香菸致癌物透過AKT/GSK3β/βTrCP訊息路徑影響DNA甲基轉移酵素穩定性

Cigarette-Specific Carcinogen Induces Stabilization of DNA methyltransferases through AKT/GSK3β/βTrCP Pathway in Lung Cancer

謝翊萱(Yi-Shuan Hsieh)

指導教授 ： 王憶卿 ； 蘇銘燦

國立臺灣師範大學/理學院/生命科學研究所/碩士(2007年)

摘要

研究背景:台灣地區???性或男性肺癌皆高居癌症死亡?之首位，而肺癌的發生與長期??於環境中的致癌物質有關，尤其是香菸中的成份nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone，簡稱NNK，被認為是造成肺癌主要的致癌物?型之一。NNK除?會導致DNA 的損害 (DNA damage) 外，近?研究也發現NNK 容?造成癌症形成過程中外顯基因變? (epigenetic alteration)，使抑癌基因的啟動子上被過?甲基化。而造成啟動子CpG 位置上過?甲基化的DNA 甲基轉移酵素 (DNA methyltransferase, DNMT)DNMT1、DNMT 3a 及DNMT 3b，目前也已發現在癌細胞中有過?表現的情形。

研究目的: 本實驗室先前針對肺癌做?許多與抑癌基因CpG 過?甲基化相關的研究，並發現DNMTs 的過?表達與抽煙的肺癌病人有顯著的相關性，然而造成此現象的詳細機制仍?清楚。因此本篇研究目的為以細胞、?床及動物模式探討香菸致癌物NNK 是透過何種機制而誘導DNMTs 表達，進而導致許多抑癌基因啟動子CpG位置過?甲基化的現象。

研究方法及結果: 首先在細胞模式由西方轉漬法 (Western Blot)發現處?香菸中的尼古丁 (nicotine) 6 小時後，會促使DNMT1 蛋白表現增加；而由nicotine 所衍生出?的致癌物NNK 則是在隨其處?濃?及時間增加，DNMT1 蛋白表現也隨之增加，尤其在10 μM NNK處?2 小時即有很明顯誘導效果；同時藉由外送DNMT1 載體表現分析實驗也得知NNK 會誘導外生性DNMT1 蛋白表現增加；然而透過反轉?聚合酵素鏈反應 (RT-PCR) 得知NNK 處?2 小時並?會影響DNMT1 mRNA 的表達改變。進一步，處?可以抑制新蛋白質生成的轉譯抑制劑Cycloheximide 得知DNMT1 蛋白的半衰期大約6小時，但是同時受到NNK ?激後，DNMT1 蛋白的半衰期增長為24
小時。本研究結果亦顯示nicotine 及NNK 在2 小時內與p-NFκB、p-AKT、p-ERK1/2 及p-p38 的訊息蛋白活化有關；?進一步由處?AKT抑制劑 (LY294002) 及AKT knock down 實驗得知NNK會透過AKT 訊息?徑影響DNMT1 蛋白表現的增加。由免疫?澱法(Immunoprecipitation)、蛋白質?解抑制劑 (MG132) 處?等實驗證明NNK透過AKT訊息?徑影響泛素 (ubiquitin) 調節的蛋白質體?解系統而增加DNMT1 蛋白的穩定性；此外，?進一步?用GSK3β抑制劑及分別外送GSK3β、βTrCP 載體表現?驗證GSK3β/βTrCP?徑會促使DNMT1 蛋白?解，但NNK 則會活化AKT 而影響
GSK3β/βTrCP ?徑使DNMT1 ??被?解。由免疫?澱法也首?證實DNMT1 蛋白會與GSK3β 及 βTrCP 蛋白結合，由此可知GSK3β/βTrCP 蛋白?解?徑的確會影響DNMT1 蛋白調控。接下?我們以染色質?澱的聚合?鏈鎖反應(chromatinimmunoprecipitation-polymerase chain reaction assay) 及聚合酵素鏈反應為基礎的甲基化分析 (methylation-specific PCR) 方法發現NNK 所誘導的DNMTs 表現會結合至抑癌基因啟動子區域，進而造成抑癌基因啟動子有過?甲基化的情形。

在動物模式實驗中，以免疫組織染色分析NNK 處?及未處?的?鼠肺組織?片，發現NNK 處?後所產生肺腫瘤組織的DNMT1、DNMT3B、p-AKT 與?活化態的p-GSK3β(ser9)蛋白表現比較高，而βTrCP 蛋白則有下?表現的情形。

在?床研究方面，我們以免疫組織染色分析(Immunohistochemistry)偵測109 位?床肺癌病人DNA 甲基轉移酵素表現?，發現曾經吸煙但後?有戒煙病人的DNMT1 蛋白過?表現情形 (31.1%) 比持續吸煙病人的DNMT1 蛋白過?表現情形 (69.4%) 明顯?的低 (P 值=0.001)。

