A Novel SR31747A-Binding Protein

[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 pharmacological 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

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. Purified antibodies were tested for had a significant negative effect on DFS when considering low-grade specific recognition on Western blotting membrane by peptide competition (10 ␮ ␮ SBR patients. g/ml) before immunoblotting and used at 1 g/ml. Analysis of the Subcellular Localization of SRBP-2 by Confocal Mi- croscopy. The MDA-MB-231 human breast cancer cell line was used for MATERIALS AND METHODS immunofluorescence analysis of the SRBP-2 protein subcellular localization. Cells were fixed overnight with 1% paraformaldehyde, washed once, and Reagents. SR31747A, (Z)N-cyclohexyl-N-ethyl-3-(3-chloro4-cyclohexyl- permeabilized for 10 min in a 0.1% saponin, 1% BSA, PBS solution. For phenyl)propen-2-ylamine hydrochloride, was produced and provided by Sanofi comparative purposes, cells were incubated in parallel with 1 ␮g/ml anti- Synthelabo Laboratories (Montpellier, France). Tamoxifen and pentazocine SRBP-1 (9), anti-HSI (13), or anti-SRBP-2 antibodies for 60 min. After two were purchased from Sigma. DTG and 3PPP were supplied by Interchim. washes, cells were incubated for 30 min with Cy5 antimouse or antirabbit 3 3 3H-tamoxifen (80 CimM) [ H]DTG (31 Ci/mM), [ H]3PPP (101 Ci/mM) conjugates (Southern Biotechnology, Inc., Birmingham, AL). The subcellular 3 3 from NEN; [ H]SR31747A (51 Ci/mM) from Amersham; [ H]Pentazocine (28 distribution of the SRBP-2 protein was analyzed using a laser scanning Ci/mM) from NEN. confocal microscope (LSM 410; Zeiss, Oberkochen, Germany) equipped with Cell Lines and Culture Conditions. Hormono-responsive breast adeno- a c-apochromat water immersion lens (ϫ63, numerical aperature ϭ 1.2). carcinoma MCF-7 cells were cultured in 50% DMEM/50% Ham’s F12 (1/1, Specificity controls were carried out by preincubation of anti-SRBP-2 anti- volume for volume) supplemented with 16 ng/ml insulin, 2 mML-glutamine, bodies with the immunizing peptides at 10 ␮g/ml. 10 mM HEPES buffer, 50 IU/ml penicillin, 50 ␮g/ml streptomycin, and 10% Quantitative Analysis of SR31747A-binding Protein Expression by heat-inactivated FCS. The hormono-unresponsive breast cancer cells, MDA- Flow Cytometry. The cell lines used in this study were hormono-responsive MB-231, were maintained in Leibovitz L15 culture medium supplemented breast cancer MCF-7, hormono-unresponsive MDA-MB-321 and BT-20, with 10 mM HEPES buffer, 6 ␮g/ml human insulin, 2 mML-glutamine, 1% hormono-responsive prostate cancer LNCaP, and hormono-unresponsive nonessential amino acids, 50 IU/ml penicillin, 50 ␮g/ml streptomycin, and DU145 and PC3. Normal cells included lymphocytes and monocytes. All cells 10% FCS. Three human prostatic cancer cell lines that were either hormono- were cultured at 37°C in a humid atmosphere of 5% CO in air, except for the responsive, LNCaP, or hormono-unresponsive, DU145 and PC3, were cultured 2 MDA-MB-231 cell line, which was cultured in the absence of CO , (as in RPMI 1640 supplemented with 10% fetal bovine serum. All cells were 2 described in Ref. 8). Cells were fixed overnight with 1% formaldehyde and cultured at 37°C in a humid atmosphere of 5% CO in air, except for the 2 permeabilized for 10 min with a solution of 0.1% saponin in PBS containing MDA-MB-231 cell line, which was cultured in the absence of CO . 2 0.1% BSA. Cells were incubated with anti-SRBP-1, anti-SRBP-2, or anti-HSI Cloning of Human SRBP-2 cDNA. The AI858023 clone was obtained antibodies for 60 min. After two washes, cells were incubated with antimouse from the IMAGE consortium. This clone, which encompasses the entire coding sequence of SRBP-2, was fully sequenced to assess the absence of any or antirabbit conjugate for 30 min. After washing, fluorescence intensity was mutation. measured with a FACSCALIBUR cytometer. Cloning of Mouse SRBP-2 cDNA. To identify the mouse homologue of Immunohistochemical Analysis of SRBP-2 Expression in PC3 Human human SRBP-2 cDNA, we defined primers specific to the human sequence and Prostate Tumor Cells. Human prostatic PC3 tumor cell sections were depar- used these primers to amplify mouse liver cDNA (Clontech). The primers used affinized in toluene and incubated in 10 mM Na-citrate buffer (pH 6), at 95°C were 5Ј ctcatctggctctgctac 3Ј (sense) and 5Ј atccagcagccatacagct 3Ј (antisense). for 40 min. After three washes in water, SRBP-2 antibodies (0.7 or 0.5 mg/ml The PCR reaction was performed at 45°C for 30 cycles. A 302-bp amplicon for Ct and Nt, respectively) were added for 25 min at room temperature. was obtained and sequenced. Immunoreactions were visualized by incubation with a biotinylated antirabbit Northern Blot Analysis. A cDNA probe containing the entire nucleotide IgG for 25 min at 37°C, followed by an indirect streptavidin-biotin method 32 sequence encoding the SRBP-2 protein was labeled with P using the using H2O2/3-amino-9-ethylcarbazole as chromogenic substrate (red label, RadPrime DNA labeling system (Life Technologies, Inc.), according to the DAKO ChemMate Detection kit, peroxidase/AEC, rabbit/mouse; DAKO A/S, manufacturer’s instructions. After labeling, the probe was purified using a Glostrup, Denmark). Labeling specificity was determined by the absence of CHROMA SPIN-200 column (Amersham Life Science). The purified labeled staining after preincubation of anti-SRBP-2 antibodies with the immunizing probe was then used to examine various human and rodent tissues for SRBP-2 peptides at 10 ␮g/ml. Negative controls were also obtained by the absence of mRNA. Human RNA Master Blots, Multiple Tissue Northern blots containing staining after omission of the primary antibody. Some slides were counter- various human tissues, human cell lines, and mouse and rat Multiple Tissue stained with hematoxylin. Sections were mounted on faramount-covered slides Northern blots were obtained from Clontech and examined with the labeled (DAKO), air-dried, and analyzed using a Leica DMLB microscope. probe using the hybridization solution (Church buffer: 1% BSA, 7% SDS, Recombinant SRBP-2 Protein Expression in Yeast. The expression

0.5 M NaH2PO4,and1mM EDTA). Membranes were prehybridized for1hat plasmid was made by cloning cDNA-encoding, full-length SRBP-2 into 65°C in hybridization solution, then hybridized overnight at 65°C using the pRS42xGAL1 expression vector containing 2␮ and URA3 or LEU2 radiolabelled SRBP-2 probe in hybridization solution containing 100 ␮g/ml as selective markers. The yeast strain producing SRBP-2 was EMY- salmon sperm DNA. Blots were washed twice with WBA (0.5% BSA, 5% 90ϩp2232(pRS425GAL1:MYC-SRBP-2). SRBP-2 expression was induced ϩ SDS, 40 mM NaH2PO4,and1mM EDTA) for 5 min at 65°C and once with by adding 2% galactose to YNB raffinose 2%. 4810

Binding Studies on Yeast Cell Membranes. Yeast cells were treated with of Pathology (Montpellier, France). Selection criteria included presentation zymolyase (ICN Biomedicals, Inc., 250 ␮g/gram cells for 25 min at 30°C) and with primary invasive breast carcinoma, no preoperative chemotherapy, endo- homogenized in Tris-HCl (pH 7.4) buffer containing protease inhibitors. crine or radiation therapy, sufficient tumor tissue remaining after diagnosis to Homogenates were centrifuged at 100,000 ϫ g for 20 min, and pellets were allow biochemical quantification of receptor status and additional immunohis- stored at Ϫ20°C until use. In competition experiments, 10 ␮g of yeast tochemical assays (tumor size Ͼ 1 cm in diameter), and long-term follow-up membranes were incubated for 60 min at 20°Cin200␮lof50mM Tris-HCl for disease recurrence and death. A total of 95 patients who met these criteria 0.1% BSA containing 1 nM radioactive ligand and the different drugs at was included. concentrations ranging from 1 nM to 1 ␮M. In the saturation experiment, Surgical treatment included radical mastectomy with axillary dissection in membranes were incubated with the radioactive ligand (0.2–50 nM), and 58% of patients and breast conservative sector resection with axillary dissec- nonspecific binding was obtained with 1 ␮M the corresponding nonradioactive tion in 42% of patients. After surgery, all patients with conservative treatment ligand. and 60% with radical mastectomy underwent combined postoperative radio- ⌬8-⌬7 Sterol Isomerase Assay. The ⌬8-⌬7 sterol isomerase activity assay therapy to eradicate all local traces of the disease. A majority (80%) of patients was performed as described previously (5). Briefly, cells were disrupted by received systemic adjuvant therapy, according to the CRLC routine assessment glass bead homogenization in 0.1 M phosphate buffer (pH 7.5) in the presence for clinical management of the disease, and depending on their age, menopau- of 1.5 mM reduced glutathione and 30 mM nicotinamide for 10 min at 4°C. sal status, steroid receptor status, and nodal status: (a) chemotherapy alone for After centrifugation at 10,000 ϫ g to remove cell debris, mitochondria, and 16 patients; (b) endocrine therapy alone (Tamoxifen) for 58 patients; and (c) nuclei, microsomes were isolated by additional centrifugation at 100,000 ϫ g combined chemotherapy and endocrine therapy for 2 patients. Patients were for 20 min and resuspended in 0.1 M phosphate buffer (pH 7.5) containing 3 reviewed for disease recurrence and death, with a median follow-up of 75 mM reduced glutathione and 20% (volume for volume) glycerol. Microsome months. suspension (400 ␮l; 2.5 mg/ml protein concentration) was incubated with Tumor Samples. At surgery, all patients had a small portion of tumor cholest-8-en-3␤-olfor3hat30°C, and the metabolite content was analyzed by removed, which was snap frozen in liquid nitrogen and stored at Ϫ80°C for GC as described previously (5). Enzyme activity was expressed as the rate of estrogen and PR analysis. The remaining part of the tumor was fixed in conversion of cholest-8-en-3␤-ol into cholest-7-en-3␤-ol in nmoles/mg pro- formalin-alcohol for 24 h, paraffin embedded, and subsequently processed tein/h. with routine techniques followed by immunohistochemical analysis. Clinical Assessment of SRBP-2 Marker Expression in Breast Cancer Histopathological Study. Tumor slides (5-␮m thick) were stained with Patients. From January 1992 to February 1993, 850 new breast cancers were H&E for the histopathological study. Tumor grading was performed according diagnosed at the Centre de Recherche et de Lutte Contre le Cancer Department to the methodology of Refs. 18 and 19. Mitosis counts were performed in 10

Fig. 1. A, sequence of human SRBP-2 cDNA and its encoded protein. The deduced protein sequence of human SRBP-2 is shown below the cDNA sequence. The numbers on the left refer to the nucleotide sequence; numbers on the right refer to the amino acid sequence. The underlined bold sequences indicate putative transmembrane domains (TM1–4). The ATG of the open reading frame the termination codon. B, sequence comparison ,ء .is in bold letters of HSI and SRBP-2 proteins. The human HSI and SRBP-2 protein sequences were compared using the Bestfit Function, GCG Software version 10.3. Both proteins share 41% overall homology and 32% identity. The swissprot accession number for the human HSI protein is Q15125. Residues important for the sterol isomerase activity of HSI are indicated by ϩ, ⅐,o,or2, which indicated residues whose mutation reduced by Ͻ34, 35–64, 65–89, and Ͼ90% of the sterol ⌬8-⌬7 isomerase activity, respectively (according to Ref. 21).

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. A NEW SR31747A-BINDING PROTEIN high power fields (ϫ400) using a Leica microscope (Leitz DMRB). Tumor size was recorded as the maximum diameter of the surgically removed tumor mass. Axillary lymph node status was assessed in each case by histopathological examination of a minimum of seven lymph nodes. Immunohistochemical analysis was done on deparaffined 2-␮m-thick slides according to methods described previously (19). Anti-SRBP-2 antibody was used at 1/300 dilution, for this antibody, the antigen retrieval was performed by heating in citrate buffer (pH 6), and anti-SRBP-1 and anti-HSI antibodies were used at 1/400 and 1/100 dilutions, respectively. Cytoplasmic staining was expressed as 0 (no staining or Ͻ10% of tumor cells), 1 (weak staining from 10 to 30% of tumor cells, 2 (intermediate staining), and 3 (intense and diffuse staining). Normal breast tissue surrounding the tumor in the same slide was considered as a positive control. For each tumor, immunodetection of HER2 protein expression, as a marker of poor prognosis, was done using the polyclonal DAKO antibody at 1/1500 dilution, according to the DAKO HercepTest procedure. Staining pattern was evaluated with the DAKO HercepTest score system: (a) HER2 overexpression assessment was considered weakly positive for Ͼ10% of the tumor cells showing a weak to moderate staining of the entire membrane and strongly positive when this staining was strong; and (b) staining of a part of the membrane cell was considered as negative. For this staining, normal breast tissue was considered as a negative control. Statistical Analysis. Correlations between the clinicopathological data and expression of three immunohistochemical markers analyzed (HSI, SRBP-1, and SRBP-2) were assessed using ␹2 tests. Median values of different variables were compared using the nonparametric Kruskal-Wallis test. Locoregional disease relapse and/or distant metastasis and death caused by cancer were considered as end points for DFS. DFS curves starting from the date of surgery were estimated using the Kaplan-Meier method. The statistical significance of each variable was evaluated for prognosis using the Log-rank test for univa- riate analyses and the Cox proportional hazards model for multivariate analyses. For all statistical analyses, P Ͻ 0.1 was considered statistically significant. The SBR used was modified by Elston and Ellis (18). Nucleotide Sequence Accession Number. The human SRBP-2 sequence is identical to the AF243433 sequence, which is called EBPR. The murine SRBP-2 sequence accession number is AF243434.

