Metronomic 5-Fluorouracil and Vinorelbine Reduce Cancer Stemness and Modulate EZH2/NOTCH-1/STAT3 Signaling in Triple-Negative Breast Cancer Spheroids

Triple Negative Breast Cancers (TNBCs) are heterogeneous and aggressive tumors with a median overall survival of less than two years. Despite the availability of new drugs, the prognosis remains poor, implicating a more aggressive clinical course in the metastatic setting. This study investigated the effects of metronomic treatment (mCHT) with 5-fluorouracil (5-FU) plus vinorelbine (VNR) on spheroids derived from two different TNBC cell lines (BT-549 and MDA-MB-231) and a patient-derived primary cell line (MS-186). mCHT significantly reduced spheroid growth and altered spheroid architecture, with a pronounced effect in second-generation spheroids, enriched in self-renewing cancer stem cells (CSCs). Expression of CSC-related markers (CD44, CD133, NOTCH-1, and MYC) was more significantly altered—both at the mRNA and protein levels—by mCHT than by standard treatment (STD). In MS-186-derived spheroids, mCHT downregulated EZH2 and STAT3, key regulators of CSC maintenance, and reduced H3K27ac, suggesting a global epigenetic reprogramming. Unlike STD, which partially and transiently reduced stemness markers, mCHT achieved sustained suppression, indicating preferential targeting of therapy-resistant CSCs. These results indicate mCHT as a promising strategy for specifically aiming at the CSC-like compartment in TNBC, underscoring a therapeutic approach that reprograms key epigenetic networks and overcomes resistance to treatment.