探討DKK1於Sorafenib治療肝細胞癌中可能的角色

Abstract

Sorafenib是用來針對晚期肝細胞癌的標靶治療藥物，也是第一個被證實可以有效提升晚期肝細胞癌病患存活率的藥物。Sorafenib最初是作為Raf磷酸激酶抑制劑而設計出來的標靶藥物，後來有許多研究證實，Sorafenib有許多「標靶外作用」（off-target effects），與其抗癌療效及抗藥性產生有密切關係存在。而我們實驗室過去在使用一些有用的標靶藥物組合劑以解決Sorafenib在肝細胞癌抗藥性問題時，發現Wnt路徑與DKK1受到有效標靶藥物組合抑制最顯著，這暗示了DKK1在肝癌中或許佔有一定角色及功能，也許可以做為肝癌的治療標的。過去亦有研究發現，佔有一定比例的肝癌病患，Wnt/beta-catenin訊息傳遞路徑及DKK1有異常活化的現象，因此本論文主要目標分三部份：（1）為了瞭解DKK1是否可以當作Sorafenib的治療生物性標記，（2）了解DKK1是否可以作為治療標的以及在肝癌細胞中利用Sorafenib及DKK1抑制劑探討調控機制；（3）了解使用Sorafenib合併DKK1抑制劑是否可以克服對Sorafenib具抗藥性肝癌細胞的問題。本篇研究利用DKK1抑制劑（WAY-262611）以及Sorafenib進行體外細胞株與小鼠生體試驗。目前在細胞實驗與小鼠實驗研究結果發現，對Sorafenib敏感性的肝癌細胞株，細胞內與分泌出細胞外，小鼠肝腫瘤治療後其血清中的DKK1蛋白會隨之減少，顯示DKK1表現量與Sorafenib抑制肝癌細胞程度呈反比，與腫瘤大小呈正比。另外利用DKK1抑制劑與干擾核酸(si-DKK1)發現對Sorafenib抗藥性的肝癌細胞株（Huh-7R）比Sorafenib敏感性的肝癌細胞株（Huh-7）有更強的能力促使肝癌細胞走入細胞凋亡，而DKK1抑制劑與干擾核酸也可以有效增強Sorafenib促使所有肝癌細胞株走向細胞凋亡的能力。最後利用DKK1抑制劑、干擾核酸(si-DKK1)與過度表現DKK1技術探討Sorafenib抑制DKK1機制，結果發現在Sorafenib敏感性的肝癌細胞株，Sorafenib會透過c-Jun與beta-catenin競爭DKK1活化的調控，達到抑制DKK1表現。目前結果顯示DKK1有潛力作為治療標的並且提供解決Sorafenib抗藥性的問題。

Sorafenib is a drug for standard systemic therapy in patients with advanced hepatocellular carcinoma (HCC), and it is also the first drug with survival benefits. Although Sorafenib was originally designed as a specific Raf kinase inhibitor, we and other investigators have found many off-target effects of Sorafenib that may have significant implications regarding its anti-tumor activity and the resistance mechanism of Sorafenib in HCC cells. In the past, our laboratory had tried to treat some effective targeted drugs combination solving Sorafenib resistance problems in HCC cells. In Addition, we found that Wnt pathway and DKK1 are effectively marked the most significant inhibiting in drug combinations treatment. This suggests that DKK1 may play certain roles in HCC, and may thus be a good therapeutic target for treating liver cancer. Past studies have also found that in a certain proportion of patients with liver cancer, the Wnt/beta-catenin signaling pathway and DKK1 exhibit abnormal activation. Therefore, the present study had 3 primary specific aims: (1) to clarify whether DKK1 is a Sorafenib therapeutic biomarker; (2) to clarify whether DKK1 is a good therapeutic target and to clarify the regulatory mechanisms in HCC cells treated with Sorafenib and DKK1 inhibitor; and (3) to clarify whether the combination of Sorafenib and DKK1 inhibitor could overcome Sorafenib resistance in HCC cells. To these ends, we used DKK1 inhibitor (WAY-262611) and Sorafenib in in vitro and in vivo tests. In the mice tests and cell line results, we found that the proliferation inhibition of HCC cells and DKK1 expression were decreased after Sorafenib treatment. The results further showed that the expression of DKK1 were inversely correlated with the effect of Sorafenib, but correlated with tumor volume. Furthermore, WAY-262611 and siDKK1 did not exhibit only more activity in apoptosis induction in a Sorafenib-resistant HCC cell line, Huh-7R, than in a Sorafenib-sensitive cell line, Huh-7, but also enhance effectiveness of Sorafenib in inducing all HCC cell lines into apoptosis. Moreover, we investigated the mechanism of Sorafenib inhibition of DKK1 via WAY-26261, siDKK1, and overexpression of DKK1. The results showed that Sorafenib inhibits DKK1 by up-regulating c-Jun levels, as c-Jun, in turn, competes with the regulation of beta-catenin activating DKK1. These results so far indicated that DKK1 might be a good therapeutic target for providing a solution to Sorafenib resistance problems.