- 學位論文
探討Kelch-Like ECH-Associated Protein 1( Keap1)抑制肺腺癌移動之分子機制
The Mechanism of Kelch-Like ECH-Associated Protein 1( Keap1) in Lung Adenocarcinoma Migration
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摘要
Kelch-like ECH-associated protein 1(KEAP1)是一個負責調控細胞內氧化還原的銜接蛋白，功能為將cul3 ubiquitin ligase 上的ubiqutin 銜接到KEAP1 下游結合的蛋白而進行蛋白降解，而根據研究NRF2 為KEAP1 的主要下游調控蛋白，因此若細胞中的KEAP1 減少會造成NRF2 增加並累積在細胞核中，NRF2 本身是一個轉錄分子，累積的NRF2 會結合到具有antioxidant responsive element(ARE)的啟動子上，進而增加抗氧化酵素或phase II 酵素的表現量，另外研究指出，NRF2 下游基因PRDX1 的表現會促進肺癌細胞生長、轉移、以及抗化療能力，因此在我們觀察到KEAP1 的表現量和lung cancer 的轉移有相反的趨勢時，我們便猜測或許 KEAP1是藉由降解調控NRF2 進而使下游基因改變而影響肺癌細胞的轉移能力。 在本篇研究中，我們首先將238 名以及167 名病患組織進行KEAP1 以及NRF2 免疫組織染色，統計分析結果發現，高KEAP1 表現的病患具有較好的存活率並且較常在早期的肺癌中發現，相反的，高NRF2 表現的病患具有較差的存活率並且在晚期的肺癌中表現量越高，而在in vitro 實驗當中我們發現KEAP1 的表現會抑制肺癌的轉移能力，並且在單獨抑制NRF2 表現量時也會抑制肺癌的轉移能力，因此我們進一步結合兩種shRNA 來證明KEAP1 是藉由調控NRF2 表現量來調控轉移能力，接著藉由microarray 來分析過量KEAP1 表現以及抑制NRF2 表現的兩組肺癌細胞中交集的基因，我們發現S100P 表現量同時在兩組別都受到抑制，S100P在研究中已被證實對多種癌細胞具有促進轉移能力，最後我們嘗試使用GST-pulldown 方法發現NRF2 可能是藉由和TBPA 的連接一起結合到S100P 的啟動子上，進而促使S100P 的過量表現而造成癌細胞轉移能力的增加，結論地來說，我們的實驗結果發現KEAP1 會調控癌細胞的轉移，並且在肺癌機制上我們發現是藉由降解NRF2 以及轉錄調控S100P 來達成調控轉移的目的。
並列摘要
Kelch-loke ECH-associated protein 1(KEAP1) is a redox-dependent substrate adaptor protein which interact with cul3 ubiquitin ligase complex to degradate NRF2(NF-E2-related factor 2). The decreased expression of KEAP1 will enhance NRF2 expression and accumulate in the nucleus, where binding with antioxidant responsive element(ARE) and activate the antioxidative enzyme expression. Here we observed the KEAP1 expression had inversely correlation with cell migration ability. So we suggesting that the NRF2 target gene may provide advantage for cancer cell progression and may control by KEAP1. In this study, KEAP1 and NRF2 protein expression in 238 and 167 lung cancer specimens was investigated immunohiistochemically and was significantly coorelated with survival and stage. We found KEAP1 high expression in early stage whether NRF2 is high expression in late stage. Also, KEAP1 had ability to inhibit many cancer cells migration and NRF2 had inversely correlation with KEAP1 in migration ability. Further we confirm the degradation interaction between KEAP1 and NRF2 in lung cancer cells and confirm the KEAP1 inhibit cancer cell migration is caused by degradation of NRF2. By microarray analysis, we first found KEAP1-NRF2 signaling pathway to connect with S100P protein which facilitate cancer metastasis in many cancer type. Moreover, the NRF2 protein may combine with TBPA which a binding protein of S100P promoter region. In conclusion, our data suggested that KEAP1 may mediate lung cancer migration ability through degradation of NRF2 which transcriptional control the S100P expression.
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