Effects of the induction of activation and inhibition of the Wnt/beta-catenin pathway by short hairpin RNA (shRNA) in adrenocortical tumor cell lines and treatment of adrenocortical tumor xenograft model with PNU-74654 compound

Abstract

Background: there is no effective adjuvant treatment for patients with advanced or recurrent adrenocortical tumors (ACT). The activation of Wnt/beta-catenina is the most frequent alteration in these tumors. It can be due to CTNNB1 mutations or alterations in inhibitors of the Wnt/beta-catenin pathway. We had previously shown activation of the Wnt/beta-catenin pathway in pediatric ACT due to down regulation of the Wnt/beta-catenin inhibitors DKK3, SFRP1 and AXIN1.Aim: To evaluate the effects of CTNNB1 and DKK3 silencing through short hairpin RNA (shRNA) over the Wnt/beta-catenin pathway, as well as treatment with PNU-74654, a Wnt inhibitor compound in cell line and pediatric primary culture of adrenocortical tumor cells and xenograft mouse model.Material and Methods: we will perform CTNNB1 and DKK3 silencing in the cell line NCI-H295 using shRNA and treatment with PNU-74654 of NCI-H295 and pediatric primary cultures of adrenocortical tumor cells, a Wnt/beta-catenin inhibitor. We will evaluate: (A) expression of beta-catenin and beta-catenin target genes by qPCR, immunofluorescence e western blot; (B) cell viability by MTS-based assay; (C) apoptosis by Annexin V (flow citometry); (D) epithelial-mesenchymal transition and cell invasion by migration assay; (E) steroidogenesis by measurement of cortisol, testosterone, androstenedione and aldosterone concentrations by RIA and ELISA, and (F) the expression of steroidogenic enzymes and SF1 (STAR, CYP11B2, CYP21A2 e NR5A1/SF-1) by qPCR and western blot. Additionally, we will engineer a xenograft murine model with the tumoral cell line NCI-H295 to evaluate in vivo effect of the inhibition of the Wnt/beta-catenin pathway with PNU-74654. Perspectives: The results of this study will contribute for the knowledge of adrenocortical tumorigenesis and the role of the Wnt/beta-catenin in adrenocortical tumor development and steroidogenesis including potential new targeted therapies.