Mechanisms of Glioblastoma resistance to antitumoral temozolomide in cells cultured as three-dimensional (3D) multicellular tumor spheroids in vitro: the role of translesion synthesis polymerases

Abstract

Glioblastoma Multiforme (GBM) is the most aggressive and undifferentiated tumor type that affects the Central Nervous System (CNS). In chemotherapy, the antitumoral Temozolomide (TMZ) demonstrates the best clinical benefits by easily penetrating the blood-brain barrier. TMZ promotes DNA damage by adding methyl radicals (-CH3) in nitrogenous bases, which can cause nucleotide substitutions, strand breaks, or blockages in the replication machinery, triggering cell death. However, the appearance of resistant cells to TMZ is one of the biggest problems in GBM treatment. Among the related factors, DNA repair mechanisms, including Translesion Synthesis (TLS), appear as important candidates for allowing cancer cells to remove and/or tolerate lesions in the DNA molecule. More specifically, TLS DNA polymerases (TLS pols) allow cells to tolerate genomic damage by bypassing lesions, thereby avoiding replicative stress and ensuring cell survival. Besides, this process can develop genomic instability, increasing the repertoire of tumor cells to the development of resistance. In this project, we intend to focus studies on TLS pols of the Y family [Pol Eta (Pol ·), Kappa (Pol º), Iota (Pol ¹) and Rev1] as potential enzymes of resistance to TMZ in GBM, through studies in depleted cells of these enzymes by genomic editing tools. Initial results previously obtained by our group identified TLS pols (Pol ¹ and Pol º) associated with TMZ resistance in glioma cell lines in vitro. In this project, we intend to extend these studies with cells cultured in three-dimensional (3D) models in vitro. In these conditions, the cells share properties of the tumor microenvironment corresponding to the in vivo and can help to understand better the mechanisms that these TLS pols act in resisting to this chemotherapy. Specifically, we propose to cultivate GBM nocautes tumor cells (U-251) for TLS pols of Y family (Pol ¹ and Pol º) in 3D Multicellular Tumor Spheroids (MTS) and, in parallel, generate other GBM cell lines (U-251) nocautes (Pol · and Rev1) by CRISPR-Cas9, in order to analyze the effects of treatments with the antitumoral TMZ. These assessments intend to investigate the responses of deficient cells to treatment with TMZ by volume/integrity analysis and cell migration/invasion assays in MTS; application of cytotoxicity tests; studies of cell cycle kinetics, ability to overcome S phase, and cell death types; execution of genotoxicity assays, and elongation measurement tests in DNA replication fork. Additionally, assessment in animal models (in vivo) with xerographic tumors induced by GBM cell lines will be performed for comparative analyzes of response to TMZ. Finally, we aimed to carry out Whole-Exome Sequencing (WES) to identify mutations induced by chemotherapy by comparing the data of deficient and complemented cells for TLS pols for both 3D cell culture (in vitro) and animals (in vivo) models. With the results, it is expected to understand better the role of TLS pols of the Y family in the resistance to damages caused by TMZ, indicating potential paths for the therapy of patients with GBM. (AU)