Abstract 2655: Novel stress-induced extracellular regulatory mechanism leads to decreased cellular invasion in prostate cancer

Abstract Background: Cancer cells are exposed to multiple stressors in their environment. The ability to overcome stress involves mechanisms that drive aggressive behavior such as cell motility and subsequent metastasis. Expression and secretion of HSP90α has been linked to many forms of cell stress and plays an important role in cancer cell migration and invasion. It's been shown to do so through co-chaperone mediated activation of extracellular proteases such as MMPs, which are responsible for degradation of the extracellular matrix allowing for cell migration/invasion. Yet how MMPs respond to cellular stress and subsequent levels of extracellular HSP90α is not well understood. We conducted a comprehensive evaluation of the above factors focusing on human prostate cancer (PCa). Methods: Cell stress was induced in PCa cell lines via heat shock (43°C) and serum starvation (SS). Cell lysate and conditioned media (CM) were probed separately to examine intracellular vs. extracellular proteins by Western blot. MMP-2 activation was measured by zymography and gelatin dequenching assays. Cell invasion was measured using scratch wound and Matrigel drop assays. HSP90α knockout (KO) was performed in PC3 cells utilizing CRISPR-Cas9 transient transfection and verified by genomic sequencing and Western blot. Results: We examined human PCa cells in vitro, demonstrating that under stress conditions (i.e., increased temperature and SS), cytosolic and extracellular HSP90α increased. In contrast to the findings of others, we demonstrate that this increase is associated with a decrease in MMP-2 activity. We went on to corroborate the functional relevance of MMP-2 decrease by demonstrating that CM from stressed cells decreased PCa cell invasion compared to media from non-stressed cells. Investigating further we found that decreased MMP-2 activity is mediated by processes dependent on extracellular protease/phosphatase activity. To examine the primary role of HSP90α in these processes, we created HSP90α KO PCa cells. KO cells exhibit normal proliferation and decreased invasion. We are currently using HSP90α KOs to investigate the direct role of extracellular HSP90α in stress-induced MMP-2 responses. Conclusion: While others have examined separate components of this complex regulatory process, to our knowledge this is the first comprehensive evaluation integrating cell stress, the composition and function of secreted factors, how they are regulated and the resultant impact on PCa cell motility. Cell stress in all PCa cells tested led to increased expression and secretion of HSP90α. Surprisingly, this is associated with a decrease in MMP-2 activity. Decreased MMP-2 is due to a novel regulatory mechanism involving extracellular protease/phosphatase activity. HSP90α is involved in PCa cell invasion. We are currently testing its role in stress-related changes of MMP-2. Citation Format: Katelyn L. O'Neill, Johnny Zigmond, Raymond Bergan. Novel stress-induced extracellular regulatory mechanism leads to decreased cellular invasion in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2655.