[CANCER RESEARCH 64, 3538–3544, May 15, 2004] The Phosphoprotein StarD10 Is Overexpressed in Breast Cancer and Cooperates with ErbB Receptors in Cellular Transformation Monilola A. Olayioye,1 Peter Hoffmann,2 Thomas Pomorski,3 Jane Armes,4 Richard J. Simpson,5 Bruce E. Kemp,2 Geoffrey J. Lindeman,1 and Jane E. Visvader1 1The Walter and Eliza Hall Institute of Medical Research and Bone Marrow Research Laboratories, Royal Melbourne Hospital, Parkville, Victoria, Australia; 2St. Vincent’s Institute of Medical Research, St. Vincent’s Hospital, Victoria, Australia; 3Humboldt-University Berlin, Institute of Biology and Biophysics, Berlin, Germany; 4Molecular Pathology Laboratory, Victorian Breast Cancer Research Consortium, Department of Pathology, University of Melbourne, Victoria, Australia; and 5Joint Proteomics Laboratory, Ludwig Institute for Cancer Research and The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia ABSTRACT tion of the phosphatidylinositol 3-kinase (PI3K) pathway (15, 16). Signaling through PI3K plays an important role in cellular survival by We have identified that StarD10, a member of the START protein phosphorylating and inactivating growth-inhibitory and proapoptotic family, is overexpressed in both mouse and human breast tumors. proteins, including the FOXO transcription factors (17). In addition, StarD10 was initially discovered on the basis of its cross-reactivity with a phosphoserine-specific antibody in mammary tumors from Neu/ErbB2 the ErbB2 and ErbB3 receptors recruit the adaptor proteins Shc and transgenic mice and subsequently isolated from SKBR3 human breast Grb2 (16, 18, 19), resulting in stimulation of the Ras-Raf-mitogen- carcinoma cells using a multistep biochemical purification strategy. We activated protein kinase pathway (20, 21), which has been implicated have shown that StarD10 is capable of binding lipids. StarD10 was found in mammary tumor progression (22, 23). Furthermore, Src kinase to be overexpressed in 35% of primary breast carcinomas and 64% of activity is enhanced in both mammary tumors from Neu transgenic human breast cancer cell lines, correlating with their ErbB2/Her2 status. mice (24) and human breast tumors (25, 26), and Src cooperates with Coexpression of StarD10 with ErbB1/epidermal growth factor receptor in ErbB1 in the transformation of mouse fibroblasts (27). Thus, aberrant murine fibroblasts enhanced anchorage-independent growth in soft agar, expression of ErbB receptors triggers the activation of multiple down- providing evidence for functional cooperation between StarD10 and ErbB stream effectors, in addition to synergizing with other proto-onco- receptor signaling. Taken together, these data suggest that overexpression genes to transform mammary epithelium. of this lipid-binding protein contributes to breast oncogenesis. The Neu mouse model has served as a valuable model for the identification of intracellular mediators of ErbB2-induced tumor de- INTRODUCTION velopment. Although ErbB receptor signaling has been studied exten- The ErbB family of receptor tyrosine kinases plays a critical role in sively, little is known about the deregulation of transcription factors in the pathogenesis of breast cancer. Amplification and/or overexpres- ErbB2-induced tumorigenesis. In the course of analyzing the expres- sion of HER2/ErbB2 occurs in 20–30% of human breast cancers and sion and activation of the forkhead transcription factor FKHR/FOXO1 correlates with poor prognosis (1). Deregulated expression of HER1/ in Neu transgenic mice, we observed a novel protein band that was ErbB1/epidermal growth factor (EGF) receptor has also been ob- specifically recognized by a phospho-FKHR antibody. Interestingly, served in breast cancer, often in association with aberrant expression this Mr 35,000 protein was abundant in tumors derived from Neu of ErbB2 (2, 3). ErbB1 and ErbB2, together with HER3/ErbB3 and transgenic mice but not adjacent normal tissue. Here, we describe the HER4/ErbB4, constitute the ErbB family of receptor tyrosine kinases. isolation and characterization of this phosphoprotein, StarD10, a These receptors signal cooperatively by forming ligand-induced com- member of the START domain family of lipid-binding proteins. binations of homo- and hetero-dimers (4, 5). The complex network of StarD10 was found to be coexpressed with ErbB2 in many breast ErbB receptor-ligand combinations provides precise signaling along carcinoma lines and cooperated with the ErbB pathway in cellular pathways underlying diverse developmental processes (6–8). transformation. Finally, StarD10 overexpression was observed in a The importance of ErbB2 signaling in mammary tumorigenesis has high proportion of primary breast cancers, supporting a role for this been established using transgenic mouse models. Overexpression of lipid-binding protein in deregulated cell growth and tumorigenesis. neu, the rat homologue of ErbB2, from the mouse mammary tumor virus promoter induces focal mammary tumors which frequently MATERIALS AND METHODS metastasize to the lung (9). The long tumor latency period in these mice suggested that additional molecular events were required for Reagents. Antibodies used were polyclonal antiphospho-FKHR (S256) antibody (New England Biolabs, Beverly, MA), mouse anti-␣-tubulin mono- mammary tumors to develop. Further analysis of these tumors iden- clonal antibody (clone B-5-1-2; Sigma, St. Louis, MO), mouse anti-ErbB2 tified somatic mutations in neu, resulting in constitutive activation of monoclonal antibody (Ab-10; Neomarkers, Fremont, CA), rabbit anti-ErbB2 the receptor (10). Up-regulation of ErbB3 protein in these tumors polyclonal antibody (A0485; DAKO, Carpinteria, CA), mouse anti-Neu mono- suggested that ErbB2 synergizes with ErbB3 in oncogenesis (11). In clonal antibody (Ab-3; Calbiochem, Darmstadt, Germany), mouse anti-Flag human breast carcinoma cells that overexpress ErbB2, down-regula- monoclonal antibody (Sigma), and rabbit anti-Pctp-l polyclonal antiserum tion of ErbB3 using an artificial erbB3-specific transcriptional repres- (kindly provided by Y. Nishimune, Osaka University, Osaka, Japan). sor revealed that ErbB3 is essential for ErbB2-mediated proliferation Cell Culture and Mouse Strains. The majority of breast epithelial cell (12). The ErbB2/3 heterodimer appears to have potent mitogenic and lines was maintained in RPMI containing 10% fetal bovine serum (Common- transforming properties in vitro (13, 14) and leads to efficient activa- wealth Serum Laboratory). MCF10A cells were grown in DMEM:F12, sup- plemented with 10% fetal bovine serum, 10 ng/ml EGF (Sigma), 5 ␮g/ml insulin (Sigma), and 1 ␮M dexamethasone (Sigma). HER14 (NIH3T3 cells Received 11/30/03; revised 2/20/04; accepted 3/3/04. transfected with human ErbB1) was a gift from R. Daly (Garvan Institute, Grant support: The Victorian Breast Cancer Research Consortium, Inc. M. Olayioye was supported by EMBO and HFSPO fellowships. Sydney, Australia). HER14, 293T, and Bosc cell lines were grown in DMEM The costs of publication of this article were defrayed in part by the payment of page containing 10% fetal bovine serum. HER14 StarD10 transductants were gen- charges. This article must therefore be hereby marked advertisement in accordance with erated by infection with a pBabe retrovirus encoding hemagglutinin (HA)- 18 U.S.C. Section 1734 solely to indicate this fact. tagged StarD10 into Bosc packaging cells. Virus was collected, then used to Requests for reprints: Jane E. Visvader, The Walter and Eliza Hall Institute of ␮ Medical Research, 1G Royal Parade, Victoria 3050, Australia. Phone: 61-3-9342-8633; infect HER14 NIH3T3 cells, and selected with 1 g/ml puromycin (Sigma), Fax: 61-3-9347-0852; E-mail: [email protected]. thus generating HER14-StarD10 transductants. For transient transfections, 3538 STARD10 IN BREAST CANCER 293T cells were transfected with Fugene reagent (Roche, Penzberg, Germany) washed with acetone, dissolved in sample buffer, and loaded onto a large 10% according to the manufacturer’s instructions. Mouse mammary tumor virus- SDS-PAGE gel. The gel was fixed and stained with Coomassie Phast-gel Blue neu mice have been described previously (9). Nulliparous female mice were R (Amersham Pharmacia). aged for 8–12 months in standard animal facilities at The Walter and Eliza In-Gel Tryptic Digest and Mass Spectrometry. Coomassie-stained bands Hall Institute and sacrificed when tumors developed. were destained with 50 mM NH4HCO3/50% acetonitrile (1:1) and digested Cloning of StarD10. Total RNA was extracted from SKBR3 cells using with modified porcine trypsin (Promega, Madison, WI). The sample was then RNAzol (Tel-Test, Friendswood, TX). First-strand cDNA synthesis was per- concentrated on a Zip Tip ␮-C18 (Millipore) and eluted into nanospray formed, and StarD10 cDNA was amplified by PCR using primers based on the capillaries (MDS Proteomics, Charlottesville, VA) with 20–60% methanol CGI-52 sequence (accession no. AF151810; Ref. 28). StarD10 cDNA was containing 5% formic acid. Mass spectrometry was performed on a Pulsar ion cloned into Flag-pEFrPGKpuro and pBabePuro vectors for expression in quadruple Time of Flight (TOF) mass spectrometer (Applied Biosystems, mammalian cells. Foster City, CA) with a nanoelectrospray ion source (MDS Proteomics). Protein Extraction of Cells and Tissues. For cytosolic protein extraction, Product ion scans were acquired using collision energies that retained 10% of cells were lysed in hypotonic buffer [10 mM HEPES (pH 7.9), 133 mM sorbitol,