PO-276 Histone acetylation readers BET in hypoxia adaptation in triple negative breast cancer (TNBC)

Abstract

Introduction Hypoxia (low oxygen) is associated with worst prognosis and therapy resistance in most solid tumours. Hypoxia occurs in ~50% of breast cancer, most frequently in triple negative breast cancer (TNBC), the most aggressive subtype. Hypoxia drives tumour aggressiveness through HIF, which activates genes that promote adaptation to hypoxia, including CA9, VEGFA, LOX, that promote respectively pH regulation, angiogenesis and metastasis. Epigenetic factors mediate gene expression changes that occur in pathological conditions, like hypoxia. BET proteins read histone-acetylation and recruit transcription factors. BET inhibition has anti-tumour effects. Thus we investigated BET proteins as regulators of TNBC hypoxic adaptation. Material and methods Cell lines were investigated in 2D in normoxia and hypoxia (0.1%-1% O2), spheroids and as orthotopic xenografts with BET inhibitors (BET-i: JQ1, iBET151 and iBET762). The impact of BET-i on transcriptional regulation and hypoxic phenotypes was investigated using a variety of molecular and cell biology techniques. Results and discussions BET-i reduced growth in hypoxia and normoxia in vitro (2D and 3D) and in vivo where xenograft vascularisation was reduced (reduced CD31 staining). JQ1 modulated 44% of hypoxia-induced genes (including CA9, VEGFA and LOX), but did not alter HIF expression. Ongoing investigations have identified that hypoxia increases H4ac and H3K27ac, associated with transcription and BET protein binding, in the promoters of HIF targets CA9, VEGFA and LOX. BET-i also reduced HIF binding to the CA9 promoter. Silencing of individual BET proteins (BRD 2, 3 and 4) downregulates CA9 in hypoxia, while only BRD4 knockdown reduces VEGFA expression. The knockdown of individual BET proteins reduced colony formation in hypoxia and normoxia, but not as comprehensively as JQ1, suggesting there could be a compensatory role of the BET proteins. Conclusion We identified histone acetylation and BET proteins play an important role in tumour adaptation to hypoxia. This provides evidence to support the development of BET-i as a therapeutic option for hypoxic TNBC.