RESULTS In Silico Identification of SRBP-2. The human SRBP-2 coding sequence (Fig. 1A) was identified in silico by sequence homology search using Blast and PSI-Blast programs on the GenBank database. SRBP-2 exhibited 42% homology with HSI at the nucleic acid level

Table 1 Accession numbers and alternative protein names of SR31747A- binding proteins GenBank AN Swissprot AN Other names SRBP-1 U79528 AAB51238 ␴-1 SRBP-2 AF243433 Q9BY08 EBRP HSI Z37986 Q15125 EBP ERG2 M74037 P32352 ␴-2 Unknown Unknown

Table 2 Sequence comparison within the sterol isomerase protein familya Fig. 2. A, phylogenetic tree among the family of SR31747 binding protein family. B, multiple sequence alignment of proteins of the sterol isomerase family. The phylogenetic Cobaye Mouse Rat A. Thaliana Human Mouse tree and alignment were done using the Pileup function, GCG software version 10.3. In HSI SI SI SI SI SRBP-2 SRBP-2 B, identical amino acid residues conserved in four or more sequences are in bold letters; the transmembrane domains (TM) are indicated. The corresponding sequence accession HSI 100 numbers are: hSI: Q15125, Cobaye SI (Cob SI): Q60490, Mouse SI (mSI): P70245, Rat Cobaye SI 79 100 SI: Q9JJ46, A. thaliana SI: O48962, Human SRBP-2: Q9BY08, Mouse SRBP-2: Q9D0P0, Mouse SI 82 75 100 Mouse ␴-1 receptor: AAC33306, Rat ␴-1 receptor: AAF08342, Cavia Porcellus ␴-1 Rat SI 84 76 90 100 receptor: CAA91441, S. cerevisiae ERG2: P32352, ERG2 from the rice blast fungus A. thaliana SI 48 47 53 52 100 Magnaportae grisea, P33281, ERG2 from the maize smut pathogen Ustilago maydis: Human SRBP-2 41 38 38 38 47 100 P32360, human SRBP1: AAB51238. Mouse SRBP-2 51 46 43 41 53 78 100 a Percentage similarity is indicated. Sequence homologies at the amino acid levels are indicated. The corresponding sequence accession numbers are: HSI: Q15125, Cobaye SI: and 41% homology at the amino acid level (Fig. 1B). SRBP-2 did not Q60490, Mouse SI: P70245, Rat SI: Q9JJ46, Arabidopsis thaliana: O48962, Human show significant homology with either SRBP-1 or ERG2, which is the SRBP-2: Q9BY08, Mouse SRBP-2: Q9D0P0. S. cerevisiae ERG2 (P32352) does not show significant homology with these sequences (i.e., homology percentage on the entire functional equivalent of HSI in yeast. Human SRBP-2 cDNA is 1142 protein sequence is Ͻ20%). bp long. The open reading frame is of 621 bp and encodes a 206 4812

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. A NEW SR31747A-BINDING PROTEIN amino acid polypeptide (Fig. 1A). On the basis of the sequence, the script exhibiting broad ubiquitous expression among human tissues corresponding protein was estimated to have a molecular weight of Mr (Fig. 4, A and B). The highest signal was obtained in liver, followed 23,000 and a isoelectric point of 6.25. Sequence analysis highlighted by pancreas, stomach, small intestine, kidney, and adrenal and sali- the presence of four putative transmembrane domains and a reticulum vary glands. SRBP-2 mRNA was also found to be present at a slightly retention signal at the COOH-terminal end of the protein (KKXX). lower level in heart, colon, placenta, and ovary tissues. The brain and Some of the amino acid required for the in vivo sterol isomerase other central nervous system tissues showed the lowest expression activity of HSI are conserved between the two sequences (Fig. 1B). level (Fig. 4B). All human cell lines tested expressed SRBP-2, with Subsequent database searches revealed that the nucleic SRBP-2 se- the Burkitt’s lymphoma cell line Raji showing the highest signal (Fig. quence is identical to the AF243433 sequence, which has been as- 4C). A similar transcript size and predominant expression in the liver signed to the human emopamil binding related protein (Table 1). were also observed in mouse and rat (Fig. 4D). Identification of the Mouse Homologue of Human SRBP-2. We Characterization of SRBP-2 Protein Expression. Rabbits were sought the murine SRBP-2 sequence during this study. Primers se- immunized with synthetic peptides corresponding to the NH2-or lected in the human sequence were used to amplify mouse liver COOH-terminal part of SRBP-2. Specific SRBP-2 recognition was cDNA. The mouse 302-bp amplicon exhibited 85.7% homology and first assessed in Western blot experiments. Immunopurified antibodies

