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[Cancer Research 63, 4809-4818, August 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Identification and Pharmacological Characterization of SRBP-2

A Novel SR31747A-binding Protein

Hubert Vidal, Guillaume Mondesert, Sylvaine Galiègue, Dominique Carrière, Pascal-Henri Dupuy, Pierre Carayon, Thérèse Combes, Estelle Bribes, Joëlle Simony-Lafontaine, Andrew Kramar, Gérard Loison and Pierre Casellas1

Immunology-Oncology Department, Sanofi~Synthelabo, F-34184 Montpellier cedex 04 [H. V., S. G., D. C., P. C., T. C., E. B., P. C.]; Molecular and Functional Genomics Department, Sanofi~Synthelabo, F-31676 Labège 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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SR31747A is a {sigma} ligand with potent antiproliferative activity against tumor cells and for which three binding proteins have been identified to date: (a) SRBP-1 (also called {sigma}1); (b) HIS; and (c) {sigma}2. In this study, we characterized an additional SR31747A binding site, i.e., SRBP-2 (SR31747A-binding protein 2). Using an in silico screening approach, we identified this novel sequence, which exhibits 41% homology with HSI. The 1142-bp cDNA was found to encode a 206 amino acid protein not related to SRBP-1. Northern blot analysis of SRBP-2 mRNA expression revealed a single 1.1-kb transcript that was widely expressed in organs; the liver was particularly enriched, and the brain showed the lowest abundance. A murine homologue that exhibited a similar expression pattern was also characterized. Subcellular localization analysis using specific polyclonal antibodies revealed that SRBP-2 had the same nuclear membrane and endoplasmic reticulum localization as other members of the SR31747A-binding protein family. Considering SRBP-2-binding properties, pharmacological analysis clearly highlighted that SRBP-2 was distinct from {sigma}2. Scatchard plot analysis revealed Kd values of 10 and 3 nM for SR31747A and Tamoxifen, respectively. In contrast with HSI, the protein also did not exhibit detectable isomerase activity. When analyzing SRBP-2 expression in human breast cancer biopsies, we obtained evidence that SRBP-2 expression, together with SRBP-1 and HSI, may be of interest as a prognostic marker. These findings demonstrated that SRBP-2 represents an additional molecular target for SR31747A, which could help to understand the immunosuppressive and antiproliferative effects of the molecule.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SR31747A is a selective peripheral {sigma}-binding site ligand exhibiting immunosuppression and able to inhibit cell proliferation both in vitro and in vivo. The immunomodulatory properties of the SR31747A drug were first demonstrated by the inhibition of the mitogen-induced mouse and T-cell proliferation elicited by nanomolar concentrations of SR31747A (1) . In vitro SR31747A was shown to inhibit staphylococcal enterotoxin B-driven lymphocyte proliferation (2) , whereas in vivo, in mice, SR31747A treatment confers potent protection against the lethal effects of staphylococcal enterotoxin B and {Delta}–galactosamine and prevents both graft-versus-host disease and delayed type hypersensitivity granuloma formation (1, 2, 3) . SR31747A was also shown to modulate proinflammatory and anti-inflammatory cytokine responses (2 , 4) . The antiproliferative properties of nanomolar concentrations of SR31747A were shown in yeast (5 , 6) and against human tumor cell lines, both in vitro and in vivo (7 , 8) . Precisely nanomolar concentrations of SR31747A inhibited cell proliferation of either hormono-responsive or hormono-unresponsive human cancer cell lines in vitro and in vivo; a treatment with SR31747A significantly reduced both human breast or prostatic cancer cell line-derived tumor development in the mouse xenograft model (8) . In 2003, after a positive Phase IIa, SR31747A entered Phase IIb clinical trials in prostate cancer. Three high affinity SR31747A-binding proteins that may mediate SR31747A properties have been identified to date in humans: (a) SRBP-1; (b) {sigma}2; and (c) HSI (7, 8, 9, 10, 11, 12) . SRBP-1 and HSI have been molecularly characterized, whereas {sigma}2 has not yet been cloned. SRBP-1, which stands for SR31747A-binding protein 1 and is also called {sigma}1 (9 , 10) , is related to the yeast Saccharomyces cerevisiae C8-C7 sterol isomerase encoded by the ERG2 gene; there is 35% sequence identity between the two proteins (5) . Despite this identity percentage, SRBP-1 receptor expression does not complement an erg2 defect in yeast, and no sterol isomerase activity has ever been demonstrated for SRBP-1 (9) . The emopamil-binding protein, HSI, was first described as a high affinity binding protein for emopamil, the anti-ischemic phenylalkylamine Ca2+ antagonist (11) . HSI is the human counterpart of the yeast ERG2 (12) . In mammals, HSI belongs to the sterol biosynthesis pathway, and the enzyme catalyzes the conversion of 5{alpha}-cholesta-8,24-dien-3ß-ol (zymosterol) and 5{alpha}-cholesta-8-en-3ß-ol (zymostenol, {Delta}8-cholestenol) to their corresponding {Delta}7-isomers. HSI and SRBP-1 have been expressed in yeast, and their expression and pharmacological profiles have been both characterized. The two proteins are colocalized and associated with the endoplasmic reticulum and outer and inner membranes of the nuclear envelope. They also delocalize during the cell cycle at the mitosis step when the nuclear membranes disappear (13) . Only the pharmacological properties of {sigma}2 have been unraveled. {sigma}2 has been identified in rat spleen using tritiated DTG,2 with an estimated protein size of Mr 21,000. Although the {sigma}2 sequence has not yet been identified, some important information has been reported on the basis of the protein expression in tumors. Indeed, the {sigma}2 receptor is considered to be a potential biomarker of proliferation in cancer, and radiolabeled ligands specific to the {sigma}2 receptor were demonstrated to be useful for assessing the proliferative status of tumors and normal tissues (14 , 15) . Identification of {sigma}2 is a critical issue in this context.

