Elucidating the molecular interaction between RELT and Filamin A and the physiological role of this interaction in breast cancer cells

Receptor Expressed in Lymphoid Tissues (RELT) is a member of the Tumor Necrosis Factor Receptor Superfamily that was previously shown to be upregulated in breast cancer. A proteomic screen identified Filamin A (FLNA), an actin-binding scaffold protein involved in various cell processes, including DNA repair, signal transduction, and cell migration, as a potential RELT-interacting partner. FLNA is implicated to play a vital role in breast cancer, as its expression correlates with tumor grade and stage, and it has been shown to bind both BRCA1 and BRCA2. This study aimed to further explore the novel interaction between RELT and FLNA and to characterize its physiological significance. A yeast two-hybrid screen was utilized to identify a carboxy-terminal fragment of FLNA (C-FLNA) as a potential RELT-binding partner. Transient overexpression of C-FLNA and deletion mutants of RELT in HEK-293 cells, followed by co-immunoprecipitation (co-IP) and western blotting, was performed to identify the binding site on RELT for FLNA. Immunofluorescence (IF) microscopy was used to evaluate RELT-FLNA subcellular colocalization in MDA-MB-231 (231) breast cancer cells. To examine the impact of C-FLNA on RELT's ability to induce cell death, 231 cells, a model for triple-negative breast cancer, were transiently transfected with varying combinations of RELT, FLNA mutants, and empty vector control plasmids. Consequently, these cells were analyzed for apoptosis via flow cytometry, using Annexin V/Propidium Iodide (AV/PI) staining. Co-IP experiments performed with deletion mutants demonstrated that C-FLNA binds within amino acids 340-400 of RELT's intracellular domain. IF studies confirmed cytosolic colocalization of RELT and FLNA, supporting a direct interaction between these two proteins. Flow cytometry data revealed that co-expression of RELT and C-FLNA induced significantly higher AV/PI staining in 231 cells, indicating greater levels of apoptosis, compared to cells expressing either protein alone. These findings define an intracellular binding region on RELT for FLNA binding. Furthermore, co-expression of RELT and C-FLNA resulted in significantly enhanced apoptosis in breast cancer cells, suggesting a potentially synergistic effect. Current studies are examining whether RELT expression influences breast cancer cell migration given FLNA's established role in cytoskeletal dynamics and motility. Collectively, these results enhance our understanding of the interaction between RELT, a protein that is upregulated in breast cancer, and FLNA, a protein whose importance for breast cancer has been well established.