SERPINE1: Role in Cholangiocarcinoma Progression and a Therapeutic Target in the Desmoplastic Microenvironment.

A heterogenous population of inflammatory elements, other immune and nonimmune cells and cancer-associated fibroblasts (CAFs) are evident in solid malignancies where they coexist with the growing tumor mass. In highly desmoplastic malignancies, CAFs are the prominent mesenchymal cell type in the tumor microenvironment (TME), where their presence and abundance signal a poor prognosis. CAFs play a major role in the progression of various cancers by remodeling the supporting stroma into a dense, fibrotic matrix while secreting factors that promote the maintenance of cancer stem-like characteristics, tumor cell survival, aggressive growth and metastasis and reduced sensitivity to chemotherapeutics. Tumors with high stromal fibrotic signatures are more likely to be associated with drug resistance and eventual relapse. Identifying the molecular underpinnings for such multidirectional crosstalk among the various normal and neoplastic cell types in the TME may provide new targets and novel opportunities for therapeutic intervention. This review highlights recent concepts regarding the complexity of CAF biology in cholangiocarcinoma, a highly desmoplastic cancer. The discussion focuses on CAF heterogeneity, functionality in drug resistance, contributions to a progressively fibrotic tumor stroma, the involved signaling pathways and the participating genes.

Cancer-Associated Fibroblasts: Mechanisms of Tumor Progression and Novel Therapeutic Targets.

Cancer-associated fibroblasts (CAFs) are a heterogenous population of stromal cells found in solid malignancies that coexist with the growing tumor mass and other immune/nonimmune cellular elements. In certain neoplasms (e.g., desmoplastic tumors), CAFs are the prominent mesenchymal cell type in the tumor microenvironment, where their presence and abundance signal a poor prognosis in multiple cancers. CAFs play a major role in the progression of various malignancies by remodeling the supporting stromal matrix into a dense, fibrotic structure while secreting factors that lead to the acquisition of cancer stem-like characteristics and promoting tumor cell survival, reduced sensitivity to chemotherapeutics, aggressive growth and metastasis. Tumors with high stromal fibrotic signatures are more likely to be associated with drug resistance and eventual relapse. Clarifying the molecular basis for such multidirectional crosstalk among the various normal and neoplastic cell types present in the tumor microenvironment may yield novel targets and new opportunities for therapeutic intervention. This review highlights the most recent concepts regarding the complexity of CAF biology including CAF heterogeneity, functionality in drug resistance, contribution to a progressively fibrotic tumor stroma, the involved signaling pathways and the participating genes.

TGF-ß1/p53 signaling in renal fibrogenesis.

Fibrotic disorders of the renal, pulmonary, cardiac, and hepatic systems are associated with significant morbidity and mortality. Effective therapies to prevent or curtail the advancement to organ failure, however, remain a major clinical challenge. Chronic kidney disease, in particular, constitutes an increasing medical burden affecting >15% of the US population. Regardless of etiology (diabetes, hypertension, ischemia, acute injury, urologic obstruction), persistently elevated TGF-ß1 levels are causatively linked to the activation of profibrotic signaling networks and disease progression. TGF-ß1 is the principal driver of renal fibrogenesis, a dynamic pathophysiologic process that involves tubular cell injury/apoptosis, infiltration of inflammatory cells, interstitial fibroblast activation and excess extracellular matrix synthesis/deposition leading to impaired kidney function and, eventually, to chronic and end-stage disease. TGF-ß1 activates the ALK5 type I receptor (which phosphorylates SMAD2/3) as well as non-canonical (e.g., src kinase, EGFR, JAK/STAT, p53) pathways that collectively drive the fibrotic genomic program. Such multiplexed signal integration has pathophysiological consequences. Indeed, TGF-ß1 stimulates the activation and assembly of p53-SMAD3 complexes required for transcription of the renal fibrotic genes plasminogen activator inhibitor-1, connective tissue growth factor and TGF-ß1. Tubular-specific ablation of p53 in mice or pifithrin-α-mediated inactivation of p53 prevents epithelial G2/M arrest, reduces the secretion of fibrotic effectors and attenuates the transition from acute to chronic renal injury, further supporting the involvement of p53 in disease progression. This review focuses on the pathophysiology of TGF-ß1-initiated renal fibrogenesis and the role of p53 as a regulator of profibrotic gene expression.

SERPINE1: A Molecular Switch in the Proliferation-Migration Dichotomy in Wound-"Activated" Keratinocytes.

Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds. Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments. Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment. Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with deficient or excessive PAI-1 levels.

PAI-1: An Integrator of Cell Signaling and Migration.

Cellular migration, over simple surfaces or through complex stromal barriers, requires coordination between detachment/re-adhesion cycles, involving structural components of the extracellular matrix and their surface-binding elements (integrins), and the precise regulation of the pericellular proteolytic microenvironment. It is now apparent that several proteases and protease inhibitors, most notably urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1), also interact with several cell surface receptors transducing intracellular signals that significantly affect both motile and proliferative programs. These events appear distinct from the original function of uPA/PAI-1 as modulators of the plasmin-based proteolytic cascade. The multifaceted interactions of PAI-1 with specific matrix components (i.e., vitronectin), the low-density lipoprotein receptor-related protein-1 (LRP1), and the uPA/uPA receptor complex have dramatic consequences on the migratory phenotype and may underlie the pathophysiologic sequalae of PAI-1 deficiency and overexpression. This paper focuses on the increasingly intricate role of PAI-1 as a major mechanistic determinant of the cellular migratory phenotype.

Plasminogen activator inhibitors regulate cell adhesion through a uPAR-dependent mechanism.

Binding of type-1 plasminogen activator inhibitor (PAI-1) to cell surface urokinase (uPA) promotes inactivation and internalization of adhesion receptors (e.g., urokinase receptor (uPAR), integrins) and leads to cell detachment from a variety of extracellular matrices. In this report, we begin to examine the mechanism of this process. We show that neither specific antibodies to uPA, nor active site inhibitors of uPA, can detach the cells. Thus, cell detachment is not simply the result of the binding of macromolecules to uPA and/or of the inactivation of uPA. We further demonstrate that another uPA inhibitor, protease nexin-1 (PN-1), also stimulates cell detachment in a uPA/uPAR-dependent manner. The binding of both inhibitors to uPA leads to the specific inactivation of the matrix-engaged integrins and the subsequent detachment of these integrins from the underlying extracellular matrix (ECM). This inhibitor-mediated inactivation of integrins requires direct interaction between uPAR and those integrins since cells attached to the ECM through integrins incapable of binding uPAR do not respond to the presence of either PAI-1 of PN-1. Although both inhibitors initiate the clearance of uPAR, only PAI-1 triggers the internalization of integrins. However, cell detachment by PAI-1 or PN-1 does not depend on the endocytosis of these integrins since cell detachment was also observed when clearance of these integrins was blocked. Thus, PAI-1 and PN-1 induce cell detachment through two slightly different mechanisms that affect integrin metabolism. These differences may be important for distinct cellular processes that require controlled changes in the subcellular localization of these receptors.

Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signaling activity: generation of a new integrin inhibitor.

We investigated the interaction between the urokinase receptor (uPAR) and the integrin alphavbeta3. Vitronectin (VN) induces cell migration by binding to alphavbeta3, but expression of the uPAR boosts its efficacy. Thus, uPAR may regulate VN-induced cell migration by interacting laterally with alphavbeta3. In contrast, cells expressing a uPAR mutant lacking domain 2 do not migrate in response to VN. This effect is overcome by D2A, a synthetic peptide derived from the sequence of domain 2. In addition, D2A has chemotactic activity that requires alphavbeta3 and activates alphavbeta3-dependent signaling pathways such as the Janus kinase/Stat pathway. Moreover, D2A disrupts uPAR-alphavbeta3 and uPAR-alpha5beta1 co-immunoprecipitation, indicating that it can bind both of these integrins. We also identify the chemotactically active epitope harbored by peptide D2A. Mutating two glutamic acids into two alanines generates peptide D2A-Ala, which lacks chemotactic activity but inhibits VN-, FN-, and collagen-dependent cell migration. In fact, the GEEG peptide has potent chemotactic activity, and the GAAG sequence has inhibitory capacities. In summary, we have identified an integrin-interacting sequence located in domain 2 of uPAR, which is also a new chemotactic epitope that can activate alphavbeta3-dependent signaling pathways and stimulate cell migration. This sequence thus plays a pivotal role in the regulation of uPAR-integrin interactions. Moreover, we describe a novel, very potent inhibitor of integrin-dependent cell migration.

