[CANCER RESEARCH 63, 4809–4818, August 15, 2003] Identification and Pharmacological Characterization of SRBP-2: A Novel SR31747A-binding Protein Hubert Vidal, Guillaume Mondesert, Sylvaine Galie`gue, Dominique Carrie`re, Pascal-Henri Dupuy, Pierre Carayon, The´re`se Combes, Estelle Bribes, Joe¨lle Simony-Lafontaine, Andrew Kramar, Ge´rard Loison, and Pierre Casellas1 Immunology-Oncology Department, SanofiϳSynthelabo, F-34184 Montpellier cedex 04 [H. V., S. G., D. C., P. C., T. C., E. B., P. C.]; Molecular and Functional Genomics Department, SanofiϳSynthelabo, F-31676 Labe`ge Innopole cedex [G. M., P-H. D., G. L.]; and Departments of Pathology [J. S-L.] and Biostatistics [A. K.], Montpellier Cancer Institute, Montpellier cedex, France ABSTRACT derived tumor development in the mouse xenograft model (8). In 2003, after a positive Phase IIa, SR31747A entered Phase IIb clinical ␴ SR31747A is a ligand with potent antiproliferative activity against trials in prostate cancer. Three high affinity SR31747A-binding pro- tumor cells and for which three binding proteins have been identified to ␴ ␴ teins that may mediate SR31747A properties have been identified to date: (a) SRBP-1 (also called 1); (b) HIS; and (c) 2. In this study, we ␴ characterized an additional SR31747A binding site, i.e., SRBP-2 date in humans: (a) SRBP-1; (b) 2; and (c) HSI (7–12). SRBP-1 and (SR31747A-binding protein 2). Using an in silico screening approach, we HSI have been molecularly characterized, whereas ␴2 has not yet identified this novel sequence, which exhibits 41% homology with HSI. been cloned. SRBP-1, which stands for SR31747A-binding protein 1 The 1142-bp cDNA was found to encode a 206 amino acid protein not and is also called ␴1 (9, 10), is related to the yeast Saccharomyces related to SRBP-1. Northern blot analysis of SRBP-2 mRNA expression cerevisiae C8-C7 sterol isomerase encoded by the ERG2 gene; there revealed a single 1.1-kb transcript that was widely expressed in organs; is 35% sequence identity between the two proteins (5). Despite this the liver was particularly enriched, and the brain showed the lowest identity percentage, SRBP-1 receptor expression does not comple- abundance. A murine homologue that exhibited a similar expression ment an erg2 defect in yeast, and no sterol isomerase activity has ever pattern was also characterized. Subcellular localization analysis using specific polyclonal antibodies revealed that SRBP-2 had the same nuclear been demonstrated for SRBP-1 (9). The emopamil-binding protein, membrane and endoplasmic reticulum localization as other members of HSI, was first described as a high affinity binding protein for emo- 2ϩ the SR31747A-binding protein family. Considering SRBP-2-binding prop- pamil, the anti-ischemic phenylalkylamine Ca antagonist (11). HSI erties, pharmacological analysis clearly highlighted that SRBP-2 was dis- is the human counterpart of the yeast ERG2 (12). In mammals, HSI ␴ tinct from 2. Scatchard plot analysis revealed Kd values of 10 and 3 nM belongs to the sterol biosynthesis pathway, and the enzyme catalyzes for SR31747A and Tamoxifen, respectively. In contrast with HSI, the the conversion of 5␣-cholesta-8,24-dien-3␤-ol (zymosterol) and 5␣- protein also did not exhibit detectable isomerase activity. When analyzing cholesta-8-en-3␤-ol (zymostenol, ⌬8-cholestenol) to their correspond- SRBP-2 expression in human breast cancer biopsies, we obtained evidence ing ⌬7-isomers. HSI and SRBP-1 have been expressed in yeast, and that SRBP-2 expression, together with SRBP-1 and HSI, may be of their expression and pharmacological profiles have been both charac- interest as a prognostic marker. These findings demonstrated that SRBP-2 represents an additional molecular target for SR31747A, which could help terized. The two proteins are colocalized and associated with the to understand the immunosuppressive and antiproliferative effects of the endoplasmic reticulum and outer and inner membranes of the nuclear molecule. envelope. They also delocalize during the cell cycle at the mitosis step when the nuclear membranes disappear (13). Only the pharmacolog- ical properties of ␴2 have been unraveled. ␴2 has been identified in rat INTRODUCTION 2 spleen using tritiated DTG, with an estimated protein size of Mr SR31747A is a selective peripheral ␴-binding site ligand exhibiting 21,000. Although the ␴2 sequence has not yet been identified, some immunosuppression and able to inhibit cell proliferation both in vitro important information has been reported on the basis of the protein and in vivo. The immunomodulatory properties of the SR31747A drug expression in tumors. Indeed, the ␴2 receptor is considered to be a were first demonstrated by the inhibition of the mitogen-induced potential biomarker of proliferation in cancer, and radiolabeled li- mouse and T-cell proliferation elicited by nanomolar concentrations gands specific to the ␴2 receptor were demonstrated to be useful for of SR31747A (1). In vitro SR31747A was shown