- 學位論文
p53 失去活性轉錄活化 Nrf2 轉錄表現促進肺腫瘤發展以及患者有較差疾病復發之存活率
Nrf2 transcriptional activation by p53 dysfunction promotes tumor progression and associated with poor relapse free survival in lung cancer
摘要
Nrf2-Keap1 訊息傳遞路徑所調控的抗氧化與 Phase II 解毒基因對於腫瘤形成可能扮演很重要的角色。有研究顯示肺癌患者之 Nrf2 和 Keap1 基因會發生突變，這些突變促進了 Nrf2 往核內的轉移並活化 ARE 調控之下游基因的表現。這些研究也顯示 Nrf2-Keap1 路徑的活化可能參與腫瘤的發展，並增加腫瘤細胞對 cisplatin 的抗性且使得患者有較差的預後。本研究之初步結果顯示， HPV16 E6-positive TL-1 肺腺癌細胞之 Nrf2 和 GPx1 mRNA 表現較 E6-negative TL-4 細胞為高。推測 HPV透過 E6 降解 p53 增加 Nrf2 轉錄上的表現活化 Nrf2-Keap1 路徑，進而促進了腫瘤的發展與較差的臨床結果。為了證實此一假設，本研究首先在 TL-1 與 TL-4細胞中，分別轉染 E6 shRNA 和 E6 cDNA plasmid 來抑制或過度表現 E6 。發現 Nrf2 的 mRNA 與蛋白表現量在 TL-1 抑制 E6 時有顯著的上升；在 TL-4表現 E6 時則有顯著的減少。而 Nrf2-Keap1 下游調控的 GPx1 基因也有相同的結果。這表示 E6 參與了 Nrf2 在轉錄上的表現並且能活化 Nrf2-Keap1 下游的訊息傳遞路徑。為了進一步的探討 E6 如何調控 Nrf2 ，本研究設計了三段不同的啟動子片段載體，並且轉染入 TL-1 細胞與抑制 E6 之 TL-1 細胞。 Luciferase reporter assay 結果發現若將 -1036/-1啟動子片段定義有100% 之轉錄活性，則 -740/-1啟動子片段的活性約為80%，相對於 -229/-1明顯較高 (84.2% vs. 33.4% for TL-1 cells; 87.4% vs. 37.5% for E6-knockdown TL-1 cells) 。此結果顯示 p53 與 Sp1 所在的 -740~-229 啟動子片段在 Nrf2 的轉錄調控上可能扮演了很重要的角色。進一步的了解 p53 對於 Nrf2 的調控，本研究在 A549 和 p53 null 的 H1299 肺癌細胞株中分別抑制 p53 或轉染正常 (wild-type) p53 cDNA 載體。Luciferase reporter assay 結果發現 -740/-1 啟動子片段的活性在過度表現 p53 的 H1299 細胞中明顯的下降，而在抑制 p53 表現的 A549 細胞中則有明顯的上升。ChIP 的結果顯示將 p53轉染至 H1299 細胞中，p53 蛋白的確會與 Nrf2啟動子上的結合位結合。並且 Sp1 的結合能力在 H1299 轉染 p53 的細胞中則明顯的減少。此一結果顯示 p53 能透過減少 Sp1 對 Nrf2 啟動子的結合能力來抑制 Nrf2 的轉錄表現。為了進一步證實 E6 透過降解 p53增加 Nrf2 表現而促進腫瘤的生長，本研究利用細胞倍增時間分析與群落形成分析結果發現當 TL-1 分別轉染 1 μg 或 5 μg 的 shNrf2 時，其倍增時間從21.8小時增加為 25.5 與 32.8 小時。相反的，在TL-4 分別轉染 1 μg 或 5 μg 的 Nrf2 載體時，其倍增時間從 28.3 小時減少為 25.7 與 30.8 小時。在菌落形成分析中也有類似結果，細胞形成群落的能力會因轉染 shE6 和 E6 載體而改變。本研究結果與過去的研究相符，因此推測 Nrf2 轉錄活化會促進肺癌細胞生長。在肺癌患者的 Nrf2 的組織免疫染色實驗中，結果發現在 p53 突變之患者較 p53 沒有突變之患者，有較高之 Nrf2 表現的趨勢。在本研究室過去的研究發現，在167個肺癌患者中 Nrf2 蛋白的表現在 OS 中有較差的預後。為了進一步了解 E6 與 p53 突變對於這167個肺癌病患在 OS 上的影響，本研究利用多變相分析結果發現； Nrf2 表現同時 E6 表現與 p53 突變之患者較 Nrf2 沒有表現同時 E6 不表現與 p53 沒有突變之患者，有顯著較高的風險 (RR = 2.052, 95% CI = 1.110-3.793, P = 0.022；RR = 2.014, 95% CI = 1.127-3.600, P = 0.018) 。為了進一步驗證 Nrf2 在 RFS 上的影響，本研究利用167位患者中131 個具有 RFS 資料的患者經由多變相分析發現， Nrf2 可以做為肺癌中 RFS 上的獨立指標 (RR = 1.609, 95% CI = 1.077-2.433, P = 0.040) 。總之，本研究首次發現 p53 能透過降低 Sp1 與 Nrf2 啟動子的結合能力抑制 Nrf2 之轉錄表現。因此本研究推測E6 會經由抑制 p53 蛋白活性而活化 Nrf2-Keap1 路徑和促進肺癌細胞的生長，因而導致肺癌患者有較差的 OS 和 RFS。
並列摘要
Antioxidant and Phase II detoxification genes regulated by Nrf2-Keap1 signaling pathway may play an important role in lung tumorigenesis. Mutations of Keap1 and Nrf2 genes have been found in lung tumors and both gene mutations increased Nrf2 nuclear translocation to upregulate ARE-response gene expressions. These studies also showed that activation of Nrf2-Keap1 signaling pathway may promote tumor progression, and patients with poorer prognosis and resistance to cisplatin. Our preliminary data showed that Nrf2 and GPx1 mRNA expression levels in HPV16 E6-positive TL-1 lung cancer cells were higher than in HPV16 E6-negative TL-4 cells. This observation prompts us to question whether HPV16 E6 could upregulate Nrf2 transcription via p53 dysfunction by E6 to activate Nrf2-Keap1 signaling pathway and then promote tumor progression and patients with poor clinical outcome. To verify the hypothesis, TL-1 and TL-4 cells were knocked down and overexpressed E6 by transfected with E6 shRNA and E6 cDNA plasmid, respectively. Nrf2 mRNA expression levels were significantly decreased in E6-knockdown TL-1 cells and increased in E6-overexpressed TL-4 cells. Meanwhile, Nrf2-Keap1 signaling pathway regulated GPx mRNA expression levels were concomitantly in decreased TL-1 cells and increased in TL-4 cells, suggesting that E6 is involved in Nrf2 trnascription and may activate Nrf2-Keap1 signaling pathway. To further explore E6 how to regulate Nrf2 transcription, three promoter fragments of Nrf2 were constructed by PCR and deletion mutation and transfected into TL-1 cells with or without E6 RNAi. Luciferase reporter assay indicated that -740/-1 fragment was about 80% reporter activity of -1036/-1 full length fragment (100%), however the reporter activity of -740/-1 was significantly higher than that of -229/-1 fragment (84.2% vs. 33.4% for TL-1 cells; 87.4% vs. 37.5% for E6-knockdown TL-1 cells). This result suggests that p53 and Sp1 binding site on -740~-229 promoter region may play an important role in Nrf2 transcription. To verify whether p53 may play a role in Nrf2 transcription, p53 null H1299 and A549 lung cancer cells were transfected with wild-type p53 cDNA plasmid and p53 shRNA, respectively. Luciferase reporter assay showed that the reporter activity of -740/-1 was significantly decreased by wild-type p53 overexpressed H1299 cells and increased by p53-knockdwon in A549 cells. ChIP assay further indicated that the binding ability of p53 on its binding site of -740~-229 promoter region was decreased in p53-overexpressed H1299 cells. Additionally, Sp1 binding ability was markedly decreased by p53 overexpression in H1299 cells. These results suggest that p53 may suppress Nrf2 transcription via decreased Sp1 biniding ability on Nrf2 promoter. To further verify whether Nrf2 upregulated by E6 via p53 derepression to promote cell growth, doubling time and colony formation assay were performed. Our data showed that the doubling time of TL-1 cells was increased from 21.8 hr to 25.5 and 32.8 hr after the cells were transfected with 1μg and 5 μg shNrf2. Conversely, the doubling time of TL-4 cells were decreased from 28.3 hr to 25.7 and 20.8 hr after the cells were transfected with Nrf2 cDNA plasmid. Similar observations were also seen in colony formation efficacy in both cells changed by shE6 and E6 cDNA plasmid transfection. These results were consistent with previous reports indicating that Nrf2 may promote lung cancer cell growth. Immunohistochemical data showed that tumors with p53 mutation tended to have higher Nrf2 expression compared with those with wild-type p53. Our previous data showed that Nrf2 protein expression may be associated with OS 167 lung cancer patients. To further verify whether the combination effects of E6 or p53 mutation on OS 167 lung cancer patient, multivariate Cox regression analysis was performed, and our data indicated that E6 or p53 mutation in lung tumors synergistically increased the hazard ratio to 2.052 (95% CI = 1.110-3.793, P = 0.022) and 2.014 (95% CI = 1.127-3.600, P = 0.018) as compared with those with Nrf2 negative + E6 negative or p53 wild-type. To further verify whether Nrf2 could predict RFS in lung cancer, 131 of 167 patients were obtained their information of tumor recurrence and/or metastasis. Mutivariate Cox regression analysis showed that Nrf2 may act an independent indicator of RFS in lung cancer (RR = 1.609, 95% CI = 1.077-2.433, P = 0.040). However, the combination effects of E6 and p53 on RFS was not observed. In summary, to our best our knowledge, there are for the first time to provide the evidence that Nrf2 transcription is deregulated by p53 via decreased Sp1 binding ability on Nrf2 promoter. Therefore, we suggest that Nrf2-Keap1 pathway activated by E6 via p53 inactivation may promote lung tumor growth and consequently resulted in patients with poorer OS and RFS.