以動態磷酸化蛋白體學探討異位表達ATP合成酶抑制劑黃綠青黴素在艾瑞莎抗藥性肺癌細胞中所扮演的角色

Temporal Phosphoproteome Dynamics Reveals the Role of ATP Synthase Inhibitor Citreoviridin in Gefitinib-resistant Lung Cancer Cells

陳湘鈞(Xiang-Jun Chen)

指導教授 ： 阮雪芬

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601742

摘要

非小細胞肺癌是全世界癌症死亡的首要原因，艾瑞莎(gefitinib)是表皮生長因子受體酪氨酸激酶的抑製劑作為非小細胞癌患者的第一線治療藥物，然而，許多患者最終卻對藥物產生抗藥性。因此，有效地開發藥物去治療具抗藥性的患者正迫在眉睫。我們先前發現黃綠青黴素，從真菌物種提煉的毒素之一，可以通過抑制異位表達三磷酸腺苷合成酶的活性進而抑制肺癌細胞生長，但對正常細胞的影響有限。在老鼠模型與CL1-0 (艾瑞莎非抗性肺癌細胞株)證實黃綠青黴素會藉由降低熱休克蛋白中特定絲氨酸(S255)的磷酸化而去抑制絲裂原活化蛋白激酶所參與的訊息傳遞。我們好奇黃綠青黴素在治療具艾瑞莎獲得性抗藥性肺癌細胞的潛在信號傳遞是否不同。在這項研究中，我們發現黃綠青黴素會抑制NCI-H1975(EGFR T790M突變艾瑞莎獲得性抗藥性)細胞生長與群落形成。此外，我們藉由時間性偵測磷酸化蛋白質體學來探討動態分子反應共鑑定出738個磷酸化蛋白，1476條磷酸化胜肽鏈以及1901個磷酸化位置。其中有顯著調控的磷酸化蛋白有174條對應274個磷酸化位置。功能富集分析發現黃綠青黴素會影響染色質的組織，細胞週期和細胞凋亡。有趣的是，我們發現黃綠青黴素藉由磷酸化拓撲異構酶的特定絲氨酸1106位置進而抑制細胞生長，而黃綠青黴素誘發DNA雙股斷裂，進而造成DNA損傷反應(DNA damage response)。DNA受損會促使細胞進行細胞週期停滯在S期去進行修復或是走向細胞凋亡。而所有資料指出黃綠青黴素可能作為治療具艾瑞莎抗藥性的有效藥物。

關鍵字

艾瑞莎獲得性抗藥性肺癌細胞 ； 黃綠青黴素 ； 磷酸化蛋白質體學 ； 拓撲異構酶 ； DNA損傷反應

並列摘要

Non-small cell lung cancer is the leading cause of cancer death worldwide. Gefitinib, epidermal growth factor receptor tyrosine kinase inhibitor, is the first-line treatment of NSCLC, however, many patients eventually become resistant and experience progressive disease. Therefore, development of efficient therapeutic agents to overcome resistance is urgent. We previously found that citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. Citreoviridin suppresses mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 in gefitinib non-resistant lung cancer CL1-0 cells and xenograft model. We are curious whether signaling pathways underlying citreoviridin-treated gefitinib-acquired resistant lung cancer cells are different. In this study, we showed that citreoviridin inhibited cell proliferation and anchorage-dependent growth of gefitinib-acquired resistance NCI-H1975 cells with EGFR T790M mutation. Furthermore, we explored the dynamic molecular response by temporal phosphoproteomic approach. We identified 1476 phosphopeptides corresponding to 738 phosphoproteins and quantified 1901 phosphorylation sites. There were 274 phosphosites corresponding to 174 phosphorylated proteins significantly differential expressed. Functional enrichment analysis demonstrated that citreoviridin treatment affected chromatin organization, cell cycle and apoptosis. Interestingly, we found that citreovirdin suppressed cell proliferation by site-specific phosphorylation of topoisomerase on serine 1106. Citreovirdin induced double strands breaks, and then leaded to DNA damage response. The DNA lesions triggered cells to cell cycle arrest at S phase for repairing or apoptosis for cell death. The results indicated that citreoviridin could potentially be a therapeutic agent against gefitinib-resistant NSCLC.

並列關鍵字

gefitinib-acquired resistant lung cancer cell ； citreoviridin ； phosphoproteome ； topoisomerase ； DNA damage response

參考文獻

1. Siegel, R. L., Miller, K. D., and Jemal, A. (2015) Cancer statistics, 2015. CA Cancer J Clin 65, 5-29

2. Nawaz, K., and Webster, R. M. (2016) The non-small-cell lung cancer drug market. Nat Rev Drug Discov 15, 229-230

3. Lemjabbar-Alaoui, H., Hassan, O. U., Yang, Y. W., and Buchanan, P. (2015) Lung cancer: Biology and treatment options. Biochim Biophys Acta 1856, 189-210

4. Li, J., Deng, H., Hu, M., Fang, Y., Vaughn, A., Cai, X., Xu, L., Wan, W., Li, Z., Chen, S., Yang, X., Wu, S., and Xiao, J. (2015) Inhibition of non-small cell lung cancer (NSCLC) growth by a novel small molecular inhibitor of EGFR. Oncotarget 6, 6749-6761

6. Zhang, X., Gureasko, J., Shen, K., Cole, P. A., and Kuriyan, J. (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125, 1137-1149