鑑定蕾莎瓦抗藥性肝癌細胞株中酪胺酸激酶EphA2受體之新型致效劑

Identification novel agonists of EphA2 in sorafenib-resistant HCC cells

黃詠璇(Yung-Hsuan Huang)

指導教授 ： 周綠蘋

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.10291

摘要

肝癌(liver cancer)是全球發生率及死亡率高的癌症之一。而其中最常見的原發性肝癌為肝細胞癌 (Hepatocellular carcinoma, HCC)，而晚期肝癌病患的整體存活期小於8個月。蕾莎瓦(Sorafenib)是目前唯一經過美國食品藥物管理局 (Food and Drug Administration, FDA)認證核可針對晚期肝癌病患的標靶治療藥物。但是，近年來，根據越來越多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。
　　在本實驗室之前的研究，已證實不論是在動物模型或是細胞模型上，EphA2都有高度表達的現象。而我們發現在HuH7R細胞中，EphA2的S897磷酸化位點會因為Akt的高度磷酸化而被活化。為了雙向證明EphA2的對蕾莎瓦抗藥性的重要，我們利用RNA干擾使EphA2的基因表現沉默化，發現在無法表達EphA2情形下的HuH7R細胞可以回復對蕾莎瓦藥物的敏感度。
　　所以我們假設EphA2的高度表達對HuH7R細胞產生蕾莎瓦抗性是很重要的角色。我們藉由對小量喹唑啉衍生物的篩選，經過細胞存活率的測試後，發現三個具有潛力的小分子藥物：prazosin, DZ-50 和 DZ-3。接著，我們想探討小分子藥物對HuH7R細胞的影響。發現prazosin抑制EphA2和Akt的磷酸化的程度最劇。在功能方面，三個小分子藥物都能引起癌細胞凋亡並且抑制細胞移動的能力。
　　根據引起EphA2內化的實驗中，prazosin對HuH7R細胞EphA2的表現量降低最多，顯示prazosin最有潛力與EphA2產生直接的交互作用，並引起EphA2路徑的細胞死亡，進而克服蕾莎瓦的抗藥性。我們接著進行藥物共同處理測試，從結果顯示，在HuH7R細胞中只有prazosin能與蕾莎瓦產生協同作用，表示prazosin可以回復對蕾莎瓦的靈敏度，並且逆轉在抗藥性肝癌細胞株中的蕾莎瓦抗藥性。我們更進一步探討此三種小分子藥物對EphA2結合力的偵測。利用電腦接合模擬及表面電漿共振生物感測儀(SPR)分析，從兩個實驗結果發現DZ-50和DZ-3針對EphA2的結合能力沒有比prazosin來得好。
　　綜合以上研究，我們知道對於抗癌研究中，EphA2是個很好的標的蛋白，但是透過細胞存活率測試的方式篩選出來的小分子藥物對EphA2的專一性並不是很好，如DZ-50和DZ-3。為了更優化我們的篩藥系統，我們建立了高通量藥物篩選平台來找尋針對EphA2專一性高的小分子藥物。而我們利用先前篩選過的少量喹唑啉(Quinazoline)衍生物進行測試，發現prazosin仍是針對抑制EphA2最有潛力的小分子藥物，並且能引發透過EphA2路徑的細胞死亡，並回復具抗藥性肝癌細胞株其對蕾莎瓦的敏感性。

關鍵字

肝細胞癌 ； 酪胺酸激?EphA2受體 ； ?唑? ； 高通量細胞影像分析 ； 篩藥

並列摘要

Liver cancer is one of the most malignant cancers in the world. The most frequent liver cancer is hepatocellular carcinoma (HCC), and the overall survival rate of advanced HCC patient is shorter than 8 months. Sorafenib is the only drug that U.S. FDA approved to treat advanced HCC patients. However, many clinical researches showed that most of the HCC patients who received sorafenib therapy eventually resulted in drug resistant to sorafenib.
From previous study of our lab, we found that EphA2 overexpressed in HuH7R cells both in vitro and in vivo. We also found that EphA2 S897 phosphoylated by AKT kinase in HuH7R cells. To prove the important relevance between EphA2 and sorafenib drug resistant, we found that knockdown of EphA2 recovers the sensitivity of HuH7R cells to sorafenib.
We hypothesized the EphA2 up-regulation is important to sorafenib resistance in HuH7R cells. We screened several Quinazoline-based compounds by MTT assay and revealed that prazosin, DZ-50 and DZ-3 are the three most potential candidates. Next, we treated potential candidates to examine the efficacy in HuH7R cells. We found that the phosphorylation of EphA2 (Ser897) and Akt (Ser473) decreased by Prazosin comparing than DZ-50 and DZ-3. All of prazosin, DZ-50 and DZ-3 can induce cell apoptosis and inhibit cell migration ability.
According to the internalization assay, prazosin decreased EphA2 expression on HuH7R cell membrane significantly, indicated that prazosin potentially involved in direct interaction with EphA2, activated ligand-dependent pathway and induced EphA2-mediated cell death to overcome sorafenib drug resistance.We also examine the anti-cancer effect of drug combined treatment. As the result, combined treatment of prazosin and sorafenib has the best synergistic effect on HuH7R cells, suggests that prazosin can recover the sensitivity to sorafenib and overcome drug resistance in sorafenib-resistant HCC model. To further detect the binding affinity of 3 compounds and EphA2, we analyze their interaction by molecular docking and SPR assay. Both results indicated that the binding affinity of DZ-50 and DZ-3 to EphA2 show no significant improvement compared to that of prazosin.
Based on the above result, EphA2 is a good target of anticancer treatment, but the candidates selected by cell viability screening are not that specific to EphA2, such as DZ-50 and DZ-3. To optimize the screening system, we establish a high-content drug screening platform by high-content analysis ( HCA ) to discover EphA2 specific small molecules. And HCA of Quinazoline-based compounds reveals prazosin is a best potential candidate to induce EphA2-mediated cell death and recovers the sensitivity of sorafenib in resistant Huh7 cell lines.

並列關鍵字

HCC ； EphA2 ； Quinazoline ； High-content analysis ； Drug screening

參考文獻

1 Laursen, L. A preventable cancer. Nature 516, S2-3, doi:10.1038/516S2a (2014).

2 Pellicoro, A., Ramachandran, P., Iredale, J. P. Fallowfield, J. A. Liver fibrosis and repair: immune regulation of wound healing in a solid organ. Nature reviews. Immunology 14, 181-194, doi:10.1038/nri3623 (2014).

3 Farazi, P. A. DePinho, R. A. Hepatocellular carcinoma pathogenesis: from genes to environment. Nature reviews. Cancer 6, 674-687, doi:10.1038/nrc1934 (2006).

4 Bruix, J., Sherman, M. American Association for the Study of Liver, D. Management of hepatocellular carcinoma: an update. Hepatology 53, 1020-1022, doi:10.1002/hep.24199 (2011).

5 Sherman, M. et al. Multidisciplinary Canadian consensus recommendations for the management and treatment of hepatocellular carcinoma. Current oncology 18, 228-240 (2011).