Epigenetic control of Wnt/β-catenin signaling pathway in the accumulation of chemoresistant cancer stem cells in oral carcinoma

Author(s)

Thaís Moré Milan

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-09-12

Examining board members

Luciana Oliveira de Almeida; Carlos Henrique Viesi do Nascimento Filho

• Advisor: Luciana Oliveira de Almeida

Abstract

Oral carcinoma is responsible for many deaths around the world by causing recurrences and metastasis due failures in therapy. The conventional treatments destroy the differentiated cancer cells; however, the population of cancer stem cells are resistant and repopulates the tumor. The Wnt/β-catenin signaling pathway is involved in the maintenance, survival, self-renewal, and differentiation of cancer stem cells, and its signaling can be regulated by epigenetic modifications. The aim of the project was to identify epigenetic modifications involved in the control of the Wnt/β-catenin signaling pathway and its targets and investigate the participation of this pathway in the accumulation of cancer stem cells and in the chemoresistance of oral carcinoma cell lines. Three wild-type oral carcinoma cell lines (Cal27 WT; SCC9 WT; SCC25 WT) and resistant to cisplatin (Cal27 CisR; SCC9 CisR; SCC25 CisR) and their populations of cancer stem cells (CSC+) and non-cancer stem cells (CSC-) were investigated. Analysis of qPCR were performed to evaluate the gene expression, and western blot to evaluate protein levels; the IC50 dose of the inhibitors was determined through cellular viability assay. Flow cytometry and spheres formation assay identified the CSC+. Chromatin immunoprecipitation was performed to identify the epigenetic regulation of the pathway. The xenograft model was performed to investigate the potential of the Wnt/β-catenin signaling pathway as therapeutical target. We observed an increase in the expression of genes that regulate the epigenetic machinery as BRD7, EZH2, KDM4C, MLL1 and the gene CTNNB1 that encode β-catenin, in the cell lines resistant to cisplatin. The upstream genes of the Wnt/β-catenin signaling pathway APC and GSK3β were decreased in the 3 chemoresistant cell lines, and the downstream genes FGF18 and MMP7 were increased. The CSC+ population demonstrated high expression of genes involved in the histones methylation. β-catenin and the methylated histones H3K27me3 and H3K9me2 were increased in the cell lines resistant to cisplatin and in the CSC+ as well. The EZH2 inhibitor (UNC1999) and the β-catenin inhibitors (ICG-001 and FH535) diminished the population of CSC+ and diminished the proteins β-catenin and EZH2 in the chemoresistant cell lines. H3K27me3 was diminished after the treatment with the inhibitors as well. The treatment with UNC1999 increased the expression of the upstream genes APC and GSK3β, and the treatments with ICG-001, FH535 and UNC1999 were effective in the decrease of the downstream gene MMP7 in the CSC+. FH535 demonstrated effectiveness diminishing the population of CSC+ specially when combined with cisplatin and UNC1999. The β-catenin inhibitors in monotherapy or combined to cisplatin and UNC1999 diminished the stemness of CSC+. The tumor growth was reduced after the administration of FH535, FH535+cisplatin and UNC1999+FH535, and β-catenin, EZH2, H3K27me3 and the cancer stem cells markers OCT4 and SOX2 decreased in the tumor tissue. Chromatin immunoprecipitation in the chemoresistant cell lines and in the populations of CSC- and CSC+ demonstrated that EZH2 strongly interacts with the promotor region of the genes APC, FGF18 and VEGFA and in a less intensive way with the promotors of AXIN2, GSK3β and MMP7, increasing the presence of H3K27me3, which promotes the repression of the genes APC, GSK3β and VEGFA that participates of the Wnt/β-catenin signaling pathway. In this way, the results demonstrate that β-catenin and EZH2 are increased in the chemoresistant cell lines and in the cancer stem cells population, and the histone methylation participates in a critical way, controlling the Wnt/β-catenin signaling pathway, through the inhibition of the upstream regulators APC and GSK3β, leading to the translocation of β-catenin to the nucleus, where it binds to transcription factors and regulate the downstream genes as MMP7, participating in the cancer stem cells regulation. The administration of the pharmacological inhibitors of the Wnt/β-catenin signaling pathway and of EZH2, can be an effective strategy to the oral carcinoma treatment. (AU)

• FAPESP's process: 20/02740-5 - Epigenetic control of WNT/b-catenin signaling in chemoresistant cancer stem cells accumulation in oral carcinoma
• Grantee: Thaís Moré Milan
• Support Opportunities: Scholarships in Brazil - Master