Deciphering the role of EZH2 in the control of HIF2α signaling and its effects on cellular phenotypes in breast and lung cancer

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KruijningSalisa-2026-02-18.pdf (3.78 MB)

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2026

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DOI:
https://doi.org/10.22029/jlupub-20724

Abstract

Hypoxia is a hallmark of solid tumors and a critical driver of cancer progression, largely mediated by hypoxia-inducible factors (HIFs). While HIF1α has been extensively studied, the transcriptional regulation of HIF2α, encoded by the endothelial PAS domain protein 1 (EPAS1) gene, remains less well understood. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), is a known epigenetic regulator with canonical roles in gene repression via H3K27 tri-methylation and emerging non-canonical functions, including transcriptional activation. The interplay between EZH2 and HIF signaling, particularly regarding HIF2α, is largely unexplored. In this study, the role of EZH2 as a potential regulator of HIF2α in breast and lung cancer model systems was investigated. EZH2 knockdown decreased HIF2α protein under hypoxic conditions and EPAS1 mRNA levels under normoxic and hypoxic conditions in MDA-MB-231 and PC-9 cell lines. Restoration of EZH2 rescued EPAS1 expression. Mechanistic studies revealed that this regulation occurs independently of PRC2, EZH2’s methyltransferase activity, EZH1, Notch1 signaling, and transcriptional elongation. Chromatin immunoprecipitation demonstrated direct binding of EZH2 to a region approximately 1.7 kb downstream of the EPAS1 transcription start site, without enrichment of H3K27me3, supporting a non-canonical transcriptional activator function for EZH2. These findings indicate that EZH2 directly maintains EPAS1 transcription independent of chromatin repression and contributes to sustaining transcriptional activity within a globally repressive hypoxic environment. Functionally, EZH2 depletion impaired the expression of HIF2α target genes, including GLUT1 and PGK1, and reduced invasion capacity in MDA-MB-231 cells under hypoxia. In PC-9 cells, EZH2 knockdown decreased proliferation, which was partially rescued by transient HIF2α restoration, and anchorage-independent growth. These findings indicate that EZH2 promotes tumorigenicity at least in part through HIF2α. Clinical analyses revealed that high EZH2 and EPAS1 expression correlate with poor prognosis in breast cancer patients, underscoring the potential clinical relevance of this regulatory axis. Overall, this study establishes EZH2 as a novel non-canonical transcriptional activator of EPAS1, linking an epigenetic regulator to hypoxia signaling. These findings extend our understanding of EZH2 beyond its canonical repressive role and suggest that the EZH2-HIF2α regulatory axis may contribute to malignant phenotypes such as proliferation and invasion under hypoxic conditions. Future studies utilizing RNA-seq, in vivo models, and pharmacological inhibitors targeting EZH2 and HIF2α could provide further insight into the mechanistic and therapeutic potential of this regulatory pathway in cancer.

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