ORIGINAL ARTICLE PELO negatively regulates HER receptor signalling and metastasis K Pedersen 1 , F Canals 1 , A Prat 2 , J Tabernero 3 and J Arribas 1,4,5 The HER family is composed of four receptor tyrosine kinases, which are frequently deregulated in several types of cancer. Activated HER receptors initiate intracellular signalling pathways by attracting to the plasma membrane a plethora of adaptor and signalling molecules. Although there are more than a dozen HER-interacting proteins that regulate signal transduction and have been extensively studied, recent proteomic studies have shown the existence of many novel but largely uncharacterized factors that may bind HER receptors. In this report, we describe a cell-based identification of several new HER2-binding proteins, including HAX1, YWHAZ, PELO and ACP1. Analysis of these factors showed that one of them, PELO, binds to active HER2 and epidermal growth factor receptor and thereby attenuates phosphatidylinositol 3-kinase (PI3K)/AKT signalling, likely through regulation of the recruitment of p85-PI3K to activated receptor. Functional characterization of PELO showed that it negatively regulates cell migration and metastasis in vivo. These results reveal that PELO is a novel regulator of HER-signalling and therefore is likely to have a role in inhibiting tumour progression and invasion. Oncogene (2014) 33, 1190–1197; doi:10.1038/onc.2013.35; published online 25 February 2013 Keywords: breast cancer; EGFR; metastasis; p95HER2; PELO; PI3K INTRODUCTION The epidermal growth factor receptor (EGFR, also known as HER1 or ERBB1) is the prototype of a receptor tyrosine kinase family that comprises three additional members: HER2 (neu, ERBB2), HER3 and HER4 (ERBB3 and ERBB4). HER receptors react to the binding of epidermal growth factor (EGF)-like ligands by forming homo- or heterodimers. Despite a strong overall similarity, each of the receptors has its own unique functional properties. Remarkably, HER2 does not bind any known ligands, instead the structure of its extracellular domain is constitutively ready for heterodimerization with a ligand-bound family member. 1 The dimerization results in juxtaposition and subsequent activation of the intracellular kinase domains. Autophosphorylation of certain tyrosine residues in the carboxyl-terminal domain of the activated receptors triggers the recruitment of proteins bearing phosphotyrosine-binding domains, that is, SRC-homology-2 or phosphotyrosine-binding domains. 2 Some of these proteins, such as SHC1 or GRB2, have been extensively characterized and shown to contribute to the initiation of intracellular signalling pathways, which in turn regulate different gene expression programs. 3 Other proteins, such as the negative regulator of HER-signalling ERRFI1 (also known as MIG6 or RALT), interact with HER receptors independently of a phosphotyrosine-binding motifs. 4 HER receptors often have a causal role in malignant progres- sion. They have been found overactivated in a variety of solid tumours through different mechanisms, including over- expression or acquisition of activating mutations. 5 In fact, HER receptors are the targets of different drugs, including tyrosine kinase inhibitors and monoclonal antibodies, currently used to treat different cancers. Furthermore, signalling pathways initiated by the activated HER receptors, such as the RAS/MEK, and the phosphatidylinositol 3-kinase (PI3K)/AKT pathways are frequently overactivated in human tumours, and numerous new anti-cancer drugs are directed against components of these pathways. 6 Different in vitro studies have unveiled the existence of many novel partners of the HER receptors. 7–9 The interaction of most of these proteins with HER receptors in vivo remains to be confirmed and their functional relevance are currently unknown. Prompted by these studies, we conducted a proteomic analysis to identify new HER2-binding proteins using as a bait the HER2 fragment known as 100–115-kDa p95HER2 (also known as 611-CTF). This truncated form of HER2, hereafter referred to simply as p95HER2, lacks most of the extracellular domain but is constitutively active because of its ability to form homodimers maintained by disulphide bonds. 10 PELO, one of the novel p95HER2-interacting proteins we identified and validated, is an evolutionary conserved and pleiotropic protein. In Drosophila melanogaster males with mutations in pelota, the gene that encodes for PELO, germ cells undergo normal mitoses. However, they are not able to complete the meiotic division as they remain arrested in late prophase. In contrast, in mutant D. melanogaster females, the mitotic division is affected during oogenesis. In addition to these germline defects, the eyes of pelota mutant flies display impaired development. 11 In yeast, the accumulation of free ribosomes and a decrease in the number of polyribosomes in dom34 (the ortholog of pelota) mutants, indicates that PELO participates in the regulation of mRNA translation. 12 Mice with pelo knockout fail to develop past day 7.5 of embryogenesis due to defects in cell proliferation. 13 Interestingly, heterozygous pelo knockout mice showed an increase in the incidence of benign tumours, however, the link between PELO and cancer progression remained unexplored. 13 Here we show that PELO binds to active HER2 and EGFR in different breast cancer cell lines. The binding of PELO to these 1 Preclinical Research Program, Vall d’Hebron Institute of Oncology (VHIO), Barcelona, Spain; 2 Translational Genomics Research Program, VHIO, Barcelona, Spain; 3 Clinical Research Program, VHIO, Barcelona, Spain; 4 Department of Biochemistry and Molecular Biology, Universitat Autonoma de Barcelona, Campus de la UAB, Bellaterra, Spain and 5 Institucio ´ Catalana de Recerca i Estudis Avanc ¸ats (ICREA), Barcelona, Spain. Correspondence: Dr K Pedersen or Dr J Arribas, Preclinical Research Program, Vall d’Hebron Institute of Oncology, Psg. Vall d’Hebron 119-129, 08035 Barcelona, Spain. E-mail: kp@kim-pedersen.com (KPJ) or jarribas@vhio.net (JA) Received 13 September 2012; revised 14 December 2012; accepted 21 December 2012; published online 25 February 2013 Oncogene (2014) 33, 1190–1197 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc