Loss of RALT/MIG-6 expression in ERBB2-amplified breast carcinomas enhances ErbB-2 oncogenic potency and favors resistance to Herceptin Sergio Anastasi 1,5 , Gianluca Sala 1,5,6 , Chen Huiping 2,7 , Elisabetta Caprini 3 , Giandomenico Russo 3 , Stefano Iacovelli 4 , Fabiana Lucini 1 , Sigurdur Ingvarsson 2 and Oreste Segatto* ,1 1 Laboratory of Immunology, Regina Elena Cancer Institute, via Delle Messi d’Oro, 156/158, 00158, Rome, Italy; 2 Institute for Experimental Pathology, University of Iceland at Keldur, Reykjavik, Iceland; 3 Laboratory of Molecular Oncology, IDI, Rome, Italy; 4 Laboratory of Molecular Oncogenesis, Regina Elena Cancer Institute, via Delle Messi d’Oro, 156/158, 00158, Rome, Italy An emerging paradigm holds that loss of negative signalling to receptor tyrosine kinases (RTKs) is permis- sive for their oncogenic activity. Herein, we have addressed tumor suppression by RALT/MIG-6, a tran- scriptionally controlled feedback inhibitor of ErbB RTKs, in breast cancer cells. Knockdown of RALT expression by RNAi enhanced the EGF-dependent proliferation of normal breast epithelial cells, indicating that loss of RALT signalling in breast epithelium may represent an advantageous condition during ErbB-driven tumorigen- esis. Although mutational inactivation of the RALT gene was not detected in human breast carcinomas, RALT mRNA and protein expression was strongly and selec- tively reduced in ERBB2-amplified breast cancer cell lines. Reconstitution of RALT expression in ERBB2- amplified SKBr-3 and BT474 cells inhibited ErbB-2- dependent mitogenic signalling and counteracted the ability of ErbB ligands to promote resistance to the ErbB-2-targeting drug Herceptin. Thus, loss of RALT expression cooperates with ERBB2 gene amplification to drive full oncogenic signalling by the ErbB-2 receptor. Moreover, loss of RALT signalling may adversely affect tumor responses to ErbB-2-targeting agents. Oncogene (2005) 24, 4540–4548. doi:10.1038/sj.onc.1208658 Published online 25 April 2005 Keywords: RALT; MIG-6; ErbB-2; breast cancer; Herceptin; targeted therapies Introduction Net signal output by receptor tyrosine kinases (RTKs) depends on the dynamic equilibrium between signal generation (positive signalling) and signal attenuation (negative signalling). Perturbation of this balance has deleterious consequences on cell and tissue homeostasis, as demonstrated by developmental studies in inverte- brate organisms (Perrimon and McMahon, 1999; Moghal and Sternberg, 2003). Recently, attention has also been drawn on loss of negative signalling as a potential mechanism of oncogenic activation of RTKs. For instance, transforming mutations of MET and CSF- 1R prevent c-Cbl from binding to these RTKs (Peschard and Park, 2003). Along this line, the increased c-Src activity associated to oncogenic EGFR signalling drives degradation of c-Cbl (Bao et al., 2003). In aggregate, these events uncouple c-Cbl from RTK signalling, thus allowing oncogenic receptors to escape downregulation via the internalization/degradation pathway (Bache et al., 2004; Polo et al., 2004). Dramatic overexpression of the ErbB-2 RTK, most often caused by gene amplification, is detected in 20–30 % of human breast carcinomas and is causally linked to the aggressive clinical behaviour of this tumor subset (Slamon et al., 1989). Although unable to bind ligands directly, ErbB-2 is the hierarchically dominant receptor in the combinatorial assembly of ligand-driven hetero-dimeric complexes between ErbB family members, namely ErbB-1 (EGFR), ErbB-3 and ErbB-4 (Olayioye et al., 2000; Yarden and Sliwkowski, 2001). Among these signalling complexes, the ErbB-2.ErbB-3 combination is most remarkable in terms of oncogenic potency, due to the fact that ErbB-2 and ErbB-3 are strong activators of the Ras-ERK and PI-3K-AKT pathways, respectively (Olayioye et al., 2000; Yarden and Sliwkowski, 2001). Consistently, overexpression of ErbB-3 is linked to oncogenic activa- tion of ErbB-2 in both human and mouse mammary tumors (Siegel et al., 1999; Holbro et al., 2003). Not surprisingly, although ErbB-2 homo-dimers may form and signal in ERBB2-amplified cancer cells, genetic evidence indicates that ErbB-3 acts as an essential partner of oncogenic ErbB-2 in ERBB2-amplified breast tumors (Holbro et al., 2003), possibly in conjunction with autocrine/paracrine ligand stimulation (Mincione et al., 1996). An unresolved issue concerns negative signalling to ErbB-2-containing dimers in normal cells and its possible subversion in cancer cells. Negative signalling Received 16 November 2004; revised 11 February 2004; accepted 28 February 2005; published online 25 April 2005 *Correspondence: O Segatto; E-mail: segatto@ifo.it 5 These two authors contributed equally to this work 6 Current address: Laboratory of Molecular Oncology, Aging Research Center, Chieti, Italy 7 Current address: Washington University School of Medicine, St Louis, MO, USA Oncogene (2005) 24, 4540–4548 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc