LKB1缺失促進肺腺癌腫瘤化之機制

The molecular mechanism of LKB1 loss in lung adenocarcinoma progression

蔡隆宏(Lung-Hung Tsai)

指導教授 ： 李輝 ； 許國堂

中山醫學大學/醫學院/醫學研究所/博士(2014年)

https://doi.org/10.6834/CSMU.2014.00135

摘要

肺腺癌之LKB1缺失常因homozygous deletion 與基因突變造成。但此機制造成LKB1缺失之肺腺癌主要發現於歐美國家，較少發生在亞洲國家。因此LKB1缺失可能由其他機制造成。初步分析台灣肺腺癌患者LKB1之表現，發現LKB1蛋白與mRNA表達一致，並且LKB1缺失與p53突變呈正相關性。先前於細胞實驗發現NKX2-1可調控p53之轉錄。因此假設LKB1缺失可能經由NKX2-1改變p53而調控LKB1之轉錄。本研究以16株肺癌細胞了解NKX2-1是否會透過p53調控LKB1之轉錄而造成LKB1缺失。結果發現p53-wild-type細胞中，NKX2-1可經由促進p53表達而正調控LKB1表現。而在p53-mutated細胞中， NKX2-1則是抑制SP1對LKB1的轉錄活性。在soft agar growth 與invasion assay的分析結果得知，NKX2-1在p53-wild-type細胞會抑制細胞兩者能力，在p53-mutated細胞則有相反之結果。肺腺癌組織的immunohistochemistry和real-time RT-PCR結果顯示，在p53-wild-type和p53-mutated患者有相反的NKX2-1與LKB1之相關性，這結果與細胞實驗一致。LKB1低表現的患者較LKB1高表現者有較差的預後(OS與RFS)。在p53-wild-type患者，NKX2-1與LKB1同時低表現的患者有較差的預後。而在p53-mutated患者，NKX2-1高表現又LKB1低表現的患者則是有較差的預後。因此NKX2-1可調控p53及SP1在轉錄層次造成LKB1缺失，進而促進腫瘤惡化和患者有較差之預後。為了解LKB1缺失造成腫瘤惡化之可能路徑，本研究假設MYC在wild-type-LKB1缺失造成的腫瘤化可能扮演重要角色。細胞研究結果顯示，LKB1缺失會增強MZF1轉錄活化MYC，進而促進soft agar growth、migration與invasion之能力。MYC抑制劑 (10058-F4 and JQ1) 能有效抑制LKB1缺失引起之致腫瘤能力。在肺腺癌患者中，LKB1低表現同時MZF1高表現或同時MYC高表現的患者都有較差的預後。因此本研究首次證實wild-type-LKB1之缺失是經由NKX2-1/p53路徑調控其轉錄層次，導致MZF/MYC過度表現，造成腫瘤惡化以及肺腺癌患者較差之預後。

關鍵字

LKB1 ； NKX2-1/p53 ； 肺腺癌

並列摘要

LKB1 loss is a frequent homozygous deletion and/or gene mutation in lung adenocarcinomas. However, few cases of LKB1 loss by either deletion or mutation are seen in Asian patients including Taiwanese. Therefore, other factors may also result in the loss of LKB1. Our preliminary data showed that LKB1 loss was associated with p53 mutation in lung tumors from Taiwanese adenocarcinoma patients and p53 transcription is directly regulated by NKX2-1. Therefore, we hypothesized that LKB1 loss could occur due to aberration of p53 regulation mediated by NKX2-1. Herein, 16 lung adenocarcinoma cell lines were investigated to determine if LKB1 transcription could be deregulated by NKX2-1-mediated p53 aberration. Mechanistically, LKB1 was directly upregulated by p53 and that NKX2-1 mediated p53 expression may positively regulate LKB1 expression in p53-wild-type cells. However, in p53-mutated cells, LKB1 transcription was deregulated by NKX2-1 via suppression of SP1 binding onto the LKB1 promoter. Therefore, the action of the NKX2-1/p53 pathway on LKB1 loss differed in p53-wild-type versus p53-mutated cells. Soft agar growth and invasion capability was significantly reduced by ectopic expression of NKX2-1 in p53-wild-type cells, but it was markedly elevated by silencing NKX2-1 in p53-mutated cells. Similar reciprocal observations were also seen in lung tumors from lung adenocarcinoma patients with either wild-type or mutated p53 tumors. In p53-wild-type patients, shorter OS and RFS periods were predicted for low-NKX2-1/low-LKB1 tumors than for high-NKX2-1/high-LKB1 tumors. In patients with p53-mutated tumors, poorer OS and RFS were predicted for high-NKX2-1/low-LKB1 tumors than for low-NKX2-1/high-LKB1 tumors. We next explored the possibility that MYC expression can be increased via LKB1 loss and promoted tumor progression. Mechanistically, MYC transcription is up-regulated by LKB1 loss-mediated MZF1 expression. The LKB1 loss-mediated MZF1/MYC axis is responsible for soft-agar growth, migration, and invasion in lung adenocarcinoma cells. Consistently, LKB1 loss-induced cell invasiveness was markedly suppressed by MYC inhibitors (10058-F4 and JQ1). In conclusion, LKB1 loss at transcription levels, not gene mutations and LOH, is mediated through the NKX2-1/p53 axis. In addition, MZF1-mediated MYC expression due to LKB1 loss is responsible for tumor progression and consequently resulting in poor outcomes in lung adenocarcinoma.

並列關鍵字

LKB1 ； NKX2-1 ； p53 ； MYC ； lung adenocarcinoma

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