Urokinase-type plasminogen
activator (uPA)

Plasminogen activator, urinary
Urokinase

Gene: uPA maps to 10q24-qter; spans 7.25 kb and consists of 11 exons.
mRNA: size: 2.5 kb.
Protein: a serine protease. uPA has a molecular weight of about 54 kDa and is composed of 2 disulfide-linked chains, A and B, of molecular weights 18 kDa and 33 kDa, respectively.

Cell lines:
- Breast tumor cells were shown to secrete factor(s) other than TGF beta1 ultimately responsible for the generation of powerful uPA-producing breast fibroblasts (Sieuwerts A.M. et al., 1998).

- In MDA-MB-231 BCC stably transfected with ER, uPA was down-regulated by estradiol (E₂). The addition of exogenous uPA did not block the growth inhibition by E₂ (Levenson A.S. et al., 1998).

- In MDA-MB-231 BCC, TGF-beta1 had no effect on cell proliferation but increased uPA activity and PAI-1 antigen level (Dong-Le Bourhis X. et al., 1998).

- Studies on SK-BR3 BCC have suggested that the transcription factors Ets-1 and Ets-2 provide the link connecting epidermal growth factor (EGF) stimuli with activation of uPA and MMP9 promoters and may contribute to invasion phenotypes (Watabe T. et al., 1998).

- uPA activity was found to be higher in the culture medium from MDA-MB-231 compared to MCF-7 BCC. In MDA-MB-231 BCC, uPA release was not altered by any of the antiestrogens [Analog II (1,1-dichloro-cis-2,3-diphenyl cyclopropane), ICI-182,780, and tamoxifen], used alone or in the presence of estradiol (E₂). In contrast, in MCF-7 BCC, ICI-182,780 alone produced maximal inhibition (40%) of enzyme release, while E₂ alone produced a 120% increase in enzyme activity. When co-administered with E₂, in MCF-7 cultures, each antiestrogen reduced enzyme activity to control levels (Abidi S.M. et al., 1998).

- The absence of urokinase expression in normal mammary epithelial cells and in MCF-7 and T-47D breast cancer cells (BCC) could be due to the methylation of its gene (Xing R.H. and Rabbani S.A., 1999).

- Populations of BCC stably transfected with an osteopontin (OPN)-expression vector showed higher levels of cell invasiness than control vector transfectants. Examination of transfectants for mRNA of a number of secreted proteases showed that only urokinase-type plasminogen activator (uPA) expression was closely associated with OPN expression and cellular invasiveness (Tuck A.B. et al., 1999).

- uPA was shown to promote MCF-7 BCC migration on vitronectin-coated surfaces, in an integrin (a beta₁-integrin -probably alpha_Vbeta₁-, and alpha_Vbeta₅)-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MAP kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and myosin light chain kinase (MLCK) serve as essential downstream effectors (Nguyen D.H. et al., 1999).

- Treatment of highly invasive BT549 BCC with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these BCC to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. SB203580-induced reduction in uPA/uPAR mRNA expression was shown to result from the de-stabilization of uPA and uPAR mRNA. Finally, it was demonstrated that the p38alpha, rather than the p38beta, MAPK isoform activity was essential for uPA/uPAR expression. Thus, p38alpha MAPK signaling pathway might be important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA (Huang S. et al., 2000).

Tumors:
- The concentrations of cathepsin D, uPA, PAI-1, and PAI-2 were analysed in the cytosols of 43 benign and 87 malignant mammary tumors. The mean levels of these markers were significantly higher in malignant tumors than in benign tumors. The increases were about 4-, 5-, 74-, and 29-fold, respectively. When cathepsin D, uPA, PAI-1, and PAI-2 levels in malignant tumors were compared, positive correlations were found for all combinations (Foucré D. et al., 1991).

- In a study of breast tumors from 118 pre- and 72 postmenopausal high-risk patients, the levels of uPA and PAI-1 were strongly correlated. High uPA level was associated with grade of anaplasia in premenopausal patients and with number of tumor-positive lymph nodes in postmenopausal patients (Grøndahl-Hansen J. et al., 1993).

- uPA, its receptor uPAR, and PAI-1 were measured in breast cancer cytosol from 111 low-risk premenopausal patients and 184 low-risk postmenopausal patients with a median follow-up of 6.0 years. uPA, uPAR, and PAI-1 levels were all weakly but significantly correlated with each other in both menopausal groups. There were no significant differences in the median levels of uPA, uPAR, and PAI-1 between premenopausal and postmenopausal patients (Grøndahl-Hansen J. et al., 1997).

- 575 node-positive and node-negative breast cancer samples with an average of 10 years follow-up were studied by in situ hybridization on formalin-fixed, paraffin-embedded material using MMP2, MMP11, and uPA mRNA antisense probes. A significant correlation was found between high (more than 10%) MMP11 expression and a younger age, lymph node involvement, poor nuclear grade, ductal histology, aneuploidy, and HSP-27 expression. High MMP2 expression was significantly associated with c-erbB2, ductal histology, and HSP-27 expression. High uPA expression correlated with poor nuclear grade, ductal histology, lack of estrogen and progesterone receptors, and p53 protein accumulation. High level of expression of all three proteases correlated significantly with each other and with cathepsin D expression by reactive stromal cells. By univariate analysis, both ST3 and uPA expression significantly predicted a shorter recurrence-free survival (Têtu B. et al., 1998).

- In a prospective study of 130 patients with node-negative invasive breast cancer who underwent radical operation (the median follow-up was 52.6 months), patients with high u-PA, high PAI-1, or low t-PA had significantly higher relapse rates than did those with low u-PA, low PAI-1, or high t-PA, respectively, by the Kaplan-Meier method (P = 0.006, 0.032, and 0.028, respectively). Analyses of the combinations of both u-PA and PAI-1 or both u-PA and t-PA showed that the differences in relapse rate between the high- and low-risk groups were statistically very significant. In the univariate analysis, u-PA, PAI-1, t-PA, progesterone receptor, and tumor size (T3 versus T1) were significantly correlated with relapse. However, the multivariate analysis revealed that only u-PA (P = 0.023) was an independent prognostic factor (Kim S.J. et al., 1998).

- In 499 primary breast cancer cytosols, uPA was determined using a classical ELISA method and an automatic immunoluminometric assay. The correlation coefficient between the two methods was acceptable (r = 0.81), but the two techniques were not interchangeable. Univariate analyses confirmed the poor outcome of patients whose tumours contained large amounts of uPA, regardless of the technique used (Bouchet C. et al., 1998).

- uPA and uPAR levels were semiquantitated by immunocytochemistry in 36 primary breast carcinomas. Both were mostly present in stromal cells in invasive breast carcinomas, suggesting that stromal cells collaborate with malignant cells to mediate metastasis (Kennedy S. et al., 1998).

- The levels of p53 and uPA were measured (in cytosols prepared for estradiol receptor (ER) and progesterone receptor (PgR) assays) in 634 tumor tissues from 634 different node-negative primary breast cancer patients. p53 was elevated in 13.7% of the tumors, and uPA was elevated in 27.5% of the tumors; they were negatively related (chi 2 test) to ER and PgR and positively related to histoprognostic grading (HPG) and tumor diameter. uPA was negatively correlated to ER and PgR, and p53 and uPA were positively correlated to each other (Peyrat J.P. et al., 1998).

- In a retrospective study of 429 primary breast cancer patients with a median follow-up of 5.1 years, the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death (Knoop A. et al., 1998).

- It has been suggested that uPA family members and sex steroid receptors may be measured in the same cytosol fraction from breast tumor homogenates (Descotes F. et al., 1998).

- By substrate zymography, uPA was measured in paired tumour and normal tissue samples from 43 breast cancer patients. uPA was expressed 90% breast tumours and 56% normal breast samples (Garbett E.A. et al., 1999).

- In a study of 159 small axillary node-negative tumors, no consistent association between the immunohistochemically quantified uPA and PAI-1 and prognosis was found (Jahkola T. et al., 1999).

