The role of HDAC6 in the regulation of oxidative stress and chemoresistance of tumor stem cells in oral carcinoma

Author(s)

Marcela de Oliveira Tavares

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-01-31

Examining board members

Luciana Oliveira de Almeida; Márcio Mateus Beloti; Rogério Moraes de Castilho; Anna Lvovna Okorokova Façanha

• Advisor: Luciana Oliveira de Almeida

Abstract

Chemorresistance is a factor in enhancing metastasis development during cancer therapies. It starts with a small group of cells identified as tumor stem cells (CTTs). In oral carcinoma, the population of CTTs is accumulated after chemotherapy and epigenetic changes may be controlling this process. HDACs are a group of deacetylase enzymes that epigenetically regulate gene expression by removing acetyl groups from lysine residues in histones and non-histone proteins. HDAC6 acts on several physiological processes, including oxidative stress response, autophagy and DNA damage response, and its accumulation is associated with the development of cancer and the accumulation of CTTs. This project aimed to investigate the role of HDAC6 in CTT-mediated chemoresistance in oral carcinoma; evaluate the influence of HDAC6 in the response to DNA damage and oxidative stress and its application as a potential therapeutic target to eliminate CTTs and reverse chemoresistance. Strains of wild oral carcinoma (CAL27 and SCC9), resistant to cisplatin (CAL27 CisR and SCC9 CisR) and tumor stem cells (CTT+) and non-stem (CTT-) derived from the resistant strains were used. We identified an increase in HDAC6 expression and concentration by qPCR and Western Blot in resistant strains and in CTTs. We determined by sphere assay the use of 15nM Tubastatin A to reduce CTTs. By immunofluorescence, we identified that HDAC6 moves to the nucleus in resistant cells, which may contribute to protection against DNA damage, as observed by the reduced expression of phospho-H2A.X in resistant strains. Tubastatin A increased phospho-H2A.X levels and decreased PRDX2 in CTTs. The combined use of Tubastatin A and Cisplatin increased phospho-H2A.X. Oxidative stress was evaluated by ROS assay and antioxidant enzymes (PRDX2, PRDX6, TXN and SOD2) by qPCR. We emphasize the high expression of PRDX2 in all samples. We identified by cell apoptosis assay that the use of tubastatin A induces apoptosis in CisR and CTTs of our strains, and together with cisplatin we obtained success in apoptotic induction. Tubastatin A is still able to induce the reduction of CTTs, identified by flow cytometry. We conclude that there is a relationship between HDAC6 accumulation, cell differentiation, oxidative stress, DNA damage and apoptosis. This interaction may be influencing cellular chemoresistance, and we suggest that pharmacological inhibition of HDAC6 may be an effective therapeutic strategy to reduce tumor stem cells and reverse cisplatin resistance. (AU)

• FAPESP's process: 19/19301-7 - The role of HDAC6 in the regulation of oxidative stress and chemoresistance of cancer stem cells in oral carcinoma
• Grantee: Marcela de Oliveira Tavares
• Support Opportunities: Scholarships in Brazil - Master