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10780432ccr122505-sup-fig2.pdf (1.26 MB)

Supplementary Figure 2 from Dysregulation of the Repressive H3K27 Trimethylation Mark in Head and Neck Squamous Cell Carcinoma Contributes to Dysregulated Squamous Differentiation

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posted on 2023-03-31, 18:05 authored by Orla M. Gannon, Lilia Merida de Long, Liliana Endo-Munoz, Mehlika Hazar-Rethinam, Nicholas A. Saunders

PDF file - 1290K, Supplementary Figure 2. siRNA against EZH2 does not induce apoptosis. Detroit 562 cells and primary keratinocytes (HEK) were treated with siRNA against EZH2 for 48 hours or 96 hours and stained with Annexin V/7 aminoactinomycin D and subjected to FACS analysis to determine the proportion of apoptotic cells. a) The ability of siRNA to inhibit EZH2 was determined in the HEKs. β-actin is a loading control. b) EZH2 siRNA did not induce apoptosis after 48 hours or c) 96 hours. Data is mean � SEM, n=2

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ARTICLE ABSTRACT

Purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers diagnosed worldwide and is associated with a 5-year survival rate of 55%. EZH2, a component of the polycomb repressor complex 2, trimethylates H3K27 (H3K27me3), which has been shown to drive squamous differentiation in normal keratinocytes. This study determined whether inhibition of EZH2-mediated epigenetic silencing could induce differentiation or provide therapeutic benefit in HNSCC.Experimental Design: We determined the effects of inhibiting EZH2, by either RNA interference or pharmacologically, on HNSCC growth, viability, and differentiation in vitro. Xenografts of HNSCC cell lines were used to assess efficacy of 3-deazaneplanocin A (DZNep), an inhibitor of H3K27 trimethylation, in vivo.Results: EZH2 was highly expressed in HNSCC cell lines in vitro and tissue microarray analysis revealed high expression in (n = 59) in situ relative to normal oral epithelium (n = 12). Inhibition of EZH2 with siRNA could induce expression of differentiation genes in differentiation-refractory squamous cell carcinoma cell lines. Differentiation-refractory HNSCC cell lines displayed persistent H3K27me3 on the promoters of differentiation genes. DZNep caused cancer-cell–specific apoptosis in addition to a profound reduction in colony-forming efficiency and induction of some squamous differentiation genes. Furthermore, in vivo, DZNep attenuated tumor growth in two different xenograft models, caused intratumor inhibition of EZH2, and induction of differentiation genes in situ.Conclusions: Collectively, these data suggest that aberrant differentiation in HNSCC may be attributed to epigenetic dysregulation and suggest that inhibition of PRC2-mediated gene repression may represent a potential therapeutic target. Clin Cancer Res; 19(2); 428–41. ©2012 AACR.

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