探討在肝癌細胞株中EphA2的升高對於蕾莎瓦抗藥性及癌化的作用以及作為標靶治療的潛力

To study the role of EphA2 mediating tumorigenesis and sorafenib resistance and the potential of EphA2-targeted therapy in hepatocellular carcinoma cells

莊學群(Hsueh-Chun Chuang)

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(2020年)

https://doi.org/10.6342/NTU202003038

摘要

肝癌是全世界盛行率與死亡率名列前茅的癌症之一。原發性肝癌中最常見的是肝細胞癌 (hepatocellular carcinoma, HCC)，約占肝癌整體比例八成。當肝細胞癌發生至晚期 (advanced stage)只能依靠藥物控制。蕾莎瓦 (sorafenib) 是少數被核可使用於晚期肝細胞癌的標靶治療藥物。但許多臨床文獻指出病人在接受治療後會產生對蕾莎瓦的抗藥性。實驗室先前的研究，在質譜分析中發現EphA2會在具有抗藥性的HuH-7R中被大量表現。為了證實EphA2對於抗藥性的影響，所以使用了RNA干擾的技術抑制EphA2的表現，在此實驗中發現EphA2的抑制，降低了HuH-7R對於蕾莎瓦的抗藥性、生長、移動、侵襲等癌化現象。因此我們想了解EphA2對於其他肝細胞癌細胞株是否也有類似的影響。於是使用了Hep3B、PLC-5、SK-Hep1來做驗證，發現SK-Hep1與HuH-7R相似。兩株細胞對蕾莎瓦的耐受度都較高，並且同樣具有高度表現的EphA2與EphA2 Serine 897 (S897) 位點磷酸化。接下來，對SK-Hep1細胞進行EphA2表現的抑制。結果發現抑制EphA2後，同樣的提高細胞對於蕾莎瓦的感受度，並且抑制了細胞生長、移動、侵襲等能力。先前質譜分析的結果發現EphA2中磷酸化位點S897的大量的提高。於是我們想進一步了解這個磷酸化位點對於癌症抗藥性及癌化現象的影響。在HuH-7R細胞中以慢病毒送入帶有EphA2-wild type或EphA2-S897A的載體，發現去除S897磷酸化對於抗藥性與癌化現象與抑制EphA2表現實驗相同都有抑制抗藥性及癌症的效果。接下來我們想探討作為訊息傳遞上游的EphA2是如何傳遞訊息而影響細胞。當EphA2與配體 (ligand) Ephrin-A1結合後，會活化ligand-dependent pathway抑制Akt的磷酸化。我們也由小分子藥物篩選發現Prazosin能夠與EphA2結合並活化ligand-dependent pathway。並且發現Akt會調控一個影響HuH-7R中EMT的重要轉錄因子YB-1。後續實驗使用Ephrin-A1和Prazosin證明活化EphA2的ligand-dependent pathway會透過Akt抑制YB-1而抑制癌細胞移動、侵襲的能力。由於EphA2具有調控抑癌訊息路徑的功能，實驗室也開發了EphA2標靶胜肽 (SEK)。使用了SEK胜肽對HuH-7R進行實驗後，發現SEK能夠抑制癌細胞生長、移動的能力。綜合以上，本篇研究進一步的說明EphA2在肝癌細胞中調控對蕾莎瓦抗藥性與癌化現象的重要性，並且找出EphA2調控細胞的分子機制，最後說明標靶治療的可能性。

關鍵字

肝細胞癌 ； 蕾莎瓦sorafenib ； 酪胺酸激酶受體EphA2 ； YB-1 ； Prazosin ； 胜肽 ； 標靶治療

並列摘要

Liver cancer is one of the most malignant cancer in the world. Hepatocellular carcinoma (HCC) accounts for about 80% of liver cancer. When the HCC progresses to the advanced stage, it can only be treated with few targeted drugs. Sorafenib is one of it. However, the clinical researches showed that patients treated with sorafenib gain the acquired resistance eventually. In our previous study we found that a tyrosine receptor kinase, EphA2 was highly upregulated in the sorafenib-resistant HuH-7R cell line. The EphA2 was found to play a critical role in sorafenib resistance and HCC malignancy by shRNA-mediated knockdown functional assays. To further examine the relationship between EphA2 and HCC. Three HCC cell lines (Hep3B, SK-Hep1, PLC-5) were examined for their sensitivities to sorafenib and the EphA2 expression level. From the results, SK-Hep1 showed similar IC50 to HuH-7R and expressed high level of EphA2 as HuH-7R cells does. According to the data from mass spectrometry analysis, the phosphorylation of S897 were also highly elevated in HuH-7R cells. Next, the importance of S897 were investigated by overexpressing the lentiviral vector containing EphA2-wild type or EphA2-S897A in HuH-7R cells. This mutation of phosphorylation suppressed the resistance to sorafenib and the malignancy of HuH-7R cells. EphA2 ligand-dependent pathway was activated to inhibit the phosphorylation of Akt when EphA2 binds to its ligand, Ephrin-A1. Previously, we also found prazosin, a small molecule drug, can bind with EphA2 and activate the ligand-dependent pathway. Additionally, our lab found a transcription factor associated with EMT, YB-1, which were regulated by Akt. The Ephrin-A1 and the Prazosin were adopted to treat the HuH-7R cells respectively and YB-1 was inhibited via EphA2 ligand-dependent pathway. According to the ability of EphA2 to inhibit the malignancy of cancer, we developed a peptide, SEK, specifically binds to EphA2. The cell proliferation and motility of HuH-7R cells were inhibited by SEK treatment. To summarize, this study confirmed the relationship between EphA2 and sorafenib resistance and malignancy of HCC and found a potential pathway for EphA2-targeted therapy to treat sorafenib resistant HCC.

並列關鍵字

Hepatocellular carcinoma (HCC) ； sorafenib ； receptor tyrosine kinase EphA2 ； YB-1 ； Prazosin ； peptide ； targeted therapy

參考文獻

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