Deregulation of p53 and RB Transcriptional Control Leads to Overexpression of DNA Methyltransferases in Lung Cancer

p53及RB異常轉錄調控導致DNA甲基轉移酵素過度表現

湯硯安(Yen-An Tang) ； 蔡侑庭(Yo-Ting Tsai) ； 林若凱(Ruo-Kai Lin) ； 許瀚水(Han-Shui Hsu) ； 陳志毅(Chih-Yi Chen) ； 王憶卿(Yi-Ching Wang)

《台灣癌症醫學雜誌》 1卷1期 (2014/06) Pp. 14-27

https://doi.org/10.6323/JCRP.2014.1.1.02

摘要

背景：抑癌基因的啟動子被過度甲基化時，會導致基因不表達進而使腫瘤生成；此甲基化現象主要是由DNA甲基轉移酵素（DNA methyltransferases, DNMTs）所調控，DNMTs主要有DNMT1、DNMT3A以及DNMT3B，文獻指出，在很多癌症中發現DNMTs有過度表達的情形，然而造成此現象的詳細機制仍不清楚。目的：文獻顯示p53及RB抑癌蛋白可以負調控許多基因表現，其上游蛋白MDM2則可抑制p53及RB蛋白表現，因此本篇研究以肺癌細胞及臨床模式探討p53/RB/MDM2調控路徑參與DNMT其轉錄調控異常之機制。方法：在細胞層次上，由報導基因冷光酵素活性分析（luciferase assay）與染色質免疫沈澱（chromatin immunoprecipitation）觀察抑癌蛋白p53及RB是否對DNMTs扮演負轉錄調控因子的角色。在臨床研究上，以免疫組織染色法（immunohistochemistry）及DNA定序分析非小細胞肺癌病人組織中DNMT與RB蛋白及p53突變之相關性。結果：在癌細胞株中大量表現p53及RB後，會抑制DNMT1及DNMT3A啟動子的活性及mRNA表現量；以siRNA降低p53及RB的表現量後，DNMT1及DNMT3A?動子的活性及mRNA表現量上升，顯示p53及RB的確能負轉錄調控DNMT3A的基因表現。在臨床研究上，以免疫組織染色法及DNA定序分析100位非小細胞肺癌病人組織，發現DNMT1、DNMT3A及DNMT3B蛋白過度表現且RB蛋白低表現或p53突變有顯著負相關性（P值為0.016~0.024），且其抑癌基因多為?動子過度甲基化；此外，病人存活率分析資料指出：DNMT3A、RB與MDM2蛋白皆正常表現的病人，與其他病人相較，有較好的預後（P值為0.049）。結論：由以上細胞及臨床模式實驗結果顯示，p53及RB蛋白會負轉錄調控DNMT的啟動子，進而降低DNMT的mRNA及蛋白質表現，但此p53及RB轉錄負調控DNMT的作用可被MDM2蛋白所拮抗。臨床資料顯示DNMT3A、RB與MDM2蛋白的表現程度可做為肺癌病人預後的指標。

關鍵字

DNA甲基轉移酵素 ； 轉錄調控 ； 肺癌

並列摘要

Background: Overexpression of DNA 5'-cytosine-methyltransferases (DNMTs), which silence genes including tumor suppressor genes, is involved in many cancers. However, the mechanism of DNMT overexpression remains mostly unclear. Objectives: The tumor suppressor p53 and RB proteins, which repress gene transcription, are often inactivated by gene mutation or overexpression of the E3 ubiquitin ligase MDM2 in cancer cells. Therefore, this study aims to examine whether the transcriptional regulation of DNMT1, 3A and 3B genes is controlled by p53/RB/MDM2 pathway in lung cancer using cell and clinical studies. Methods: The interaction and regulation between p53 or RB with DNMT1, 3A and 3B promoters were detected by promoter activity assay and chromatin immunoprecipitation. Transcriptional repression of DNMT1, 3A and 3B by p53 and RB was confirmed using siRNA-mediated knockdown or overexpression of p53/RB. Immunohistochemistry for p53, RB, MDM2, and DNMTs protein expression and DNA sequencing for p53 mutation were performed on tumor tissues from lung cancer patients. Results: p53 and RB suppressed the promoter activity and mRNA/protein expression of DNMT1, 3A and 38 through binding to their promoter. The suppression could be attenuated by knocking down of RB and p53. Importantly, co-transfection of both p53 and RB expression constructs showed additional inhibition of DNMT protein expression. In clinical study, functional RB and p53 inversely correlated with DNMT1, 3A and 3B expressions in samples from lung cancer patients (P = 0.016-0.024). Lung cancer patients with low RB expression or p53 mutation resulting in DNMTs overexpression showed promoter hypermethylation in multiple tumor suppressor genes. Patients with normal expression of DNMT3A, RB and MDM2 was associated with better prognosis compared to other patients (P = 0.049). Conclusions: This study provides cell and clinical evidence that p53 and RB pathways transcriptionally repress DNMT expression. Normal expression of DNMT3A, RB and MDM2 proteins can be a biomarker for good prognosis in lung cancer.

並列關鍵字

DNMT ； p53 ； RB ； transcription ； lung cancer