Hydroxytyrosol Reprograms the Tumor Microenvironment in 3D Melanoma Models by Suppressing ERBB Family and Kinase Pathways

Malignant cutaneous melanoma is among the most aggressive forms of skin cancer, characterized by high metastatic potential and frequent resistance to standard therapies. Hydroxytyrosol, a phenolic compound derived from extra virgin olive oil, has shown promising anticancer properties in various models, yet its effects in 3D melanoma systems remain poorly understood. In this study, we used paired 3D spheroid models of non-tumorigenic (HEMa) and melanoma (C32) to assess the therapeutic potential of hydroxytyrosol. To evaluate the anti-tumoral effect of hydroxytyrosol, we performed cytotoxicity, metastasis, invasiveness, cell cycle arrest, apoptotic, and proteomic assays. Hydroxytyrosol treatment significantly impaired spheroid growth, reduced cell viability, and induced cell cycle arrest and apoptosis in C32 spheroids, with minimal cytotoxicity observed in HEMa models. Proteomic profiling further demonstrated that hydroxytyrosol selectively downregulated a network of oncogenic proteins, including ERBB2, ERBB3, ERBB4, VEGFR-2, and WIF-1, along with suppression of downstream PI3K-Akt and MAPK/ERK signaling pathways. In conclusion, compared to dabrafenib, hydroxytyrosol exerted a broader range of molecular effects and was more selective toward tumor cells. These findings support the use of hydroxytyrosol as a multi-targeted agent capable of attenuating melanoma progression through suppression of kinase signaling and tumor-stromal interactions.