85.7% identity with human SRBP-2 (data not shown). At the protein revealed that c-myc-SRBP-2-transformed yeast cells expressed a Mr level, the 302-bp amplicon may be translated into a 100 amino acid ϳ25,000 hybrid protein (Fig. 5, lines 2 and 3). The labeling obtained polypeptide with 86% homology and 84% identity. In addition, this with anti-SRBP-2 antibodies is identical to the signal revealed with polypeptide exhibited 59% similarity relative to the mouse sterol the anti-c-myc antibody (Fig. 5, line 1) and corresponded to the isomerase (data not shown). Database searches revealed that this expected molecular weight of Mr 23,000 for SRBP-2. This labeling portion of the murine SRBP-2 is identical to the AF243434 sequence. was reversed when purified antibodies were preincubated with the The complete murine SRBP-2 sequence showed 78% homology with corresponding immunogenic peptide (data not shown). This result the human SRBP-2 protein sequence (Table 2). confirmed that the cDNA sequence encoded the SRBP-2 protein. Characteristics of the HSI Family. A phylogenetic tree evi- The subcellular distribution of SRBP-2 was examined in different denced that SRBP-2 belongs to the HSI protein family, which is human cancer cell lines by immunohistochemistry, confocal, and distinct from the ␴1 receptor family (Fig. 2A). Comparing mammal electronic microscopy using the two anti-SRBP-2 antibodies. Both SRBP-2 (human and mouse) with the known mammal HSI and plant antibodies gave similar results so that representative images obtained sterol isomerase (Arabidopsis thaliana), we noted 35–50% sequence only with the antibody targeting the COOH-terminal part of SRBP-2 homology between homologues and 75% between orthologues (Ta- are shown. First, immunohistochemical analysis was performed on bles 1 and 2). Although the global sequence homology was interme- human prostatic tumor PC3 cells. We found that SRBP-2 expression diary, the structural features (transmembrane domains) were main- prevailed in the cytoplasmic region. The highest SRBP-2 expression tained among members of the HSI protein family (Fig. 2B). was observed with both antibodies in some cell aggregates, whereas in In Situ Hybridization and FISH Detection. To address the chro- tumor sections, we noted that a few cells did not express SRBP-2 (Fig. mosomal localization of the human SRBP-2 gene, we performed in 6A). Positive SRBP-2 staining was not observed in the absence of situ hybridization and FISH detection using the human full-length primary antibody or with the addition of immunogen peptide (Fig. SRBP-2 cDNA as a probe. In 100 metaphase cells examined after in 6B). Similar results were obtained by confocal analysis on MDA-MB- situ hybridization, 51 mitotic figures showed signals on one pair of 231 cells, where strong cytoplasmic labeling was observed, along with chromosomes. DAPI banding identified chromosome 13 and assigned slight labeling on the nuclear envelope (Fig. 7A). When MDA-MB the probe signal to a single locus, i.e., the q14.3-q21.1 region on the 231 cells were simultaneously labeled with anti-HSI antibody, the long arm of chromosome 13. These results mapped the SRBP-2 probe merged image demonstrated that both proteins were colocalized in the to the 13q14.3-q21.1 region on the long arm of human chromosome cytosol and on the nuclear envelope (Fig. 7A). A similar localization 13. (Fig. 3). was observed with LNCaP, MCF7, and PC3 (data not shown). The Characterization of SRBP-2 mRNA Expression. Northern blot electron microscopic analysis clearly revealed the nuclear localization experiments indicated that SRBP-2 mRNA is a single 1.1-kb tran- of SRBP-2 and also that the protein was expressed on the endoplasmic

Fig. 3. Chromosomal localization of the human SRBP-2 gene. A, FISH mapping of the human SRBP-2 gene. Left panel, the FISH signals on chromosome. Arrow, the specific site of hybridization to chromosome 13. Right panel, the same mitotic figure stained with DAPI to identify chromosome 13. B, ideogram of the human chromosome 13 illustrating the distribution of labeled sites for the human SRBP-2 probe. Each dot represents the double FISH signals detected on human chromosome 13.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. A NEW SR31747A-BINDING PROTEIN reticulum (Fig. 7B). Finally, human breast carcinoma tumors were positively stained for SRBP-2 and showed cytoplasmic staining of various intensity very often dramatically increased around the nu- cleus. Malignant structures as well as normal structures were involved. In the same positive structure, 10–90% of the cells expressed SRBP-2 (Fig. 8). Comparative Analysis of SRBP-2, HIS, and SRBP-1 Expression Levels. The SRBP-2 expression level was compared with that of HSI and SRBP-1 in several breast and prostate cancer cell lines by flow cytometry (Fig. 9). Every cell line tested expressed SRBP-2. Interest- ingly, the three proteins were expressed at a high level in tumor cell lines as compared with normal cells, lymphocytes, and monocytes. The SRBP-2 expression level was higher than that of SRBP-1 and HSI in the panel of tumor cell lines tested except the breast cancer hormono-dependent MCF-7 cell line. Fig. 5. Western Blot analysis. Lysates of yeast transformed with the cDNA-encoding full-length SRBP-2-containing plasmid were subjected to SDS-PAGE, transferred to Pharmacological Characterization of SRBP-2. The binding nitrocellulose membranes, and then analyzed by a Western blot using antibodies targeting: properties of the human SRBP-2 protein were characterized in the line 1, c-myc; line 2, NH2-terminal SRBP-2; line 3, COOH-terminal SRBP-2. The ϳ yeast ERG2 disruptant strain EMY90, which produces SRBP-2. The Western blot yielded a band with a size of Mr 25,000 for c-myc-SRBP-2, which indicates a size of M ϳ23,000 for SRBP-2. binding activity of 3H SR31747 was only detected on membrane r homogenates and absent in the supernatants, indicating that SRBP-2 is localized in the membranes when expressed in yeast (Fig. 10). As a only Tamoxifen inhibited 3H-SR31747A binding, whereas no ␴ li- control, mock-transformed yeast did not show any binding activity gands, such as haloperidol Ϯ pentazocine or DTG, were shown to (Fig. 10). Scatchard plot analysis of those membrane extracts express- compete with 3H-SR31747A at SRBP-2 sites (Fig. 11). Binding ing SRBP-2 using 3H-SR31747A revealed a single high affinity site experiments were then performed with 3H-DTG, 3H-pentazocine, or ϳ Ϯ 3 with a Kd of 10 nM and a Bmax of 208 21 fmoles/mg protein. As H-3PPP and confirmed the absence of a binding site for those ligands a control, no specific binding was observed with a different erg2 gene on the SRBP-2 protein (data not shown). As Tamoxifen has been disruptant (data not shown). Interestingly, in competitive experiments, described to strongly inhibit 3H-SR31747A binding to HSI (20), we

