EFFECTS OF THE TYROSINE-KINASE INHIBITOR GELDANAMYCIN ON LIGAND-INDUCED HER-2/NEU ACTIVATION, RECEPTOR EXPRESSION AND PROLIFERATION OF HER-2-POSITIVE MALIGNANT CELL LINES Frank HARTMANN 1,6 , Eva M. HORAK 1 , Cheryl CHO 3 , Ruth LUPU 4 , Joseph B. BOLEN 5 , Mary A. STETLER-STEVENSON 2 , Michael PFREUNDSCHUH 6 , Thomas A. WALDMANN 1 and Ivan D. HORAK 1,7 1 Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 2 Department of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 3 Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, Washington, DC 20007, USA 4 Lawrence Berkeley National Laboratory, University of California Berkeley, Berkeley, CA 94720, USA 5 Department of Cellular Signaling, DNAX Research Institute, Palo Alto, CA 94304, USA 6 Innere Medizin I, Universita ¨tskliniken des Saarlandes, D-66421 Homburg/Saar, Germany Geldanamycin belongs to the family of benzoquinoid ansa- mycin tyrosine-kinase inhibitors. W e have examined its ef- fects on Her-2/neu kinase activity, protein expression level, and proliferation of H er-2 1 malignant cells. In SK-BR-3 breast- cancer cells, short-time treatment with geldanamycin com- pletely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treat- ment of intact cells with geldanamycin induced decreased steady-state H er-2 autophosphorylation activity, which corre- lated with reduction of H er-2 protein expression and phospho- tyrosine content of several proteins. T he decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduc- tion of the Her-2 protein level probably resulted from in- creased degradation, since the Her-2 mRN A level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of H er-2 (SK-BR-3 G SK-OV-3 G OVCAR3 G MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [ 3 H ] -thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concen- trations only in SK-BR-3 (IC 50 2nM) and MCF7 (IC 50 20nM), while O VCAR3 was only moderately sensitive (IC 50 2mM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansa- mycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced. Int. J. Cancer, 70:221–229, 1997. r 1997 Wiley-Liss, Inc. The receptor protein tyrosine kinases (PTKs) appear to play a pivotal role in the initiation of growth-factor-induced signal- transduction pathways. Despite the diversity of receptor PTKs, ligand-induced receptor dimerization followed by increased tyro- sine-kinase activity and protein phosphorylation of several sub- strates is common and essential for eliciting biological responses, including proliferation, differentiation and cell survival (Fantl et al., 1993). Her-2 (Neu or c-erbB-2, p185 erbB-2 ) belongs to the epidermal-growth-factor-receptor (EGFR) family, showing exten- sive homology to EGFR (p170 erbB-1 ) itself. Her-2 is the non- mutated, human homologue proto-oncogene of the oncogenic mutated form of the rat protein p185 neu , which, due to a point mutation within the transmembrane region, exhibits ligand- independent homodimerization leading to constitutive signalling and cell transformation. Amplification and/or over-expression of Her-2 has been observed in a variety of human adenocarcinomas, particularly breast cancer, and has been correlated with poor prognosis. Structure, functional properties, and signal-transduction pathways of Her-2 have been reviewed by Dougall et al. (1994). Although the exact mechanisms of Her-2-mediated cellular transformation remain elusive, physical and functional interaction between Her-2 and EGFR by heterodimerization and a high degree of autophosphorylation activity appear to play important roles. The involvement of PTKs as oncoproteins and proto-oncopro- teins in abnormal proliferative processes identifies them as poten- tial targets for anti-tumor therapy with tyrosine-kinase inhibitors (Levitzki and Gazit, 1995). Geldanamycin is a benzoquinoid ansamycin antibiotic structurally related to herbimycin A. For both, pleiotropic effects on different tyrosine kinases has been noted, although geldanamycin has been studied less extensively than herbimycin A. Specifically, herbimycin A blocks the intracellular PTKs src (Uehara et al., 1989), and bcr-abl (Okabe et al., 1992) as well as receptor PTKs such as EGFR (Murakami et al., 1994), and the insulin-like growth-factor receptor (Sepp-Lorenzino et al., 1995) or Her-2 (Miller et al., 1994). PTK inhibition has been shown to coincide with decreased PTK-protein-level expression, probably caused by increased protein degradation and reduction of total phosphotyrosine levels. However, the exact mechanism of PTK inhibition by benzoquinoid ansamycins remains undeter- mined. In particular, the ability to directly inhibit the kinase activity and the relationship between PTK inhibition and PTK protein decrease is still a matter of debate. In addition, herbimycin A has been shown to diminish substrate protein-tyrosine phosphorylation and abrogate T-cell-antigen-receptor signalling pathways, includ- ing inhibition of lck and fyn (June et al., 1990). It inhibits growth and reverses the morphology of cells trans- formed by the PTK oncogenes src, yes, fps, and erbB-1 but not the non-PTK oncogenes ras, raf, and myc (Murakami et al., 1988; Uehara et al., 1988) and causes growth inhibition and differentia- tion in leukemic cells expressing the bcr-abl fused gene product with augmented PTK activity (Okabe et al., 1992). Geldanamycin has been shown to inhibit src and cause suppression of c-myc expression and enhanced expression of hyperphosphorylated pRB along with G 2 /M cell-cycle arrest (Yamaki et al., 1989, 1995). Furthermore, both geldanamycin and herbimycin A have been shown to have anti-tumor effects in vitro and in vivo on malignant cells of neural origin probably unrelated to PTK inhibition (Whitesell et al., 1992). In order to further characterize the tyrosine-kinase inhibitory and anti-tumoral capacity of geldanamycin, we examined its effects on spontaneous and ligand-induced Her-2-kinase activity and protein expression in SK-BR-3 cells and looked for a correlation between 7 Present address: Department of Clinical Research and Development, Janssen Research Foundation, Titusville, NJ 08560-0200, USA. *Correspondence to: Innere Medizin I, Universita ¨tskliniken des Saarlan- des, D-66421 Homburg/Saar, Germany. Tel.: (06841) 16-3002. Fax: (06841) 16-3101. Received 5 June 1996; revised 7 October 1996. Int. J. Cancer: 70, 221–229 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer