Nrf2 transcriptional activation by p53 dysfunction promotes tumor progression and associated with poor relapse free survival in lung cancer

• Main Authors: Po-Lin, 林伯霖
• Other Authors: 李輝
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/60962745338242321124

碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 98 === 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.