Extracellular Hsp90 Binds to and Aligns Collagen-1 to Enhance Breast Cancer Cell Invasiveness

SIMPLE SUMMARY: Breast cancer cells secrete Hsp90, a protein that, inside of cells, regulates the function of hundreds of proteins, but outside of cells, extracellular Hsp90 (eHsp90) can activate a subset of proteins that promote invasion, the first step of metastasis. Blocking eHsp90 in mouse models inhibits metastasis, and we sought to understand how this occurs. Prior studies have predominantly focused on eHsp90 in cancer invasion within the immediate vicinity of the primary tumor, specifically its role in invading outside the epithelial compartment. However, eHsp90’s role in cancer invasion across the extended connective tissue after the cells have crossed the boundary of the epithelial compartment remains unknown. We show here that eHsp90 directly binds to and aligns Collagen-1 fibers, a major structural component of connective tissues, which, when aligned, form highways that allow efficient cancer migration. Our study suggests that the Hsp90 dimer, in its open state, binds to Collagen-1 molecules to align the fibers, which results in enhanced breast cancer invasion through the Collagen-1 matrix. Knowing this could help us propose experiments to test eHsp90 inhibitors for therapeutically targeting metastatic breast cancer. ABSTRACT: Cancer cell-secreted eHsp90 binds and activates proteins in the tumor microenvironment crucial in cancer invasion. Therefore, targeting eHsp90 could inhibit invasion, preventing metastasis—the leading cause of cancer-related mortality. Previous eHsp90 studies have solely focused on its role in cancer invasion through the 2D basement membrane (BM), a form of extracellular matrix (ECM) that lines the epithelial compartment. However, its role in cancer invasion through the 3D Interstitial Matrix (IM), an ECM beyond the BM, remains unexplored. Using a Collagen-1 binding assay and second harmonic generation (SHG) imaging, we demonstrate that eHsp90 directly binds and aligns Collagen-1 fibers, the primary component of IM. Furthermore, we show that eHsp90 enhances Collagen-1 invasion of breast cancer cells in the Transwell assay. Using Hsp90 conformation mutants and inhibitors, we established that the Hsp90 dimer binds to Collagen-1 via its N-domain. We also demonstrated that while Collagen-1 binding and alignment are not influenced by Hsp90’s ATPase activity attributed to the N-domain, its open conformation is crucial for increasing Collagen-1 alignment and promoting breast cancer cell invasion. These findings unveil a novel role for eHsp90 in invasion through the IM and offer valuable mechanistic insights into potential therapeutic approaches for inhibiting Hsp90 to suppress invasion and metastasis.