Midasin inhibition disrupts the sumoylation axis to impair mammosphere stability through alteration of transcriptional network

Endocrine resistance remains a major barrier to effective treatment of estrogen receptor-positive (ER+) breast cancer, necessitating identification of molecular drivers that sustain tumor growth, plasticity, and cancer stem cell (CSC) associated phenotypes. Midasin (MDN1), a key regulator of ribosome biogenesis, is elevated in endocrine-resistant tumors, yet its contribution to aromatase inhibitor (AI) resistance is not well defined. Here, we examined differential responses to MDN1 inhibition in AI-sensitive AC-1 and AI-resistant LTLT-Ca cells cultured in 2D and 3D. Basal characterization showed higher aromatase (1.2-fold) and ERalpha (1.6-fold) expression in AC-1 cells, while LTLT-Ca cells exhibited 7-fold and 3.85-fold increase in EGFR and HER2, respectively, with further EGFR and HER2 amplification (up to 4-fold) in 3D mammosphere. Although MDN1 levels were higher in AC-1 cells under 2D conditions, MDN1 was enriched in LTLT-Ca mammosphere, linking elevated ribosome biogenesis to the resistant, stem-like phenotype. Treatment with the MDN1 inhibitor, Rbin-2 (18 µM) significantly reduced MDN1 expression and disrupted spheroid architecture in both models. AC-1 spheroids displayed moderate size reduction (1010.5 µm² to 731.7 µm² by day 12), whereas larger LTLT-Ca spheroids (1204.5 µm²) became fragmented and irregular (1539.3 µm²), indicating structural collapse rather than shrinkage. Acridine Orange/Ethidium Bromide staining revealed a 65% decrease in the live-to-dead fluorescence ratio in AC-1 versus 14% in LTLT-Ca, consistent with extensive degeneration in sensitive cells and instability in resistant spheroids. Spheroid invasion assays showed modest reduction in matrix outgrowth in both AC-1 and LTLT-Ca after Rbin-2 exposure. LTLT-Ca mammospheres expressed elevated OCT4 and SOX2, which decreased by 49% and 45% respectively, following MDN1 inhibition, implicating MDN1 in sustaining CSC transcriptional networks. Analysis of SUMOylation dynamics revealed intrinsically higher SUMO2/3 in resistant cells, with a further increase upon Rbin-2 treatment, consistent with decreased midasin associated cell death. Conversely, the deSUMOylase SENP3, also elevated in resistant cells, was reduced by Rbin-2 in both lines. This increase in SUMO2/3 coupled with decreased SENP3 reflects MDN1-dependent disruption of homeostasis and aligns with reduced ALDH1A1 and loss of CSC properties. Collectively, these findings identify a SUMO2/3-SENP3-MDN1 regulatory axis that supports, stemness and invasiveness in AI-resistant breast cancer, and demonstrate that MDN1 inhibition disrupts this axis to impair mammosphere integrity, attenuate CSC programs, and suppress invasion. This work establishes MDN1 as a promising therapeutic vulnerability for overcoming AI resistance.