結?: 由以上細胞、?床及動物模式實驗結果顯示，香菸中致癌物NNK 的確會誘導DNMTs 蛋白表現的增加；進一步由細胞實驗結果也知NNK 會透過AKT 訊息?徑削弱GSK3β/βTrCP 調控DNMT1 蛋白?解作用，進而使DNMT1 蛋白質穩定性增加；而這些NNK 所誘導的DNMTs 蛋白也會結合到抑癌基因的啟動子位置上，進而導致抑癌基因啟動子產生過?甲基化的情形，因此成為導致肺癌發生的原因之一。

關鍵字

NNK ； DNA甲基轉移酵素1 穩定性 ； 肺癌 ； AKT/GSK3β/βTrCP訊息路徑

並列摘要

Background: Most lung cancer cases are caused by cigarette-specific carcinogens, such as nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Previous studies have shown that NNK induces promoter hypermethylation of several tumor suppressor genes (TSGs) in mouse models. However, the mechanism involved in the
promoter hypermethylation induced by NNK remains unclear. DNA methylation is carried out by DNA methyltransferases (DNMTs), which have been shown to be overexpressed in human cancers including lung cancer.
Purpose: Our previous study showed that smoking lung cancer patients have a significant high level of DNMTs expression. Therefore, the present study aims to investigate what mechanisms involved in DNMTs overexpression induced by a cigarette carcinogen, NNK, in
lung cancer by cell, animal, and clinical models.

Results: Western blot assays indicated that DNMT1 and DNMT3b increased after treated with pro-carcinogen, nicotine for 6 h in IMR90, A549, and H1299 lung cells. In addition, nicotine-derived carcinogen, NNK treatment for 2 h increased endogenous and exogenous DNMT1 protein levels but not mRNA expression level in A549 and H1299 lung
cells, suggesting that NNK-induced DNMT expression occurs at the post-translational level. Treatment with translation inhibitor cycloheximide with or without combining treatment of NNK confirmed that NNK indeed prolonged DNMT1 protein half-life. In addition, NNKactivated phosphorylation of AKT, NFκB, ERK1/2, and p38 pathways in IMR90 and A549 cells. Note that AKT pathway was significantly
stimulated by NNK between 15 and 120 min in these cells. To evaluate the involvement of AKT signaling pathway in DNMT1 protein accumulation after NNK treatment, cells were treated with AKT inhibitor, LY294002, or AKT siRNA knock down oligos. The results indicated that NNK increased DNMT1 protein level can be abolished by both AKT inhibition treatments. In addition, immunoprecipitation assay suggested that NNK increased DNMT1 protein stability through AKT signaling pathway and was associated with ubiquitination protein degradation system. This conclusion was validated by combining treatment of AKT inhibitor and proteasome inhibitor MG132 in A549 and IMR90, which abolished the DNMT1 degradation induced by AKT inhibitor.
Since AKT downstream GSK3β/βTrCP ubiqutin-proteasome
pathway has been implicated in degradation of many proteins. We investigated whether GSK3β/βTrCP ubiqutin-proteasome pathway was involved in NNK-induced DNMT1 protein stability. We treated the cells with GSK3β inhibitor, SB415286, in the presence of NNK. In addition,
cells overexpressing different levels of GSK3β or βTrCP construct were analyzed for DNMT1 protein level. The data indicated that NNK induced DNMT1 protein stability resulted from attenuation of GSK3β/βTrCP-mediated DNMT1 protein degradation system. In addition, we found that DNMT1 protein interacted with GSK3β and βTrCP by
immunoprecipitation assay. Furthermore, chromatin-immunoprecipitationPCR and methylation-specific PCR assays showed that increased DNMT1 indeed bound to methylated TSG promoters after treated with NNK.

In animal model data, immunohistochemical staining assay showed that NNK increased protein expression level of DNMT1, DNMT3B, p-AKT, and inactive form of p-GSK3β (ser9) in mice adenoma lung tissue treated with NNK. However, protein expression level of βTrCP was reduced in NNK-treated mice adenoma lung tissue.

In clinical data, immunohistochemical staining for the DNMT1 protein expression was performed on 109 NSCLC tumor samples with smoking status data available. The results indicated that DNMT1 protein expression level was significantly higher in smoking patients compared to non-smoking patients including ex-smokers and never smokers
(P<0.001). Interestingly, the DNMT1 protein of ex-smoke patients were expressed at a significantly lower level in tumor nuclear compared to that found in still-smoking patients (P=0.001).

Conclusion: These data suggest that deregulation of DNMTs is associated with the NNK-induced DNMT stability by
AKT/GSK3β/βTrCP pathway and results in epigenetic alteration of target TSGs and ultimately leads to lung cancer.

並列關鍵字

NNK ； DNA methyltransferase 1 stability ； lung cancer ； AKT/GSK3β/βTrCP pathways

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