研究背景:台灣地區不論女性或男性肺癌皆高居癌症死亡率之首位，而肺癌的發生與長期暴露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類型之一。NNK除了會導致DNA 的損害 (DNA damage) 外，近來研究也發現NNK 容易造成癌症形成過程中外顯基因變異 (epigenetic alteration)，使抑癌基因的啟動子上被過度甲基化。而造成啟動子CpG 位置上過度甲基化的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度表現的情形。

研究目的: 本實驗室先前針對肺癌做了許多與抑癌基因CpG 過度甲基化相關的研究，並發現DNMTs 的過度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不清楚。因此本篇研究目的為以細胞、臨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位置過度甲基化的現象。

研究方法及結果: 首先在細胞模式由西方轉漬法 (Western Blot)發現處理香菸中的尼古丁 (nicotine) 6 小時後，會促使DNMT1 蛋白表現增加；而由nicotine 所衍生出來的致癌物NNK 則是在隨其處理濃度及時間增加，DNMT1 蛋白表現也隨之增加，尤其在10 μM NNK處理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；然而透過反轉錄聚合酵素鏈反應 (RT-PCR) 得知NNK 處理2 小時並不會影響DNMT1 mRNA 的表達改變。進一步，處理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 刺激後，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；更進一步由處理AKT抑制劑 (LY294002) 及AKT knock down 實驗得知NNK會透過AKT 訊息路徑影響DNMT1 蛋白表現的增加。由免疫沈澱法(Immunoprecipitation)、蛋白質降解抑制劑 (MG132) 處理等實驗證明NNK透過AKT訊息路徑影響泛素 (ubiquitin) 調節的蛋白質體降解系統而增加DNMT1 蛋白的穩定性；此外，更進一步利用GSK3β抑制劑及分別外送GSK3β、βTrCP 載體表現來驗證GSK3β/βTrCP路徑會促使DNMT1 蛋白降解，但NNK 則會活化AKT 而影響
GSK3β/βTrCP 路徑使DNMT1 不易被降解。由免疫沈澱法也首度證實DNMT1 蛋白會與GSK3β 及 βTrCP 蛋白結合，由此可知GSK3β/βTrCP 蛋白降解路徑的確會影響DNMT1 蛋白調控。接下來我們以染色質沈澱的聚合酶鏈鎖反應(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度甲基化的情形。

在動物模式實驗中，以免疫組織染色分析NNK 處理及未處理的老鼠肺組織切片，發現NNK 處理後所產生肺腫瘤組織的DNMT1、DNMT3B、p-AKT 與不活化態的p-GSK3β(ser9)蛋白表現比較高，而βTrCP 蛋白則有下降表現的情形。

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

結論: 由以上細胞、臨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 訊息路徑削弱GSK3β/βTrCP 調控DNMT1 蛋白降解作用，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度甲基化的情形，因此成為導致肺癌發生的原因之一。

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.

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