Abstract
摘要:分子標靶藥物已成為現今新的癌症治療方法,然而癌細胞為了克服標靶藥物的治療而產生抗藥性,顯示抗藥性是目前克不容緩尚待解決的健康議題。蕾莎瓦(Sorafenib),是一種多重性抑制劑,且是唯一用於肝癌末期病人的標準治療藥物。不幸地,大多數的病人最終產生抗藥性。近來,我們透過穩定同位素標記胺基酸(SILAC)的蛋白體磷酸化定量方法,從抗藥性細胞株 HuH-7R 和親代 HuH-7 細胞株的磷酸化變化量中,鑑定到884個蛋白質和968個磷酸化胜肽片段具有差異的表現量,得知在Akt 訊息及其相關受質中,EphA2和YB-1發生了異常調控。進一步發現(1) EphA2和YB-1高度磷酸化表現和蕾莎瓦的抗藥性有高度關聯。(2)在蕾莎瓦處理之下,YB-1蛋白被磷酸化後會從細胞質進入細胞核。(3) YB-1會調節HuH-7 R 細胞的移行,侵襲以及絲狀偽足的形成。(4) EphA2也會調節HuH-7 R細胞的生長,移行以及侵襲的能力。(5) Akt藉著協同EphA2磷酸激酶而促進了HuH-7 R 細胞的轉移和侵襲能力。但當EphA2結合了配位體,接受了訊息則會導致細胞粘附、凋亡或抑制血管新生和腫瘤的形成;因此,在癌症的治療過程中,EphA2極具潛力成為抗藥性的新穎標的蛋白。在這三年計劃中,我們將試著去了解YB-1和EphA2的功能及其如何促成抗藥性的演進,並著手研究其致病機制去釐清是否透過Akt-EphA2的訊息傳遞調節並主導腫瘤細胞的移行,侵襲和轉移能力。我們也進一步地闡明YB-1可以從細胞質進入細胞核後,使得HuH-7R細胞具有移動,侵襲和轉移的能力。這些分子在腫瘤細胞中,透過EphA2的訊息傳遞途徑所導致抗藥性的相關結果,將提供我們去設計更好的策略,來達到控制癌症的進展。最後,我們將驗證具有潛力的小分子以促進EphA2受器的活性,透過干擾致癌的訊息途徑,進而測試其在肝癌HuH-7R細胞中的成效。更以功能研究和活體實驗,再次確認標的蛋白在生物中所扮演的重要角色。期待本計劃的研究結果,可以增加我們對於EphA2和YB-1在蕾莎瓦抗藥性的肝癌細胞中,它是如何調節其相關功能和抗藥機制有更多的認識,以便於發展新的肝癌治療策略。
Abstract: Current molecularly targeted therapies for cancer have generated promising new treatments. However, cancer cells overcame acquired resistance to targeted therapy, indicating that drug-resistance is a major health issue awaiting for being solved. Sorafenib, a multikinase inhibitor, is the only standard drug treatment for HCC patients with advance-stage. Unfortunately, most of patients eventually develop acquired resistance. Recently, SILAC-based quantitative phosphoproteomics was applied to quantify the changes of phosphoproteome between the resistant HuH-7R and the parental HuH-7 cells. We have observed that 884 of proteins and 968 phosphopeptides were differentially expressed between HCC HuH-7R and HuH-7 cells, Akt signaling and their substrates were dysregulated significantly including EphA2 and YB-1. Our further studies revealed that (1) EphA2 and YB-1 abnormally highly phosphorylated and associated with sorafenib resistance. (2) Upon sorafenib treatments, YB-1 protein phosphorylated and translocated from the cytoplasm to the nucleus. (3) YB-1 regulated the migration, invasion and filopodia formation of HuH-7R cells. (4) EphA2 regulated proliferation, migration and invasion of HuH-7R cells, (5) Akt may promote cell migration and invasion by co-opting EphA2 kinase in HuH-7R cells. While binding to its ligand, EphA2-bearing cells receive the forward signaling, leading to cell adhesion, apoptosis or inhibition of angiogenesis and preventing tumor formation. Therefore, EphA2 has the potential as the therapeutic target for drug resistance in cancer.In this 3-year project, we will try to gain insight into the function of YB1 and EphA2 and how they help drive drug-resistance. We will investigate if Akt-EphA2 signaling axis is a driver mechanism in regulating tumor cell migration, invasion and metastasis. We also elucidate the role of YB-1 translocation from the cytoplasm to the nucleus in HuH-7R cell motility, invasion, and formation of metastases. The understanding of the molecules through which EphA2 confers drug resistance in tumor cells, will allow us to design better strategies to control cancer progression. Finally, we will identify potential small molecule agonists activator of EphA2 receptor that disrupt oncogenic Akt / EphA2 signaling and to test their efficacy in resistant HuH-7R cells. Following functional studies and in vivo validation will be performed to confirm the biologic significances. We expect that the results obtained from this project should improve our understanding about the molecular mechanisms how EphA2 and YB-1 can regulate the functions of sorafenib resistant HCC cells and new therapies that may be developed for sorafenib resistance in HCC.
Keyword(s)
肝細胞癌(HCC)
蕾莎瓦
抗藥性
轉移
YB-1
EphA2
Hepatocellular carcinoma (HCC)
sorafenib
drug resistance
metastasis
YB-1
EphA2