Relationships of protein biomarkers of the Urokinase Plasminogen Activator System with expression of their cognate genes in primary breast carcinomas.

Background: Urokinase plasminogen activator (uPA), its receptor uPAR and serine protease inhibitors PAI-1 or PAI-2 play key roles in tissue membrane remodeling and invasion of basement membranes by induction of a fibrinolytic pathway. Earlier studies reported that uPA and PAI-1 protein levels in breast carcinomas assist in prediction of response to chemotherapy. Our goal is to develop molecular signatures of candidate genes and identify novel relationships with these four protein biomarkers that demonstrate clinical utility for assessment of breast carcinoma outcomes. Methods: This retrospective study used de-identified biomarker results and clinical outcomes from primary breast cancers that were stored in an IRB-approved Database. ELISA analyses of uPA, uPAR and PAI-1 performed using IMUBIND kits (American Diagnostica Inc.) used cutoff values previously reported. Estrogen (ER) and progestin receptor (PR) assays were performed either by EIA (Abbott Labs) or by radioligand binding (NEN/DuPont). Relative expression levels of 22,000 genes were determined by microarray using RNA extracted and amplified from Laser-Capture Microdissection (LCM) procured breast carcinoma cells. Violin plots, scatter grams, univariable Cox regression and survival analyses were performed by R Studio version 3.4.1. Results: Patient age was significantly associated with level of either uPA, uPAR or PAI-1 protein, while Violin plots showed protein content of biomarkers was related to either ER or PR status of the primary. Examination of expression revealed that either ER- or PR- breast cancers expressed elevated levels of uPA, uPAR and PAI2 genes compared to that of ER+ or PR+ carcinoma cells. Scatter plots revealed inverse relationships between ER/PR protein levels and expression of uPA, uPAR and PAI-2, whereas HER2 status was unrelated to either protein content or gene expression for each relationship examined. Interrelationships analysis revealed an elevation of uPAR content in pre-menopausal cancer and increased PAI-1 expression in node negative cancers. When carcinomas were sorted by urokinase biomarker levels, qPCR expression of RERG and NQO-1 genes were elevated in uPA- lesions while CD34 and EDG-1 genes were elevated in uPAR- cancers. However, ERBB4 gene expression was elevated in PAI-1+ carcinomas. Furthermore, Cox regression analyses revealed relationships of ER/PR status and uPA system members with regard to clinical outcomes of breast cancer. Conclusions: Expression of either uPA, uPAR, PAI1 or PAI2 genes was significantly elevated in either ER- or PR- carcinomas similar to observed elevation of protein content in ER-/PR- carcinomas, strongly suggesting relationships between sex-hormone activity and regulation of urokinase plasminogen activator system in breast cancer. This was supported by results showing protein content of uPA system members was related to ER/PR status of the primary lesion. Use of LCM-procured breast carcinoma cells uncovered relationships between expression of several known cancer-associated genes and protein content of uPA system members. Collectively, results indicate evaluation of ER and PR protein of the primary breast cancer biopsy combined with analyses of uPA, uPAR and PAI-1 protein content improve assessment of clinical outcomes