(831) Molecular Insights into the RELT–FLNA Interaction and its Role in Breast Cancer Cells

Introduction/Rationale: Receptor Expressed in Lymphoid Tissues (RELT), member of the Tumor Necrosis Factor Receptor Superfamily, is upregulated in breast cancer (BC). A proteomic screen identified Filamin A (FLNA), an actin-binding scaffold protein involved in many cell processes, as a potential RELT-interacting partner. This study aimed to reveal the interaction between RELT and FLNA and to characterize its physiological significance. FLNA has an established role in BC, where its expression correlates with tumor grade and stage, and has been shown to bind Breast Cancer Gene 1/2.

Methods: Co-immunoprecipitation (co-IP) with western blotting was performed to assess interactions between recombinant RELT and FLNA proteins expressed in HEK293 cells. Deletion mutants of RELT were utilized in co-IP assays to identify the FLNA binding site, using a carboxy-terminal fragment of FLNA (C-FLNA) identified in the proteomic screen. Immunofluorescence (IF) microscopy was used to evaluate RELT–FLNA subcellular colocalization. To examine the effect of co-expression, MDA-MB-231 (231) triple-negative BC cells were transiently transfected with differing combinations of plasmids for RELT, C-FLNA and empty vector control, and analyzed for apoptosis via Annexin V/Propidium Iodide (AV/PI) flow cytometry and X-gal morphology assays.

Results: Co-IP experiments demonstrated that C-FLNA binds within amino acids 340–400 of RELT’s intracellular domain. IF confirmed cytosolic colocalization of RELT and FLNA, supporting a direct interaction. Co-expression of RELT and C-FLNA induced significantly higher AV/PI staining in 231 cells compared to cells expressing either protein alone, corroborated by X-gal assays.

Conclusion: These results define a binding region on RELT for C-FLNA and show that co-expression of RELT and C-FLNA enhances apoptosis in BC cells, suggesting a synergistic effect. Ongoing studies are probing whether RELT mutant expression influences BC cell migration, given FLNA’s established role in cytoskeletal dynamics and motility.