以蕾莎瓦抗藥性的肝癌細胞株為平台尋找具濳力的胜肽促進EphA2 訊號以抑制腫瘤增生

Abstract

肝癌是世界上最常見的癌症之一，其中最常見的是肝細胞癌（Hepatocellualr carcinoma, HCC）。在台灣，肝癌的盛行率是所有癌症的第二名，僅次於肺癌。根據巴賽隆納臨床肝癌分期方法，晚期的肝癌病人常使用唯一的口服肝癌標靶藥物蕾莎瓦（Sorafenib）治療。蕾莎瓦是一種多重激脢抑制劑，能藉由Raf抑制MAPK細胞傳遞路徑，且是目前唯一美國FDA核可上市的口服肝癌標靶藥物。但是近年來，根據越來越多的臨床報導及文獻指出，接受蕾莎瓦治療的病人逐漸有抗藥性的產生。本實驗室過去的研究，不論是動物或是細胞模型上，EphA2在抗藥性的肝細胞癌細胞都有大量表達的現象。 EphA2是一種細胞膜上的受體酪氨酸激酶（Receptor tyrosine kinase），最早是在神經科學與發育生物學中被提及，具有引導軸突 （Axon）生長的作用，但近年來EphA2在癌症中扮演的角色已經越來越多文獻報導。當EphA2缺乏其配體 （Ligand） ephrin時，EphA2會傾向以單體 (Monomer)形式存在，伴隨著本身高度的Ser897磷酸化，EphA2會與其他受體酪氨酸激酶一同使Akt的Ser473位點高度磷酸化，使得癌細胞侵襲及轉移的能力上升，這或許也解釋了高度表達EphA2的許多癌症患者其癒後情況也相當差。而當其配體結合上EphA2時，受體的Ser897去磷酸化和連帶導致的Akt Ser473位點去磷酸化則抑制了癌細胞移動及增生的能力。 所以我們是希望能夠尋找能夠作為EphA2受體的胜肽分子。首先純化出EphA2 LBD ( Ligand binding domain)重組蛋白，也進行了表面電漿共振的分析，確定蛋白活性，未來可以作為針對EphA2的篩藥平台。利用分子模擬的方法計算出結合自由能，期望能找到一段適合的胜肽分子具有和EphA2良好的結合能力。並且在細胞實驗中進行驗證。綜合以上研究，我們能夠利用分子模擬的方法，尋找EphA2合適的受體促效劑，日後也能夠優化並開發出針對EphA2的抗藥性癌症藥物分子。

Hepatocellular carcinoma, HCC, is the most common liver cancer in the world. In Taiwan, it ranks on top two deadly cancers. Barcelona Clinic Liver Cancer（BCLC） has been suggested to be the best treatment guidance. According to the BCLC system, Sorafenib is the only anticancer drug with proven prognostic efficacy in HCC. Sorafenib is a multikinase inhibitor of several growth factor signaling pathways. It inhibits the VEGF pathway by inhibiting VEGF receptors, and the MAPK pathway by inhibiting Raf. In our previous study, we found that EphA2 is overexpressed in Huh7R cells. The Eph receptor family is the largest of the 20 receptors tyrosine kinase family. A member of the Eph receptor family, EphA2, is highly expressed in tumors. It can promote cell migration/invasiveness and tumor malignancy in a ligand-independent manner. Its tumorigenic activity has been linked to high levels of Ser897 phosphorylation and low levels of tyrosine phosphorylation. EphA2 appears to behave as an oncoprotein in the absence of ligand binding, which may explain why its overexpression is associated with poor prognosis in many cancers. Previously, when ligand bound to EphA2 resulted de-phosphorylation of Ser897 and suppressed tumor progression. In this study, we want to find a potential ligand to bind EphA2 and activate downstream signaling which suppress tumorigenic activity. First, we expressed ligand-binding domain (LBD) of EphA2 protein using E.coli system and identified this protein using LC-MS/MS and western blotting. It is also important for us to screen the best condition to express EphA2 LBD soluble protein since the protein is membrane protein and majority exist in inclusion body. After induction with 0.5 mM IPTG overnight in 16o C, the soluble native protein can be enriched. Recombinant His-tag LBD proteins were purified by Ni2+ affinity column. The recombinant proteins further purified by gel filtration using Hiload Superdex FPLC column. We also investigated the binding affinity between LBD of EphA2 and the ligand ephrin by surface plasmon resonance（SPR）. To find the potential binding peptides, molecular dynamic simulation (MD simulation) is used to calculate the binding free energy. It is expected to find a suitable peptide with a good binding ability to EphA2. And in vitro experiments will be validate the effect of the peptide on Huh7R cells. Based on the above studies, we will able to use molecular modeling methods to find suitable receptor agonists for EphA2. In the future, we will be able to optimize and develop drug-resistant cancer drug molecules against EphA2.