Zigmond, J. W. | bioRxiv Science

bioRxiv2026

Integrative Proteomic Analysis Implicates Inhibition of Intracellular Protein Trafficking in Therapy-Induced Migrastasis in Prostate Cancer

BackgroundDysregulated cell migration leading to metastasis remains the primary cause of cancer-related mortality. It has been challenging to understand how cells regulate migration. We have previously created the first selective inhibitor of cell migration, KBU2046. Here, we use it as a probe to identify regulatory processes. MethodsMetastatic and primary human prostate cancer cells were treated for different times and at different concentrations with KBU2046. Immunofluorescent microscopy examined protein localization in cells. Label-free mass spectrometry (MS) was performed on total cell proteins, Tandem Mass Tag (TMT) labeling MS was used on membrane fractions, and temporal phosphoproteomic profiling. Results were analyzed with a suite of bioinformatic tools. ResultsKBU2046-induced migrastasis is associated with the accumulation of activated integrin {beta}1 into focal adhesions. Whole-cell proteomics demonstrated suppression of processes that mediate intracellular protein trafficking and increases in mitochondrial energy-generation signatures. Evaluation of the membrane fraction identified increases in membrane repair and maintenance processes and decreases in those that drive motility. Temporal- and concentration-dependent phosphoproteomic profiling revealed that KBU2046 initiates a dynamic, cascading sequence of transient signaling waves rather than a static block. ConclusionsKBU2046-induced migrastasis appears to operate through spatial decoupling rather than structural degradation. By restricting the intracellular trafficking machinery required for receptor recycling, KBU2046 limits focal adhesion turnover, providing a correlative framework to inhibit metastatic dissemination independent of direct cytotoxicity. O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/736165v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1efd74dorg.highwire.dtl.DTLVardef@1202106org.highwire.dtl.DTLVardef@581ed3org.highwire.dtl.DTLVardef@14fdcfb_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract C_FIG

cancer biology↗

bioRxiv2025

HSP90α and KLK6 Co-Regulate Stress-Induced Prostate Cancer Cell Motility

BackgroundProstate cancer (PCa) metastasis is reliant on the activity of proteases, such as matrix metalloproteinase-2 (MMP-2). While increased extracellular heat shock protein 90 (eHSP90) has been linked to increased MMP-2 activity, this has not been examined in the context of cellular stress. MethodsWe examined stress-induced eHSP90 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. HSP90 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. ResultsStress increased eHSP90 in 4 of 4 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 HSP90 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. ConclusionWe 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 eHSP90 is required for the rescue of MMP-2 activity once KLK6 is neutralized.

cancer biology↗