Background <p>Among the diverse mechanisms driving cancer progression, dysregulated kinase signaling and immune evasion mechanisms are particularly critical, necessitating the development of therapies that simultaneously target kinase-dependent tumor survival pathways and reinvigorate antitumor immunity. Oleuropein (OP), a natural polyphenol with established chemopreventive activity, exhibits limited potency and bioavailability. To overcome these limitations, a series of structurally optimized semi-synthetic OP analogs were developed and screened for their anticancer activity. The most prominent, Ole-4, was further evaluated for its antitumor mechanism of action.</p> Methods <p>Ten OP analogs were screened against a panel of human and mouse cancer cell lines. The most active, Ole-4 was further assessed for its cytostatic and pro-apoptotic effects in breast cancer cells using proliferation assays, cell-cycle analysis, and apoptosis quantification. Kinome profiling was performed to identify alterations in protein tyrosine (PTK) and serine/threonine (STK) kinase activity induced by Ole-4. C57BL/6J mice bearing subcutaneous B16.F1 melanoma tumors and BALB/c mice bearing CT26 colon and D2F2/E2 breast carcinomas, were treated intraperitoneally with Ole-4 to evaluate its in vivo efficacy. Immune activation and tumor infiltration by effector cells were analyzed by flow cytometry and immunohistochemistry.</p> Results <p>In vitro, the OP analog Ole-4 exhibited potent cytotoxicity against breast, ovarian, melanoma, cervical and colon carcinoma cell lines. Specifically in HER2⁺ breast cancer cells, Ole-4 significantly inhibited proliferation by inducing S-phase arrest and triggering apoptosis. These effects correlated with kinome profiling data, indicating sustained functional suppression of the STKs Akt and P70S6KB, key regulators of cell survival and growth. Likewise, Ole-4 reduced the activity of Src, ROCK1, and EphA2 kinases which are critically implicated in tumor metastasis, and enhanced the RAF/ERK and Jak1b signaling axes, closely linked to stress-adaptive responses. In vivo, Ole-4 suppressed melanoma, colon and breast carcinoma growth in mice subcutaneously inoculated with syngeneic B16.F1, CT26 and D2F2/E2 cancer cells. Additionally, Ole-4 generated immune cell-mediated antitumor cytotoxicity in treated mice, and importantly, increased tumor infiltration by cytotoxic T and natural killer cells.</p> Conclusions <p>Our findings suggest that Ole-4 exerts direct anticancer effects, likely mediated via STK-dependent mechanisms, while indirectly it enhances immune effector responses, sensitizing tumor cells to immune-mediated apoptosis. Based on these tumor inhibitory and immunomodulatory properties, Ole-4 emerges as a promising candidate for designing next-generation anticancer drugs.</p>