Anticancer and Immunomodulatory Effects of a Thiazolyl Benzodiazepine Targeting HSP90 in ER+ Breast Cancer

<b>Background:</b> Heat shock protein 90 (HSP90) is a molecular chaperone that stabilizes numerous oncogenic proteins and supports tumor survival. Small molecules targeting HSP90 offer a novel approach to overcome drug resistance and immune suppression in breast cancer. <b>Methods:</b> A novel thiazolyl benzodiazepine (TB) containing a hydrazone moiety was evaluated in breast cancer cell lines (ER⁺ MCF-7, TNBC MDA-MB-231, and HER2⁺ SK-BR-3). Cytotoxicity was assessed using the CCK-8 assay, followed by PCR sequencing, flow cytometry, RT-qPCR, protein profiling, and HSP90 binding assays. <b>Results:</b> TB showed the strongest activity in MCF-7 cells (IC₅₀ = 7.21 µM) compared to MDA-MB-231 (IC₅₀ = 28.07 µM) and SK-BR-3 (IC₅₀ = 12.8 µM) cells. Mechanistic studies showed that TB binds to HSP90 (Kd = 3.10 µM), leading to disruption of the oncogenic signal. TB induced G2/M cell cycle arrest, promoted apoptosis via Bax and Caspase-3 activation, and suppressed cancer stem cell markers (<i>NANOG</i>, <i>OCT4</i>, <i>SOX2</i>). Additionally, TB activated immune-related pathways via ERK/MAPK signaling and upregulated genes such as <i>SMAD2, SMAD3,</i> and <i>JUN.</i><b>Conclusions:</b> TB functions as an HSP90 inhibitor with dual anticancer and immunomodulatory properties in Estrogen Receptor-Positive (ER⁺) breast cancer cells. These findings suggest that TB represents a promising scaffold for the development of multi-targeted breast cancer therapies.