探討肝腫瘤細胞中酪胺酸激酶EphA2受體升高對於蕾莎瓦抗藥性之影響

Abstract

肝細胞癌是最常見的肝癌，常見的成因有病毒感染，過量飲酒以及肝硬化引發肝癌。癌症末期常常發現病患預後不良以及腫瘤轉移，整體存活期少於八個月。蕾莎瓦為肝細胞癌末期唯一核可使用的標靶藥物，能抑制細胞中酪胺酸激酶的活性，達到抑制癌細胞的功用，但臨床使用結果顯示不到10%的病患在用藥後還能有良好的效果，原因是癌細胞對於蕾莎瓦產生抗藥性。 為了解決蕾莎瓦抗藥性的問題，本實驗室建立了在蕾莎瓦長期篩選後所得到的Huh7抗藥性細胞株 (Huh7R)，發現其細胞移動以及侵襲能力相較於Huh7細胞株高，顯示其細胞轉移能力強。進一步利用差異蛋白質體學分析 (Stable Isotope Labeling by Amino acids in Cell Culture，SLIAC)，發現酪氨酸激酶EphA2受體在Huh7R細胞中表現量顯著高於Huh7細胞。已知EphA2與癌症發生和轉移有關，將EphA2基因降低發現Huh7R失去移動和侵襲能力。進一步用定量磷酸化蛋白質體學分析，發現EphA2受體在Ser 897位置絲氨酸的磷酸化顯著高於Huh7。並由文獻搜尋發現此磷酸化位置和Akt激酶的活性呈正相關。 我們推測抗藥性的產生是由於EphA2受體的過度表現。為了要解決這個問題，本實驗室篩選出Compound A，並發現Huh7R在加入Compound A可以抑制EphA2 受體Ser 897和Akt Ser 473的磷酸化，並引起細胞凋亡。另外發現Compound A可以藉由降低FAK/Paxillin的活性抑制細胞移動能力。為了進一步了解Compound A和蕾莎瓦合併使用是否效率更高，利用細胞存活實驗發現Compound A可以增加細胞對蕾莎瓦感受性，此外合併使用兩種藥物可以顯著減弱細胞移動和侵襲能力。另外利用西方墨點法，我們發現Compound A和蕾莎瓦單獨使用對EphA2受體和FAK/Paxillin有抑制效果，經由合併使用下抑制現象更顯著。並且這個現象在活化Caspase 3和抑制Cyclin D1上也有相同趨勢。此外，合併使用兩種藥物可以顯著提升細胞凋亡以及抑制細胞群落形成。從以上結果我們發現合併使用Compound A與蕾莎瓦可以抑制EphA2受體訊號傳遞，並且提升Huh7R對蕾莎瓦的感受性。 未來我們將會以Compound A為先導化合物，去尋找更有效且更專一能抑制EphA2受體活性的小分子藥物，並且期望能夠找到更合適的化合物來解決抗藥性難題。

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Hepatocarcinogenesis is linked tightly to viral infection, alcoholic liver disease, and hepatic cirrhosis. Advanced HCC is a metastatic and poorly prognosis disease. The overall survival rate of advanced HCC patient is shorter than 8 months. Sorafenib is a small molecule drug which targets multi-kinases. Evidences showed that sorafenib can improve the survival of patient with advanced HCC. However, more and more clinical studies showed that most patients who received sorafenib therapy eventually resulted in drug resistant to sorafenib. To understand the mechanism of drug resistance, our lab established a sorafenib-resistant cell line, Huh7R, from Huh7 cells (HCC cell line) by long-term exposure to sorafenib. We found that the markers of cell migration and invasion such as E-cadherin and β-catenin were decreased and vimentin was increased in Huh7R, which indicated that sorafenib resistant cells tend to metastasis. By proteomic analysis, we also found that EphA2 is overexpression in sorafenib resistant cells both in vitro and in vivo. It has been reported that Eph receptor A2 (EphA2) was linked to tumor initiation and metastatic progression. After knockdown EphA2 in Huh7R, we found that cells loss ability to migration. We further used quantitative phosphoproteomic approach to compare Huh7 and Huh7R cells. We found that EphA2 Ser 897 phosphorylation was significant higher in Huh7R. It has been shown that high level of EphA2 Ser 897 phosphorylation is correlated with Akt activation and associated with cancer development. We hypothesize that EphA2 up-regulation is involved in acquired resistance to sorafenib in Huh7 R cell. In order to inhibit EphA2 activity, we screened several compounds to block EphA2 activity. The compound A had the best inhibitory effect to EphA2 S897 phosphorylation. We found that compound A inhibited Akt S473 phosphorylation and induced cell apoptosis. In addition, cell migration ability and FAK/Paxillin activity was decreased in compound A treatment. To further examine the efficiency of combined treatment to overcome drug resistance, Huh7R were treated with Compound A and sorafenib. By cell viability assay, we found that the Huh7R cells are more sensitive to sorafenib in combined treatment with compound A. Furthermore, combined treatment also reduced cell migration and invasion ability. Western blotting indicated that sorafenib and compound A individually inhibited phosphorylation of EphA2 and FAK/Paxillin, whereas the combined treatment significantly inhibited the phosphorylation of EphA2 and FAK/Paxillin in Huh7R cell. In addition, cleavage form caspase 3 was increased and cyclin D1 was decreased in combined treatment. Similarly, the cell apoptosis percentage was 41% and 23% by compound A and sorafenib, and by 56% by the combined treatment. In conclusion, combined treatment compound A and sorafenib can increase Huh7R cell sensitivity to sorafenib by inhibiting EphA2 signaling pathway. In the future, we will further investigate new compounds by using compound A as a lead compound and examine the anti-cancer effect in HCC model.