ABSTRACT
BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.
Subject(s)
Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Plasminogen Activator Inhibitor 1/biosynthesis , Protein C Inhibitor/biosynthesis , RNA, Messenger/metabolism , Urokinase-Type Plasminogen Activator/biosynthesis , Adult , Aged , Aged, 80 and over , Disease-Free Survival , Female , Humans , Immunohistochemistry , In Situ Hybridization , Middle AgedABSTRACT
BACKGROUND: The plasminogen activator inhibitor type 1 (PAI-1) 4G/5G polymorphism may have significance for PAI-1 expression. High levels of PAI-1 in breast cancer patients are associated with a poor prognosis. In this study, we analyzed the influence of the PAI-1 4G/5G polymorphism on tissue PAI-1 levels and its association with tumor severity in women with breast cancer. MATERIAL AND METHODS: We studied 104 women with breast carcinoma (patient group) and 104 healthy age-matched women (control group). In patients and controls, the PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. In patients, PAI-1 levels were quantified in breast cancer tissue by using an ELISA. RESULTS: The frequency of the PAI-1 4G allele tended to be higher in patients than in controls (p=0.062). The presence of the 4G allele (4G/5G plus 4G/4G genotypes) was significantly higher among patients with histological grade 3 tumors than among those with grade 1 tumors (p=0.026). Furthermore, patients with the 4G/4G genotype had significantly higher tissue PAI-1 levels than those with the 5G/5G genotype. Moreover, tissue PAI-1 antigen levels were significantly and positively correlated with tumor severity (p=0.003) and tumor size (p=0.009). However, no significant differences in PAI-1 level were observed in relation to menopause, hormone receptor or nodal status. CONCLUSION: Tissue PAI-1 antigen levels and tumor severity seem to be associated with the PAI-1 4G/5G polymorphism. Further studies with a larger number of patients are needed to clarify the influence of this polymorphism in breast cancer.
Subject(s)
Breast Neoplasms/genetics , Plasminogen Activator Inhibitor 1/genetics , Polymorphism, Genetic , Severity of Illness Index , Adult , Aged , Aged, 80 and over , Alleles , Breast Neoplasms/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Genotype , Humans , Middle Aged , Plasminogen Activator Inhibitor 1/metabolism , Prognosis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: The plasminogen activation system and matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, cancer cell invasion, and metastasis. METHODS: Quantitative real-time reverse-transcription-PCR (RT-PCR) assays were developed to quantify urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue metalloproteinase inhibitor type 1 (TIMP-1) mRNA in 54 breast cancer tissues. Gene fragments were amplified in a LightCycler real-time PCR system using gene-specific primers and SYBR Green I. The results were normalized to beta-actin mRNA. We also quantified antigen and functional concentrations of these components. RESULTS: The intra- and interassay variabilities for mRNA quantification showed mean SDs for the crossing point of 0.12 and 0.15 cycles, respectively. PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and PAI-1 and uPA functional concentrations increased with tumor severity; the increase was statistically significant for PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and for uPA functional concentrations. Node-positive patients showed significantly higher PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations than those who were node negative. CONCLUSIONS: Quantitative real-time RT-PCR is a highly sensitive, reproducible, and fast method for measuring gene expression of PAI-1, uPA, and TIMP-1 in breast cancer. These components may be involved in breast cancer development, and increased mRNA expression may be associated with a worse prognosis.
