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Ras proteins play a causal role in human cancer by activating multiple pathways that promote cancer growth and invasion. However, little is known about how Ras induces the first diagnostic features of invasion in solid tumors, including loss of epithelial integrity and breaching of the basement membrane (BM). In this study, we found that oncogenic Ras strongly promotes the activation of hepsin, a member of the hepsin/TMPRSS type II transmembrane serine protease family. Mechanistically, the Ras-dependent hepsin activation was mediated via Raf-MEK-ERK signaling, which controlled hepsin protein stability through the heat shock transcription factor-1 stress pathway. In Ras-transformed three-dimensional mammary epithelial culture, ablation of hepsin restored desmosomal cell-cell junctions, hemidesmosomes, and BM integrity and epithelial cohesion. In tumor xenografts harboring mutant KRas, silencing of hepsin increased local invasion concomitantly with accumulation of collagen IV. These findings suggest that hepsin is a critical protease for Ras-dependent tumorigenesis, executing cell-cell and cell-matrix pathologies important for early tumor dissemination. SIGNIFICANCE: These findings identify the cell-surface serine protease hepsin as a potential therapeutic target for its role in oncogenic Ras-mediated deregulation of epithelial cell-cell and cell-matrix interactions and cohesion of epithelial structure., (©2021 American Association for Cancer Research.)

Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.