Dissecting the role of FLNA cleavage and signaling in mediating calpeptin effects in neuroendocrine tumors

Abstract Background Neuroendocrine tumors (NETs) are rare malignancies with a rising incidence and heterogeneous clinical behavior, posing significant challenges to diagnosis and treatment. The urgent need for novel therapeutic strategies is increasingly evident. Filamin A (FLNA), a cytoskeletal protein involved in cancer progression and inflammatory signaling, is cleaved by calpain to generate a 90 kDa C-terminal fragment (FLNA CT ), whose functional role in NETs remains unclear. The calpain inhibitor calpeptin prevents FLNA cleavage. Methods This study investigates the role of FLNA cleavage and the effects of calpeptin in NET progression using western blotting, RNA sequencing, functional assays, and patient-derived NET organoid models. Results We found that NET cells express both full-length FLNA and FLNA CT , with both localized in the cytoplasm. Calpeptin reduces FLNA CT expression and inhibits proliferation and viability in NET models. Its inhibitory effect on cell migration was observed specifically in H727 cells. RNA sequencing identified numerous calpeptin-regulated genes. Comparing the top 500 most significantly altered genes in QGP-1 and H727 cells, we identified 115 genes consistently downregulated and 148 upregulated. Among them, CEACAM5, CDC6, and EGR1 were affected by FLNA silencing in QGP1, while CCNE1, CDC6, ATF3, and EGR1 were influenced in H727. KEGG pathway analysis highlighted cytokine–cytokine receptor interactions in QGP-1 cells, cell cycle regulation in H727 cells, and TGF-β signaling in both models. Calpeptin downregulated TGFBR2 and TGFB3 in both cell lines, with FLNA mediating this effect only in H727 cells. Additionally, it reduced IL5RA in QGP1 and IL17RB in H727, with FLNA involvement limited to H727 cells. Expression of a calpain-resistant FLNA mutant indicated that most transcriptional effects of calpeptin were not fully recapitulated by inhibition of FLNA cleavage alone. In patient-derived NET organoids, calpeptin reduced viability and arrested organoid growth, without inducing a significant increase in caspase-3/7 activity, supporting a predominantly cytostatic rather than pro-apoptotic effect. Conclusions These findings highlight the complex interplay between calpain activity, FLNA function, and FLNA proteolysis in NETs and support further preclinical investigation of calpain inhibition as a potential therapeutic approach.