Laminin-332 Is a Substrate for Hepsin, a Protease Associated with Prostate Cancer Progression

Abstract

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the ␤3 chain of Ln-332. N-terminal sequencing identified the cleavage site at ␤3 Arg 245 , in a sequence context (SQLR 245 2LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.Prostate cancer is the second leading cause of cancer death in men in the United States; according to the American Cancer Society, 186,320 new prostate cancer cases and 28,660 deaths from prostate cancer are projected to occur in 2008 (1). This high rate of mortality is largely due to metastasis of the primary tumor (2). For metastasis to occur, primary tumor cells must breach the basement membrane (BM) 3 by degrading extracellular matrix (ECM) molecules to initiate the invasion process (3). Escaped tumor cells interact with neighboring ECM molecules to promote this activity. This interaction sometimes promotes remodeling of the ECM to create a more conducive environment for tumor cell migration and invasion and thereby aids in cancer progression (4). The remodeling of certain ECM molecules has been reported to occur as a direct result of protease processing, which results in increased tumor cell migration and invasion (5-7).Laminin-332 (Ln-332; previously known as laminin-5), an ECM molecule, is an important component of BM (8). It is a trimeric glycoprotein consisting of disulfide-bonded subunits: ␣3, ␤3, and ␥2 polypeptide chains (9). The importance of Ln-332 in BM assembly was established by the discovery of the occurrence of a lethal skin blistering disorder, jun...

ZEB1 Coordinately Regulates Laminin-332 and β4 Integrin Expression Altering the Invasive Phenotype of Prostate Cancer Cells*

“…Hao et al reported that LAMB3 and LAMC2 mRNA could be detected in prostate cancer tissue, suggesting that the mechanism is post-transcriptional (23). Cleavage of ␤3 and ␥2 proteins by matrix metalloproteases and hepsin has also been described (7,24,25). These results may appear at odds with those reported here; however, more than one mechanism may account for the reduction of laminin-332 immunoreactivity in prostate cancer.…”

Proteolytic Activation of Pro-Macrophage-Stimulating Protein by Hepsin

“…Hepsin activates pro-HGF by cleavage at KQLR#V (19,34). The scissile peptide sequence of pro-MSP, SKLR#V, has strong similarity to that of pro-HGF as well as to the SQLR#L cleavage sequence of the recently identified hepsin substrate laminin-332 (36), suggesting that pro-MSP could potentially be a hepsin substrate. Here, we provide several lines of evidence to show that hepsin is an efficient pro-MSP activator that generates the biologically active 2-chain a/b-heterodimeric MSP signaling molecule.…”

“…Moreover, gene expression analyses have also implicated hepsin in ovarian cancer (30), renal cell carcinoma (31,32), and endometrial cancer (33). A putative function of hepsin in tumor progression could be related to its enzymatic activity toward the macromolecular substrates pro-HGF (19,34), pro-uPA (35), and laminin-332 (36). Additional substrates that have been identified are coagulation factors (37) and pro-prostasin (38).…”

Type II Transmembrane Serine Proteases

“…Overexpression of hepsin in basal prostate epithelial cells of mice leads to the disorganization of the basement membrane (47). Substrates linked to carcinogenesis that can be cleaved by hepsin ex vivo are coagulation factor VII, pro-hepatocyte growth factor, laminin 332, and pro-urokinase plasminogen activator (46,49).…”