Abstract 11: Overexpression of midasin drives cancer stem cell dynamics in <i>ESR1</i> mutant breast cancer

Abstract While most cases of estrogen receptor-positive (ER+) breast cancer initially respond to endocrine therapy, many women acquire resistance. Two major hurdles hampering the response of metastatic breast cancer patients to endocrine therapy is constitutively active somatic mutations in the ER ligand binding domain (LBD) and the presence of cancer stem cells (CSCs). We previously identified midasin, a novel ribosomal protein, which was significantly overexpressed in letrozole-resistant MCF-7 cells enriched with CSCs. However, the consequences of midasin (MDN1) expression, stemness, and ESR1 mutations are unclear. We hypothesize that ESR1 mutations along with MDN1 cooperate to alter the microenvironment and position breast cancer cells for a survival advantage through increased stemness. Here, the objective is to evaluate the significance of midasin inhibition on CSCs formation in MCF-7 cells with ESR1 LBD point mutations. RNA-Seq studies were conducted in MCF-7 parental cells (MCF-7LUC) and cells containing ESR1 LBD point mutations (MCF-7Y537S and MCF-7D538G) and MDN1 levels were similar. Western blots were conducted and MCF-7D538G cells expressed the highest level of MDN1 followed by MCF-7Y537S and MCF-7LUC. ER levels were highest in the MCF-7D538G followed by the MCF-7LUC and MCF-7Y537S. Viability studies were performed using 1-12 µM ribozinoindole-2 (Rbin-2) (ie., midasin inhibitor) and while all cells were responsive, the MCF-7LUC and MCF-7D538G cells were more sensitive. After 48 hours of Rbin-2 treatment MDN1 protein expression was decreased in all cell lines. Wound healing assays were conducted and the MCF-7D538G cells were the most migratory followed by the MCF-7LUC and MCF-7Y537S (ie., motility was inhibited by 48-55%). As there is a functional link between stemness and motility, mammosphere formation assay were performed. Interestingly, the MCF-7D538G cells formedlarge, dense spheres with an average length of (480.3 ± 7.4 µm), followed bythe MCF-7Y537S which formed large and hollow spheres (435.7 ± 0.52 µm), and the MCF-7LUC cells which formed small, dense spheres (372. ± 2.7µm). Rbin-2 treatment induced a 57%decrease in MCF-7LUC mammosphere formation, while the ESR1mutant mammospheres disintegrated, and sizes ranged from (101.8 ± 1.52 - 112.5 ±2.2 µm). Western blot studies were conducted in the mammospheres, and both MCF-7D538G and MCF-7LUC cells were CD44high/CD24low and the MCF-7Y537S cells were CD44low/CD24low. The cells were treated with Rbin-2 and CD44 expression was decreased while CD24 was unaltered. Likewise Rbin-2 treatment inhibited the mammosphere expression of MDN1 by 69-86%. Based on the preliminary results, higher levels of midasin and ER are associated with denser mammosphere that exhibit more stemness as indicated by the CD44high/CD24low profile. Together, this may contribute to the highly migratory phenotype and survival advantaged which may be reversed by targeting midasin. Citation Format: Mounika Pamukuntla, Manasa Kotina, Bipika Banjara, Afia Ohemeng, Alicia Hudson, A Michael Davidson, Jullian Pope, Selina Darlin-Reed, Simak Ali, Syreeta L. Tilghman. Overexpression of midasin drives cancer stem cell dynamics in ESR1 mutant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 11.