The serine protease hepsin is frequently overexpressed in human prostate cancer (PCa) and is associated with matrix degradation and PCa progression in mice. Curiously, low expression of hepsin is associated with poor survival in different cancer types, and transgenic overexpression of hepsin leads to loss of viability in various cancer cell lines. Here, by comparing isogenic transfectants of the PCa cell line PC-3 providing inducible overexpression of wild-type hepsin (HPN) vs. the protease-deficient mutant HPN
S353A
, we were able to attribute hepsin-mediated tumor-adverse effects to its excess proteolytic activity. A stem-like expression signature of surface markers and adhesion molecules, Notch intracellular domain release, and increased pericellular protease activity were associated with low expression levels of wild-type hepsin, but were partially lost in response to overexpression. Instead, overexpression of wild-type hepsin, but not of HPN
S353A
, induced relocalization of the protein to the cytoplasm, and increased autophagic flux in vitro as well as LC3B punctae frequency in tumor xenografts. Confocal microscopy revealed colocalization of wild-type hepsin with both LC3B punctae as well as with the autophagy cargo receptor p62/SQSTM1. Overexpression of wild type, but not protease-deficient hepsin induced expression and nuclear presence of CHOP, indicating activation of the unfolded protein response and ER-associated protein degradation (ERAD). Whereas inhibitors of ER stress and secretory protein trafficking slightly increased viability, combined inhibition of the ubiquitin-proteasome degradation pathway (by bortezomib) with either ER stress (by salubrinal) or autophagy (by bafilomycin A1) revealed a significant decrease of viability during overexpression of wild-type hepsin in PC-3 cells. Our results demonstrate that a precise control of Hepsin proteolytic activity is critical for PCa cell fate and suggest, that the interference with ERAD could be a promising therapeutic option, leading to induction of proteotoxicity in hepsin-overexpressing tumors.
Smart nanocarriers for the transport of drugs to tumor cells are
nowadays of great interest for treating cancer. The use of enzymatic
stimuli to cleave peptide-based drug nanocapsules for the selective
release of nanocapsule cargo in close proximity to tumor cells opens
new possibilities in cancer research. In the present work, we demonstrate
a methodology for finding and optimizing cleavable substrate sequences
by the type II transmembrane serine protease hepsin, which is highly
overexpressed in prostate cancer. The design and screening of combinatorial
libraries in silico against the binding cavity of hepsin allow the
identification of a panel of promising substrates with high-calculated
docking scores. In vitro screening verifies the predictions and showed
that all substrates are cleaved by hepsin with higher efficiency than
the literature known hepsin substrate RQLR↓VVGG. The introduction
of d-amino acids on a selected peptide with the highest catalytic
efficiency (k
cat/K
m) renders it resistant to cleavage by plasma or serum while
maintaining their susceptibility to hepsin.
An efficient nanoparticulate drug carrier intended for chemotherapy based on intravenous administration must exhibit a long enough blood circulation time, a good penetrability into the tumour volume, as well as...
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