PRSS3

PRSS3 (mesotrypsin) is a serine protease that drives tumor cell invasion, metastasis, and survival primarily through proteolytic activation of protease-activated receptors and degradation of extracellular inhibitors. Its catalytic activity is required for invasion: active PRSS3 cleaves PAR1 and PAR2, engaging ERK/VEGF signaling to promote proliferation and metastasis, and directly degrades the Kunitz-type inhibitor TFPI-2 to facilitate endothelial cell migration (PMID:20947888, PMID:23258495, PMID:27161730, PMID:37395651). PRSS3-V1 interaction with PAR2 suppresses ferroptosis through maintenance of SLC7A11/GPX4, while the opposing isoform PRSS3-V2 is oncogenic and PRSS3-V1/-V3/-V4 are tumor-suppressive in hepatocellular carcinoma, with isoform-specific expression governed by intragenic CpG methylation controlled by the UHRF1/DNMT1–MZF1 axis (PMID:41046946, PMID:35480112, PMID:37250150). In keratinocytes, mesotrypsin processes pro-filaggrin and remodels cell junctions and the actin cytoskeleton, indicating physiological roles beyond cancer (PMID:38811772).

1. 2010 Medium

   Establishing that PRSS3 promotes pancreatic cancer through a defined signaling axis answered how a secreted trypsin isoform could drive metastasis: PRSS3 upregulates VEGF via PAR1-mediated ERK activation.

   Evidence PRSS3 overexpression/silencing in pancreatic cancer cells with VEGF ELISA, ERK inhibitor treatment, and in vivo metastasis models

   PMID:20947888

   Open questions at the time

   • Whether PRSS3 directly cleaves PAR1 or acts indirectly was not resolved
   • Contribution of individual PRSS3 splice variants was not examined
   • In vivo specificity of the PAR1-ERK-VEGF axis was not validated with receptor-null models
2. 2012 High

   Demonstrating that catalytic activity is required for PRSS3-driven invasion resolved whether the protease acts enzymatically or as a scaffold: a catalytically dead mutant failed to promote invasion, and a mesotrypsin-specific inhibitor blocked it.

   Evidence Catalytically inactive mutant, recombinant active mesotrypsin, mesotrypsin-specific inhibitor, Matrigel invasion and 3D culture in prostate cancer cells, orthotopic mouse model

   PMID:23258495

   Open questions at the time

   • The direct proteolytic substrate mediating invasion was not identified
   • Whether the same catalytic requirement applies in other cancer types was untested
3. 2016 Medium

   Identification of TFPI-2 as a direct substrate revealed how PRSS3 remodels the tumor microenvironment: cleavage of this extracellular matrix-associated inhibitor liberates endothelial cell migration.

   Evidence Cell-free proteolytic cleavage assay plus PRSS3 siRNA in tumor-derived endothelial cells with wound healing migration readout

   PMID:27161730

   Open questions at the time

   • Cleavage site on TFPI-2 was not mapped
   • In vivo relevance of TFPI-2 degradation to angiogenesis was not tested
   • Other extracellular substrates were not surveyed
4. 2017 Medium

   Paradoxically, PRSS3 overexpression in hepatocellular carcinoma suppressed proliferation and invasion via G1/S arrest and MEK/ERK inhibition, raising the question of context-dependent or isoform-dependent function.

   Evidence PRSS3 overexpression and siRNA knockdown in HCC cell lines with cell cycle, cyclin/CDK, MMP2, and pERK western blot readouts

   PMID:28844099

   Open questions at the time

   • Which PRSS3 isoform(s) mediated tumor suppression was not delineated
   • How PRSS3 both activates and suppresses ERK in different contexts was unexplained
5. 2022 Medium

   Resolution of the oncogene-versus-tumor-suppressor paradox came from dissecting individual splice variants: PRSS3-V2 is oncogenic while V1/V3/V4 are tumor-suppressive, with expression governed by site-specific CpG methylation at the PRSS3 promoter.

   Evidence CRISPR/Cas9 deletion of PRSS3 followed by individual isoform re-expression in HCC cells, methylation analysis

   PMID:35480112

   Open questions at the time

   • Structural basis for opposing isoform functions was not determined
   • Whether isoform-specific methylation patterns hold across cancer types was untested
6. 2023 Medium

   The epigenetic machinery controlling isoform-specific expression was mapped: UHRF1/DNMT1-mediated intragenic CpG methylation blocks MZF1 binding, selectively silencing tumor-suppressive PRSS3-V3 in lung cancer.

   Evidence Site-specific methylation in BALF and tumor tissues, ChIP for MZF1 binding, UHRF1/DNMT1 complex characterization, demethylating agent treatment with isoform-specific qPCR

   PMID:37250150

   Open questions at the time

   • Whether other transcription factors co-regulate isoform expression was not explored
   • In vivo rescue of V3 expression by demethylation and its effect on tumor growth was not shown
7. 2023 High

   Direct cleavage of PAR2 by PRSS3 was demonstrated with temporal resolution, connecting mechanical shear stress to a PRSS3→PAR2→Gαi→Src-ERK/p38/JNK→EMT positive feedback loop in circulating tumor cells.

   Evidence Microfluidic shear stress system, PAR2 N-terminal cleavage assay, transcriptomics, Gαi pathway dissection, in vivo metastasis assays in lung cancer

   PMID:37395651

   Open questions at the time

   • The precise PAR2 cleavage site for PRSS3 versus other trypsin-like proteases was not compared
   • Whether PAR1 and PAR2 are cleaved with different kinetics or specificity by PRSS3 was not addressed
8. 2024 Medium

   A non-cancer physiological role emerged: mesotrypsin processes pro-filaggrin in keratinocytes, remodeling the actin cytoskeleton, enhancing E-cadherin adhesion, and promoting tight junction formation.

   Evidence Venus-mesotrypsin fusion expression in HaCaT keratinocytes, immunofluorescence for junctional markers, proliferation assays

   PMID:38811772

   Open questions at the time

   • Whether filaggrin processing is the primary substrate event or a bystander cleavage is unclear
   • Relevance to in vivo epidermal barrier function was not tested
   • Catalytic requirement was not confirmed with an inactive mutant in this system
9. 2025 Medium

   The PRSS3-V1/PAR2 interaction was shown to suppress ferroptosis by maintaining SLC7A11 and GPX4 levels, linking protease-activated receptor signaling to redox homeostasis and iron metabolism in breast cancer.

   Evidence Co-immunoprecipitation of PRSS3-V1 and PAR2, ferroptosis markers (MDA, ROS, lipid peroxidation, labile iron), rescue by PAR2 agonist and ferrostatin, NLRP3 inflammasome activation, in vivo xenograft

   PMID:41046946

   Open questions at the time

   • Whether PRSS3-V1 cleaves PAR2 or signals through a non-catalytic interaction was not resolved
   • The mechanism linking PAR2/ERK to SLC7A11/GPX4 transcription or stability was not dissected
   • Whether other PRSS3 isoforms similarly regulate ferroptosis was not tested

• Key unresolved questions include the full substrate repertoire of mesotrypsin, the structural basis for opposing isoform functions, and whether PRSS3's physiological role in epithelial barrier maintenance connects to its tumor biology.
• No unbiased substrate profiling (e.g., TAILS, N-terminomics) has been performed
• Structural models for individual PRSS3 splice variants are lacking
• Genetic models (PRSS3-knockout mice) with phenotypic characterization have not been reported in this timeline