Blonanserin suppresses epithelial-mesenchymal transition and stemness in glioblastoma cells by targeting PIM1

Abstract

Abstract

Glioblastoma (GBM) is the most prevalent and aggressive primary malignancy of the central nervous system. Epithelial-mesenchymal transition (EMT) and tumor cell stemness are recognized as critical contributors to GBM malignancy and therapeutic resistance. PIM1, a serine/threonine kinase, has been implicated in tumor proliferation, apoptosis resistance, and EMT promotion across multiple cancer types. Blonanserin (BNS), a small molecule with known neuroregulatory and antitumor activities, has not yet been explored in the context of GBM. The effects of BNS on cell viability, migration, invasion, and apoptosis were evaluated in the GBM cell lines SF126 and LN-229. Direct binding between BNS and PIM1 was assessed through molecular docking and cellular thermal shift assays (CETSA). Western blotting and quantitative PCR were employed to analyze the expression of PIM1, EMT markers (Snail, Vimentin, E-cadherin, N-cadherin), and stemness-associated markers (CD44, CD133, Nestin). Tumorsphere formation assays and limiting dilution assays (LDA) were performed to quantify alterations in self-renewal capacity. The role of PIM1 was validated through overexpression rescue experiments. Furthermore, an in vivo xenograft model using LN-229 cells was established to evaluate the therapeutic efficacy of BNS, with tumor tissues analyzed via immunohistochemistry (Ki-67 and Cleaved Caspase-3) and Western blot. BNS treatment markedly suppressed the viability, migration, and invasion of SF126 and LN-229 cells while inducing apoptosis. Molecular docking and CETSA confirmed a direct interaction between BNS and PIM1. BNS administration led to the downregulation of PIM1, which subsequently inhibited EMT (decreased Snail, Vimentin, and N-cadherin; increased E-cadherin) and reduced stemness markers (CD44, CD133, and Nestin). LDA revealed a significant reduction in stem cell frequency following BNS treatment. Overexpression of PIM1 partially reversed these effects, confirming PIM1 as a functional target. In vivo, BNS significantly inhibited tumor growth, characterized by a reduction in Ki-67-positive proliferative cells and an increase in Cleaved Caspase-3-mediated apoptosis. Western blot analysis of tumor tissues further confirmed the modulation of PIM1, EMT, and stemness markers in vivo. This study identifies BNS as a novel inhibitor of glioblastoma EMT and stemness by targeting PIM1. The validation of its efficacy in both in vitro (multiple cell lines) and in vivo models highlights PIM1 as a promising therapeutic target and suggests BNS as a potential candidate for drug repurposing in GBM treatment. 1. Blonanserin (BNS) inhibits epithelial-mesenchymal transition (EMT) and stemness in glioblastoma (GBM) cells, significantly reducing their migratory, invasive, and tumorsphere-forming capacities. 2. BNS directly binds to and downregulates PIM1 expression, providing mechanistic insights into GBM progression inhibition and supporting the potential of PIM1 as a novel therapeutic target and BNS as a candidate for drug repurposing in GBM treatment.