The yeast S. cerevisiae has been used as a model to study the mechanism of the antiproliferative effect of SR31747A (5) : (a) in this model, we showed that SR31747A binds Erg2 and blocks cell proliferation by inhibiting the sterol biosynthesis pathway; and (b) using the DNA chip strategy, we also demonstrated that ERG2 is the only target that mediates the antiproliferative SR31747A property in yeast (6) . A similar inhibitory mechanism has been demonstrated in animal cell lines grown in sterol-free medium (7) . However, although candidate targets have been identified in mammals, the mechanism that prevails in the sterol starvation-induced cell proliferation inhibition is not yet fully understood. In addition, the analysis of the expression of both receptors indicated that the sensitivity of human tumor cell lines to SR31747A is not correlated with either HSI or SRBP-1 expression (8) . These data support that additional binding sites may exist.

To elucidate the SR31747A mode of action, we searched for additional SR31747A binding sites that could account for its reported properties in mammals. With this aim, we used an in silico technique to screen for the protein that exhibits high homology for human SRBP-1 or HSI. In the present study, we characterized SRBP-2 (SR31747A-binding protein 2), an original protein that exhibits 43% identity with HSI and binds SR31747A with high nanomolar affinity. SRBP-2, based on its binding profile, is distinct from {sigma}2. SRBP-2 is thus a novel human SR31747A binding site. Here, we characterized this new protein and its binding properties, and we addressed whether the protein exhibits sterol isomerase activity. In addition, we analyzed its expression both at the mRNA and protein levels, the latter using immunohistochemical and electronic microscopy approach. Interestingly, we evidenced that SRBP-2 expression level is higher than HSI and SRBP-1 expression levels in several breast and prostate cancer cell lines. Finally, we tested the interest of SRBP-2 as a potential breast cancer marker, and our results showed that SRBP-2 expression had a significant negative effect on DFS when considering low-grade SBR patients.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
SR31747A, (Z)N-cyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)propen-2-ylamine hydrochloride, was produced and provided by Sanofi Synthelabo Laboratories (Montpellier, France). Tamoxifen and pentazocine were purchased from Sigma. DTG and 3PPP were supplied by Interchim.

3H-tamoxifen (80 CimM) [3H]DTG (31 Ci/mM), [3H]3PPP (101 Ci/mM) from NEN; [3H]SR31747A (51 Ci/mM) from Amersham; [3H]Pentazocine (28 Ci/mM) from NEN.

