EXPRESSION AND FUNCTIONS OF EGF, FGF AND TGFb-GROWTH-FACTOR FAMILY MEMBERS AND THEIR RECEPTORS IN INVASIVE HUMAN TRANSITIONAL-CELL-CARCINOMA CELLS Willem I. DE BOER 1 *, Adriaan B. HOUTSMULLER 2 , Vali IZADIFAR 1 , Be ´atrice MUSCATELLI-GROUX 1 , Theodorus H. V AN DER KWAST 2 and Dominique K. CHOPIN 1 1 GETU-Centre des Recherches Chirurgicales, Universite ´ Paris XII, Cre ´teil, France 2 Department of Pathology, Erasmus University, Rotterdam, The Netherlands Studies on epidermal-growth-factor-like-, fibroblast- and transforming growth factors suggested their implication in tumorigenesis involving effects on tumour-cell proliferation and migration. In human transitional-cell carcinomas (T CC), enhanced expression of TGFa and EGF receptors correlated with an aggressive phenotype. H owever, little is known about functions of these growth factors in invasive TCCs. In this study, we performed protein- and RN A-expression studies on a set of growth factors and their receptors on the newly established invasive human TCC cell line designated 1207. The data were correlated with functional proliferation and migration studies. Similar expression patterns of many cellu- lar markers, growth factors and their receptors were noted both in the original TCC tissue and in its derivative cell line, indicating the relevance of this cell line to the investigation of growth factor functions on TCC cells. The proliferation induction by EGF, TGFa, amphiregulin, heregulin a, FGF-1 and FGF-7 correlated with the presence of EGF receptors, c-erbB4 and FGFR2 (IIIb), respectively. Amphiregulin and heregulin a induced the most proliferation. In conformity with the low expression of TGFb receptors I and II, TGFb 1 barely inhibited proliferation, while T GFa induced invasion of 1207 cells into Matrigel. These data support the notion that notably EGF-like proteins mediate T CC growth and invasion through autocrine pathways which can be reinforced by loss of TGFb 1 regulation. Int. J. Cancer 71:284–291, 1997. r 1997 Wiley-Liss, Inc. Growth factors such as transforming growth factor beta(TGFb), epidermal growth-factor(EGF) and fibroblast-growth-factor- like(FGF) proteins have been reported to be implicated in wound healing, tumorigenesis and embryogenesis. These proteins are capable of modulating, e.g., cellular proliferation, differentiation, migration or protein synthesis. EGF-like proteins, including EGF, TGFa, amphiregulin and heregulins (HRG), are expressed in various normal epithelial and carcinoma cells (Plowman et al., 1990; Johnson et al., 1992; Danilenko et al., 1995; Salomon et al., 1995). In several carcinomas, such as breast and renal carcinomas, over-expression of TGFa and/or amphiregulin has been observed (Mydlo et al., 1989; LeJeune et al., 1993; Salomon et al., 1995). Normal human bladder urothelium is reported to express EGF, TGFa and amphiregulin, while high levels of EGF have been found in urine of normal individuals (Kimball et al., 1984; Lau et al., 1988; Cilento et al., 1994). However, the urine of patients with transitional-cell carcinoma (TCC) contains enhanced levels of TGFa (Kimball et al., 1984). Expression of the EGF receptor (EGFR) which binds EGF, TGFa and amphiregulin, was demon- strated to be augmented in human TCCs, notably in the more aggressive TCCs (Messing et al., 1987; Neal et al., 1990). Functional effects of EGF-like proteins include stimulation of proliferation and migration of different normal epithelial and carcinoma cells, including murine urothelial cells (De Boer et al., 1993, 1994; Danilenko et al., 1995). Both expression and func- tional data suggest a direct function for EGF-like molecules in TCC development or progression. While HRG were shown to enhance proliferation, migration or differentiation of normal skin and breast epithelial cells (Danilenko et al., 1995; Marikovsky et al., 1995; Yang et al., 1995), less is known about the functions of HRG in tumour growth. The expression of their receptors, c-erbB3 (HER3) and c-erbB4 (HER4), was observed to be enhanced in several tumours, including breast and bladder carcinomas (Lemoine et al., 1992; Plowman et al., 1993; Rajkumar et al., 1996), suggesting an implication of HRG in these tumours. While c-erbB3 was also reported to be present in the normal urogenital tract, little is known about functions of HRG or their receptors in human urothelium or TCCs. FGF have also been suggested to be involved in human TCC growth. Chopin et al. (1993) demonstrated enhanced FGF-1, but not FGF-2, expression in TCCs. Furthermore, FGF-1 was shown to induce in vitro proliferation, motility and invasion of murine bladder-carcinoma cells (Valle ´s et al., 1990; Tucker et al., 1991; De Boer et al., 1993). These studies suggest a direct role for FGF-1 in TCC-cell invasion. Finally, mis-sense mutations in TGFb receptors(TGFbR) types I and II have been shown in colon-, gastric- and prostate-carcinoma cells (Myeroff et al., 1995; Kim et al., 1996; Lee et al., 1996), resulting in TGFb-non-responsive cells (Kim et al., 1996). Although functional TGFb receptors(TGFbR) types I and II are expressed in normal human urothelium (De Boer et al., 1996), little is known about the expression of TGFb receptors in human TCCs. Since little is known about EGF-like, FGF-like or TGFb-growth- factor functions in human TCCs, we provided a model for examining the direct functional effects of EGF and FGF family members, as well as HRGa and TGFb 1 and their receptors on invasive human TCC cells. We established a new human TCC cell line derived from an invasive poorly differentiated TCC, desig- nated 1207. We determined the expression of these growth factors and their receptors by immunocytochemistry where possible, or on the RNA level by RT-PCR, on early passages of the cell line and on the original tissue. This was correlated with functional analysis of the cell line with regard to growth-factor-modulated proliferation or invasion into a 3-dimensional gel comprised of growth-factor- reduced Matrigel. MATERIAL AND METHODS Chemicals Biochemicals were purchased as follows: human fibroblast growth factors, human TGFa, and human transferrin (Boehringer, Mannheim, Germany); culture medium additives, human EGF and TGFb 1 (Sigma, St. Louis, MO); amphiregulin and HRGa (R&D Contract grant sponsors: l’Association Claude Bernard, la De ´le ´gation a ` la Recherche Clinique (DRC), l’Assistance Publique des Ho ˆpitaux de Paris (AP-HP), le Ministe `re de la Sante ´, le Comite ´ du Val de Marne de la Ligue Contre le Cancer; contract grant sponsor: l’Universite ´ Paris XII; contract grant number: EA418; contract grant sponsor: Re ´publique Franc ¸aise; contract grant number: AOA94015. *Correspondence to: GETU-Centre de Recherches Chirurgicales, Medical Faculty, University Paris XII, 8, rue du Ge ´ne ´ral Sarrail, 94010 Cre ´teil, France. Fax: 33 1 49 81 35 52. E-mail: deboer@pathology.medfac.leidenuniv.nl Received 26 September 1996; revised 14 November 1996 Int. J. Cancer: 71, 284–291 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer