PSMD2 Suppression Disrupts Autophagy Turnover and Creates an Autophagy-Dependent Vulnerability in ER+ Breast Cancer

Background: The ubiquitin-proteasome system (UPS) and autophagy cooperate to maintain protein homeostasis, yet how these pathways interact to regulate breast cancer stem cells (BCSCs) remains unclear. PSMD2, a 19S regulatory subunit of the proteasome, has been linked to poor outcomes in several cancers, but its role within the autophagy-proteostasis network in ER⁺ breast cancer has not been defined. Here, we examined how PSMD2 influences CSC maintenance and drug response through UPS-autophagy crosstalk and explored how its suppression alters autophagy regulation and CSC vulnerability in ER⁺ breast cancer. Methods: Stable PSMD2 knockdown (ShPSMD2) lines were generated in ER⁺ breast cancer cells (MCF-7, ZR-75-1). Autophagy was modulated using hydroxychloroquine (HCQ). Western blotting assessed CSC-related markers (SOX2, OCT4, NANOG), EMT markers (E-cadherin, N-cadherin, Vimentin), and autophagy proteins (LC3B-II, p62, p-mTOR). Functional assays, including MTT viability, colony formation, migration, and invasion, were performed to evaluate PSMD2-dependent phenotypes. Mammosphere and viability assays assessed CSC formation and HCQ sensitivity. Tumor growth and metastasis were tested using mammary fat-pad and tail-vein xenografts in NSG mice. Results: PSMD2 depletion reduced CSC frequency, mammosphere formation, and colony formation, with decreased stemness markers (SOX2, OCT4, NANOG) and reversal of EMT features, shown by lower N-cadherin, Vimentin, and Snail. Loss of PSMD2 resulted in LC3B-II and p62 accumulation with mTOR suppression, indicating impaired autophagy turnover and disrupted proteostasis. PSMD2-deficient cells displayed reduced migration and invasion and were more sensitive to HCQ-induced cytotoxicity. HCQ treatment further lowered PSMD2 protein levels in a dose-dependent manner, implying feedback destabilization of the proteasome under lysosomal stress. This correlated with loss of CD44⁺/CD24⁻ CSC-like populations and reduced sphere-forming ability. In xenografts, PSMD2 knockdown suppressed tumor growth and lung, liver metastasis, suggesting that PSMD2 is a critical node linking proteostasis to metastatic progression. Conclusions: PSMD2 depletion disturbs UPS-autophagy coordination, leading to loss of proteostasis and CSC depletion in ER⁺ breast cancer. This insight reveals PSMD2 as a key regulator of proteolytic balance and a potential target for autophagy-based therapeutic strategies.