Papel do WT161, inibidor específico da hdac6, sobre a progressão tumoral e instabilidade cromossômica em glioblastoma

Abstract

Glioblastoma (GBM) is considered the most aggressive and common form of brain tumors in adults, with the worst prognosis, and an average survival of approximately fifteen months. Currently, GBM treatment involves surgical resection followed by radiotherapy concomitant with temozolomide (TMZ) administration. However, the genetic and cellular heterogeneity and/or DNA repair pathway failure in this tumor type confer high resistance to chemotherapy. Thus, improving conventional therapeutic strategies is necessary. Aberrant epigenetic control is an early event in tumor progression, where the histone deacetylase 6 (HDAC6) enzyme plays a crucial role by modulating the function of histone proteins, as well as non-histone proteins and transcription factors such as E2F, p53, c-Myc, and NF-κB. In this context, HDAC6 inhibitors have emerged as potential antitumor agents, such as the compound WT161, whose antiproliferative potential has been demonstrated in certain cancers, including retinoblastoma, osteosarcoma, multiple myeloma, and breast cancer. This research aims to investigate the antitumor effects of WT161, either alone or in combination with TMZ, on tumor progression and chromosomal instability (CIN) in GBM cell lines U251, U87 (TMZ-sensitive), and T98G (TMZ-resistant). The antiproliferative efficiency of WT161 was evaluated using the MTT assay. For drug combination analysis, different administration strategies (simultaneous and sequential) were employed, based on the Chou-Talalay method. WT161 significantly decreased the proliferation of GBM cell lines on its own and synergistically when combined simultaneously with TMZ in all investigated cells. Clonogenic assays showed that the cells' ability to form colonies was efficiently inhibited by the treatment. Interestingly, these results were further supported in three-dimensional (3D) culture models of U251 and T98G cells. Moreover, WT161 induced apoptosis and cell cycle arrest in the G2/M phase, as well as significantly reduced the migratory (2D and 3D) and invasive activity of GBM cell lines. These findings were accompanied by an increase in acetylated α-tubulin protein expression, confirming effective HDAC6 inhibition, an increase in cleaved PARP (a marker of apoptosis), and a reduction in β-catenin levels following WT161 exposure. From a chromosomal perspective, WT161 significantly increased the occurrence of micronuclei, nucleoplasmic bridges, misaligned metaphases, and multipolar spindles, in addition to promoting aneuploidy in T98G cells. These were identified through cytokinesis-block micronucleus assays, mitotic error assays, and fluorescence in situ hybridization (FISH), demonstrating an increase in CIN in these cells after HDAC6 inhibition. Finally, WT161 altered the metabolic dynamics of the T98G cell line. Thus, we highlight the important biological role of HDAC6 in the neoplastic phenotype, particularly affecting tumor progression and CIN profiles, and point to the therapeutic potential of WT161 as a promising anti-GBM agent.