变成了细胞自噬性死亡。特异性抑制AKT提高耐药细胞株对索拉菲尼的敏感性，诱导细胞自噬性死亡。4）三氧化二砷抑制AKT活性，协同索拉非尼通过抑制增殖和诱导肝细胞癌细胞凋亡。5) 索拉菲尼耐药细胞株高表达miR-21。MiR-21通过PTEN/Akt通路抑制细胞自噬参与索拉菲尼耐药机制。miR-21是潜在的克服索拉菲尼耐药和治疗肝癌的靶点。6）通过miRNA芯片筛选出在耐药细胞处于表达水平最高的六种miRNAs ，并构建靶向这些miRNA的人工长链非编码RNA (AlncRNA)。这种人工lncRNA含有六个拷贝的串联反义核苷酸序列，可以特异性结合6种miRNA。 腺病毒载体Ad5-AlncRNA阻断了miRNA的功能，增强索拉非尼对耐药细胞的抑制作用。另外，课题组与国内外其他研究人员建立广泛的合作关系。因此，在完成计划书的同时，我们针对癌症耐药、细胞信号传导理，以及器官缺血再灌注损伤和器官保护等方面进行了研究。因为实验试剂的使用与其他基金存在重叠，发表的论文上也注明了国家自然基金资助和项目编码。本项目资助下，课题组共发表论文18篇，参加国际学术会议并发言一人次，培养博士研究生7名。