Dissecting ROBO4 function in regulating breast cancer cell plasticity

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/beta-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.