Fig. 4. Northern blot analysis of SRBP-2 mRNA expression: A, C, D, commercial blots (Clontech) containing 2 ␮g of poly(A)ϩ mRNAs from human tissues (A), cell lines (C), or rodent tissues (D) shown above each lane were hybridized with human SRBP-2 cDNA as described in “Materials and Methods.” Blots were stripped and rehybridized with actin probe to assess levels of RNA between lanes (data not shown). In B, a human master blot (Clontech) containing 50 different human tissues immobilized in separate dots was hybridized with human SRBP-2 cDNA as described in “Materials and Methods.” The diagram shows the nature and position of poly(A)ϩ RNAs and controls. 4814

Fig. 6. Immunohistochemical analysis of SRBP-2 expression in human prostate PC3 cell line using anti-COOH-terminal SRBP-2 antibody (A). The specificity of the labeling was tested in B using anti-COOH-terminal SRBP-2 antibody plus immunogenic COOH-terminal peptide. A representative image is shown as similar results are obtained with anti-NH2-terminal SRBP-2 antibody. Magnifica- tion: ϫ400.

tested the affinity of SRBP-2 for 3H-Tamoxifen, which was found to Associations among SRBP-1, SRBP-2, and HSI Expression with be2nM. Considering their pharmacological profiles, our data indi- Clinicopathological Variables and Prognostic Relevance. When cated that HSI and SRBP-2 have similar binding properties. analyzing the potential of SRBP-1 and HSI as prognostic markers in Sterol Isomerase Activity. Sterol isomerase activity was assayed a population of 95 patients with operable primary breast cancer, we using cholest-8-en-3␤-ol and microsomes from ⌬erg2 and ⌬erg2Ϫ showed previously that the presence of HSI and absence of SRBP-1 SRBP-2-transformed cells. Despite the overall homology with HSI expression were associated with poorer DFS (P ϭ 0.007; Ref. 20). and the fact that some important residues required for sterol isomerase This analysis was extended to SRBP-2 using the COOH terminus- activity are conserved between the two sequences, SRBP-2 did not targeting antibody. No correlation was shown between SRBP-2 label- exhibit any sterol isomerase activity (Table 3), which was also con- ing and age, tumor grade, and tumor size. In addition, there was no firmed by the absence of complementation in ERG-2-disrupted yeast correlation between the SRBP-2 immunoreactivity and PR or estrogen (data not shown; see protocol in Ref. 12). receptor status. Although a slight correlation was noted between

Fig. 7. A, subcellular analysis of SRBP-2 expression by confocal microscopy in human MDA-MD-231 breast cancer. Comparison with HSI. The green (fluorescein isothiocyanate) left side corresponds to labeling of SRBP-2, the red (Cy5) central part to labeling of HSI, the right side represents the merged images. B, subcellular analysis of SRBP-2 expression by electron microscopy in human MDA-MD-231 breast cancer. Magnification: ϫ125,000. 4815

Fig. 8. Immunostaining of infiltrating breast cancer with anti-SRBP-2 antibody. Magnification: ϫ250 (A) and ϫ400 (B).

