IMPORTIN EXPRESSION AND INHIBITION IN BREAST CANCER: A POTENTIAL MODEL TO STUDY XIAP NUCLEAR LOCALIZATION

INTRODUCTION AND OBJECTIVES: Breast cancer is a neoplasm that has high incidence and mortality rates among women, mainly due to the development of resistance to treatment. Among the most well-known resistance mechanisms described, we have the evasion from apoptosis. XIAP (X-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis (IAP) protein whose canonical functions are the direct inhibition of caspases and the ubiquitination of pro-apoptotic targets. XIAP is mainly expressed at the cytoplasm, but some studies have provided evidence on its expression at the nucleus. We have demonstrated in a previous work that nuclear expression of XIAP was associated with poor prognosis in a subgroup of patients with breast cancer. In addition, classical nucleocytoplasmic transport is often modified in cancer, which may result in altered subcellular localization of XIAP. In this context, the aim of the study is to investigate the expression and role of importins α and β1 in the mechanisms of nuclear translocation of proteins by using their respective inhibitors, ivermectin and INI-43, in breast cancer cells.MATERIAL AND METHODS: To start this study, we analyzed gene and protein expression of α1, α2 and β1 importins in breast cancer cell lines by real time PCR and Western Blotting. Subsequently, we performed MTT and clonogenic assays with nuclear import inhibitors of α/β and β1 importins, Ivermectin and INI-43, respectively, in MCF-7 and MDA-MB-231 cell lines. Furthermore, we assessed cytotoxicity of inhibitors with lymphocytes isolated from peripheral blood of healthy individuals. In parallel, we are currently working on the generation of a doxorubicin resistant model (MCF-7 DoxR) as a potential model to study aberrant subcellular localization of proteins, including XIAP. RESULTS AND CONCLUSION: We confirmed gene expression of the α1, α2 and β1 importins and verified differential protein expression in breast cancer cell lines exhibiting distinct drug resistance phenotypes. We also observed that MCF-7 and MDA-MB-231 cell lines were sensitive to importin inhibitors and could determine the subtoxic concentrations of the pharmacological inhibitors, that did not sensitize non-neoplastic cells. Furthermore, the MCF-7 DoxR cell line showed overexpression of XIAP when compared to parental cells, indicating a potential cellular model for studying nuclear XIAP. In future experiments, the subtoxic concentrations of INI-43 and ivermectin will be used to investigate the potential modulation in XIAP subcellular localization in breast cancer models exhibiting endogenous and exogenous nuclear XIAP.