- uPA and PAI-1 were measured by ELISA in samples derived from 892 patients with primary breast cancer (median follow-up 99 months). The assays were performed in cytosolic extracts as well as in corresponding detergent extracts of pellets obtained after ultracentrifugation, which was carried out when preparing the cytosolic fractions for routine steroid hormone receptor determination. High cytosolic levels of uPA or PAI-1 were significantly associated with increased rates of relapse and death. The levels of uPA and PAI-1 in the pellet extracts also provided prognostic information, although to a lesser extent compared with the cytosolic extracts (de Witte J.H. et al., 1999).

- An ELISA has been described for the assessment of complexes between the urokinase-type (uPA) and the tissue-type plasminogen (tPA) activators with their inhibitor type-1 (PAI-1) in cell-culture medium and cytosolic extracts of breast tumours (Grebenschikov N. et al., 1999). - A kinetic sandwich format immunoassay for specific quantification of the uPA:PAI-1 complex was developed, validated, and applied to plasma from 19 advanced-stage breast cancer patients, 39 age-matched healthy women, and 31 men. 18 cancer patients had a measurable complex concentration (median, 68 ng/L; range, <16 to 8700 ng/L), whereas for healthy females and males the median signal values were below the detection limit (median, <16 ng/L; range, <16 to 200 ng/L; P <0.0001). For patient plasma, a comparison with total uPA and PAI-1 showed that the complex represented a variable, minor fraction of the uPA and PAI-1 concentrations of each sample (Pedersen A.N. et al., 1999).

- The prognostic value of uPA and p53 levels in a series of the prognostic value of urokinase plasminogen activator (uPA) and p53 levels in a series of 1245 primary breast cancer (median follow-up time of patients: 75 months), using an automatic quantitative luminometric method. In univariate analysis, high levels of uPA and p53 were associated with shorter disease-specific survival, disease-free interval, and distant recurrence-free interval. In multivariate analysis, the risk of disease-related death increased with uPA levels after adjustment for tumour size, histological grade, lymph node involvement, and estrogen receptor status. A high level of uPA was also related to a shorter disease-free interval and distant recurrence-free interval. In node-negative patients, a high level of uPA remained strongly related to the three outcomes. It was also shown that the prognostic value of p53 accumulation is limited when uPA is included in multivariate analysis (Broët P. et al., 1999).

