PRSS23 promotes ovarian cancer peritoneal dissemination independent of protease activity

Ovarian cancer metastasizes via peritoneal dissemination, requiring tumor cells to resist detachment-induced cell death (anoikis) in suspension and to reinitiate proliferation after seeding. Because disseminating ovarian cancer cells persist in malignant ascites enriched in extracellular S1 family serine proteases, we surveyed S1 protease gene expression across ovarian cancer cohorts to identify those most associated with poor outcomes. High expression of multiple family members was associated with poor survival, with HTRA3, TMPRSS12, and PRSS23 among the strongest hits. TMPRSS12 transcripts were below the limit of detection in our cell line panel, while HTRA3 depletion showed modest, histotype-dependent effects. In contrast, PRSS23 knockdown reduced proliferation and increased anoikis sensitivity in high-grade serous (HGSOC) and clear cell (OCCC) ovarian carcinoma cell lines, and diminished tumor establishment, dissemination, and ascites in intraperitoneal xenograft models. RNA-seq of PRSS23-depleted cells revealed a conserved program of reduced cell-cycle/DNA repair gene expression with induction of inflammatory and adhesion/epithelial-mesenchymal transition pathways. Endogenous epitope tagging demonstrated that PRSS23 is synthesized as a precursor and secreted as a processed, glycosylated protease homology domain that retains the catalytic triad, yet lacks the canonical Ile16-Asp194 zymogen activation switch. In complementary biochemical assays, PRSS23 showed no detectable serine hydrolase activity in either activity-based probe labeling of conditioned media or chromogenic peptide substrate assays using the recombinant protease domain. Furthermore, pro-tumorigenic phenotypes persisted after mutation of the putative catalytic serine. Together, these findings demonstrate protease-independent PRSS23 function in ovarian cancer peritoneal dissemination, and suggest that PRSS23 may ultimately warrant reclassification as a serine pseudoprotease.