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.

The Genomic Response to TGF-ß1 Dictates Failed Repair and Progression of Fibrotic Disease in the Obstructed Kidney.

Tubulointerstitial fibrosis is a common and diagnostic hallmark of a spectrum of chronic renal disorders. While the etiology varies as to the causative nature of the underlying pathology, persistent TGF-ß1 signaling drives the relentless progression of renal fibrotic disease. TGF-ß1 orchestrates the multifaceted program of kidney fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery or re-differentiation, capillary collapse and subsequent interstitial fibrosis eventually leading to chronic and ultimately end-stage disease. An increasing complement of non-canonical elements function as co-factors in TGF-ß1 signaling. p53 is a particularly prominent transcriptional co-regulator of several TGF-ß1 fibrotic-response genes by complexing with TGF-ß1 receptor-activated SMADs. This cooperative p53/TGF-ß1 genomic cluster includes genes involved in cellular proliferative control, survival, apoptosis, senescence, and ECM remodeling. While the molecular basis for this co-dependency remains to be determined, a subset of TGF-ß1-regulated genes possess both p53- and SMAD-binding motifs. Increases in p53 expression and phosphorylation, moreover, are evident in various forms of renal injury as well as kidney allograft rejection. Targeted reduction of p53 levels by pharmacologic and genetic approaches attenuates expression of the involved genes and mitigates the fibrotic response confirming a key role for p53 in renal disorders. This review focuses on mechanisms underlying TGF-ß1-induced renal fibrosis largely in the context of ureteral obstruction, which mimics the pathophysiology of pediatric unilateral ureteropelvic junction obstruction, and the role of p53 as a transcriptional regulator within the TGF-ß1 repertoire of fibrosis-promoting genes.

The TGF-ß1/p53/PAI-1 Signaling Axis in Vascular Senescence: Role of Caveolin-1.

Stress-induced premature cellular senescence is a significant factor in the onset of age-dependent disease in the cardiovascular system. Plasminogen activator inhibitor-1 (PAI-1), a major TGF-ß1/p53 target gene and negative regulator of the plasmin-based pericellular proteolytic cascade, is elevated in arterial plaques, vessel fibrosis, arteriosclerosis, and thrombosis, correlating with increased tissue TGF-ß1 levels. Additionally, PAI-1 is necessary and sufficient for the induction of p53-dependent replicative senescence. The mechanism of PAI-1 transcription in senescent cells appears to be dependent on caveolin-1 signaling. Src kinases are upstream effectors of both FAK and caveolin-1 activation as FAKY577,Y861 and caveolin-1Y14 phosphorylation are not detected in TGF-ß1-stimulated src family kinase (pp60c-src, Yes, Fyn) triple-deficient (SYF-/-/-) cells. However, restoration of pp60c-src expression in SYF-null cells rescued both caveolin-1Y14 phosphorylation and PAI-1 induction in response to TGF-ß1. Furthermore, TGF-ß1-initiated Src phosphorylation of caveolin-1Y14 is critical in Rho-ROCK-mediated suppression of the SMAD phosphatase PPM1A maintaining and, accordingly, SMAD2/3-dependent transcription of the PAI-1 gene. Importantly, TGF-ß1 failed to induce PAI-1 expression in caveolin-1-null cells, correlating with reductions in both Rho-GTP loading and SMAD2/3 phosphorylation. These findings implicate caveolin-1 in expression controls on specific TGF-ß1/p53 responsive growth arrest genes. Indeed, up-regulation of caveolin-1 appears to stall cells in G0/G1 via activation of the p53/p21 cell cycle arrest pathway and restoration of caveolin-1 in caveolin-1-deficient cells rescues TGF-ß1 inducibility of the PAI-1 gene. Although the mechanism is unclear, caveolin-1 inhibits p53/MDM2 complex formation resulting in p53 stabilization, induction of p53-target cell cycle arrest genes (including PAI-1), and entrance into premature senescence while stimulating the ATM→p53→p21 pathway. Identification of molecular events underlying senescence-associated PAI-1 expression in response to TGF-ß1/src kinase/p53 signaling may provide novel targets for the therapy of cardiovascular disease.

TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

Chronic kidney disease affects >15% of the U.S. population and >850 million individuals worldwide. Fibrosis is the common outcome of many chronic renal disorders and, although the etiology varies (i.e., diabetes, hypertension, ischemia, acute injury, and urologic obstructive disorders), persistently elevated renal TGF-ß1 levels result in the relentless progression of fibrotic disease. TGF-ß1 orchestrates the multifaceted program of renal fibrogenesis involving proximal tubular dysfunction, failed epithelial recovery and redifferentiation, and subsequent tubulointerstitial fibrosis, eventually leading to chronic renal disease. Recent findings implicate p53 as a cofactor in the TGF-ß1-induced signaling pathway and a transcriptional coregulator of several TGF-ß1 profibrotic response genes by complexing with receptor-activated SMADs, which are homologous to the small worms (SMA) and Drosophilia mothers against decapentaplegic (MAD) gene families. The cooperative p53-TGF-ß1 genomic cluster includes genes involved in cell growth control and extracellular matrix remodeling [e.g., plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor, clade E, member 1), connective tissue growth factor, and collagen I]. Although the molecular basis for this codependency is unclear, many TGF-ß1-responsive genes possess p53 binding motifs. p53 up-regulation and increased p53 phosphorylation; moreover, they are evident in nephrotoxin- and ischemia/reperfusion-induced injury, diabetic nephropathy, ureteral obstructive disease, and kidney allograft rejection. Pharmacologic and genetic approaches that target p53 attenuate expression of the involved genes and mitigate the fibrotic response, confirming a key role for p53 in renal disorders. This review focuses on mechanisms whereby p53 functions as a transcriptional regulator within the TGF-ß1 cluster with an emphasis on the potent fibrosis-promoting PAI-1 gene.-Higgins, C. E., Tang, J., Mian, B. M., Higgins, S. P., Gifford, C. C., Conti, D. J., Meldrum, K. K., Samarakoon, R., Higgins, P. J. TGF-ß1-p53 cooperativity regulates a profibrotic genomic program in the kidney: molecular mechanisms and clinical implications.

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.

A small molecule PAI-1 functional inhibitor attenuates neointimal hyperplasia and vascular smooth muscle cell survival by promoting PAI-1 cleavage.

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and it's cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

The basic helix-loop-helix/leucine zipper transcription factor USF2 integrates serum-induced PAI-1 expression and keratinocyte growth.

Plasminogen activator inhibitor type-1 (PAI-1), a major regulator of the plasmin-dependent pericellular proteolytic cascade, is prominently expressed during the tissue response to injury although the factors that impact PAI-1 induction and their role in the repair process are unclear. Kinetic modeling using established biomarkers of cell cycle transit (c-MYC; cyclin D1; cyclin A) in synchronized human (HaCaT) keratinocytes, and previous cytometric assessments, indicated that PAI-1 transcription occurred early after serum-stimulation of quiescent (G0) cells and prior to G1 entry. It was established previously that differential residence of USF family members (USF1→USF2 switch) at the PE2 region E box (CACGTG) characterized the G0 → G1 transition period and the transcriptional status of the PAI-1 gene. A consensus PE2 E box motif (5'-CACGTG-3') at nucleotides -566 to -561 was required for USF/E box interactions and serum-dependent PAI-1 transcription. Site-directed CG → AT substitution at the two central nucleotides inhibited formation of USF/probe complexes and PAI-1 promoter-driven reporter expression. A dominant-negative USF (A-USF) construct or double-stranded PE2 "decoy" attenuated serum- and TGF-ß1-stimulated PAI-1 synthesis. Tet-Off induction of an A-USF insert reduced both PAI-1 and PAI-2 transcripts while increasing the fraction of Ki-67(+) cells. Conversely, overexpression of USF2 or adenoviral-delivery of a PAI-1 vector inhibited HaCaT colony expansion indicating that the USF1 → USF2 transition and subsequent PAI-1 transcription are critical events in the epithelial go-or-grow response. Collectively, these data suggest that USF2, and its target gene PAI-1, regulate serum-stimulated keratinocyte growth, and likely the cadence of cell cycle progression in replicatively competent cells as part of the injury repair program.

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.

SERPINE1 expression discriminates site-specific metastasis in human melanoma.

Depth of invasion, a quantifier of vertical growth, is a major cutaneous melanoma staging factor. Stromal penetrance requires pericellular proteolysis regulated by the serine protease and matrix metalloproteinase cascades. The serine protease inhibitor SERPINE1, a poor prognosis biomarker in various cancers, promotes tumor progression likely by titrating the extent and local of plasmin-initiated matrix remodelling. SERPINE1 in human melanoma was assessed using tissue arrays that included primary/metastatic tumors and normal skin. SERPINE1 was basal layer-restricted in the normal epidermis. SERPINE1 immunoreactivity was evident in 27/28 primary (96%) and 24/26 metastatic tumors (92%); cutaneous metastases (80%) had significantly elevated SERPINE1 levels compared with low signals characteristic of lymph node lesions. Moderate SERPINE1 expression was a general finding in primary melanoma, whereas reduced or increased SERPINE1 immunolocalization typified metastatic deposits. The amplitude of SERPINE1 expression may impact melanoma site-specific dissemination, with cutaneous metastases representing a high-SERPINE1 tumor subtype.

Complex Regulation of the Pericellular Proteolytic Microenvironment during Tumor Progression and Wound Repair: Functional Interactions between the Serine Protease and Matrix Metalloproteinase Cascades.

Spatial and temporal regulation of the pericellular proteolytic environment by local growth factors, such as EGF and TGF-ß, initiates a wide repertoire of cellular responses coupled to a plasmin/matrix metalloproteinase (MMP) dependent stromal-remodeling axis. Cell motility and invasion, tumor metastasis, wound healing, and organ fibrosis, for example, represent diverse events controlled by expression of a subset of genes that encode various classes of tissue remodeling proteins. These include members of the serine protease and MMP families that functionally constitute a complex system of interacting protease cascades and titrated by their respective inhibitors. Several structural components of the extracellular matrix are upregulated by TGF-ß as are matrix-active proteases (e.g., urokinase (uPA), plasmin, MMP-1, -3, -9, -10, -11, -13, -14). Stringent controls on serine protease/MMP expression and their topographic activity are essential for maintaining tissue homeostasis. Targeting individual elements in this highly interactive network may lead to novel therapeutic approaches for the treatment of cancer, fibrotic diseases, and chronic wounds.

Novel Combinatorial Therapeutic Targeting of PAI-1 (SERPINE1) Gene Expression in Alzheimer's Disease.

Accumulation of neurotoxic amyloid peptides (Aß) in the brain, generated by ß-site proteolytic processing of the amyloid precursor protein (APP), is the hallmark pathophysiologic feature of Alzheimer's disease. The plasmin-activating cascade, in which urokinase (uPA) and tissue-type (tPA) plasminogen activators convert plasminogen to the broad-spectrum protease plasmin, appears to serve a protective, Aß-clearing, role in the central nervous system. Plasmin degrades Aß and catalyzes α- site APP proteolysis generating nontoxic peptides. Plasmin activation in the brain is negatively regulated by the fast-acting clade E serine protease inhibitor (SERPIN) plasminogen activator inhibitor type-1 (PAI-1; SERPINE1) resulting in Aß accumulation. PAI-1 and its major physiological inducer TGF-ß1, moreover, are both increased in Alzheimer's disease models and implicated in the etiology and progression of human neurodegenerative disorders. Current findings support the hypothesis that targeting of PAI-1 function (by small molecule drugs) and/or gene expression (by histone deacetylase inhibitors) may constitute a clinically-relevant molecular approach to the therapy of neurodegenerative diseases associated with increased PAI-1 levels.

PAI-1 Expression Is Required for HDACi-Induced Proliferative Arrest in ras-Transformed Renal Epithelial Cells.

Malignant transformation of mammalian cells with ras family oncogenes results in dramatic changes in cellular architecture and growth traits. The generation of flat revertants of v-K-ras-transformed renal cells by exposure to the histone deacetylase inhibitor sodium butyrate (NaB) was previously found to be dependent on transcriptional activation of the PAI-1 (SERPINE1) gene (encoding the type-1 inhibitor of urokinase and tissue-type plasminogen activators). NaB-initiated PAI-1 expression preceded induced cell spreading and entry into G(1) arrest. To assess the relevance of PAI-1 induction to growth arrest in this cell system more critically, two complementary approaches were used. The addition of a stable, long half-life, recombinant PAI-1 mutant to PAI-1-deficient v-K-ras-/c-Ha-ras-transformants or to PAI-1 functionally null, NaB-resistant, 4HH cells (engineered by antisense knockdown of PAI-1 mRNA transcripts) resulted in marked cytostasis in the absence of NaB. The transfection of ras-transformed cells with the Rc/CMVPAI expression construct, moreover, significantly elevated constitutive PAI-1 synthesis (10- to 20-fold) with a concomitant reduction in proliferative rate. These data suggest that high-level PAI-1 expression suppresses growth of chronic ras-oncogene transformed cells and is likely a major cytostatic effector of NaB exposure.

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.

Redox-induced Src kinase and caveolin-1 signaling in TGF-ß1-initiated SMAD2/3 activation and PAI-1 expression.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1), a major regulator of the plasmin-based pericellular proteolytic cascade, is significantly increased in human arterial plaques contributing to vessel fibrosis, arteriosclerosis and thrombosis, particularly in the context of elevated tissue TGF-ß1. Identification of molecular events underlying to PAI-1 induction in response to TGF-ß1 may yield novel targets for the therapy of cardiovascular disease. PRINCIPAL FINDINGS: Reactive oxygen species are generated within 5 minutes after addition of TGF-ß1 to quiescent vascular smooth muscle cells (VSMCs) resulting in pp60(c-src) activation and PAI-1 expression. TGF-ß1-stimulated Src kinase signaling sustained the duration (but not the initiation) of SMAD3 phosphorylation in VSMC by reducing the levels of PPM1A, a recently identified C-terminal SMAD2/3 phosphatase, thereby maintaining SMAD2/3 in an active state with retention of PAI-1 transcription. The markedly increased PPM1A levels in triple Src kinase (c-Src, Yes, Fyn)-null fibroblasts are consistent with reductions in both SMAD3 phosphorylation and PAI-1 expression in response to TGF-ß1 compared to wild-type cells. Activation of the Rho-ROCK pathway was mediated by Src kinases and required for PAI-1 induction in TGF-ß1-stimulated VSMCs. Inhibition of Rho-ROCK signaling blocked the TGF-ß1-mediated decrease in nuclear PPM1A content and effectively attenuated PAI-1 expression. TGF-ß1-induced PAI-1 expression was undetectable in caveolin-1-null cells, correlating with the reduced Rho-GTP loading and SMAD2/3 phosphorylation evident in TGF-ß1-treated caveolin-1-deficient cells relative to their wild-type counterparts. Src kinases, moreover, were critical upstream effectors of caveolin-1(Y14) phosphoryation and initiation of downstream signaling. CONCLUSIONS: TGF-ß1-initiated Src-dependent caveolin-1(Y14) phosphorylation is a critical event in Rho-ROCK-mediated suppression of nuclear PPM1A levels maintaining, thereby, SMAD2/3-dependent transcription of the PAI-1 gene.

PAI-1 mediates the TGF-beta1+EGF-induced "scatter" response in transformed human keratinocytes.

Cooperative interactions between growth factor signaling pathways are important elements in carcinoma progression. A model system combining transforming growth factor-beta1 (TGF-beta1) and EGF was developed to investigate mechanisms underlying induced epithelial-to-mesenchymal transition (EMT) in ras-transformed human (HaCaT II-4) keratinocytes. Dual stimulation with TGF-beta1+EGF resulted in keratinocyte "plasticity" and pronounced colony dispersal. The most highly expressed transcript, identified by mRNA profiling, encoded plasminogen activator inhibitor-1 (PAI-1; SERPINE1). PAI-1 negatively regulates plasmin-dependent matrix degradation, preserving a stromal scaffold permissive for keratinocyte motility. Mitogen-activated extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) and p38 signaling were required for maximal PAI-1 upregulation and TGF-beta1+EGF-stimulated cell locomotion, as pharmacologic disruption of MEK/p38 activity ablated both responses. Moreover, PAI-1 knockdown alone effectively inhibited TGF-beta1+EGF-dependent cell scattering, indicating a functional role for this SERPIN in the dual-growth factor model of induced motility. Moreover, EGFR signaling blockade or EGFR knockdown attenuated TGF-beta1-induced PAI-1 expression, implicating EGFR transactivation in TGF-beta1-stimulated PAI-1 expression, and reduced colony dispersal in TGF-beta1+EGF-treated cultures. Identification of such cooperative signaling networks and their effect on specific invasion-promoting target genes, such as PAI-1, may lead to the development of pathway-specific therapeutics that affect late-stage events in human tumor progression.

TGF-beta1-Induced Expression of the Poor Prognosis SERPINE1/PAI-1 Gene Requires EGFR Signaling: A New Target for Anti-EGFR Therapy.

Increased transforming growth factor-beta (TGF-beta) expression and epidermal growth factor receptor (EGFR) amplification accompany the emergence of highly aggressive human carcinomas. Cooperative signaling between these two growth factor/receptor systems promotes cell migration and synthesis of stromal remodeling factors (i.e., proteases, protease inhibitors) that, in turn, regulate tumor invasion, neo-angiogenesis and inflammation. ranscript profiling of transformed human cells revealed that genes encoding wound healing, matrix remodeling and cell cycle proteins (i.e., the "tissue repair" transcriptome) are significantly up-regulated early after growth factor stimulation. The major inhibitor of plasmin generation, plasminogen activator inhibitor-1 (PAI-1), is among the most highly induced transcripts during the phenotypic transition initiated by TGF-beta maximal expression requires EGFR signaling. PAI-1 induction occurs early in the progression of incipient epidermal squamous cell carcinoma (SCC) and is a significant indicator of poor prognosis in epithelial malignancies. Mouse modeling and molecular genetic analysis of complex systems indicates that PAI-1 regulates the temporal/spatial control of pericellular proteolysis, promotes epithelial plasticity, inhibits capillary regression and facilitates stromal invasion. Defining TGF-beta1-initiated signaling events that cooperate with an activated EGFR to impact the protease-protease inhibitor balance in the tumor microenvironment is critical to the development of novel therapies for the clinical management of human cancers.

Differential requirement for MEK/ERK and SMAD signaling in PAI-1 and CTGF expression in response to microtubule disruption.

Colchicine and nocodazole, both established microtubule disruptors, are useful tools to investigate cytoskeletal-dependent signaling cascades and the associated downstream transcriptional targets. Since cytoskeletal events impact pathophysiologic consequences in the vascular system, the signaling requirements underlying colchicine-stimulated expression of PAI-1 and CTGF, two prominent cell deformation-sensitive fibrosis-initiating proteins, were evaluated in vascular smooth muscle cells. Microtubule disruption rapidly induced EGFR transactivation (at the src kinase-sensitive EGFR(Y845) site) in a ROS-dependent manner. Genetic deficiency of EGFR, inhibition of EGFR signaling with AG1478 or introduction of a kinase-deficient EGFR construct effectively blocked colchicine-stimulated PAI-1 and CTGF expression. MEK/ERK involvement downstream of ROS generation was critical for PAI-1, but not CTGF, expression following cytoskeletal perturbation suggesting bifurcation of signaling pathways downstream of EGFR activation. Colchicine also stimulated SMAD2/3 phosphorylation by a Rho/ROCK-dependent mechanism independent of TGF-beta1 release or receptor activity. Rho/ROCK signaling initiated by tubulin network collapse was required for both CTGF and PAI-1 induction. Colchicine-initiated SMAD3 phosphorylation, however, was essential for PAI-1, but not CTGF, expression further highlighting divergence of signaling events downstream of Rho/ROCK that mediate microtubule deformation-associated changes in profibrotic gene transcription.

PAI-1 Regulates the Invasive Phenotype in Human Cutaneous Squamous Cell Carcinoma.

The emergence of highly aggressive subtypes of human cutaneous squamous cell carcinoma (SCC) often reflects increased autocrine/paracrine TGF-beta synthesis and epidermal growth factor receptor (EGFR) amplification. Cooperative TGF-beta/EGFR signaling promotes cell migration and induces expression of both proteases and protease inhibitors that regulate stromal remodeling resulting in the acquisition of an invasive phenotype. In one physiologically relevant model of human cutaneous SCC progression, TGF-beta1+EGF stimulation increases the production of several matrix metalloproteinases (MMPs), among the most prominent of which is MMP-10-an MMP known to be elevated in SCC in situ. Activation of stromal plasminogen appears to be critical in triggering downstream MMP activity. Paradoxically, PAI-1, the major physiological inhibitor of plasmin generation, is also upregulated under these conditions and is an early event in progression of incipient epidermal SCC. One testable hypothesis proposes that TGF-beta1+EGF-dependent MMP-10 elevation directs focalized matrix remodeling events that promote epithelial cell plasticity and tissue invasion. Increased PAI-1 expression serves to temporally and spatially modulate plasmin-initiated pericellular proteolysis, further facilitating epithelial invasive potential. Defining the complex signaling and transcriptional mechanisms that maintain this delicate balance is critical to developing targeted therapeutics for the treatment of human cutaneous malignancies.