SRBP-1 and SRBP-2 (P ϭ 0.073), as well as between nodal status genome sequencing project, sequence databases provide a wealth of and SRBP-2 (P ϭ 0.09), the correlation was stronger between information that should be investigated. Our strategy was thus to HSI and SRBP-2 (P ϭ 0.008). There was also a significant correlation explore these sequence databases to identify the ␴2 protein sequence. between HER2 and SRBP-2 (P ϭ 0.042). As far as the prognosis Homology searches indicated that SRBP-1 did not exhibit significant of SRBP-1, HIS, and SRBP-2 on DFS is concerned, we noted that sequence homology with any known human proteins. However, it SRBP-2 had a significant effect on DFS when considering the sub- showed 35% identity with ERG2, the yeast sterol isomerase that is group of patients with SBR 1 and 2 [4.34 (95% confidence interval: also known to bind SR31747A with nanomolar affinity. Despite its ϭ 0.79–23.84), P 0.067, stratified on nodal status], whereas SRBP-1 sequence homology with ERG2, SRBP-1 did not exhibit detectable ϭ ϭ (P 0.38) and HSI (P 0.56) were not significant in this subgroup sterol isomerase activity. HSI is the human counterpart to the yeast of patients. This effect was essentially seen in node-positive SBR 1 or sterol isomerase. This M 25,000 protein bound ␴ ligands and espe- 2 patients where the relative risk of failure was equal to 8.7 (95% r cially SR31747A with nanomolar affinity. No significant sequence confidence interval: 0.9–85.1) for SRBP-2-positive patients. Al- homology was noted when comparing human SRBP-1 and HSI. To though this latter subgroup included a few patients, there were 13 identify additional novel proteins that could bind SR31747A, we used versus 75% failures for SRBP-2-negative and -positive patients, respectively. both human SRBP-1 and HSI sequences as bait to screen available sequence databases and looked for proteins having high sequence homology with any of these two SR31747A receptors. These screen- DISCUSSION ings led to the identification of SRBP-2, an original Mr 23,000 protein Cloning of ␴2 would be warranted on the basis of the ␴2 expression that exhibited 41% homology with HSI at the protein level. In our profile in human tumors, its potential use as a marker of proliferation, database search, we also found the mouse homologue of human and by the fact that the antiproliferative effect of SR31747A is not SRBP-2. The human SRBP-2 gene was localized on chromosome fully understood in mammals. With the completion of the human 13q14.3-q21.1. The other corresponding SR31747A-binding protein

Fig. 9. Measurement of HSI, SRBP-1, and SRBP-2 sites in breast or prostatic cancer epithelial cell lines. HSI, SRBP-1, and SRBP-2 expression were determined by flow cytometric analysis of immun- ofluorescent stained cells, as described in “Materials and Methods.” For comparative purposes, HSI, SRBP-1, and SRBP-2 sites were also evaluated on normal cells, lymphocytes, and monocytes. Dis- crepancies of expression levels between tumoral and normal cells were statistically tested using the Wilcoxon test and found significant with P ϭ 0.045 Ͻ 0.05, for each protein.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. A NEW SR31747A-BINDING PROTEIN genes were localized on different human chromosomes. Human SRBP-1 and HSI genes were located on chromosome 9 and X, respectively. As SRBP-2 was initially identified as a sequence, we first assessed whether this protein is expressed in cells and performed Northern and Western blot experiments. Northern blot experiments showed that the SRBP-2 transcript expression pattern was similar to that of HSI or SRBP-1. To analyze SRBP-2 protein expression, we produced specific polyclonal antibodies targeted against specific SRBP-2 peptides selected within the SRBP-2 sequence. When expressed in yeast, the ϳ protein was revealed as a Mr 23,000 protein, which is in accordance with the size expected on the basis of the sequence. The subcellular protein expression analysis performed on human tumor cell lines revealed that SRBP-2 was expressed on the nuclear membrane and endoplasmic reticulum, like SRBP-1 and HSI. In addition, when analyzing SRBP-2 expression on different breast carcinoma biopsies, we observed that not all of the cells of the same structure expressed SRBP-2 in normal breast cells as well as in malignant structures. This may indicate a possible cycle-related status of the protein, which has to be investigated. In this context, it would be also interesting to test whether the three proteins interact or form a multiprotein complex, i.e., because these three proteins share the ability to bind SR31747A and are colocalized on the same organelles, they might interact to 3 regulate the SR31747A effect. Additional studies are required to test Fig. 11. Competitive binding experiments on binding of H SR31747A to SRBP-2 expressing yeast membrane. Membranes were incubated with 3H SR31747A in the this hypothesis. presence of various concentrations of unlabelled SR31747A (F), Tamoxifen (E), (ϩ) To a functional point of view, although numerous critical residues pentazocine (), (Ϫ) pentazocine (ƒ), 3PPP (f), DTG (Ⅺ), or haloperidol (ࡗ). Mem- branes were filtered, and radioactivity was measured by scintillation counting. Data points for sterol isomerase activity in HSI are conserved in SRBP-2, we did represent mean of the percentage of 3H SR31747A bound using triplicate measurements, not evidence any significant sterol isomerase activity for SRBP-2. bars Ϯ SE. This may suggest either that some residues are more important than expected for sterol isomerase activity or that SRBP-2 may interact Table 3 Measurement of ⌬8-⌬7 sterol isomerase activitya with an additional partner that is lacking in yeast and would contribute ⌬ ⌬ to the sterol isomerase activity of this protein. Additional studies are 8- 7 sterol isomerase activity warranted to address this issue. Yeast strains (nmoles/mg/h) The pharmacological profile of SRBP-2 was assessed to further EMY-90 (pRS426GAL1(URA3)ϩpRS425GAL1(LEU) UDb characterize this protein. We found that SR31747A bound to SRBP-2 EMY-90ϩp2208(pRS425GAL1:MYC-HSI) ϩ pRS425GAL1(LEU2) 5.01 Ϯ 0.10 EMY-90ϩp2232(pRS425GAL1:MYC-SRBP-2) UD witha10nM affinity. Although this binding potency was lower ⌬erg2ϩpEMR1235 (yeast SI) 3.1 Ϯ 0.6 compared with HSI, both SRBP-2 and HSI proteins exhibited similar a Yeast sterol isomerase activity was assayed as described in “Materials and Methods”. binding properties when considering other ␴ ligands. Neither halo- Enzyme activity is expressed as the rate of conversion of cholest-8-en-3␤-ol into cholest- 7-en-3␤-ol in nmoles/mg protein/h. b UD, undetectable.

peridol, DTG, nor pentazocine competed with SR31747A binding. Tamoxifen was the only ligand that displaced SR31747A with high ϭ potency (Kd 3nM). Altogether, these pharmacological data (and specifically the absence of binding of DTG on SRBP-2) indicate that SRBP-2 and ␴2 are two different proteins. On the basis of the identification of SRBP-2, it would now be crucial to determine the specific roles of each SR31747A-binding site in the light of the antiproliferative effect of the molecule. When analyzing the expression of SRBP-1, SRBP-2, and HSI in different human cell lines, we noticed that cell lines could be distinguished according to the respec- tive expression levels of the three receptors. Correlation studies of

IC50 and expression levels may help to understand which receptor or combination of receptors preferentially mediates the antiproliferative effect of SR31747A. In addition, we have not yet detected a negative cell line for SRBP-2. Such a tool could also be very informative. Finally, apart from the HSI enzyme belonging to the cholesterol biosynthesis pathway, the identification of ligands specific to either SRBP1 or SRBP-2 could also be helpful for characterizing the func- Fig. 10. Assessment of SR31747A binding in SRBP-2 expressing yeast. Membrane tion of these binding sites. homogenates and supernatants from yeast ERG2 disruptant strain EMY90 transformed Recently, SRBP-1 and HSI expression were immunocytochemi- with SRBP-2 full-length cDNA were prepared as indicated in “Materials and Methods” and tested for SR31747A binding. Control are mock-transformed yeast that do not express cally investigated relative to a series of clinicopathological and im- SRBP-2. munocytochemical prognostic factors in a trial involving 95 patients 4817

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. A NEW SR31747A-BINDING PROTEIN with operable primary breast cancers. The combined analysis of their effects of the immunosuppressant SR31747A on gene expression in Saccharomyces expression revealed that the presence of HSI and absence of SRBP-1 cerevisiae. Gene Expr., 10: 213–230, 2002. 7. Labit-Lebouteiller, C., Jamme, M. F., David, M., Silve, S., Lanau, C., Dhers, C., receptor expression were associated with poorer DFS (19). As we Picard, C., Rahier, A., Taton, M., Loison, G., Caput, D., Ferrara, P., and Lupker, J. found that SRBP-1, HIS, and SRBP-2 were expressed in various Antiproliferative effects of SR31747A in animal cell lines are mediated by inhibition human tumor cell lines, especially in breast cancer cells, the above- of cholesterol biosynthesis at the sterol isomerase step. Eur. J. Biochem., 256: 342–349, 1998. mentioned study has been pursued with SRBP-2 to assess the potential 8. Berthois, Y., Bourrie´, B., Galie`gue, S., Vidal, H., Carayon, P., Martin, P. M., and use of this newly described protein as a prognostic marker in human Casellas, P. SR31747A is a sigma ligand exhibiting antitumoral activity both in vitro breast cancer. Despite the absence of any correlation between SRBP-2 and in vivo. Br. J. Cancer, 88: 438–446, 2003. 9. Jbilo, O., Vidal, H., Paul, R., DeNys, N., Bensaid, M., Silve, S., Carayon, P., Davi, and the receptor status (PR or estrogen receptor), SRBP-2 expression D., Galie`gue, S., Bourrie´, B., Guillemot, J. C., Ferrarra, P., Loison, G., Maffrand, seems to correlate with poorly differentiated and highly aggressive J. P., Le Fur, G., and Casellas, P. Purification and characterization of the human SR31747A-binding protein. 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H., Labit-Lebouteiller, C., Kaghad, M., Chalon, P., Rahier, A., done, our results suggest that the expression of SRBP-2 might be a Taton, M., Lupker, J., Shire, D., and Loison, G. Emopamil-binding protein, a potential breast cancer marker. mammalian protein that binds a series of structurally diverse neuroprotective agents, In conclusion, we have identified and characterized an additional exhibits delta8-delta7 sterol isomerase activity in yeast. J. Biol. Chem., 271: 22434– 22440, 1996. binding protein for SR31747A. The family of human proteins that 13. Dussossoy, D., Carayon, P., Belugou, S., Feraut, D., Bord, A., Goubet, C., Roque, C., binds SR31747A now comprises four members. Among these, three Vidal, H., Combes, T., Loison, G., and Casellas, P. Colocalization of sterol isomerase are molecularly characterized. They share some structural features and and sigma(1) receptor at endoplasmic reticulum and nuclear envelope level. Eur. J. 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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. Identification and Pharmacological Characterization of SRBP-2: A Novel SR31747A-binding Protein

Hubert Vidal, Guillaume Mondesert, Sylvaine Galiègue, et al.

Cancer Res 2003;63:4809-4818.

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