以質譜定量技術分析磷酸化蛋白體研究Sorafenib後天抗藥性機制

Quantitative Phosphoproteomics to Investigate Sorafenib-Acquired Resistance in Hepatocellular Carcinoma

黃靖雅(Ching-Ya Huang)

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(2013年)

https://doi.org/10.6342/NTU.2013.11170

摘要

肝細胞癌為最常見的肝癌種類之一，在原發性肝癌中佔了九成比例。肝癌的成因與肝炎病毒的感染與肝硬化最有關係。其中B型肝炎病毒是肝癌形成之最重要元兇；在另一方面酗酒引發的肝硬化，也會使肝癌的機會提高。末期的肝細胞癌更成為具有高侵襲性的癌症，病人的預後較差，且對於一般臨床肝癌用藥也無感受性，近年來在臨床上唯一核可用於治療晚期肝細胞癌的標靶藥物為sorafenib，是一種多重激酶的抑制物（multi-kinase inhibitor），可延長末期病人數月的壽命，但是僅10%的病患對藥物有感受性。
　　本研究中，我們利用原始肝細胞癌細胞Huh7建立了一株經sorafenib長期篩選培養後，對sorafenib產生抗藥性的細胞（acquired-sorafenib resistance--Huh7R），並在測試藥物導致的細胞凋亡實驗中，與原先細胞相比達到顯著性差異，我們進一步利用質譜定量技術「細胞培養中標記穩定同位素胺基酸」（Stable Isotope Labeling of Amino acids in Culture－SILAC），將含同位素的氨基酸標記在細胞蛋白質上，並透過後續的磷酸化胜肽鏈純化技術，結合質譜儀(mass spectrometry)來對Huh7和Huh7R細胞進行磷酸化蛋白的相對定量分析，透過線上軟體STRING分析後，指向於Huh7R細胞中高表現的蛋白多數參與在與AKT訊息傳遞路徑與下游mTOR系統中，並且於後續西方墨點法驗證了質譜分析結果，顯示出也許AKT訊息傳遞路徑以及其下游的mTOR系統在Huh7R細胞的抗藥性中扮演了重要的角色。
　　基於以上結果，為了克服Huh7R細胞的抗藥性，我們進一步利用mTOR抑制劑rapamycin結合sorafenib治療，有效抑制了抗藥性細胞的生長情形，mTOR下游的磷酸化蛋白質表現量也降低了，同時我們也去探討受mTOR調控的下游基因之蛋白產物表現，經由文獻探討找尋到若干標的蛋白，並於Huh7R
細胞中的高表現量得到驗證，其中一種下游蛋白為低氧誘導因子 (Hif1-α)，為低氧下才會誘發表現的轉錄因子，藉由仿製低氧的實驗中，我們看到了Huh7R細胞Hif1-α的顯著活化，同時也發現Hif1-α下游調控的基因蛋白產物VEGF, survivin, PFKP和Hexokinase II等，於低氧時在的Huh7R細胞中表現量的升高，而這些蛋白的功能使得Huh7R細胞於低氧下具備血管新生與醣解代謝產生能量的能力，說明了mTOR的活化將一連串導致下游各種基因表現使Huh7R細胞產生抗藥機制。

關鍵字

肝細胞癌 ； sorafenib抗藥性 ； mTOR系統 ； rapamycin ； 低氧誘導因子 (Hif1-α)

並列摘要

Hepatocellular carcinoma (HCC) is the most common liver cancer, accounting for 90% of primary liver cancers. Most cases of HCC are secondary to either a viral hepatitis (hepatitis B or C virus infection), alcoholism, or other causes resulting in liver cirrhosis. Advanced HCC is a highly aggressive tumor with a very poor prognosis. Currently, the only approved systemic therapy for advanced HCC is sorafenib, a multi-kinase inhibitor, which has showed to increase the survival in patients with advanced HCC. Sorafenib induced disease control in 34%~43% of advanced HCC patients. However, none of them have durable diseae control and would eventually develop drug resistance to sorafenib.
In this study, we established sorafenib-resistant cell (Huh7R) from Huh7 cell (HCC cell line) by long-term exposure to sorafenib at low doses escalating to higher doses for a long period of time, and found out that Huh7R cells had significant resistance to sorafenib-induced apoptosis at the clinical relevant concentrations (up to 10 μM). We used quantitative phosphoproteomics approach, Stable Isotope Labeling of Amino acids in Cell Culture (SILAC), to in vivo incorporate labeled amino acid into proteins, phosphopeptide enrichment, and mass spectrometry (MS)-based identification/quantitation. We analyzed the phosphoproteome difference between Huh7 and Huh7R cells. By using STRING analysis, we built up the protein networks based on the phosphoproteins with significant changes and revealed that in AKT pathway and downstream mTOR pathway were heavily involved. We next confirmed that the activation of AKT and mTOR pathways maybe the major contributor mediating the resistance to sorafenib in cultured HCC cells.
　　We went on testing whether the combination of rapamycin, a mTOR inhibitor, with sorafenib can overcome the acquired resistance to sorafenib in Huh7R cells. In doing so, viability of Huh7R cells and activities of mTOR pathway were both reduced by rapamycin. We next investigate the significant of downstream molecules regulated by mTOR, such protein is Hif1-α, which is a transcription factor and only activated under hypoxia condition. In a CoCl2-mimicing experiment, Huh7R cells showed higher expression level of Hif1-α than Huh7 cells. Further, some of the Hif1-α-regulating proteins, are also up-regulated in Huh7R cells, many of which are involved in angiogenesis and metabolic pathway. Our data suggested that mTOR and downstream Hif1-α activation contribute resistance to sorafenib in Huh7R cells.

並列關鍵字

Hepatocellular carcinoma ； sorafenib-resistant ； mTOR pathway ； rapamycin ； hypoxia-inducing factor 1-α (Hif1-α)

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