Cell Lines and Culture Conditions.
Hormono-responsive breast adenocarcinoma MCF-7 cells were cultured in 50% DMEM/50% Ham’s F12 (1/1, volume for volume) supplemented with 16 ng/ml insulin, 2 mM L-glutamine, 10 mM HEPES buffer, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% heat-inactivated FCS. The hormono-unresponsive breast cancer cells, MDA-MB-231, were maintained in Leibovitz L15 culture medium supplemented with 10 mM HEPES buffer, 6 µg/ml human insulin, 2 mM L-glutamine, 1% nonessential amino acids, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 10% FCS. Three human prostatic cancer cell lines that were either hormono-responsive, LNCaP, or hormono-unresponsive, DU145 and PC3, were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. All cells were cultured at 37°C in a humid atmosphere of 5% CO2 in air, except for the MDA-MB-231 cell line, which was cultured in the absence of CO2.

Cloning of Human SRBP-2 cDNA.
The AI858023 clone was obtained from the IMAGE consortium. This clone, which encompasses the entire coding sequence of SRBP-2, was fully sequenced to assess the absence of any mutation.

Cloning of Mouse SRBP-2 cDNA.
To identify the mouse homologue of human SRBP-2 cDNA, we defined primers specific to the human sequence and used these primers to amplify mouse liver cDNA (Clontech). The primers used were 5' ctcatctggctctgctac 3' (sense) and 5' atccagcagccatacagct 3' (antisense). The PCR reaction was performed at 45°C for 30 cycles. A 302-bp amplicon was obtained and sequenced.

Northern Blot Analysis.
A cDNA probe containing the entire nucleotide sequence encoding the SRBP-2 protein was labeled with 32P using the RadPrime DNA labeling system (Life Technologies, Inc.), according to the manufacturer’s instructions. After labeling, the probe was purified using a CHROMA SPIN-200 column (Amersham Life Science). The purified labeled probe was then used to examine various human and rodent tissues for SRBP-2 mRNA. Human RNA Master Blots, Multiple Tissue Northern blots containing various human tissues, human cell lines, and mouse and rat Multiple Tissue Northern blots were obtained from Clontech and examined with the labeled probe using the hybridization solution (Church buffer: 1% BSA, 7% SDS, 0.5 M NaH2PO4, and 1 mM EDTA). Membranes were prehybridized for 1 h at 65°C in hybridization solution, then hybridized overnight at 65°C using radiolabelled SRBP-2 probe in hybridization solution containing 100 µg/ml salmon sperm DNA. Blots were washed twice with WBA (0.5% BSA, 5% SDS, 40 mM NaH2PO4, and 1 mM EDTA) for 5 min at 65°C and once with WBB (1% SDS, 40 mM NaH2PO4, and 1 mM EDTA) for 10 min at 65°C. Finally, blots were autoradiographed using Kodak X-ray film for 18 h at -70°C and developed according to standard procedures.

In Situ Hybridization and FISH Detection.
The chromosomal assignment for the human SRBP-2 gene was performed by in situ hybridization and FISH detection according to a procedure published previously (16 , 17) . Gene mapping was carried out on chromosome preparations obtained from phytohemagglutinin-stimulated human lymphocytes cultured for 72 h. The lymphocyte cultures were synchronized with 5-bromodeoxyuridine (0.18 mg/ml) treatment. The entire coding sequence of SRBP-2 was used as cDNA probe. FISH signals and the DAPI-banding pattern were recorded separately on photographs, whereas the FISH mapping data were assigned to chromosomal bands by superimposing FISH signals with DAPI-banded chromosomes.

Anti-SRBP-2 Antibody Production.
Polyclonal anti-SRBP-2 antibodies were raised in rabbit against a synthetic peptide corresponding to amino acid residues 1–17 (Nt) and 187–206 (Ct) (Neosystem, Strasbourg, France). Rabbit sera were purified by immunoaffinity on a Sepharose column (Bio-Rad) to which the peptides were covalently coupled. Purified antibodies were tested for specific recognition on Western blotting membrane by peptide competition (10 µg/ml) before immunoblotting and used at 1 µg/ml.

Analysis of the Subcellular Localization of SRBP-2 by Confocal Microscopy.
The MDA-MB-231 human breast cancer cell line was used for immunofluorescence analysis of the SRBP-2 protein subcellular localization. Cells were fixed overnight with 1% paraformaldehyde, washed once, and permeabilized for 10 min in a 0.1% saponin, 1% BSA, PBS solution. For comparative purposes, cells were incubated in parallel with 1 µg/ml anti-SRBP-1 (9) , anti-HSI (13) , or anti-SRBP-2 antibodies for 60 min. After two washes, cells were incubated for 30 min with Cy5 antimouse or antirabbit conjugates (Southern Biotechnology, Inc., Birmingham, AL). The subcellular distribution of the SRBP-2 protein was analyzed using a laser scanning confocal microscope (LSM 410; Zeiss, Oberkochen, Germany) equipped with a c-apochromat water immersion lens (x63, numerical aperature = 1.2). Specificity controls were carried out by preincubation of anti-SRBP-2 antibodies with the immunizing peptides at 10 µg/ml.

Quantitative Analysis of SR31747A-binding Protein Expression by Flow Cytometry.
The cell lines used in this study were hormono-responsive breast cancer MCF-7, hormono-unresponsive MDA-MB-321 and BT-20, hormono-responsive prostate cancer LNCaP, and hormono-unresponsive DU145 and PC3. Normal cells included lymphocytes and monocytes. All cells were cultured at 37°C in a humid atmosphere of 5% CO2 in air, except for the MDA-MB-231 cell line, which was cultured in the absence of CO2, (as described in Ref. 8 ). Cells were fixed overnight with 1% formaldehyde and permeabilized for 10 min with a solution of 0.1% saponin in PBS containing 0.1% BSA. Cells were incubated with anti-SRBP-1, anti-SRBP-2, or anti-HSI antibodies for 60 min. After two washes, cells were incubated with antimouse or antirabbit conjugate for 30 min. After washing, fluorescence intensity was measured with a FACSCALIBUR cytometer.

Immunohistochemical Analysis of SRBP-2 Expression in PC3 Human Prostate Tumor Cells.
Human prostatic PC3 tumor cell sections were deparaffinized in toluene and incubated in 10 mM Na-citrate buffer (pH 6), at 95°C for 40 min. After three washes in water, SRBP-2 antibodies (0.7 or 0.5 mg/ml for Ct and Nt, respectively) were added for 25 min at room temperature. Immunoreactions were visualized by incubation with a biotinylated antirabbit IgG for 25 min at 37°C, followed by an indirect streptavidin-biotin method using H2O2/3-amino-9-ethylcarbazole as chromogenic substrate (red label, DAKO ChemMate Detection kit, peroxidase/AEC, rabbit/mouse; DAKO A/S, Glostrup, Denmark). Labeling specificity was determined by the absence of staining after preincubation of anti-SRBP-2 antibodies with the immunizing peptides at 10 µg/ml. Negative controls were also obtained by the absence of staining after omission of the primary antibody. Some slides were counterstained with hematoxylin. Sections were mounted on faramount-covered slides (DAKO), air-dried, and analyzed using a Leica DMLB microscope.

Recombinant SRBP-2 Protein Expression in Yeast.
The expression plasmid was made by cloning cDNA-encoding, full-length SRBP-2 into the pRS42xGAL1 expression vector containing 2µ and URA3 or LEU2 as selective markers. The yeast strain producing SRBP-2 was EMY-90+p2232(pRS425GAL1:MYC-SRBP-2). SRBP-2 expression was induced by adding 2% galactose to YNB + raffinose 2%.

Binding Studies on Yeast Cell Membranes.
Yeast cells were treated with zymolyase (ICN Biomedicals, Inc., 250 µg/gram cells for 25 min at 30°C) and homogenized in Tris-HCl (pH 7.4) buffer containing protease inhibitors. Homogenates were centrifuged at 100,000 x g for 20 min, and pellets were stored at -20°C until use. In competition experiments, 10 µg of yeast membranes were incubated for 60 min at 20°C in 200 µl of 50 mM Tris-HCl 0.1% BSA containing 1 nM radioactive ligand and the different drugs at concentrations ranging from 1 nM to 1 µM. In the saturation experiment, membranes were incubated with the radioactive ligand (0.2–50 nM), and nonspecific binding was obtained with 1 µM the corresponding nonradioactive ligand.

{Delta}8-{Delta}7 Sterol Isomerase Assay.
The {Delta}8-{Delta}7 sterol isomerase activity assay was performed as described previously (5) . Briefly, cells were disrupted by glass bead homogenization in 0.1 M phosphate buffer (pH 7.5) in the presence of 1.5 mM reduced glutathione and 30 mM nicotinamide for 10 min at 4°C. After centrifugation at 10,000 x g to remove cell debris, mitochondria, and nuclei, microsomes were isolated by additional centrifugation at 100,000 x g for 20 min and resuspended in 0.1 M phosphate buffer (pH 7.5) containing 3 mM reduced glutathione and 20% (volume for volume) glycerol. Microsome suspension (400 µl; 2.5 mg/ml protein concentration) was incubated with cholest-8-en-3ß-ol for 3 h at 30°C, and the metabolite content was analyzed by GC as described previously (5) . Enzyme activity was expressed as the rate of conversion of cholest-8-en-3ß-ol into cholest-7-en-3ß-ol in nmoles/mg protein/h.

Clinical Assessment of SRBP-2 Marker Expression in Breast Cancer Patients.
From January 1992 to February 1993, 850 new breast cancers were diagnosed at the Centre de Recherche et de Lutte Contre le Cancer Department of Pathology (Montpellier, France). Selection criteria included presentation with primary invasive breast carcinoma, no preoperative chemotherapy, endocrine or radiation therapy, sufficient tumor tissue remaining after diagnosis to allow biochemical quantification of receptor status and additional immunohistochemical assays (tumor size > 1 cm in diameter), and long-term follow-up for disease recurrence and death. A total of 95 patients who met these criteria was included.

Surgical treatment included radical mastectomy with axillary dissection in 58% of patients and breast conservative sector resection with axillary dissection in 42% of patients. After surgery, all patients with conservative treatment and 60% with radical mastectomy underwent combined postoperative radiotherapy to eradicate all local traces of the disease. A majority (80%) of patients received systemic adjuvant therapy, according to the CRLC routine assessment for clinical management of the disease, and depending on their age, menopausal status, steroid receptor status, and nodal status: (a) chemotherapy alone for 16 patients; (b) endocrine therapy alone (Tamoxifen) for 58 patients; and (c) combined chemotherapy and endocrine therapy for 2 patients. Patients were reviewed for disease recurrence and death, with a median follow-up of 75 months.

Tumor Samples.
At surgery, all patients had a small portion of tumor removed, which was snap frozen in liquid nitrogen and stored at -80°C for estrogen and PR analysis. The remaining part of the tumor was fixed in formalin-alcohol for 24 h, paraffin embedded, and subsequently processed with routine techniques followed by immunohistochemical analysis.

Histopathological Study.
Tumor slides (5-µm thick) were stained with H&E for the histopathological study. Tumor grading was performed according to the methodology of Refs. 18 and 19 . Mitosis counts were performed in 10 high power fields (x400) 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-µm-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 <10% 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 >10% 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 {chi}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 univariate analyses and the Cox proportional hazards model for multivariate analyses. For all statistical analyses, P < 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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In SilicoIdentification of SRBP-2.
The human SRBP-2 coding sequence (Fig. 1A)Citation 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 and 41% homology at the amino acid level (Fig. 1B)Citation . SRBP-2 did not show significant homology with either SRBP-1 or ERG2, which is the functional equivalent of HSI in yeast. Human SRBP-2 cDNA is 1142 bp long. The open reading frame is of 621 bp and encodes a 206 amino acid polypeptide (Fig. 1A)Citation . On the basis of the sequence, the corresponding protein was estimated to have a molecular weight of Mr 23,000 and a isoelectric point of 6.25. Sequence analysis highlighted the presence of four putative transmembrane domains and a reticulum retention signal at the COOH-terminal end of the protein (KKXX). Some of the amino acid required for the in vivo sterol isomerase activity of HSI are conserved between the two sequences (Fig. 1B)Citation . Subsequent database searches revealed that the nucleic SRBP-2 sequence is identical to the AF243433 sequence, which has been assigned to the human emopamil binding related protein (Table 1)Citation .



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  		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 is in bold letters. *, the termination codon. B, sequence comparison 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 +, ·, o, or {downarrow}, which indicated residues whose mutation reduced by <34, 35–64, 65–89, and >90% of the sterol {Delta}8-{Delta}7 isomerase activity, respectively (according to Ref. 21 ).

