Dual Epigenetic and Chaperone Inhibition Disrupts Hypoxia Signaling and Tumor Progression in 3-D Models of Triple-Negative Breast Cancer.

Therapeutic resistance remains a major barrier to treating aggressive breast cancers, particularly triple-negative breast cancer (TNBC), in which hypoxia-associated stress adaptation within the tumor microenvironment limits treatment efficacy. Here, we investigated a multi-targeted therapeutic strategy combining histone deacetylase (HDAC) inhibition with blockade of hypoxia-associated chaperones HSP90β and mitochondrial TRAP1 in 3-D breast cancer models grown from TNBC human cells. HDAC inhibitors (HDACi; vorinostat [SAHA; pan-HDACi], valproic acid [VPA; HDAC1i], and CAY10603 [CAY; HDAC6i]; showed greater efficacy than paclitaxel at reducing viability and mammosphere formation in 3-D cultures, and induced apoptosis and G2/M cell cycle arrest in TNBC cells. Combined inhibition of HDACs with our novel agents targeting HSP90β (NDNB-25) or TRAP1 (NDNT-34) synergistically reduced mammosphere viability and disrupted spheroid architecture. Mechanistically, HSP90β and TRAP1 inhibition attenuated hypoxia-associated adaptive signaling by suppressing HIF-1α, VEGFA, HSP90β, and TRAP1 gene and protein expression. In co-cultures with HUVEC cells, these effects were accompanied by impaired endothelial network formation, tumor cell migration and invasion, mitotic progression, and clonogenic growth. Following Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analysis, genome-wide HIF-1α occupancy analysis revealed extensive reprogramming of HIF-1α-associated regulatory programs under HDAC, HSP90β, and TRAP1 inhibition, including pathways linked to innate immunity, interferon signaling, redox balance, autophagy, mitochondrial function, and metabolic adaptation. Together, these findings identify hypoxia-associated stress adaptation as a therapeutically actionable vulnerability in aggressive breast cancer and support coordinated targeting of epigenetic regulation, proteostasis, and mitochondrial stress adaptation to enhance treatment response.