Boric Acid Modulates Hippo, TGF-β/SMAD, and Epigenetic Signaling Pathways in MDA-MB-231 Triple-Negative Breast Cancer Cells

Background: The Hippo signaling pathway plays a critical role in the regulation of cell proliferation, apoptosis, and tumor progression, and its dysregulation has been strongly associated with triple-negative breast cancer (TNBC). Boric acid has been reported to exhibit anti-proliferative and pro-apoptotic effects in various cancer models; however, its effects on Hippo signaling and related molecular pathways in TNBC remain poorly understood. This study aimed to investigate the time-dependent effects of boric acid on Hippo pathway-related gene expression and associated protein markers in MDA-MB-231 breast cancer cells.Methods: MDA-MB-231 cells were treated with boric acid, and cell viability was assessed using the MTT assay. The half-maximal inhibitory concentration (IC₅₀) was determined and used for subsequent analyses. Expression levels of Hippo pathway-related genes (YAP, TAZ, TEAD1–4), TGF-β/SMAD pathway components (SMAD2, SMAD3), the chromatin remodeling factor CHD2, and extracellular matrix remodeling genes (COL1A1, MMP2, TIMP1–3) were evaluated by quantitative real-time PCR after 24, 48, and 72 h of treatment. Immunocytochemical analyses were performed to assess Cleaved Caspase-3, TNF-α, and TGFβR1 expression.Results: Boric acid reduced cell viability in a concentration-dependent manner, with an IC₅₀ value of approximately 240 μg/mL after 48 h of treatment. Gene expression analyses revealed significant downregulation of YAP and TEAD2 at 24 h, CHD2 at 48 h, and TAZ, YAP, SMAD2, and CHD2 at 72 h. In contrast, MMP2 expression was significantly increased at 72 h. Immunocytochemical evaluation demonstrated a time-dependent increase in Cleaved Caspase-3 immunoreactivity, moderate alterations in TNF-α expression, and reduced TGFβR1 staining intensity following boric acid treatment.Conclusions: These findings indicate that boric acid modulates multiple interconnected signaling pathways in TNBC cells, including Hippo, TGF-β/SMAD, apoptotic, inflammatory, and extracellular matrix remodeling pathways. The observed suppression of YAP/TAZ-associated signaling together with CHD2 downregulation suggests that both transcriptional and epigenetic mechanisms contribute to the anti-proliferative effects of boric acid. Boric acid may therefore represent a promising modulatory agent for targeting key regulatory networks in aggressive breast cancer subtypes.