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.
|Effective start/end date||8/1/12 → 7/31/13|
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