MODULATION OF BASE EXCISION REPAIR PATHWAY FOR BREAST CANCER THERAPY

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Modulation of human apurinic/apyrimidinic endonuclease 1 (APE1) functions for breast cancer therapy

Publication . Guerreiro, Patrícia S.; Oliveira, Nuno Guerreiro de, 1970-; Miranda, Joana Paiva Gomes

DNA repair is required for the maintenance of genome stability. In the last years DNA repair pathways have emerged as important targets for cancer therapy. Since standard anticancer agents are mainly DNA-damaging drugs, its combination with DNA repair inhibitors may contribute to improve treatment outcomes. Among the multiple effectors involved in DNA repair, the multifunctional base excision repair (BER) protein apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most attractive druggable targets in this field. APE1 is the major endonuclease in BER participating in the repair of different DNA lesions including toxic abasic sites. In addition to the DNA repair activity, APE1 also acts independently as a reduction/oxidation signalling protein modulating the activation and DNA binding ability of several transcription factors implicated in cell survival and tumour promotion and progression. In this context, this thesis is focused on the combination of APE1 pharmacological inhibitors with conventional anticancer agents in the highly aggressive human breast cancer MDA-MB-231 cell line. Endonuclease activity has been the most studied function of APE1 in cancer therapy. Methoxyamine (MX), a commercially available indirect inhibitor of APE1 DNA repair function, was evaluated in combination with doxorubicin (Dox) in MDA-MB-231 cells. The chemotherapeutic drug Dox is widely used in the treatment of advanced breast cancer and may act, in part, by inducing oxidative DNA damage. MX had little effects in viability and colony formation of MDA-MB-231 cells. However, a significant increase in the frequency of micronucleated cells and an alteration in the pattern of micronuclei distribution were observed suggesting an increase in Dox genotoxicity. The differential results obtained in terms of cytotoxicity and genotoxicity showed that a therapeutic strategy based on APE1 inhibition is likely to have no relevance for the improvement of outcomes of Dox treatment. Although several putative inhibitors of APE1 endonuclease activity have been reported they still lack potential to be translated to the clinical setting. Therefore, in this thesis a structure-based virtual screening (SBVS) study based on molecular docking analysis of National Cancer Institute (NCI) database of compounds was performed to identify novel small-molecule inhibitors of APE1. The evaluation of SBVS study most promising compounds in a fluorescence-based APE1 endonuclease activity assay revealed three APE1 inhibitors. Compound 22 was a potent APE1 inhibitor showing inhibitory effects at nanomolar concentrations, while compounds 37 and 41 inhibited the enzyme in the micromolar range. These novel scaffolds for the design of more potent APE1 inhibitors did not affect the viability of non-tumourigenic human breast epithelial MCF10A cell line highlighting their promising features. The importance of APE1 modulation is beyond its functions in DNA repair. Therefore, E3330, a commercially available inhibitor of APE1 redox function, was also evaluated as single agent and in combination with the taxane drug docetaxel (DTX) in MDA-MB- 231 cells. DTX has anti-migratory and anti-angiogenic effects and is frequently used in advanced breast cancer refractory to anthracycline-based regimens. Consequently, relevant endpoints of cell migration and invasion were studied in addition to cell viability, proliferation and cell cycle profile assessment. Minor effects were observed in cell proliferation. However, E3330 alone significantly reduced the collective cell migration evaluated by the wound-healing assay without affecting chemotaxis and chemoinvasion. The combination of E3330 with DTX significantly decreased invasion of MDA-MB-231 cells suggesting a potential therapeutic role in metastatic breast cancer. The results described in this work emphasise the importance of preclinical studies of APE1 functions in cancer therapy and highlight the potential of novel drug combinations based on APE1 inhibitors reinforcing the role of targeting DNA repair in cancer treatment.

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Fundação para a Ciência e a Tecnologia

Número da atribuição

SFRH/BD/70293/2010