Abidi S.M. et al. (1998) The influence of antiestrogens on the release of plasminogen activator (uPA) by MDA-MB-231 and MCF-7 breast cancer cells. Clin. Exp. Metastasis 16, 235-241.
Bouchet C. et al. (1998) Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods. Br J Cancer 1998 May 77:9 1495-501.
Broët P. et al. (1999) Prognostic value of uPA and p53 accumulation measured by quantitative biochemical assays in 1245 primary breast cancer patients: a multicentre study. Br. J. Cancer 80, 536-545.
Descotes F. et al. (1998) Tissue extraction procedures for investigation of urokinase plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in human breast carcinomas. Breast Cancer Res. Treat. 49, 135-143.
de Witte J.H. et al. (1999) Prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) in cytosols and pellet extracts derived from 892 breast cancer patients. Br. J. Cancer 79, 1190-1198.
Dong-Le Bourhis X. et al. (1998) Transforming growth factor beta 1 and sodium butyrate differentially modulate urokinase plasminogen activator and plasminogen activator inhibitor-1 in human breast normal and cancer cells. Br. J. Cancer 77, 396-403.
Duffy M.J. et al. (1999) Urokinase plasminogen activator: a prognostic marker in multiple types of cancer. J. Surg. Oncol. 71, 130-135 (Review).
Foucré D. et al. (1991) Relationship between cathepsin D, urokinase, and plasminogen activator inhibitors in malignant vs benign breast tumours. Br. J. Cancer 64, 926-932.
Garbett E.A. et al. (1999) Proteolysis in human breast and colorectal cancer. Br. J. Cancer 81, 287-293.
Grebenschikov N. et al. (1999) ELISA for complexes of urokinase-type and tissue-type plasminogen activators with their type-1 inhibitor (uPA-PAI-1 and tPA-PAI-1). Int. J. Cancer 81, 598-606.
Grøndahl-Hansen J. et al. (1993) High levels of urokinase-type plasminogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis. Cancer Res. 53, 2513-2521.
Grøndahl-Hansen J. et al. (1997) Plasminogen activator inhibitor type 1 in cytosolic tumor extracts predicts prognosis in low-risk breast cancer patients. Clin. Cancer Res. 3, 233-239.
Huang S. et al. (2000) Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity. J. Biol. Chem. 275, 12266-12272.
Jahkola T. et al. (1999) Cathepsin-D, urokinase plasminogen activator and type-1 plasminogen activator inhibitor in early breast cancer: an immunohistochemical study of prognostic value and relations to tenascin-C and other factors. Br. J. Cancer 80, 167-174.
Kennedy S. et al. (1998) Semi-quantitation of urokinase plasminogen activator and its receptor in breast carcinomas by immunocytochemistry. Br. J. Cancer 77, 1638-1641.
Kim S.J. et al. (1998) Prognostic impact of urokinase-type plasminogen activator (PA), PA inhibitor type-1, and tissue-type PA antigen levels in node-negative breast cancer: a prospective study on multicenter basis. Clin. Cancer Res. 4, 177-182.
Knoop A. et al. (1998) Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer. Br. J. Cancer 77, 932-940.
Levenson A.S. et al. (1998) Oestradiol regulation of the components of the plasminogen-plasmin system in MDA-MB-231 human breast cancer cells stably expressing the oestrogen receptor. Br. J. Cancer 78, 88-95.
Nagai M. et al. (1985) Molecular cloning of cDNA coding for human preprourokinase. Gene 36, 183-188.
Nguyen D.H. et al. (1999) Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner. J. Cell. Biol. 146, 149-164.
Pedersen A.N. et al. (1999) Determination of the complex between urokinase and its type-1 inhibitor in plasma from healthy donors and breast cancer patients. Clin. Chem. 45, 1206-1213.
Peyrat J.P. et al. (1998) Prognostic value of p53 and urokinase-type plasminogen activator in node-negative human breast cancers. Clin. Cancer. Res. 4, 189-196.
Rajput B. et al. (1985) Chromosomal locations of human tissue plasminogen activator and urokinase genes. Science 230, 672-674.
Riccio A. et al. (1985) The human urokinase-plasminogen activator gene and its promoter. Nucleic Acid Res. 13, 2759-2771.
Sieuwerts A.M. et al. (1998) Urokinase-type-plasminogen-activator (uPA) production by human breast (myo) fibroblasts in vitro: influence of transforming growth factor-beta(1) (TGF beta(1)) compared with factor(s) released by human epithelial-carcinoma cells. Int. J. Cancer 76, 829-835.
Têtu B. et al. (1998) Prognostic significance of stromelysin 3, gelatinase A, and urokinase expression in breast cancer. Hum. Pathol. 29, 979-985.
Tripputi P. et al. (1985) Human urokinase gene is located on the long arm of chromosome 10. Proc. Nat. Acad. Sci. USA 82, 4448-4452.
Tuck A.B. et al. (1999) Osteopontin induces increased invasiveness and plasminogen activator expression of human mammary epithelial cells. Oncogene 18, 4237-4246.
Watabe T. et al. (1998) The Ets-1 and Ets-2 transcription factors activate the promoters for invasion-associated urokinase and collagenase genes in response to epidermal growth factor. Int. J. Cancer 77, 128-137.
Xing R.H. and Rabbani S.A. (1999) Transcriptional regulation of urokinase (uPA) gene expression in breast cancer cells: role of DNA methylation. Int. J. Cancer 81, 443-450.

Plasminogen activator inhibitor, type 1 (PAI-1), plasminogen activator inhibitor, type 2 (PAI-2), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator receptor (uPAR)