Dependence on Endocytic Receptor Binding via a Minimal Binding Motif Underlies the Differential Prognostic Profiles of SerpinE1 and SerpinB2 in Cancer

Abstract

Tumor overexpression of urokinase-type plasminogen activator (uPA) and its specific inhibitor SerpinE1 (plasminogen activator inhibitor type-1) correlates with poor prognosis and increased metastatic potential. Conversely, tumor expression of uPA and another specific inhibitor, SerpinB2 (plasminogen activator inhibitor type-2), are associated with favorable outcome and relapse-free survival. It is not known how overexpression of these uPA inhibitors results in such disparate outcomes. A possible explanation may be related to the presence of a proposed low density lipoprotein receptor (LDLR)-binding motif in SerpinE1 responsible for mitogenic signaling via ERK that is absent in SerpinB2. We now show that complementation of such a LDLR-binding motif in SerpinB2 by mutagenesis of two key residues enabled high affinity binding to very LDLR (VLDLR). Furthermore, the VLDLR-binding SerpinB2 form behaved in a manner indistinguishable from SerpinE1 in terms of enhanced uPA-SerpinB2 complex endocytosis and subsequent ERK phosphorylation and cell proliferation; that is, the introduction of the LDLR-binding motif to SerpinB2 was necessary and sufficient to allow it to acquire characteristics of SerpinE1 associated with malignancy. In conclusion, this study defines the structural elements underlying the distinct interactions of SerpinE1 versus SerpinB2 with endocytic receptors and how differential VLDLR binding impacts on downstream cellular behavior. This has clear relevance to understanding the paradoxical disease outcomes associated with overexpression of these serpins in cancer.SerpinE1 (plasminogen activator inhibitor type-1, PAI-1) 4 and SerpinB2 (plasminogen activator inhibitor type-2, PAI-2) are both efficient inhibitors of the urokinase-type plasminogen activator (uPA), a key enzyme in the tissue remodeling process. Combined tumor overexpression of uPA, its receptor (uPAR), and SerpinE1 (serine protease inhibitor E1) is strongly correlated with poor patient prognosis (1-3) and metastatic potential (4 -8). Consequently, uPA and SerpinE1 expression is recommended as an independent prognostic indicator in node-negative breast cancer (9). On the contrary, tumor expression of SerpinB2 is correlated with increased relapse-free survival in breast cancer (and possibly other cancer types) (10). Further, the relationship between outcome and high SerpinB2 levels in node-negative breast cancer is only relevant if uPA levels are also elevated (1, 11), suggesting that the inhibitory relationship between uPA and SerpinB2 mediates this functionality.Binding of uPA to uPAR has been reported to induce the activation of numerous cell type-specific signaling pathways, such as MAPK/ERK, JAK/STAT, Src, focal adhesion kinase, and Rac, thereby affecting cell adhesion, migration, differentiation, and apoptosis (12). Because uPAR is not a transmembrane protein, being linked to the cell surface by a glycosylphosphatidylinositol anchor, uPA/uPAR signaling is mediated by integrins and several other adaptor molecules, including members of the lo...

SerpinB2 regulates stromal remodelling and local invasion in pancreatic cancer

“…As the canonical role of SerpinB2 is the specific, rapid inhibition and clearance of cell surface-bound uPA, 19 , 40 , 41 , 42 this function may also be relevant to the observed effects on PDAC invasion in vivo . To this end we measured uPA proteolytic activity in tumour allograft homogenates.…”

“… 18 Negative regulation of this pathway occurs at several levels, including inhibition and clearance of protease activity by naturally occurring inhibitors, such as SerpinB2 and SerpinE1 (Plasminogen Activator Inhibitors, PAI-2 and PAI-1, respectively). 15 , 19 Elevated SerpinB2 expression has been linked with prolonged survival, decreased metastasis or decreased tumour growth in a number of cancer types, 15 including three small-scale studies in human PDAC. 20 , 21 …”

Plasminogen Activator Inhibitor-2 Plays a Leading Prognostic Role among Protease Families in Non-Small Cell Lung Cancer

“…PAI-2 overexpression was reported to reduce metastasis in xenograft models and decrease the level of uPA and migration in vitro [ 38 , 39 ]. It has recently been demonstrated that the low binding affinity of PAI-2 for endocytic receptors facilitates the clearance of uPA without evoking downstream signaling events, providing a possible explanation for the inhibitory role of PAI-2 in cancer progression [ 28 , 40 ]. The physiological function of PAI-2 may further provide clues regarding its role in cancer progression.…”

“…When either PAI-1 or PAI-2 is present, the ability of uPA to activate plasmin is inhibited, and in turn, ECM degradation is also inhibited. However, uPA-PAI-1 complexes have also been reported to increase MMP-2 and MMP-9 expression level through downstream signaling pathway [ 27 ], whereas the uPA-PAI-2 complex has been shown to facilitate the clearance of uPA without activating downstream signaling [ 28 ]. Moreover, MMP-9 can enhance uPA activity by regulating protease nexin-1 cleavage [ 29 ].…”

Different radiolabelling methods alter the pharmacokinetic and biodistribution properties of Plasminogen Activator Inhibitor Type 2 (PAI-2) forms

“…Active, receptor-bound uPA then efficiently converts any cell surface-bound zymogen plasminogen into the highly active, serine protease plasmin, which promotes tissue degradation and remodelling of the local extracellular environment [4]. The specific uPA inhibitor human plasminogen activator inhibitor type-2 (PAI-2, serpinB2) efficiently inhibits uPAR-bound uPA resulting in proteolytic inhibition and rapid and specific receptor-mediated endocytosis of the uPAR/uPA:PAI-2 complex [5][6][7][8]. PAI-2 is thus able to efficiently and specifically deliver and concentrate attached cytotoxins within targeted cells [5,9].…”