Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes’ expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.

Modulating the expression of a coding or noncoding gene is a key tool in scientific research. This strategy has evolved methodologically due to advances in cloning approaches, modeling/algorithms in short hairpin RNA (shRNA) design for knockdown efficiency, and biochemical modifications in RNA synthesis, among other developments. Overall, these modifications have improved the ways to either reduce or induce the expression of a given gene with efficiency and facility for implementation in the lab. Allied with that, the existence of [...] ABSTRACT Modulating the expression of a coding or noncoding gene is a key tool in scientific research. This strategy has evolved methodologically due to advances in cloning approaches, modeling/algorithms in short hairpin RNA (shRNA) design for knockdown efficiency, and biochemical modifications in RNA synthesis, among other developments. Overall, these modifications have improved the ways to either reduce or induce the expression of a given gene with efficiency and facility for implementation in the lab. Allied with that, the existence of

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer type harboring TP53, TERT promoter and MAPK signaling alterations. Additionally, EZH2 overexpression leads to epigenetic silencing of tumor suppressor and cell differentiation genes. Here, we investigate the mechanism of EZH2 transcriptional activation in ATC. We used several luciferase reporter constructs with deletion of transcription factor (TF) binding sites identified in silico to determine EZH2 minimal promoter in ATC. MAPK signaling blockage with U0126, TF overexpression and knock-down strategies were used to evaluate EZH2 expression and reporter plasmid response, and the cross talk with TFs. As a result, we observed that EZH2 transcription is regulated by a minimal promoter of 107 bp (E3/4 region), that contains binding sites for TF NFYA, YY1 and FOXM1, highly expressed in ATC, that when deleted reduced EZH2 promoter activation. MAPK blockage reduced EZH2 and influenced YY1 and FOXM1 TF levels, while overexpression of NFYA, YY1 and FOXM1 resulted in pro-tumoral effects and EZH2 upregulation in papillary thyroid cancer cells. On the other hand, FOXM1 knock-down reduced EZH2 activation in ATC cells. Thus, we identified the minimal promoter region essential for EZH2 activation in ATC that is controlled by MAPK signaling in crosstalk with TFs.