Abstract 2964: Chromatin silencing complex EZH2/PRC2 modulates aggressive anaplastic thyroid cancer biology
Authors: Mello Diego C. , Cristovao Marcella M. , Saito Kelly C. , Kimura Edna T. , Fuziwara Cesar Seigi 
Published: June 2022

Introduction: Anaplastic thyroid cancer (ATC) is a rare and lethal undifferentiated thyroid malignancy, with rapid growth, invasion of adjacent tissues and distant metastasis (&gt;50% of cases), leading to survival of ~ 6 months. Understanding the molecular evolution of ATC is a current challenge in thyroid cancer field that may lead to identification of new targets for treatment. In this extent, deregulation of epigenetic regulators and chromatin modifiers may contribute to ATC aggressiveness by altering chromatin state. High levels of EZH2, a component of Polycomb Repressive Complex 2 (PRC2) lead to deposition of H3K27me3 in histones, heterochromatin formation and repression of target genes expression such as tumor suppressors and differentiation genes.

Objective: Investigate the role of PRC2/EZH2 in ATC biology, using strategies to block EZH2 function, either pharmacologically or with CRISPR/Cas9-mediated gene editing.

Methods: We analyzed the expression of PRC2/EZH2, thyroid differentiation genes and EMT in human data from ATC microarray repository. In vitro, we analyzed protein levels of EZH2 in a panel of thyroid cancer cell lines using Western Blot. In order to block EZH2 function in ATC cells, we used two strategies: A) temporary blockade of EZH2 in KTC2 cells using GSK126, an allosteric inhibitor of EZH2, at 0.5 uM - 5.0 uM, for 6 days; B) permanent blockage of EZH2 using CRISPR/Cas9-induced gene editing in KTC2 cells with PX459 plasmid that contains sgRNA to target EZH2 start codon region. Gene expression of thyroid differentiation genes was evaluated by qPCR. Cell viability was evaluated by MTT assay.

Results: The expression of EZH2/PRC2 components is upregulated in ATC while thyroid differentiation genes, transcription factors and epithelial genes are reduced, and EMT genes are induced. EZH2 function temporary blockage with GSK126 induced thyroid differentiation with dose dependent increase in NIS, TG, TPO (8-fold at 5uM), genes related to iodine metabolism in KTC2 cells. Moreover, thyroid transcription factors NKX2-1 (7-fold at 5uM) and PAX8 (5-fold at 5uM), and the epithelial gene E-cadherin (5-fold at 5uM) are induced by GSK126 treatment. CRISPR/Cas9-mediated EZH2 gene editing efficiently reduced EZH2 protein levels, while induced a more epithelial-like cell morphology and reduced cell viability in KTC2-CRISPR-Cl5. Moreover, disruption of EZH2 gene with CRISPR/Cas9 increased TPO (6-fold), NKX2-1 (1.8-fold) and E-cadherin (4-fold) mRNAs, however did not induce NIS, TG and PAX8 as observed in GSK126 treatment.

Conclusion: The over-expression and over-activation of EZH2/PRC2 pathway in ATC may contribute to tumor aggressiveness by reducing thyroid cell differentiation and inducing EMT; These results indicate a potential benefit for EZH2 blockage as a neoadjuvant approach to induce differentiation and radioiodine uptake in aggressive thyroid cancer.

Citation Format: Diego C. Mello, Marcella M. Cristovao, Kelly C. Saito, Edna T. Kimura, Cesar Seigi Fuziwara. Chromatin silencing complex EZH2/PRC2 modulates aggressive anaplastic thyroid cancer biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2964.