Breast Cancer Research and Treatment 56: 91–97, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands. Report Association of in vitro invasiveness and gene expression of estrogen receptor, progesterone receptor, pS2 and plasminogen activator inhibitor-1 in human breast cancer cell lines Dan Tong 1 , Klaus Czerwenka 2 , Jan Sedlak 3 , Christian Schneeberger 4 , Ingrid Schiebel 1 , Nicole Concin 1 , Sepp Leodolter 1,5 , and Robert Zeillinger 1 1 Division of Gynecology, Molecular Oncology Group; 2 Department of Clinical Pathology, University of Vienna, Vienna, Austria; 3 Cancer Research Institute, Bratislava, Slowakia; 4 Division of Gynecological Endocrinology and Reproductive Medicine, Department of Obstetrics and Gynecology, Vienna, Austria; 5 Ludwig Boltzmann Institute for Gynecological Oncology and Reproductive Medicine, A-1090 Vienna, Austria Key words: breast cancer cell lines, gene expression pattern, invasiveness Summary The invasive potential of tumor cells is usually tested either by in vitro invasion assays which evaluate cell spreading ability in basement membrane-like matrices or by in vivo invasion assays in nude mice. Both methods are laborious and time-consuming. Tumor invasiveness is accompanied by the changes in expression of various genes. The invasive behavior of cells is therefore represented by certain gene expression patterns. The purpose of this study was to investigate whether expression patterns of several genes are characteristic for the invasiveness of cultured cells. We examined the mRNA levels of estrogen receptor (ER), progesterone receptor (PR), estrogen inducible pS2 and plasminogen activator inhibitor-1 (PAI-1) in 23 cell lines derived from benign and malignant breast tissues using a competitive reverse transcription-polymerase chain reaction (cRT-PCR) system. We also evaluated the invasiveness of these cell lines by their ability to penetrate into a collagen-fibroblast matrix. We demonstrate that the gene expression pattern of breast cell lines is clearly associated with their in vitro invasiveness. In general, cells with ER, PR, pS2 but no PAI-1 expression showed a non-invasive phenotype, while cells expressing PAI-1 mRNA but not ER mRNA are invasive. Our study indicates that the invasiveness of breast cancer cell lines is characterized by PAI-1 gene expression and the lack of ER mRNA. This suggests that PAI-1 may participate in the invasive process. Introduction Metastasis causes death in approximately 50% of breast cancer patients [1]. The process of metastasis involves a complex series of cellular alterations and is influenced by different steroid hormones, their recept- ors, growth factors, oncogenes and tumor suppressor genes. Various enzymes also play important roles in this process by degrading basement membranes and extracellular matrix components. Estrogen receptor (ER) and progesterone receptor (PR) inversely cor- relate with tumor size, histological grade and lymph node involvement [2–4]. They are indicators of longer disease-free intervals and better overall survival for patients with primary breast cancer [5]. It has been documented that breast cancer cells without ER are generally less differentiated and more aggressive than those containing functional ER [6]. Moreover, activ- ation of ER by transfecting it into a receptor-negative breast cancer cell line decreases the metastatic and in- vasive potential of the cells [7]. The protein pS2 was first identified during a search for estrogen receptor- associated markers [8]. pS2 has been found to correl- ate with ER and PR status and to correlate inversely with tumor size and histological grade [2, 9, 10]. Like ER and PR, pS2 is also a marker for good prognosis [11, 12]. The urokinase-type plasminogen activator (uPA) is a serine protease that catalyzes the conver-