Abstract
摘要:Sorafenib 是一個多重激酶抑制劑,可抑制細胞Raf 激酶以及血管內皮生長因子接受器,sorafenib 目前是治療晚期肝細胞癌病患的標準用藥。然而,目前sorafenib治療肝細胞癌有效的分子機制未被完全釐清,我們在先前的研究已證實,除了已知的藥物標的(即Raf 激酶及VEGFR)外,其他的非標的蛋白(off-target)在sorafenib 治療肝細胞癌的療效扮演重要的角色。過去已有許多研究針對肝細胞癌的基因體(genome)、mRNA 及蛋白質,於腫瘤組織與非腫瘤組織之間表現差異進行分析研究;然而,這些研究並未發現特殊的標的,可應用於發展成肝細胞癌的標靶治療。定量磷酸蛋白質體學(quantitativephosphoproteomics)方法如SILAC(Stable Isotope Labeling with Amino acids in Cellculture)與iTRAQ(Isobaric Taq for Relative and Absolute Quantitation)標定法,近來已成為全面化研究磷酸蛋白改變及細胞訊息傳導最有利的研究策略。已經有許多文獻報導應用此一方法來深入研究標靶藥物(如imatinib與gefitinib的藥物及抗癌機轉),並且發現新的藥物作用機制。我們也利用SILAC標定法研究sorafenib治療Huh7肝癌細胞株後細胞內磷酸化蛋白質的變化。我們的初步研究證實,sorafenib除了調控RAS/MEK/ERK 訊息傳導路徑,也導致細胞內其他訊息路徑,如PI3K/Akt、JAK/STAT3和NF-kappa B等的變化。所以在本研究計畫中,我們則提出另一種迴異於過去研究的假說,即我們將利用己知可能對肝細胞癌治療有效、但其抗癌機制仍不十分清楚的藥物或化合物,如sorafenib,用定量磷酸蛋白質體學平台來全面分析這些藥物在肝癌細胞所造成的分子變化。我們相信此一全面磷酸蛋白質的定量分析將有助於闡明sorafenib作用機制,並有助於發現可運用於肝細胞癌治療而尚未被確認的分子標的。在本計畫中,我們將藉由SILAC 與iTRAQ 方法來多元性的分析肝癌細胞株與動物組織,經sorafenib 或其他具潛力藥物處理後的磷酸蛋白分子變化,並且尋找重要目標分子與新的治療標的,並將利用功能性研究來探討證明這些新發現的目標分子的生物重要性與臨床意義。我們期望經由整合性的研究,達成以下的研究目標:1. 全面比較分析對 sorafenib 不同敏感度的肝癌細胞株其藥物處理前後磷酸化蛋白質變化概況(與共同主持人徐志宏醫師合作);2. 全面比較分析動物實驗中組織檢體對 sorafenib 其藥物處理前後蛋白質變化概況(與共同主持人徐志宏醫師合作);3. 分析上述研究成果來探尋並驗證其 sorafenib 療效相關的分子機轉;4. 利用上述研究結果找尋可預測 sorafenib 療效相關的預後生物標記,並驗證其臨床意義。我們預此一研究將有助以下長期目標的達成:(1)開發新的治療策略來提昇sorafenib在肝癌患者的治療療效;(2) 發現可以預測sorafenib 於肝癌病人治療效果並具臨床應用價值的預後生物標記;(3)研發可運用於治療肝癌嶄新的分子標的!
Abstract: Sorafenib, a multi-kinase inhibitor against Raf kinase and vascular endothelial growth factorreceptor (VEGFR), has become standard therapy for patients with advanced hepatocellularcarcinoma (HCC). However, the molecular mechanisms underlying the efficacy of sorafenib inadvanced HCC are not fully understood. Previously, we and others have identified several“off-target” effects independent of inhibiting Raf kinase or VEGFR, are critically important inthe anti-HCC effect of sorafenib.Quantitative phosphoproteomic approaches such as SILAC (Stable Isotope Labeling withAmino acids in Cell culture) and iTRAQ (Isobaric Tag for Relative and Absolute Quantitation)have become the most powerful strategies to globally investigate cellular phosphorylatedproteins and their signaling pathways. These approaches have been demonstrated to elucidate thecomplex changes of the phosphoproteins in cancer cells treated with various types of kinaseinhibitors such as imatinib and gefitinib, and helped reveal novel mechanisms of action for thesekinase inhibitors. Using SILAC approach, we have found that several signaling pathways otherthan RAS/MEK/ERK pathway, such as PI3K/Akt, JAK/STAT3, and NF-kappa B, aresignificantly altered and dynamically changed in Huh 7 HCC cells treated with sorafenib.We hypothesize that delineation of the phosphoproteomic changes induced by sorafenibwill shed light on the molecular mechanisms underlying the effectiveness of sorafenib, andmay contribute to the identification of the yet-unidentified molecular targets for thetreatment of HCC.This study will compare the quantitative changes of phospho-proteomes in a panel of HCCcells treated with sorafenib, by SILAC approach and LC-MS/MS analysis. Furthermore, we willalso conduct orthotopic and subcutaneous liver cancer models for determining possible effects ofsorafenib to treat liver cancer in vivo. To elucidate its molecular mechanisms, proteome ofcancer-bearing mice treated with sorafenib was performed by iTRAQ labeling and theLC-MS/MS analysis. The combination results from in vitro and in vivo assays will then beconducted for the identified mechanisms to prove their biologic and clinical significance. Furtherclinical validation studies will then be performed for the identified mechanisms to prove theirbiologic and clinical significance.The specific aims of this study proposal include:(1) To identify the phosphoproteome differentially expressed in a panel of HCC cells withdifferent sensitivities to sorafenib (in vitro profilings).(2) To identify the proteomes in tissues and serum/plasma of HCC-xenograft and orthotopicmouse models treated with sorafenib (in vivo profiling).(3) To verify the molecular mechanisms related to effectiveness of sorafenib,revealed in thedifferentially expressed (phospho-)proteome profilings in HCC cells and in cancer-bearingmice during treatment with sorafenib.(4) To discover the potential prognosis biomarkers in treatment of advanced HCC and validateForm C010 Page 2 of 2 pagesthe relevance in clinical samples.The long-term objectives of the study are to develop (1) new strategies that could improve theefficacy of sorafenib, (2) potential biomarkers predictive of the efficacy of sorafenib, and (3)novel therapeutic targets in HCC.With a worldwide HCC-related death of nearly 700,000 per year, any improvement in drugtreatment for HCC will represent huge health market revenues. Results of this project willimprove therapy for HCC patients and make major breakthroughs in new drug and/or biomarkerdevelopment for HCC.
Keyword(s)
肝細胞癌(HCC)
sorafenib
定量磷酸蛋白質體學
穩定同位素胺基酸標定
(SILAC)
同重元素相對與絕對定量標定(iTRAQ)
Hepatocellular carcinoma (HCC)
sorafenib
quantitative phosphoproteomics
SILAC
iTRAQ