Inhibition of oncogenic phenotypes by hexanoate in triple-negative breast cancer

Background: Triple-Negative Breast Cancer (TNBC) is an invasive molecular subtype of breast cancer defined by the lack of estrogen and progesterone receptors, and the absence of HER2 amplification. This molecular profile limits current treatment options contributing to poor patient outcomes. Recent evidence features the microbiome as a key factor in cancer biology, modulating tumor progression through metabolites and bacterial products. Among these products, medium-chain fatty acids (MCFAs), such as hexanoate, are produced by microbial fermentation within the microbiome. Previous studies from our laboratory demonstrated that hexanoate reduces the viability and proliferative capacity in oral cancer cells, which may suggest hexanoate as a promising candidate target for alternative cancer treatments. Therefore, the aim of this study was to evaluate the impact of hexanoate on oncogenic phenotypes including cell viability, proliferation, and migration of the TNBC cell line MDA-MB-231. Methods: MDA-MB-231 cells were cultured at 37°C, 5% CO 2 in RPMI-media. Cell viability was measured using AlamarBlue after 24, 48 and 72 hours (h) of treatment with sodium hexanoate at 9 different concentrations starting at 800mM (1:2 serial dilution) to measure the half-minimal inhibitory concentration (IC50). Cell proliferation was measured by seeding 100,000 cells per well and counting with TrypanBlue the change in cell number after treatment with hexanoate at 5mM for 24h, 48h and 72h. Cell migration was evaluated using a scratch assay; after inflicting a wound, its width was measured at 0h, 24h, and 48h to calculate the percentage of wound closure. Results: Hexanoate reduced cell viability at 24h, 48h, and 72h with a consistent IC50 of 29mM. Additionally, no significant effects on the proliferation rate were observed at 24h and 48h when treated with hexanoate, whereas it decreased significantly at 72h (p=0.004). Furthermore, hexanoate showed a decreased percentage of wound closure at both time points, showing significance at 48h (p = 0.002). Conclusions: Overall, hexanoate exhibited inhibitory effects on viability, proliferation, and migration in TNBC cells. As a microbiome-associated compound, hexanoate may modulate cancer cell behavior in a time-dependent manner and represent a potential alternative target for TNBC treatment, although additional studies are required for validation.