PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the sternness of breast cancer stern cells

Objective: Glycogen synthase kinase 3 beta (GSK3 beta) has been recognized as a suppressor of Wnt/beta-catenin signaling, which is critical for the stem ness maintenance of breast cancer stem cells. However, the regulatory mechanisms of GSK3 beta protein expression remain elusive. Methods: Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3 beta, and to characterize the interactions of GSK3 beta, heat shock protein 90 (Hsp90), and co-chaperones. The role of PGK1 in Hsp90 chaperoning GSK3 beta was evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90 alpha, and by performing a series of binding assays with bacterially purified proteins and clinical specimens. The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays. Results: We showed that GSK3 beta was a client protein of Hsp90. Hsp90, which did not directly bind to GSK3 beta, interacted with phosphoglycerate kinase 1 via its C-terminal domain, thereby facilitating the binding of GSK3 beta to Hsp90. GSK3 beta-bound PGK1 interacted with Hsp90 in the "closed" conformation and stabilized GSK3 beta expression in an Hsp90 activity-dependent manner. The Hsp90 inhibitor, 17-AAG, rather than HDN1, disrupted the interaction between Hsp90 and PGK1, and reduced GSK3 beta expression, resulting in significantly reduced inhibition of beta-catenin expression, to maintain the stemness of breast cancer stem cells. Conclusions: Our findings identified a novel regulatory mechanism of GSK3 beta expression involving metabolic enzyme PGK1-coupled Hsp90, and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3 beta expression.