Abstract 349: The role of RELTfms and FLNA in human cancer and cellular apoptosis

Abstract

Abstract Receptor Expressed in Lymphoid Tissues (RELT) is a Tumor Necrosis Factor Receptor family member implicated in several cancers. RELL1 and RELL2 (RELT-Like 1 and 2) are RELT homologs that bind RELT; the three proteins are collectively referred to as RELT family members (RELTfms). We sought to evaluate the role of the OXSR1 kinase and actin-binding protein Filamin A (FLNA) in modulating RELT-induced apoptosis of cancer cells. We also assessed RELTfm expression in human breast and lung cancer biopsies and their ability to sensitize breast cancer cells to chemotherapeutic agents. Methods: Empty vector or expression plasmids for RELTfms were transiently transfected into MDA-MB-231 (231) breast cancer cells with TransfeX reagent. Cell death was assessed by morphology assays and ATP luciferase assays. Apoptosis of 231 cells was assessed by Annexin V/PI staining using Flow cytometry (FC). Immunohistochemistry (IHC) was used to assess RELT expression in human breast and lung cancer biopsies. IHC and western blotting were utilized to determine RELT cellular localization. Results: All three RELLfms were co-immunoprecipitated by a C-terminal fragment of FLNA. RELT overexpression induced death in 231 cells, and this effect was increased with co-transfection of a FLNA S215A mutant, an important phosphorylation site that protects FLNA from cleavage. 231s transfected with RELT and RELL2 demonstrated increased Annexin V/PI signal in comparison to vector control, demonstrating that these RELTfms induce death via an apoptotic pathway. Co-transfection of a mutated plasmid for the OXSR1 kinase did not abrogate RELT-induced apoptosis. A RELT construct with a mutated binding site for the OXSR1 kinase induced apoptosis to a similar extent as wild-type RELT. An increase in doxorubicin-induced apoptosis was observed when 231 cells were transfected with RELL2 versus vector control. IHC results showed increased RELT expression in malignant breast cancer biopsies compared to patient-matched benign tissue. Interestingly, RELT was localized in the nucleus of some malignant lung cancer biopsies, and western blot analysis indicates that RELT translocates to the nucleus and interacts with chromatin in 231 cells. Conclusions: Collectively, these results demonstrate that RELTfms induce apoptosis of breast cancer cells, and potentially increase the sensitivity of breast cancer cells to chemotherapeutic agents. Furthermore, this work enhances our understanding of the interaction between RELT and FLN, a protein that is implicated in many cancers, including breast cancer. Phosphorylation by OXSR1 was previously shown to be required for RELT to activate the p38 pathway, yet the OXSR1 kinase is not required for RELT-induced death. RELT expression is enhanced in breast cancer biopsies, and interestingly, RELT can localize to the nucleus in cancerous cells. Collectively, these findings significantly enhance our understanding of RELTfms and cancer. Citation Format: Emily Chou, Esther Chang, Ihab Abed, Ashley Christensen, Kailey Nguyen, Anusri Yanumula, Yennie Shyu, Alyssa Abram, Jessa Alcaide, Gianne Cusick, Maryann Batiste, Yihui Shi, Eslam Mohammed, John Cusick. The role of RELTfms and FLNA in human cancer and cellular apoptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 349.