to inhibit staphy- assessing the proliferative status of tumors and normal tissues (14, lococcal enterotoxin B-driven lymphocyte proliferation (2), whereas 15). Identification of ␴2 is a critical issue in this context. in vivo, in mice, SR31747A treatment confers potent protection The yeast S. cerevisiae has been used as a model to study the against the lethal effects of staphylococcal enterotoxin B and ⌬– mechanism of the antiproliferative effect of SR31747A (5): (a) in this galactosamine and prevents both graft-versus-host disease and model, we showed that SR31747A binds Erg2 and blocks cell prolif- delayed type hypersensitivity granuloma formation (1–3). SR31747A eration by inhibiting the sterol biosynthesis pathway; and (b) using the was also shown to modulate proinflammatory and anti-inflammatory DNA chip strategy, we also demonstrated that ERG2 is the only target cytokine responses (2, 4). The antiproliferative properties of nanomo- that mediates the antiproliferative SR31747A property in yeast (6). A lar concentrations of SR31747A were shown in yeast (5, 6) and similar inhibitory mechanism has been demonstrated in animal cell against human tumor cell lines, both in vitro and in vivo (7, 8). lines grown in sterol-free medium (7). However, although candidate Precisely nanomolar concentrations of SR31747A inhibited cell pro- targets have been identified in mammals, the mechanism that prevails liferation of either hormono-responsive or hormono-unresponsive hu- in the sterol starvation-induced cell proliferation inhibition is not yet man cancer cell lines in vitro and in vivo; a treatment with SR31747A fully understood. In addition, the analysis of the expression of both significantly reduced both human breast or prostatic cancer cell line- receptors indicated that the sensitivity of human tumor cell lines to SR31747A is not correlated with either HSI or SRBP-1 expression Received 11/1/02; revised 4/16/03; accepted 6/13/03. (8). These data support that additional binding sites may exist. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviations used are: DTG, 1,3-Di-(o) tolylguanidine; 3PPP, HOPh-Pip-Pr; 1 To whom requests for reprints should be addressed, at Sanofi-Synthelabo, 371 rue du DFS, disease-free survival; DAPI, 4Ј,6-diamidino-2-phenylindole; PR, progesterone re- Professeur Joseph Blayac, F-34184 Montpellier cedex 04, France. Phone: (33) 4 67 10 62 90; ceptor; SBR, Bloom and Richardson grading system; FISH, fluorescence in situ hybrid- Fax: (33) 4 67 10 60 00; E-mail: [email protected]. ization. 4809 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. A NEW SR31747A-BINDING PROTEIN To elucidate the SR31747A mode of action, we searched for WBB (1% SDS, 40 mM NaH2PO4,and1mM EDTA) for 10 min at 65°C. additional SR31747A binding sites that could account for its reported Finally, blots were autoradiographed using Kodak X-ray film for 18 h at properties in mammals. With this aim, we used an in silico technique Ϫ70°C and developed according to standard procedures. to screen for the protein that exhibits high homology for human In Situ Hybridization and FISH Detection. The chromosomal assign- SRBP-1 or HSI. In the present study, we characterized SRBP-2 ment for the human SRBP-2 gene was performed by in situ hybridization and (SR31747A-binding protein 2), an original protein that exhibits 43% FISH detection according to a procedure published previously (16, 17). Gene mapping was carried out on chromosome preparations obtained from phyto- identity with HSI and binds SR31747A with high nanomolar affinity. hemagglutinin-stimulated human lymphocytes cultured for 72 h. The lympho- SRBP-2, based on its binding profile, is distinct from ␴2. SRBP-2 is cyte cultures were synchronized with 5-bromodeoxyuridine (0.18 mg/ml) thus a novel human SR31747A binding site. Here, we characterized treatment. The entire coding sequence of SRBP-2 was used as cDNA probe. this new protein and its binding properties, and we addressed whether FISH signals and the DAPI-banding pattern were recorded separately on the protein exhibits sterol isomerase activity. In addition, we analyzed photographs, whereas the FISH mapping data were assigned to chromosomal its expression both at the mRNA and protein levels, the latter using bands by superimposing FISH signals with DAPI-banded chromosomes. immunohistochemical and electronic microscopy approach. Interest- Anti-SRBP-2 Antibody Production. Polyclonal anti-SRBP-2 antibodies ingly, we evidenced that SRBP-2 expression level is higher than HSI were raised in rabbit against a synthetic peptide corresponding to amino acid and SRBP-1 expression levels in several breast and prostate cancer residues 1–17 (Nt) and 187–206 (Ct) (Neosystem, Strasbourg, France). Rabbit cell lines. Finally, we tested the interest of SRBP-2 as a potential sera were purified by immunoaffinity on a Sepharose column (Bio-Rad) to breast cancer marker, and our results showed that SRBP-2 expression which the peptides were covalently coupled.