Non-canonical Disulfide Isomerases are Strongly Selective, Understudied Targets for Cancer Therapy

Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with many cases driven by hyperactivation of the Wnt/β-catenin signaling pathway. This pathway fuels tumor growth by promoting cell proliferation, epithelial-mesenchymal transition, cancer stem cell renewal, and resistance to chemotherapy. Despite its importance in cancer progression, effective inhibitors of this pathway remain limited, highlighting an urgent need for novel therapeutic strategies. A critical component of the Wnt/β-catenin pathway is the receptor proteins LRP5 and LRP6, which must be properly folded and functional to activate the pathway. Our laboratory identified a group of compounds termed disulfide disrupting agents (DDAs), which are inhibitors of several Protein Disulfide Isomerases (PDIs), including AGR2, AGR3, ERp44, and PDIA1. DDAs also disrupt the folding of LRP5 and LRP6, effectively shutting down Wnt signaling. MESD, an ~30 kDa specialized chaperone protein, is essential for the proper folding and transport of LRP5 and LRP6. Immunoblot analysis under non-reducing conditions shows that DDA treatment increases the levels of an MESD immunoreactive band that migrates at ~80 kDa. It is therefore possible that PDIs regulate LRP5 and LRP 6 folding indirectly by regulating the disulfide bonding state of MESD. This study aims to: (1) identify the ~50 kDa protein disulfide bound to MESD, which correlates with its inactivation after DDA treatment, and (2) demonstrate the functional importance of the interaction of MESD with this unknown protein. These findings will enhance our understanding of DDA function and advance therapeutic development for Wnt-driven CRC.