HSP90α and KLK6 Coregulate Stress-Induced Prostate Cancer Cell Motility

Highlights: What are the main findings? Cellular stress leads to increased HSP90α, yet decreased MMP-2-dependent cell motility in PCa. Decreased MMP-2 activity is due to stress-induced extracellular KLK6 and can be rescued by extracellular HSP90α upon KLK6 inhibition. What are the implications of the main findings? This study identifies a novel stress-response pathway that regulates MMP-2 activity and cell motility. Enhanced understanding of cancer cell motility in the context of stress will better inform patient risk stratification, tailored care, and development of precision therapeutics. Prostate cancer (PCa) metastasis is reliant on the activity of proteases, such as matrix metalloproteinase-2 (MMP-2). While increased extracellular heat shock protein 90α (eHSP90α) has been linked to increased MMP-2 activity, this has not been examined in the context of cellular stress. We examined stress-induced eHSP90α in human prostate cell lines by immunoblot. Fluorometric gelatin dequenching and zymography assays measured MMP activity. Wound healing and Matrigel drop invasion assays were used to quantify cell motility. HSP90α knockout (KO) cells were established with CRISPR/Cas9. Proteases were profiled with molecular inhibitors and protein arrays and validated by siRNA knockdown, immunoblot, and motility assays. Stress increased eHSP90 in four out of four human prostate cell lines examined. Surprisingly, it concurrently decreased MMP-2 activity. The functional relevance of this was demonstrated when conditioned media from stressed cells decreased the motility of non-stressed cells. Screening for protease inhibitors that would rescue stress-induced decreases in MMP-2 activity identified a single serine protease inhibitor: aprotinin. Yet rescue with aprotinin was lost in HSP90α KO cells. A protease array identified stress-induced increases in kallikrein-related peptidase 6 (KLK6). Knockdown of KLK6 rescued stress-induced MMP-2 activity and cell motility. In conclusion, we identify a novel stress-induced extracellular network that regulates MMP-2 activity and cell motility. We identified KLK6 as a stress-induced extracellular protease leading to decreased MMP-2 activity and cellular invasion, while eHSP90α is required for the rescue of MMP-2 activity once KLK6 is neutralized.