Regulation of alpha5beta1 integrin conformation and function by urokinase receptor binding.

Urokinase-type plasminogen activator receptors (uPARs), up-regulated during tumor progression, associate with beta1 integrins, localizing urokinase to sites of cell attachment. Binding of uPAR to the beta-propeller of alpha3beta1 empowers vitronectin adhesion by this integrin. How uPAR modifies other beta1 integrins remains unknown. Using recombinant proteins, we found uPAR directly binds alpha5beta1 and rather than blocking, renders fibronectin (Fn) binding by alpha5beta1 Arg-Gly-Asp (RGD) resistant. This resulted from RGD-independent binding of alpha5beta1-uPAR to Fn type III repeats 12-15 in addition to type III repeats 9-11 bound by alpha5beta1. Suppression of endogenous uPAR by small interfering RNA in tumor cells promoted weaker, RGD-sensitive Fn adhesion and altered overall alpha5beta1 conformation. A beta1 peptide (res 224NLDSPEGGF232) that models near the known alpha-chain uPAR-binding region, or a beta1-chain Ser227Ala point mutation, abrogated effects of uPAR on alpha5beta1. Direct binding and regulation of alpha5beta1 by uPAR implies a modified "bent" integrin conformation can function in an alternative activation state with this and possibly other cis-acting membrane ligands.

Unexpected role of plasminogen activator inhibitor 1 in cell adhesion and detachment.

Plasminogen activator inhibitor 1 (PAI-1) is the primary physiological inhibitor of plasminogen activation in vivo, and thus it is one of the main regulators of the fibrinolytic system. In this regard, individuals with elevated PAI-1 seem to have an increased risk for thrombotic disease, whereas those lacking the inhibitor develop a lifelong bleeding diathesis. Unexpectedly, recent observations demonstrate that cancer patients with high PAI-1 levels have a poor prognosis for survival. This correlation with metastatic disease may be related to the observation that high PAI-1 levels decrease the adhesive strength of cells for their substratum, and that this de-adhesive activity of PAI-1 is not related to its role as a protease inhibitor. Initial insights into potential mechanisms by which PAI-1 regulates the attachment, detachment, and migration of cells are addressed in this review.

The low density lipoprotein receptor-related protein is a motogenic receptor for plasminogen activator inhibitor-1.

Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.

Critical role of integrin alpha 5 beta 1 in urokinase (uPA)/urokinase receptor (uPAR, CD87) signaling.

Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. alpha 5 beta 1) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to alpha 5 beta 1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-alpha 5 antibody, RGD peptide, and function-blocking mutations in alpha 5 beta 1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and alpha 5 beta 1, but alpha 5 beta 1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for alpha 5 beta 1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to alpha 5 beta 1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA.uPAR complex to alpha 5 beta 1 via uPAR, and (c) signal transduction through alpha 5 beta 1.

Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins.

The binding of urokinase plaminogen activator (uPA) to its cell surface receptor (uPAR; CD87) promotes cell adhesion by increasing the affinity of the receptor for both vitronectin (VN) and integrins. We provide evidence that plasminogen activator inhibitor (PAI)-1 can detach cells by disrupting uPAR-VN and integrin-VN interactions and that it does so by binding to the uPA present in uPA-uPAR-integrin complexes on the cell surface. The detached cells cannot reattach to VN unless their surface integrins are first activated by treatment with MnCl2. Immunoprecipitation and subcellular fractionation experiments reveal that PAI-1 treatment triggers deactivation and disengagement of uPA-uPAR-integrin complexes and their endocytic clearance by the low density lipoprotein receptor-related protein. Transfection experiments demonstrate that efficient cell detachment by PAI-1 requires an excess of matrix-engaged uPA-uPAR-integrin complexes over free engaged integrins and that changes in this ratio alter the efficacy of PAI-1. Together, these results suggest a VN-independent, uPA-uPAR-dependent mechanism by which PAI-1 induces cell detachment. This pathway may represent a general mechanism, since PAI-1 also can detach cells from fibronectin and type-1 collagen. This novel "deadhesive" activity of PAI-1 toward a variety of cells growing on different extracellular matrices may begin to explain why high PAI-1 levels often are associated with a poor prognosis in human metastatic disease.