ORIGINAL ARTICLE Flotillins as regulators of ErbB2 levels in breast cancer S Pust 1,2 , TI Klokk 1,2 , N Musa 1,2 , M Jenstad 2,3 , B Risberg 2,3,4 , B Erikstein 5 , L Tcatchoff 1,2 , K Liestøl 2 , HE Danielsen 2,3 , B van Deurs 6 and K Sandvig 1,2,7 Amplification and overexpression of the receptor tyrosine kinase ErbB2 occur in up to 30% of human breast cancers, and high ErbB2 levels are correlated with poor prognosis for breast cancer patients. In contrast to the epithelial growth factor receptor (ErbB1), ErbB2 is not downregulated by ligand-induced mechanisms. Here we show that flotillins are involved in the stabilization of ErbB2 at the plasma membrane. In SKBR3 breast cancer cells and breast cancer tissue, a positive correlation between flotillin and ErbB2 expression levels could be demonstrated. Moreover, the tissue microarray analyses of biopsies from 194 patients diagnosed with carcinomas of the breast showed that flotillin-2 emerged as a potential predictor of prognosis in breast cancer. Depletion of flotillin-1 and flotillin-2 leads to internalization and degradation of ErbB2. Furthermore, flotillin-1 and -2 were found to be in a molecular complex with ErbB2 and Hsp90. The depletion of one of these proteins results in disruption of this complex, followed by destabilization of ErbB2 at the membrane, and its internalization and degradation. As a consequence, ErbB2-triggered downstream signalling is inhibited. Our data demonstrate a novel mechanism for interfering with ErbB2 signalling, which potentially can have clinical impact. Oncogene (2013) 32, 3443–3451; doi:10.1038/onc.2012.357; published online 6 August 2012 Keywords: ErbB2 receptor tyrosine kinase; flotillin; breast cancer; endocytosis; downregulation INDRODUCTION The ligand-less receptor tyrosine kinase ErbB2 is a preferred dimerization partner for the other members (EGFR/ErbB1, ErbB3 and ErbB4) of the EGFR (epithelial growth factor receptor) family. 1 ErbB2 has been demonstrated to enable strong and constitutive signalling through different pathways that induce cell transformation, migration and proliferation. 2 In contrast to EGFR/ ErbB1, ErbB2 is endocytosed at a very slow rate and continuously recycled back to the cell surface. 3–5 The precise activation and deactivation of signalling cascades is a crucial cellular process and dysregulation can induce cancer development or progression. Thus, one of the challenges in cancer treatment is to promote silencing of signalling cascades. ErbB2 has been shown to be involved in the progression and development of a variety of different human tumours. Remarkably, in up to 20–30% of all human breast cancers, amplification and overexpression of ErbB2 can be found and high expression of ErbB2 has been demon- strated to be correlated with poor prognosis in breast cancer patients. 6–9 The inhibition of oncogenic signalling triggered by ErbB2 can be achieved by different strategies, and administration of monoclonal antibodies (trastuzumab/Herceptin) became the first-line treatment in patients with ErbB2-overexpressing metastatic breast cancer. 6,10 In breast cancer cells, ErbB2 is strongly concentrated at the plasma membrane and its stabilization at the membrane is mediated by Hsp90. Thus, another approach to abrogate oncogenic signalling is the induction of ErbB2 internalization and degradation by inhibition of Hsp90. Treatment with the Hsp90 inhibitor geldanamycin (GA) has been shown to induce ErbB2 downregulation. 11–13 Hsp90 is a known target in cancer therapy and GA analogues (for example, 17-AAG/tanespimycin) are used in the treatment and therapy of ErbB2-positive cancers in clinic. 14–16 To understand in greater detail how ErbB2 stabilization at the plasma membrane is mediated, we have investigated the involvement of the lipid raft-associated flotillins (flotillin-1 and -2) in this process. Flotillins, which are also termed reggies, 17 have been described to be localized mainly at the plasma membrane and in endosomal/ lysosomal compartments. 18–20 It has been demonstrated that flotillin proteins, among other functions, are involved in endocytic mechanisms and cellular trafficking processes. 21–23 In this study, we describe how flotillin-1 and -2 contribute to the stabilization of ErbB2 at the cell surface, and we show that ErbB2 is internalized and degraded by a GA-independent mechanism upon flotillin depletion. Importantly, flotillin and ErbB2 expression levels are positively correlated on a cellular level as well as in breast cancer tissue. RESULTS Association of flotillins with ErbB2 First, we analysed the cellular localization of ErbB2 and flotillins in SKBR3 breast cancer cells. We could show by confocal microscopy that flotillin-1 and -2 partially co-localize with ErbB2 at the plasma membrane (Figure 1a). In addition, interaction between flotillins and ErbB2 could be demonstrated by co-immunoprecipitation experiments, as immunoprecipitation of endogenous flotillin-1 or -2 resulted in pull down of ErbB2 (Figure 1b, quantification of pull down see Supplementary Figure S1a). We next analysed the impact of flotillins on the internalization and subsequent degradation of ErbB2. As described above, ErbB2 is stably 1 Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 2 Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway; 3 Institute for Medical Informatics, Oslo University Hospital, Oslo, Norway; 4 Department of Pathology, Oslo University Hospital, Oslo, Norway; 5 Oslo University Hospital, Oslo, Norway; 6 Department of Cellular and Molecular Medicine, The Panum Building, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark and 7 Department of Molecular Biosciences, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway. Correspondence: Professor K Sandvig, Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Oslo 0379, Norway. E-mail: ksandvig@radium.uio.no Received 7 March 2012; revised 29 June 2012; accepted 30 June 2012; published online 6 August 2012 Oncogene (2013) 32, 3443–3451 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc