Prognosis in adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia evaluated by uPAR-immunohistochemistry.

Adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia in humans are highly invasive tumours with poor prognosis. The localisation of urokinase-type plasminogen activator receptor (uPAR) was determined in 66 patients; 60 with adenocarcinomas and six cases with Barrett's oesophagus. uPAR was expressed in nearly all cases of invasive adenocarcinomas by populations of cancer cells, macrophages and myofibroblasts at both the invasion front and the tumour core. In areas with high-grade dysplasia or with Barrett's metaplasia adjacent to the tumour tissue, no uPAR-immunoreactivity was found. High local expression of uPAR, therefore, appears to be a characteristic marker for invasive behaviour in this tumour, suggesting that uPAR's contribution to matrix degradation during invasive growth is a late event in carcinogenesis. Using a scoring system for semiquantitative estimation of uPAR-positivity on immmunohistochemically stained specimens, a significant association was found between poor overall survival and high uPAR-score for cancer cells in the tumour core and for macrophages peripherally at the tumour invasion zone. In multivariate analysis, these two uPAR-scores were confirmed as highly significant prognostic parameters independent of Tumour, Node, Metastasis (TNM)-stage and World Health Organization (WHO) classification. The proteolytic action of these malignant and nonmalignant accessory cells thus seemed to follow two main patterns: one dominated by uPAR positive cancer cells and one by uPAR-positive macrophages. Scoring of uPAR-positivity might be a useful parameter for onset of invasion and prognosis in these adenocarcinomas.

Urokinase receptor forms in serum from non-small cell lung cancer patients: relation to prognosis.

OBJECTIVES: To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients. MATERIALS AND METHODS: Serum sampled preoperatively was available from 171 patients radically resected for NSCLC. Intact uPAR, uPAR(I-III), intact and cleaved uPAR, uPAR(I-III)+uPAR(II-III) and the liberated uPAR(I) were measured by time-resolved fluorescence immunoassays (TR-FIAs 1-3). RESULTS: High serum levels of each of the three uPAR forms were associated with short overall survival (OS). In a multivariate survival analysis uPAR(I-III) (hazard ratio (HR)=2.3, 95% confidence interval (CI): 1.2-4.5, p=0.015) and uPAR(I) (hazard ratio (HR)=1.5, 95% CI: 1.0-2.2, p=0.0497) remained significant prognostic parameters independent of stage, histology, age, performance status and therapy. CONCLUSIONS: This retrospective study shows that uPAR(I-III) and uPAR(I) in serum are independent prognostic factors in patients radically operated for NSCLC. Further prospective studies are needed to validate these markers for clinical use.

Prognostic and predictive value of intact and cleaved forms of the urokinase plasminogen activator receptor in metastatic prostate cancer.

BACKGROUND: The purpose of this study was to investigate the prognostic value of different forms of the urokinase receptor, uPAR, in serum from prostate cancer (PC) patients. PATIENTS AND METHODS: The uPAR forms were measured in samples from 131 metastatic PC patients. These constituted a subset of patients included in a randomized clinical trial of treatment with total androgen blockade (TAB) versus polyestradiol phosphate (PEP). Pre-treatment serum levels of intact uPAR (uPAR(I-III)), intact plus cleaved uPAR (uPAR(I-III) + uPAR(II-III)) and domain I (uPAR(I)) were measured using time-resolved fluorescence immunoassays (TR-FIAs). RESULTS: High serum levels of each of the uPAR forms were significantly associated with short overall survival (OS). The prognostic impact was strongest in the TAB treated patients with all uPAR forms being statistically significant. In multivariate analysis, uPAR(I-III) + uPAR(II-III) was an independent prognostic factor in TAB treated patients (HR = 5.2, 95% confidence interval (CI): 2.5-10.6, P < 0.0001) but not in PEP treated patients (P = 0.40). In the entire study population, OS was similar in the two treatment groups. The survival analysis showed significant interactions between treatment modality and the level of either uPAR(I-III) or uPAR(I-III) + uPAR(II-III). High levels of uPAR(I-III) + uPAR(II-III) were found to be predictive of effect of PEP versus TAB treatment. Patients with uPAR(I-III) + uPAR(II-III) levels above the median had significantly longer OS (median difference 11.3 months), if treated with PEP rather than with TAB (HR = 1.8, 95% CI:1.1-3.1, P = 0.03). CONCLUSION: uPAR forms are significantly associated with OS. High uPAR(I-III) + uPAR(II-III) predicts longer OS in patients treated with PEP compared to TAB. uPAR forms are promising prognostic and predictive markers in PC.

Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases.

Metastatic growth and invasion by colon cancer cells in the liver requires the ability of the cancer cells to interact with the new tissue environment. Plasmin(ogen) is activated on cell surfaces by urokinase-type PA (uPA), and is regulated by uPAR and plasminogen activator inhibitor-1 (PAI-1). To compare the expression patterns of uPA, uPAR and PAI-1 in colon cancer with that in their liver metastases, we analysed matched samples from 14 patients. In all 14 primary colon cancers, we found upregulation of uPAR, uPA mRNA and PAI-1 in primarily stromal cells at the invasive front. In 5 of the 14 liver metastases, we found intense expression of uPAR, uPA-mRNA and PAI-1 in primarily stromal cells at the metastases periphery, and in an expression pattern similar to that found in the primary tumours. In the remaining 9 liver metastases, uPAR and uPA-mRNA were only seen associated with the presence of necrosis within the liver metastases. In addition, PAI-1-immunoreactivity was in all liver metastases seen in hepatocytes at the metastases periphery. Interestingly, the former 5 liver metastases positive for uPAR, uPA mRNA and PAI-1 at the metastasis periphery all had a predominantly desmoplastic reaction, whereas 8 of the remaining 9 showed direct contact between the cancer cells and the liver parenchyma. We conclude that there are 2 distinct patterns of expression of uPAR, uPA and PAI-1 in colon cancer liver metastases and that these correlate closely with 2 morphological growth patterns. These findings may have implication for the treatment of patients with metastatic disease.

Crohn's disease but not chronic ulcerative colitis induces the expression of PAI-1 in enteric neurons.

OBJECTIVES: Chronic inflammation of the intestinal wall is the common characteristic of Crohn's disease and ulcerative colitis; disorders, which in some cases can be difficult to distinguish. The inflammation also affects the local neuronal plexuses of the enteric nervous system. It is known that plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) are upregulated in neurons after experimental peripheral nerve injury and have been linked to nerve regeneration. METHODS: The expression of PAI-1 and uPAR in neuronal cells in lesions of the gastrointestinal tract was analyzed by immunohistochemical techniques. RESULTS: PAI-1 was found in a subset of neurons primarily located in the submucosal plexus of the small and large intestine in 24 of 28 cases (86%) with Crohn's disease, but in none of 17 cases with chronic ulcerative colitis and other severe inflammatory conditions in the intestinal wall. The PAI-1 was seen in the perikarya of the neurons and a few proximal axons, whereas nerves were negative. uPAR was seen in nerves in all types of lesion varying from 21% to 88% of the cases, most frequent in colon adenocarcinomas. No uPAR-positive nerves were detected in normal colon. CONCLUSIONS: PAI-1-positive neurons in inflammatory bowel disease are linked to chronic inflammation in Crohn's disease, implying PAI-1 as a potential parameter for the differential diagnosis between Crohn's disease and ulcerative colitis. The findings also suggest that PAI-1 in neurons is related to pain and that both PAI-1 and uPAR are involved in neuronal repair in the inflamed tissue.

Metastasis is strongly reduced by the matrix metalloproteinase inhibitor Galardin in the MMTV-PymT transgenic breast cancer model.

Matrix metalloproteinases (MMP) have several roles that influence cancer progression and dissemination. However, low molecular weight metalloproteinase inhibitors (MPI) have not yet been tested in transgenic/spontaneous metastasis models. We have tested Galardin/GM6001, a potent MPI that reacts with most MMPs, in the MMTV-PymT transgenic breast cancer model. We followed a cohort of 81 MMTV-PymT transgenic mice that received Galardin, placebo, or no treatment. Galardin treatment was started at age 6 weeks with 100 mg/kg/d, and all mice were killed at age 13.5 weeks. Galardin treatment significantly reduced primary tumor growth. Final tumor burden in Galardin-treated mice was 1.69 cm3 compared with 3.29 cm3 in placebo-treated mice (t test, P = 0.0014). We quantified the total lung metastasis volume in the same cohort of mice. The median metastasis volume was 0.003 mm(3) in Galardin-treated mice compared with 0.56 mm(3) in placebo-treated mice (t test, P < 0.0001). Thus, metastasis burden was reduced more than 100-fold, whereas primary tumor size was reduced only 2-fold. We also found that primary tumors from Galardin-treated mice exhibited a lower histopathologic tumor grade, increased collagen deposition, and increased MMP-2 activity. MMPs are known to have tumor-promoting and tumor-inhibitory effects, and several clinical trials of broad-spectrum MPIs have failed to show promising effects. The very potent antimetastatic effect of Galardin in the MMTV-PymT model does, however, show that it may be possible to find broad-spectrum MPIs with favorable inhibition profiles, or perhaps combinations of monospecific MPIs, for future clinical application.

A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor.

The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can induce the same response. In this report, we show that both of these triggering mechanisms can be operative and that uPAR-dependent modulation of cell morphology can indeed occur independently of a direct vitronectin binding. Expression of wild-type uPAR on HEK293 cells led to pronounced vitronectin adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix contacts necessary to initiate the uPAR-dependent cytoskeletal rearrangements, whereas inactivation of the Rac signaling pathway in all cases suppressed the occurrence of the same events.

Murine monoclonal antibodies against murine uPA receptor produced in gene-deficient mice: inhibitory effects on receptor-mediated uPA activity in vitro and in vivo.

Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC(50) value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, an approximately 50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.

Stromal cells associated with early invasive foci in human mammary ductal carcinoma in situ coexpress urokinase and urokinase receptor.

The transition from ductal carcinoma in situ (DCIS) of the breast to invasive ductal carcinoma is facilitated by proteolytic degradation of basement membrane. The transition can be identified as microinvasive foci in a small proportion of DCIS lesions. We have previously found that MMP-13 is frequently expressed in such foci. To establish whether plasmin-directed proteolysis is likely to be involved in early invasion, we have here studied the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in human DCIS lesions with and without microinvasion. uPA mRNA was detected in periductal stromal cells in all of 9 DCIS lesions with microinvasion and in 2 of 9 DCIS lesions without microinvasion by in situ hybridization. The uPA mRNA signal was seen in numerous stromal cells in microinvasive areas together with MMP-13 mRNA expressing cells. Double immunofluorescence analyses, using emission fingerprinting, showed that the uPA expressing stromal cells included both myofibroblasts and macrophages. The early invasive carcinoma cells were negative for uPA. uPAR immunoreactivity was focally upregulated in periductal stromal cells in all of the 9 DCIS lesions with microinvasion and in only 2 of the 9 DCIS lesions without microinvasion. uPAR was seen in both macrophages and myofibroblasts in microinvasive areas, and it was evident that uPA and uPAR colocalized in both fibroblast-like cells and macrophage-like cells. We conclude that periductal macrophages and myofibroblasts are strongly involved in the initial steps of breast cancer invasion by focally upregulating the expression of the plasminogen activation system and MMP-13.

The plasminogen fibrinolytic pathway is required for hematopoietic regeneration.

Hematopoietic stem cells within the bone marrow exist in a quiescent state. They can differentiate and proliferate in response to hematopoietic stress (e.g., myelosuppression), thereby ensuring a well-regulated supply of mature and immature hematopoietic cells within the circulation. However, little is known about how this stress response is coordinated. Here, we show that plasminogen (Plg), a classical fibrinolytic factor, is a key player in controlling this stress response. Deletion of Plg in mice prevented hematopoietic stem cells from entering the cell cycle and undergoing multilineage differentiation after myelosuppression, leading to the death of the mice. Activation of Plg by administration of tissue-type plasminogen activator promoted matrix metalloproteinase-mediated release of Kit ligand from stromal cells, thereby promoting hematopoietic progenitor cell proliferation and differentiation. Thus, activation of the fibrinolytic cascade is a critical step in regulating the hematopoietic stress response.

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice.

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.

MMP-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases.

Matrix metalloproteinase-9 (MMP-9) is up-regulated in macrophages in various human cancer types. In human colon cancer, MMP-9 is expressed in a macrophage subpopulation located at the tumor edge, indicating a specific induction of MMP-9 in macrophages in direct association with cancer invasion. To test whether MMP-9 is also induced in tumor edge macrophages in metastases from colorectal adenocarcinomas, we have compared the expression pattern of MMP-9 in primary colorectal adenocarcinomas (n = 15) with that in liver metastases (n = 15) and local lymph node metastases (n = 7) from the same patients by in situ hybridization and immunohistochemistry. In all the colorectal adenocarcinomas, the expression of MMP-9 mRNA and immunoreactivity in macrophages was located at the invasive front. In contrast, only 3 of the 15 liver metastases had MMP-9 mRNA and immunoreactivity at the periphery, and this expression was confined to small foci of macrophages located either among lymphocytes or in a dense desmoplastic stroma. Expression of MMP-9 mRNA and immunoreactivity was in all liver metastases seen in macrophages located in the lumen of malignant glandular structures and in central necrotic tissue. In all the 7 lymph node metastases, MMP-9 mRNA and immunoreactivity was seen in macrophages located in the stromal tissue surrounding the metastases. We conclude that MMP-9 is not up-regulated in tumor edge macrophages in liver metastases like in their primary tumor and local lymph node metastases, suggesting that disseminating colorectal cancer cells can adopt alternative proteolytic mechanisms for invasion depending on the local microenvironment.

Enhanced discrimination of benign from malignant prostatic disease by selective measurements of cleaved forms of urokinase receptor in serum.

BACKGROUND: Early detection of prostate cancer (PCa) centers on measurements of prostate-specific antigen (PSA), but current testing practices suffer from lack of specificity and generate many unnecessary prostate biopsies. Soluble urokinase plasminogen activator receptor (uPAR) is present in blood in both intact and cleaved forms. Increased uPAR in blood is correlated with poor prognosis in various cancers, but uPAR has not been shown to be useful in PCa diagnostics. We assessed the ability of immunoassays for specific uPAR forms to discriminate PCa from benign conditions. METHODS: We measured total PSA (tPSA), free PSA (fPSA), intact uPAR [uPAR(I-III)], intact uPAR + cleaved uPAR domains II+III [uPAR(I-III) + uPAR(II-III)], and cleaved uPAR domain I [uPAR(I)] in sera from 224 men with and 166 men without PCa. We assessed differences in serum concentrations between the PCa and noncancer groups within the entire cohort and in men with tPSA concentrations of 2-10 microg/L. The diagnostic accuracy of individual analytes and analyte combinations was explored by logistic regression and ROC analyses and evaluations of sensitivity and specificity pairs. RESULTS: Serum uPAR(I) and uPAR(II-III) were higher in PCa than in benign disease. In men with tPSA between 2 and 10 microg/L, the combination of %fPSA with the ratio uPAR(I)/uPAR(I-III) had a greater area under the ROC curve (0.73) than did %fPSA (0.68). CONCLUSIONS: Specific measurements of different uPAR forms in serum improve the specificity of PCa detection. The uPAR forms may therefore be complementary to PSA for PCa detection, most importantly in men with moderately increased PSA.

Prognostic impact of liberated domain I of the urokinase plasminogen activator receptor in squamous cell lung cancer tissue.

OBJECTIVES: The purpose of this study was to determine the prognostic impact of liberated domain I of the urokinase plasminogen activator receptor, uPAR(I), in tumour extracts from patients resected for the squamous cell carcinoma (SCC) of the lung. MATERIALS AND METHODS: A recently developed time-resolved fluoroimmunoassay for uPAR(I), TR-FIA 3, was validated using a pool of SCC tumour extracts. This assay was then used to measure the levels of uPAR(I) in tumour extracts from 63 SCC patients. Overall survival of the patients was measured from the day of surgical resection until death or until a review after a median of 144 months observation. The Cox proportional hazard model was used for uni- and multivariate survival analysis. RESULTS: TR-FIA 3 was found to measure uPAR(I) in SCC tumour extracts with high precision, accuracy and specificity. Univariate analysis using log transformed uPAR(I) concentrations showed that there was an increasing risk of mortality with increasing uPAR(I) levels in SCC tumour extracts, the hazard ratio (HR) being 2.9 with a P-value of 0.003. In a multivariate analysis, including uPAR(I), gender, age, nodal status, tumour size and levels of uPAR immunoreactivity measured by ELISA, statistically significant prognostic impact was found only for levels of uPAR(I) (HR 3.7, P=0.002) and tumour size (HR 2.4, P=0.02). CONCLUSION: uPAR(I) levels in the extracts of primary tumours independently predicted overall survival of 63 patients with SCC.

Plasminogen activation and cancer.

Breakdown of the extracellular matrix is crucial for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases. The activity of each of these proteases is regulated by an array of activators, inhibitors and cellular receptors. Thus, the generation of plasmin involves the pro-enzyme plasminogen, the urokinase type plasminogen activator uPA and its pro-enzyme pro-uPA, the uPA inhibitor PAI-1, the cell surface uPA receptor uPAR, and the plasmin inhibitor alpha(2)-antiplasmin. Furthermore, the regulation of extracellular proteolysis in cancer involves a complex interplay between cancer cells and non-malignant stromal cells in the expression of the molecular components involved. For some types of cancer, this cellular interplay mimics that observed in the tissue of origin during non-neoplastic tissue remodelling processes. We propose that cancer invasion can be considered as uncontrolled tissue remodelling. Inhibition of extracellular proteases is an attractive approach to cancer therapy. Because proteases have many different functions in the normal organism, efficient inhibition will have toxic side effects. In cancer invasion, like in normal tissue remodelling processes, there appears to be a functional overlap between different extracellular proteases. This redundancy means that combinations of protease inhibitors must be used. Such combination therapy, however, is also likely to increase toxicity. Therefore for each type of cancer, a combination of protease inhibitors that is optimised with respect to both maximal therapeutic effect and minimal toxic side effects need to be identified.

Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide.

We report the crystal structure of a soluble form of human urokinase-type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor-stromal microenvironment in many human cancers. The structure was solved at 2.7 A in association with a competitive peptide inhibitor of the urokinase-type plasminogen activator (uPA)-uPAR interaction. uPAR is composed of three consecutive three-finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA-uPAR complex. This model reveals that the receptor-binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA-focalized proteolysis on the cell surface and control cell adhesion and migration.

Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas.

Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer cells. We have now used laser capture microdissection and real-time PCR to quantify the mRNA expression of components of matrix-degrading proteolytic systems in cancer and stromal areas of mouse mammary tumors genetically induced by the polyoma virus middle T (PyMT) antigen. We examined the mRNA levels of urokinase plasminogen activator, plasminogen activator inhibitor 1 and the matrix metalloproteases MMP-2, -3, -11, -13 and -14, and found that all these seven genes are predominantly expressed by stromal cells. Our results were qualitatively supported by in situ hybridization analysis of the expression of mRNAs for MMP-2, -3 and -13 in the PyMT tumors. Statistical analyses indicated that the quantitative expression patterns observed in cancer and stromal cells isolated from individual tumors from different PyMT mice are quite reproducible. The methodology described in this study provides excellent tools to study the possible interactions between cancer and stromal cells during the development of breast cancer, and the results suggest that stromal cells are involved in carcinogenesis and tumor progression, which may have important implications for the biology and therapy of cancer.

Host plasminogen activator inhibitor-1 promotes human skin carcinoma progression in a stage-dependent manner.

Angiogenesis and tumor expansion are associated with extracellular matrix remodeling and involve various proteases such as the plasminogen (Plg)/plasminogen activator (PA) system. Recently, several experimental data have implicated the plasminogen activator inhibitor-1 (PAI-1) in tumor angiogenesis in murine systems. However, little is known about PAI-1 functions in human skin carcinoma progression. By generating immunodeficient mice (in Rag-1-/- or nude background) deleted for PAI-1 gene (PAI-1-/-), we have evaluated the impact of host PAI-1 deficiency on the tumorigenicity of two malignant human skin keratinocyte cell lines HaCaT II-4 and HaCaT A5-RT3 forming low-grade and high-grade carcinomas, respectively. When using the surface transplantation model, angiogenesis and tumor invasion of these two cell lines are strongly reduced in PAI-1-deficient mice as compared to the wild-type control animals. After subcutaneous injection in PAI-1-/- mice, the tumor incidence is reduced for HaCaT II-4 cells, but not for those formed by HaCaT A5-RT3 cells. These data indicate that PAI-1 produced by host cells is an important contributor to earlier stages of human skin carcinoma progression. It exerts its tumor-promoting effect in a tumor stage-dependent manner, but PAI-1 deficiency is not sufficient to prevent neoplastic growth of aggressive tumors of the human skin.

Expression of urokinase plasminogen activator, its receptor and type-1 inhibitor in malignant and benign prostate tissue.

The plasminogen activation (PA) cascade participates in degradation of extracellular matrix during cancer invasion. We have studied the expression of urokinase-type plasminogen activator (uPA) mRNA, uPA receptor (uPAR) mRNA and immunoreactivity, and type-1 plasminogen activator inhibitor (PAI-1) mRNA and immunoreactivity in 16 prostate adenocarcinomas and 9 benign prostate hyperplasias. uPA mRNA and uPAR mRNA expression were found in 9 and 8 of the adenocarcinomas, respectively, and in 7 and 6 of the benign hyperplasias, respectively. In both malignant and benign lesions, expression of these 2 mRNAs was predominantly seen in cells identified as macrophages, which in most of the carcinomas (approximately 90%) were located in the interstitial tissue between the tumor cell islands, while in most of the benign hyperplasias they were located in the lumen of the glands and were in only a few cases (approximately 30%) found in the interstitial tissue. uPAR immunoreactivity correlated with the mRNA expression and was, in addition, found in neutrophils. PAI-1 mRNA was detected in 13 of the 16 carcinomas and in 8 of the 9 benign hyperplasias, located in scattered fibroblast-like cells in both groups, in some vascular structures and in a few macrophages located in the interstitial tissue of both malignant and benign lesions. A similar expression pattern was found for PAI-1 immunoreactivity. In 8 of the 16 carcinomas, all 3 components were present, and in several areas colocalization was observed in stromal cells in close proximity to cancer cell islands. No immunoreactivity and/or mRNA expression of uPA, uPAR or PAI-1 was observed in cancer cells or in other epithelial cells in any of the cases.

Reduced metastasis of transgenic mammary cancer in urokinase-deficient mice.

A prominent phenotype of plasmin deficiency in mice is reduced metastasis in the MMTV-PymT transgenic breast cancer model. Proteolytically active plasmin is generated from inactive plasminogen by one of 2 activators, uPA or tPA. We now find that uPA deficiency alone significantly reduces metastasis >7-fold in the MMTV-PymT model. We studied a cohort of 55 MMTV-PymT transgenic mice, either uPA-deficient or wild-type controls. Tumor incidence, latency, growth rate and final primary tumor burden were not significantly affected by uPA deficiency. In contrast, average lung metastasis volume was reduced from 1.58 mm(3) in wild-type controls to 0.21 mm(3) in uPA-deficient mice (p = 0.023). Tumor cell dissemination to brachial lymph nodes was also reduced from 53% (28/53) in wild-type controls to 31% (17/54) in uPA-deficient mice (p = 0.032). Mice without plasminogen display a severe pleiotropic phenotype. By comparison, spontaneous phenotypes are modest in uPA-deficient mice, probably because they still have active tPA. We show that metastasis is strongly and selectively decreased in uPA-deficient mice, suggesting that uPA-directed antimetastatic therapy would be efficacious and have limited side effects.