An unexpected biochemical and functional interaction between gp130 and the EGF receptor family in breast cancer cells Susan L Grant* ,1,2,3 , Annet Hammacher 2,4 , Andrea M Douglas 1,2,3 , Geraldine A Goss 1 , Rachel K Mans®eld 1,2,3 , John K Heath 5 and C Glenn Begley 1,2,3,6 1 The Rotary Bone Marrow Research Laboratories, PO Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia; 2 The Cooperative Research Centre for Cellular Growth Factors, PO Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia; 3 The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia; 4 Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, PO Box 2008, Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia; 5 Cancer Research Campaign, Growth Factors Group, School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK; 6 Centre for Child Health Research, University of Western Australia, TVW Telethon Institute for Child Health Research and Western Australian Institute for Medical Research, PO Box 855, West Perth, Western Australia Oncostatin M (OSM), an interleukin-6 type cytokine, acts via the gp130 signaling receptor to inhibit proliferation and induce dierentiation of breast cancer cells. EGF, a mitogen for breast cells, signals via EGFR/ ErbB tyrosine kinase receptors which are implicated in breast cancer pathogenesis. Here we show paradoxically that EGF enhanced the OSM-induced inhibition of proliferation and induction of cellular dierentiation in both estrogen receptor positive and negative breast cancer cells. This functional synergism was also seen with heregulin but not SCF, PDGF or IGF-1, indicating that it was speci®c to EGF-related growth factors. Immunoprecipitation experiments revealed that gp130 was constitutively associated with ErbB-2 and ErbB-3. There was a similar association between the OSMRb and ErbB-2. Furthermore, EGF unexpectedly induced tyrosine phosphorylation of gp130. We show that OSM induced phosphorylation of STAT3. Both OSM and EGF activated the p42/44 MAP kinases, but while the MEK inhibitor, PD98059, ablated the OSM-induced inhibition, it only partially ablated the inhibitory eects of OSM plus EGF. Thus, we have demonstrated that the receptors and signalling pathways of two apparently unrelated growth factors were intimately linked, resulting in an unexpected biological eect. This provides a new mechanism for generating signalling diversity and has potential clinical implications in breast cancer. Oncogene (2002) 21, 460 ± 474. DOI: 10.1038/sj/onc/ 1205100 Keywords: breast cancer; oncostatin M; epidermal growth factor; ErbB; gp130; receptors Introduction Oncostatin M (OSM) is a pleiotropic cytokine, ®rst identi®ed by its ability to inhibit growth of several cancer cell lines (Horn et al., 1990; Zarling et al., 1986). We have previously shown that OSM inhibits the proliferation of estrogen receptor (ER) positive and negative breast cancer cell lines (Douglas et al., 1997, 1998). This inhibition is associated with decreased clonogenicity, cell cycle suppression, alterations in cellular morphology and a number of other changes, all consistent with induction of dierentiation in these cells (Douglas et al., 1998). OSM is a member of the interleukin-6 (IL-6) type cytokine family which includes leukemia inhibitory factor (LIF), interleukin-11 (IL-11), cardiotrophin 1 (CT-1) and ciliary neurotrophic factor (CNTF). These cytokines mediate a number of overlapping biological activities which is in part explained by the sharing of the signal-transducing receptor subunit, gp130 (Gear- ing et al., 1992; Hibi et al., 1996; Taga, 1996). In addition, a number of these cytokines also share the LIF speci®c receptor subunit (LIFRb). The receptor complex utilized by OSM is unique in the IL-6 cytokine family in that OSM is the only member to bind gp130 directly, with low anity (Gearing et al., 1992). Subsequent to OSM binding, gp130 forms a high anity receptor complex with either the LIFRb or the OSM-speci®c receptor (OSMRb) to form the type I and II OSM receptors, respectively (Auguste et al., 1997; Grant and Begley, 1999; Mosley et al., 1996). Upon OSM binding the dimerization of receptor subunits initiates activation of intracellular signalling pathways. The JAK/STAT pathway is the most characterized pathway downstream of gp130. In response to IL-6 type cytokines, JAK1, and to a lesser extent, JAK2 and Tyk2, which associate constitutively with the cytoplasmic domains of gp130, LIFRb or OSMRb, become activated. JAKs induce phosphoryla- tion of conserved tyrosine residues on each receptor subunit, which act as docking sites for STAT molecules (Auguste et al., 1997; Guschin et al., 1995; Stahl et al., Oncogene (2002) 21, 460 ± 474 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc *Correspondence: SL Grant, Institute for Child Health Research, Subiaco, Western Australia, 6008, Australia; E-mail: suzieg@ichr.uwa.edu.au Received 24 July 2001; revised 29 August 2001; accepted 29 October 2001