| Abstract: | AP endonuclease 1 (APE1) 是base excision repair (BER) 基因，它又具有redox activity 能活化多種轉錄因子與DNA 之結合力。本研究室初步結果發現，細胞質表現之APE1 (cytoplasmic APE1) 的肺癌患者預後，較APE1 表現於細胞核(nuclear APE1) 者差，尤其是人類乳突病毒(human papillomavirus, HPV) HPV16/18 E6-positive 之患者。在肺癌細胞實驗則觀察到cytoplasmic APE1 表現較高之肺癌細胞的COX-2 表現較 cytoplasmic APE1 表現較低者高。已知NF-kB 活化是引起發炎反應的主要路徑，長期慢性發炎是引起肺癌之重要機制。本計畫擬探討cytoplasmic APE1 是否會經由活化 NF-kB 而促進肺腫瘤惡化？已知APE1 主要表現於細胞核，有研究顯示APE1 蛋白經過acetylation, phosphorylation 或nitrosation 之轉譯後修飾(posttranslational modification) 可能造成APE1 在nucleus/ cytoplasm trafficking。因此第一年將探討何種轉譯後修飾作用是引起APE1 nuclear export 的主要機制？本研究將以coimmunoprecipitation (IP), immunofluroresent staining (IF), western blot, 以及acetylation 、PKC inhibitor 、 NO-donor 、NO scavenger 等分別處理細胞，以初步找出APE1 nuclear export 之主要轉譯後修飾？進一步將APE1 已知或可能會發生acetylation, phosphorylation 或 nitrosation 之氨基酸位置做定點突變，以了解該轉譯後修飾作用是否確實會造成APE1 nuclear export？另外將以EMSA 和IP, western blot 等分析APE1 nulear export 造成 cytoplasmic APE1 表現增高，是否會促進COX-2 之表現？本計畫第二年將探討 cytoplasmic APE1 活化NF-kB 是否是促進肺腫瘤惡化之主要機轉？ 本研究將在 APE1 silencing 之肺癌細胞轉染全長APE1 (FL) 以及不同nuclear translocation sequence (NLS) 缺失之constructs (ND7, ND20, ND30, ND41)，而這些APE1 constructs 可以逃避 APE1 silencing 之small hairpin RNA (shRNA) 之干擾。培養出轉染這些constructs 之 stable clones，以了解NLS 缺失之細胞是否如預期會大多表現於細胞質，而轉染FL 者則僅會表現於細胞核中？ APE1 表現於細胞質之NLS 缺失細胞，是否會活化NF-kB 而促進COX-2 之表現？並以EMSA, IP, IF, redox reaction assay 等了解cytoplasmic APE1 redox activity 是否會還原IKK 而增加與IkBα, IkBβ之交互作用，促進IkBα, IkBβ 之磷酸化與降解。並以定點突變將可能還原之cystine 位置，以確定以上之可能性。本研究將分析細胞質表現APE1 之NLS 缺失細胞的cell proliferation, colony formation efficiency, migration/ invasion ability 和archorage-independent growth 是否確實高於FL 細胞？並注射cytoplasmic APE1 表現之NLS 缺失細胞於NOD-SCID mice 之肺臟，以了解這些APE1 高表現之細胞是否會促進肺腫瘤之形成和轉移？第三年計畫將在細胞以及動物模式中了解給予APE1 redox inhibitor 或NF-kB inhibitor 是否能有抑制肺癌細胞之致癌潛力？以及動物體內之腫瘤形成與轉移？若所有之細胞與動物實驗都能證實cytoplamic APE1 會經由活化NF-kB 而促進肺腫瘤生長與轉移，本研究將在外科手術之肺癌患者中了解cytoplasmic APE1 之外科手術後肺癌患者之腫瘤再形成(tumor recurrence) 和遠端轉移(distant metastasis)之relative ratio (RR) 是否較nuclear APE1 患者為高？這些患者之預後是否較差？NF-kB 調控之COX-2 的表現與cytoplasmic APE1 是否具有正相關性？細胞質表現之APE1 是否可做為獨立之臨床預後指標 (independent prognostic indicator)？並了解cytoplasmic APE1 表現之NLS 缺失的細胞，是否有較高之cisplatin 抗藥性，而使得患者有較差之預後？並確定multiple drug resistance (MDR)是否是造成cisplatin 抗藥性產生的主要基因？ 若給予APE1 redox inhibitor 或NF-kB inhibitor 是否能提高cisplatin 之drug sensivity？總之本計畫將由細胞模式、動物模式和肺癌檢體一系列之基礎與臨床之研究結果，以建立個人化醫療 (tailor medicine) 的一個例子，期望能有效治療細胞質表現APE1 之肺癌患者，以提高肺癌患者之存活率，尤其是HPV 引起之肺癌患者。
APE1 is not only a rate-limiting base excision repair gene but also to be an essential multiple functional protein, particularly activated several transcription factors by its redox activity including NF-kB. Our preliminary data showed that tumors with cytoplasmic APE1 have poorer prognosis than those without cytoplasmic APE1, particularly in HPV16/18-E6 positive tumors. In lung cancer cell experiments, cytoplasmic APE1 seems to be positively correlated with COX-2 induction through NF-kB activation. In the first year, we will elucidate which posttranslational modification— acetylation, phosphorylation, and nitrosation which have been shown to link with APE1 nuclear export, could be predominately responsible for APE1 nuclear export to elevated cytoplasmic APE1 