Characterization of TRIB2-mediated resistance to pharmacological inhibition of MEK

• Main Author: Henriques, Vanessa Mendes
• Publication Date: 2017
• Format: Master thesis
• Language: eng
• Source: Repositórios Científicos de Acesso Aberto de Portugal (RCAAP)
• Download full: http://hdl.handle.net/10400.1/10705
• topic: Melanoma; TRIB2; Cancro da pele

Chemoresistance and metastasis are the main reasons for treatment failure in melanoma patients. MAPK pathway is often hyperactivated in melanoma due to BRAF mutations. BRAF and MEK inhibitors revolutionized the standard-care of patients with advanced melanoma. Yet, patients develop resistance to these drugs very fast. Previous studies showed that Tribbles homolog 2 (TRIB2) is overexpressed in melanoma and confers resistance to chemotherapeutic and targeted drugs such as darcarbazin, PI3K and mTOR inhibitors. Furthermore, TRIB2 protein contains a MEK1 binding site. Taking this into account, we hypothesize that TRIB2 might confer resistance to MEK inhibition. In order to test our hypothesis, we generated isogenic melanoma cell lines with TRIB2 knockdown, using shRNA, and cells with TRIB2 depletion using CRISPR technique. Since the members of the Tribbles protein family might be functionally redundant and compensate for TRIB2 depletion, we decided to determine mRNA and protein levels of TRIB1, TRIB2 and TRIB3 using q-PCR and Western-Blot techniques, respectively, on a panel of melanoma and non-melanoma cell lines. We treated these isogenic cell lines with the MEK inhibitor Refametinib for 72h. The resistance was evaluated through cell death analysis, using cell counting based on trypan-blue and annexin V/ Propidium iodide staining. The isogenic cell lines were successfully established and determined that compensation of TRIB2 through TRIB1 or TRIB3 only plays a minor role. Importantly Refametinib treatment of melanoma cell lines with different levels of TRIB2 showed that cell death correlated with TRIB2 expression level suggesting that TRIB2 confers resistance to MEK inhibitors. Understanding the resistance mechanisms to the therapeutic agents can improve the outcomes of current therapies and contribute to the development of new therapeutic approaches.