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INTERNATIONAL CONGRESS OF

CancerGenetics

• siRNA-Mediated Knockdown of BUB1 Impairs Growth and Metastatic Potential in Pheochromocytoma Cells

• marziehsadat ahmadi,^1,* seyed jalal zargar,²
  1. university of tehran
  2. university of tehran
  
  
  
• Introduction: Pheochromocytoma and paraganglioma (PPGLs) are rare neuroendocrine tumors with limited therapeutic options in their metastatic form. While recurrent germline and somatic mutations contribute to tumorigenesis, additional vulnerabilities such as non-oncogene addiction (NOA) genes have emerged as critical dependencies in aggressive disease. Among these, budding uninhibited by benzimidazoles 1 (BUB1), a mitotic checkpoint kinase, was identified as one of the most upregulated genes in metastatic PPGL. Although BUB1 has been associated with chromosomal instability in other cancers, its functional contribution to PPGL remained unexplored. Here, we specifically investigated the role of BUB1 using RNA interference, focusing on the effects of siRNA-mediated knockdown on tumor cell viability, proliferation, apoptosis, migration, and invasion.
• Methods: The human pheochromocytoma cell line hPheo1, characterized by high BUB1 expression, was used for functional assays, whereas MTT and PC12 cells served as low-expressing comparators. Four independent siRNAs were tested, with two (siBUB1#2 and siBUB1#3) achieving reliable knockdown. Suppression was verified by Western blotting. Growth-related endpoints were assessed using sulforhodamine B (SRB) viability assays and colony formation assays. Cell death was quantified with a real-time Annexin V luminescence approach, while DNA content and cell cycle alterations were measured by propidium iodide flow cytometry. Mitotic fidelity was evaluated by micronuclei counts and phosphorylation status of histone H2A-T120. To probe metastatic properties, wound healing assays and Transwell migration/invasion assays were performed, complemented by Western blotting for epithelial-to-mesenchymal transition (EMT) markers.
• Results: Targeted depletion of BUB1 significantly reduced viability and colony-forming capacity of hPheo1 cells, demonstrating its necessity for tumor cell growth. Apoptosis was markedly increased following knockdown, aligning with impaired survival signaling. Cell cycle analysis revealed G2/M accumulation and a sub-G1 apoptotic fraction, consistent with checkpoint disruption. Micronuclei formation was enhanced, and loss of H2A-T120 phosphorylation confirmed compromised BUB1 kinase function. Beyond proliferation, silencing BUB1 strongly inhibited motility and invasiveness: scratch assays showed delayed wound closure, and Transwell experiments documented up to 90% reduction in migratory and invasive activity. Concomitantly, mesenchymal markers N-cadherin and vimentin were downregulated, implicating BUB1 in EMT regulation and metastatic competence.
• Conclusion: Our study identifies BUB1 as a central dependency in pheochromocytoma cells. Its depletion suppresses tumor cell growth, triggers apoptosis, destabilizes chromosome segregation, and curtails migration and invasion by interfering with EMT. Together with the phenotypic overlap observed under pharmacological inhibition with BAY1816032, these findings support BUB1 as a promising therapeutic target in advanced PPGL
• Keywords: Pheochromocytoma; Paraganglioma; BUB1; siRNA; Non-oncogene addiction; Apoptosis; Cell cycle; EMT;