level in lung cancer cells. EMSA, co-immunoprecipitation (IP), immunofluorescent staining (IF) and western blot will be employed to assess whether cytoplasmic APE level could be changed by the addition of the inhibitors of acetylation or phosphorylation, and NO-scavenger or NO-donor. To further verify whether APE1 nuclear export is resulted by the posttranslational modification, the possible or known amino acid residues of acetylation or phosphorylation, or nitrosation will be mutated by the site-directed mutagenesis to confirm the posttranslational modification to be responsible for APE1 nuclear export. In addition, we will examine whether COX-2 is markedly induced by cytoplasmic APE1 through NF-kB activation, not by nuclear APE1. In the second year, to establish cytoplasmic or nuclear APE1 lung cancer cells, we first establish a APE1-knockdown lung cancer cells by a small hairpin RNA (shAPE1), and then transfect with full length of APE1 construct (FL), and various deletions and/or point mutation of nuclear localizing sequence (NLS) of APE1 constructs (ND7, ND20, ND30, and ND41). The stable clones of shAPE1 with different constructs will be established to verify whether cytoplasmic APE1 is predominately expressed in shAPE1 cells with different deletion and/or point mutation constructs, not in those with FL-APE1 which will be examined by IF and western blot. We further to clarify whether COX-2 induction by cytoplasmic APE1 (ND20, ND30) through NF-kB activation is more revealed in ND20 and ND30 cells than in ND41. We next examine whether NF-kB activated by cytoplasmic APE1 is through IKK reduction by the redox activity of APE1 to enhance the interaction of IKK with IkBα and IkBβ, and then to promote the ability of phosporylation and degradation of IkBα and IkBβ. We will further elucidate whether NF-kB activated by cytoplasmic APE1 may promote the cell proliferation, cell migration/ invasion activity, anchorage-independent growth in cell in vitro, and tumor growth and metastasis in mouse in vivo. In the third year, NF-kB inhibitor will be added to verify whether NF-kB activation is responsible for the tumor aggressiveness in vivo. The cytoplasmic APE1 (ND20, ND30) or nuclear APE1 (FL) cells will be used to perform orthotopic implantation in NOD-SCID mice to verify whether cytoplasmic APE1 mice have higher tumor aggressiveness compared with those of nuclear APE1 cells. Moreover, APE1 redox inhibitor or NF-kB inhibitor will be treated to examine whether the tumor growth and metastasis could be more reduced by APE1 redox inhibitor than NF-kB inhibitor. We will enroll150 lung tumors to perform immunohistochemistry to verify (1) whether cytoplasmic APE1 is positively related with COX-2 expression, (2) whether tumors with cytoplasmic APE1 will have higher risk of tumor recurrence or metastasis compared with those without cytoplasmic APE1. To further verify whether the poor prognosis is due to cisplatin drug resistance and the role of multiple drug resistance (MDR) gene could responsibe for the drug resistance, cell and animal model experiment will be performed to examine whether APE1 redox inhibitor could improve tumor response to cisplatin chemotherapy. |
