將E2F1/GAS5/p53途徑作為標靶以治療艾瑞莎抗藥性肺癌的策略

Targeting the E2F1/GAS5/p53 Axis as a Potential Therapeutic Strategy for Gefitinib-Resistant Lung Cancer

唐承緯(Cheng-Wei Tang)

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(2018年)

https://doi.org/10.6342/NTU201803856

摘要

肺癌是世界上最普遍和致命的癌症之一。肺癌主要有兩種形式：非小型細胞肺癌(NSCLC)和小型細胞肺癌(SCLC)。近年來，一些小分子像是艾瑞莎(gefitinib)，能夠以酪胺酸激酶作為標的，被用來作為非小細胞癌患者的第一線治療藥物。然而，一些病人卻被發現再標靶治療後產生腫瘤復發最後演變成漸進的結果，因此，一些有效的治療方法是需要被發現的。在我們的研究中發現，異位表達的ATP合成酶會表現在具有艾瑞莎抗性的肺癌細胞株的細胞膜上，異位表達的ATP合成酶抑制劑：黃綠青黴素(citreoviridin)，能夠抑制肺癌細胞的細胞增生和細胞群落的形成。為了進一步的闡明黃綠青黴素的作用機制，我們做了微陣列分析。結果顯示不僅mRNA還有長鏈非編碼RNA(lncRNA)都參與了黃綠青黴素所造成的細胞死亡。其中一個長鏈非編碼RNA： 生長抑制特異性轉錄本5 (growth arrest-specific transcript, GAS5，在黃綠青黴素作用後有明顯的基因上調。為了探討GAS5上游的調控者，我們利用了染色質免疫沉澱法(ChIP)發現轉錄因子E2F1能夠結合在GAS5的啟動子上。微陣列分析和即時聚合酶鏈式反應的結果也顯示出E2F1的表達量在黃綠青黴素作用後和GAS5具有負相關的關係。這些證據顯示E2F1可能是GAS5的潛在抑制者。此外，結合微陣列分析、基因集富集分析(GSEA)和即時聚合酶鏈式反應，也證明了抑癌基因p53途徑也被活化。為了進一步了解GAS5-p53的調控網路，我們使用了核醣核酸蛋白質拉下測定試驗(RNA-protein pull down assay)，並結合液相層析串聯式質譜儀(LC-MS/MS)去剖析受到GAS5調控的蛋白質相互作用組。從結果當中我們鑑定出107個在A549和H1975細胞株和GAS5交互作用的共同蛋白質。在我們實驗室的磷酸化蛋白體分析研究中顯示拓樸異構酶2-alpha (DNA topoisomerase II-alpha, TOP2A)參與黃綠青黴素調控gefitinib抗藥性的路徑，所以我們進一步地挑選出了TOP2A。此外，我們進一步的利用西方墨點法和RNA免疫沉澱法證明TOP2A和GAS5的交互作用。綜合來說，這項研究顯示以E2F1/GAS5/p53途徑作為標的可以作為艾瑞莎抗藥性肺癌的潛在治療策略。

關鍵字

非小型細胞肺癌 ； 艾瑞莎 ； 黃綠青黴素 ； 生長抑制轉錄本 5 ； 轉錄因子E2F1 ； p53 途徑 ； 拓樸異構酶 2-alpha ； 艾瑞莎抗藥性肺癌

並列摘要

Lung cancer is one of the most common and fatal cancer worldwide. There are two major types of lung cancer, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Recently, gefitinib, a small molecule, which target tyrosine kinase, has been regarded as the first-line treatment in NSCLC patients. However, several patients have been observed tumor recurrence and eventually developed progressive outcomes after target therapy. Thus, an effective therapeutic approach need to be explored. Here, we found that ectopically expressed ATP synthase on plasma membrane exhibited gefitinib-resistance properties in lung cancer cell lines. Furthermore, we unraveled that citreoviridin, an ectopic ATP synthase inhibitor, suppressed the abilities of both proliferation and colony formation in lung cancer cell lines. To elucidate the comprehensive mechanism regulated by citreoviridin, we performed microarray analysis. The results indicated that not only mRNAs but also long non-coding RNAs (lncRNAs) are involved in citreoviridin-treated cell death. One of the well-known lncRNAs, growth arrest-specific transcript (GAS5), was robustly upregulated after citreoviridin treatment. In order to investigate the upstream modulator of GAS5, we utilized chromatin immunoprecipitation (ChIP) assay and revealed that E2F transcription factor 1 (E2F1) could bind to the promoter of GAS5. Consistently, both microarray and qPCR data showed the expression level of E2F1 was negatively correlated to GAS5 after citreoviridin treatment. The evidence suggests that E2F1 might be a potential repressor of GAS5. Furthermore, combining microarray and Gene Set Enrichment Analysis (GSEA) analysis as well as qPCR demonstrated that p53 pathway was activated. To further realize the GAS5-p53 regulating network, RNA-protein pull-down assay followed by LC-MS/MS will be utilized to dissect the GAS5-interacting proteomic profiling.From the results, we identified 107 GAS5-interacting proteins in common from A549 and H1975 cell lines. Our proteomics experiments identified topoisomerase 2-alpha (TOP2A) as the key protein involved in the citreoviridin-regulated gefitinib-resistance pathway, therefore, we picked out TOP2A for further study. Additionally, we further validated the interaction between TOP2A and GAS5 by western blot and RNA immunoprecipitation (RIP). Taken together, this study suggests targeting E2F1/GAS5/p53 axis is a potential therapeutic strategy for gefitinib-resistant lung cancer.

並列關鍵字

non-small cell lung cancer (NSCLC) ； gefitinib ； citreoviridin ； growth arrest- specific transcript (GAS5) ； E2F transcription factor 1 (E2F1) ； p53 pathway ； Topoisomerase 2-alpha (TOP2A) ； gefitinib-resistant lung cancer

參考文獻

1.Torre, L. A., Siegel, R. L. & Jemal, A. Lung Cancer Statistics. Adv Exp Med Biol 893, 1-19 (2016).

2.Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2018. CA Cancer J Clin 68, 7-30 (2018).

3.Lin, S., Chen, Y., Yang, L. & Zhou, J. Pain, fatigue, disturbed sleep and distress comprised a symptom cluster that related to quality of life and functional status of lung cancer surgery patients. J Clin Nurs 22, 1281-1290 (2013).

4.Iyer, S., Roughley, A., Rider, A. & Taylor-Stokes, G. The symptom burden of non-small cell lung cancer in the USA: a real-world cross-sectional study. Support Care Cancer 22, 181-187 (2014).

5.Maguire, R., Stoddart, K., Flowers, P., McPhelim, J. & Kearney, N. An Interpretative Phenomenological Analysis of the lived experience of multiple concurrent symptoms in patients with lung cancer: a contribution to the study of symptom clusters. Eur J Oncol Nurs 18, 310-315 (2014).