Regulation of PKR activity by microRNAs in breast cancer

Abstract

It is well known that the RNA-dependent protein kinase (PKR) plays an important role in many cellular processes such as apoptosis, differentiation and cellular proliferation. The role played by PKR in cellular proliferation is still controversial. It was found that cells from human mammary carcinoma show an increase of the expression and activity of PKR. However, the factors involved in the activation and inhibition of this protein kinase were not investigated in this type of cancer. Recently, it was observed that breast cancer cells with increase of the expression and activity of PKR were more sensitive to doxorubicin e, for this reason, PKR is considered as a biomarker for this type of chemotherapy. The main objective of this project is to investigate whether the regulation of PKR activity by the activator PACT (PKR activating protein) and by the inhibitors TRBP (TAR RNA-binding protein) and pre-miR-886 described in different tumoral cells also occurs in formalin-fixed paraffin-embedded (FFPE) tissues obtained by biopsy of patients with in situ ductal carcinoma (ISDC) invasive ductal carcinoma (IDC). The specific objectives are: 1. To evaluate, in FFPE samples of ISDC and IDC, the expression of miR-29b and miR-122 by real time PCR and their targets Sp1(Specific protein) and PACT by Western blot; 2. To investigate the expression of pre-miR-886, miR-886-3p e miR-886-5p in FFPE samples of ISDC and IDC by real time PCR; 3. To investigate the nuclear and cytoplasmic localization of total and phosphorylated PKR in FFPE samples of ISDC and IDC by immunohistochemistry. 4. To evaluate the expression of TRBP in FFPE samples of ISDC by Western blot; 5. To investigate the expression and the subcellular localization of pre-miR-886 in FFPE samples of ISDC and IDC by in situ hybridization. In summary, this study will give a general picture of the factors involved in the activation and inhibition of PKR since these factors will be analyzed in the same FFPE sample of breast cancer which will allow to establish a correlation with the results of literature obtained in vitro. It should be emphasized that the extrapolation of in vitro results to in vivo systems is a fundamental question in the biomedical area. This project will also investigate whether there are differences in the pattern of regulation of PKR activity during tumor progression since will be analyzed FFPE samples of patients with ISDC and IDC. The elucidation of the phenomenon of PKR activation may contribute with the development of new strategies to improve the chemotherapy with doxorubicin of patients with breast cancer. (AU)