Common forms of DNA damage in the genome are Apurinic/apyrimidinic (AP) sites, which can be generated either spontaneously or following treatment with chemotherapeutic drugs or radiation. Human AP-endonuclease (APE1) is the primary enzyme that repairs AP sites through the Base Excision Repair (BER) pathway. Although the overexpression of APE1 in diverse cancer types and the association of APE1 expression with chemotherapy resistance and poor prognosis are well documented, the direct evidence that APE1 contributes in promoting chemoresistance in a tumor model in vivo has not been established. We have been elucidating the regulatory roles of acetylation of multiple lysine residues in N-terminal domain of APE1 in DNA damage repair and transcriptional regulation. We discovered earlier that multiple Lys residues (Lys 6,7, 27, 31 and 32) are acetylated in cells by the histone acetyltransferase p300. We have recently shown that that these lysine residues in APE1 are acetylated (AcAPE1) at AP site damage in chromatin and that acetylation enhances damage repair activity of APE1. We further showed elevated levels of AcAPE1 in diverse cancer types and demonstrated that loss of APE1 or its acetylation results in accumulation of AP sites in the genome and increased sensitivity of cells to many chemotherapeutic drugs. Moreover, our earlier studies showed that AcAPE1 regulates expression of hundreds of genes including a key drug efflux transporter protein, MDR1. To determine the role of APE1 acetylation in inducing chemo resistance in vivo, using CRISPR/cas9 system we have established a APE1 knock-in mouse line defective in APE1 acetylation at Lys 6, 7 residues (Ape1A/A). Using this mouse model, we will test the hypothesis that acetylation of APE1 promotes 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC) in vivo.
In Aim 1, using two 5-FU-resistant CRC cell lines (HCT116 and DLD-1) expressing WT or acetylation-defective APE1 mutant, we will monitor 5-FU induced damage and repair using alkaline comet assay. We will measure sensitivity to 5-FU by colony formation assay in vitro. We will examine tumor growth and the efficacy of 5-FU on tumor growth in xenograft model in vivo.
In AIM2, we will use acetylation-defective APE1 knock-in (Ape1A/A) mouse model to probe the potential role of AcAPE1 in promoting 5-FU resistance in the context of (a) carcinogen (AOM/DSS)-induced and (b) genetic APCMin/+ (Adenomatous Polyposis Coli) colon cancer model. Our project will establish the role of APE1 acetylation in promoting 5-FU resistance in colon cancer in vivo. Furthermore, our APE1-acetylation defective knock-in genetic mouse model will be instrumental for future preclinical studies in examining whether acetylation of APE1 could be used as a novel target for sensitization of tumor cells to many chemotherapeutic drugs.
The human apurinic/apyrimidinic (AP) endonuclease (APE1), an essential multifunctional protein, is involved in the repair of spontaneously generated or drug-induced AP sites and oxidative/alkylated base damages in the human genome. APE1 is posttranslationally modified by acetylation (AcAPE1) and cancer patients? tissues have elevated levels of AcAPE1. Our project will use APE1 acetylation-defective knock-in mouse model and establish the role of APE1 acetylation in promoting chemodrug resistance in colon cancer in vivo.