Abstract 2199: Dissecting ROBO4 function in regulating breast cancer cell plasticity.

Abstract Breast cancer is the most common cancer in women and the second-leading cause of cancer-related deaths in women. Breast cancer tumors are highly heterogeneous, with small populations of stem-like tumor-initiating cells that exhibit high cellular plasticity and often display both epithelial and mesenchymal characteristics (hybrid). These plastic stem-like cells contribute to tumorigenesis, so identifying and targeting these populations can lead to improved patient survival. ROBO1-4 proteins are transmembrane receptors that, with their SLIT1-3 ligands, regulate axon guidance, cell adhesion, mammary gland development, and angiogenesis. This signaling pathway is tightly regulated and has a wide variety of roles in normal development, but cancer is defined as abnormal development and frequently has alterations in signaling pathways. SLIT-ROBO signaling has been implicated in cell adhesion regulation in breast, lung, and colorectal cancer, but there is conflicting evidence on whether it is tumor-promoting or tumor-suppressive. Previous studies have shown that ROBO4 expression in endothelial cells suppresses breast cancer growth; however, ROBO4 has not been investigated in tumor epithelial cells. We have found that ROBO4 is expressed in epithelial breast cancer cells, and a high ROBO4+ epithelial fraction in basal-like tumor cells predicts worse survival in patients. ROBO4 knockdown in breast cancer cells reduces colony formation and proliferation, and increases apoptosis. Using patient-derived xenografts (PDXs) in vivo, constitutive ROBO4 knockdown reduced tumorigenesis, and inducible knockdown halted further tumor growth in pre-established tumors. Transcriptomic and proteomic analyses reveal enrichment of cancer stem cell and plasticity markers in control cells compared to ROBO4 knockdown, including ALDH3A1, MSI2, CD44, and S100A4. Flow cytometry analysis of tumor cells shows that knocking down ROBO4 reduces hybrid cells as measured by co-expression of CD44 (mesenchymal cells) and CD104 (epithelial cells). Preliminary results from ROBO4 overexpression tumors show an increase in hybrid CD44+/CD104+ cells, suggesting that ROBO4 maintains stem and hybrid populations in breast cancer. Gene Set Enrichment Analysis (GSEA) suggests Rho GTPase, Wnt/β-catenin, and Notch signaling are downregulated in ROBO4 knockdown cells. These findings reveal a novel role for ROBO4 in maintaining stem-like and hybrid cell populations in breast cancer, with ROBO4's location on the cell surface providing an attractive target for potential therapeutic applications. Citation Format: Isobel J. Fetter, Veronica Haro Acosta, Paloma Medina, Shaheen S. Sikandar. Dissecting ROBO4 function in regulating breast cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2199.