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EZH2 regulates a SETDB1/ΔNp63α axis via RUNX3 to drive a cancer stem cell phenotype in squamous cell carcinoma

Abstract

Enhancer of zeste homolog 2 (EZH2) and SET domain bifurcated 1 (SETDB1, also known as ESET) are oncogenic methyltransferases implicated in a number of human cancers. These enzymes typically function as epigenetic repressors of target genes by methylating histone H3 K27 and H3-K9 residues, respectively. Here, we show that EZH2 and SETDB1 are essential to proliferation in 3 SCC cell lines, HSC-5, FaDu, and Cal33. Additionally, we find both of these proteins highly expressed in an aggressive stem-like SCC sub-population. Depletion of either EZH2 or SETDB1 disrupts these stem-like cells and their associated phenotypes of spheroid formation, invasion, and tumor growth. We show that SETDB1 regulates this SCC stem cell phenotype through cooperation with ΔNp63α, an oncogenic isoform of the p53-related transcription factor p63. Furthermore, EZH2 is upstream of both SETDB1 and ΔNp63α, activating these targets via repression of the tumor suppressor RUNX3. We show that targeting this pathway with inhibitors of EZH2 results in activation of RUNX3 and repression of both SETDB1 and ΔNp63α, antagonizing the SCC cancer stem cell phenotype. This work highlights a novel pathway that drives an aggressive cancer stem cell phenotype and demonstrates a means of pharmacological intervention.

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Fig. 1: SETDB1 regulates a cancer stem cell phenotype in SCC.
Fig. 2: SETDB1 regulates an SCC CSC phenotype via ΔNp63α.
Fig. 3: ΔNp63α regulates SETDB1 to influence the SCC CSC phenotype.
Fig. 4: EZH2 regulates SETDB1 and ΔNp63α, and their corresponding phenotypes.
Fig. 5: The catalytic function of EZH2 is necessary for the regulation of SETDB1 and ΔNp63α.
Fig. 6: EZH2-repressed RUNX3 represses SETDB1 and ΔNp63α, and the SCC CSC phenotype.
Fig. 7: RUNX3 is a repressor of ΔNp63α and SETDB1.
Fig. 8: EZH2, SETDB1 and p63 are overexpressed in human SCCs, and showed increased staining in cells with high SOX2 expression.

Data availability

RNA-seq and ChIP-seq data are available from the Gene Expression Omnibus database (accession no. GSE202789) Methods for CRISPR activation, CRISPR depletion, domain-focused CRISPR screening, cDNA overexpression, EPZ-6438 and GSK126 treatment, GFP depletion assays, tumor xenografts, ChIP-seq, and RNA-seq are described in the Supplementary Information. Antibodies and reagents are also listed there, as well as RT-qPCR primer sequences and sgRNA sequences.

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Acknowledgements

This work was supported by the Office of the Director, National Institutes of Health through award numbers 5P30CA045508 (Cancer Center Support Grant), CA247400 (to SB), CA225134 (to MLF), as well as R01CA190997 and R21OD018332 (to AAM). This project was also supported through the Cold Spring Harbor Laboratory and Northwell Health Affiliation.

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SB: Performed and designed experiments, paper writing. MLF: Performed and designed experiments. YH: Performed and designed experiments. CW: Performed and designed experiments. CB: Performed experiments. XS: Designed experiments. AAM: Experimental design and direction, paper writing, funding.

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Correspondence to Alea A. Mills.

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Balinth, S., Fisher, M.L., Hwangbo, Y. et al. EZH2 regulates a SETDB1/ΔNp63α axis via RUNX3 to drive a cancer stem cell phenotype in squamous cell carcinoma. Oncogene 41, 4130–4144 (2022). https://doi.org/10.1038/s41388-022-02417-4

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