 

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  		Table 1 Accession numbers and alternative protein names of SR31747A-binding proteins

 
Identification of the Mouse Homologue of Human SRBP-2.
We sought the murine SRBP-2 sequence during this study. Primers selected in the human sequence were used to amplify mouse liver cDNA. The mouse 302-bp amplicon exhibited 85.7% homology and 85.7% identity with human SRBP-2 (data not shown). At the protein level, the 302-bp amplicon may be translated into a 100 amino acid polypeptide with 86% homology and 84% identity. In addition, this polypeptide exhibited 59% similarity relative to the mouse sterol isomerase (data not shown). Database searches revealed that this portion of the murine SRBP-2 is identical to the AF243434 sequence. The complete murine SRBP-2 sequence showed 78% homology with the human SRBP-2 protein sequence (Table 2)Citation .


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  		Table 2 Sequence comparison within the sterol isomerase protein familya

 
Characteristics of the HSI Family.
A phylogenetic tree evidenced that SRBP-2 belongs to the HSI protein family, which is distinct from the {sigma}1 receptor family (Fig. 2A)Citation . Comparing mammal SRBP-2 (human and mouse) with the known mammal HSI and plant sterol isomerase (Arabidopsis thaliana), we noted 35–50% sequence homology between homologues and 75% between orthologues (Tables 1Citation and 2Citation ). Although the global sequence homology was intermediary, the structural features (transmembrane domains) were maintained among members of the HSI protein family (Fig. 2B)Citation .



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  		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 tree and alignment were done using the Pileup function, GCG software version 10.3. In 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 numbers are: hSI: Q15125, Cobaye SI (Cob SI): Q60490, Mouse SI (mSI): P70245, Rat SI: Q9JJ46, A. thaliana SI: O48962, Human SRBP-2: Q9BY08, Mouse SRBP-2: Q9D0P0, Mouse {sigma}-1 receptor: AAC33306, Rat {sigma}-1 receptor: AAF08342, Cavia Porcellus {sigma}-1 receptor: CAA91441, S. cerevisiae ERG2: P32352, ERG2 from the rice blast fungus Magnaportae grisea, P33281, ERG2 from the maize smut pathogen Ustilago maydis: P32360, human SRBP1: AAB51238.

 
In Situ Hybridization and FISH Detection.
To address the chromosomal localization of the human SRBP-2 gene, we performed in situ hybridization and FISH detection using the human full-length SRBP-2 cDNA as a probe. In 100 metaphase cells examined after in situ hybridization, 51 mitotic figures showed signals on one pair of chromosomes. DAPI banding identified chromosome 13 and assigned the probe signal to a single locus, i.e., the q14.3-q21.1 region on the long arm of chromosome 13. These results mapped the SRBP-2 probe to the 13q14.3-q21.1 region on the long arm of human chromosome 13. (Fig. 3)Citation .



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  		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.

 
Characterization of SRBP-2 mRNA Expression.
Northern blot experiments indicated that SRBP-2 mRNA is a single 1.1-kb transcript exhibiting broad ubiquitous expression among human tissues (Fig. 4, A and B)Citation . The highest signal was obtained in liver, followed by pancreas, stomach, small intestine, kidney, and adrenal and salivary glands. SRBP-2 mRNA was also found to be present at a slightly lower level in heart, colon, placenta, and ovary tissues. The brain and other central nervous system tissues showed the lowest expression level (Fig. 4B)Citation . All human cell lines tested expressed SRBP-2, with the Burkitt’s lymphoma cell line Raji showing the highest signal (Fig. 4C)Citation . A similar transcript size and predominant expression in the liver were also observed in mouse and rat (Fig. 4D)Citation .



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  		Fig. 4. Northern blot analysis of SRBP-2 mRNA expression: A, C, D, commercial blots (Clontech) containing 2 µg of poly(A)+ 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)+ RNAs and controls.

 
Characterization of SRBP-2 Protein Expression.
Rabbits were immunized with synthetic peptides corresponding to the NH2- or COOH-terminal part of SRBP-2. Specific SRBP-2 recognition was first assessed in Western blot experiments. Immunopurified antibodies revealed that c-myc-SRBP-2-transformed yeast cells expressed a Mr ~25,000 hybrid protein (Fig. 5Citation , lines 2 and 3). The labeling obtained with anti-SRBP-2 antibodies is identical to the signal revealed with the anti-c-myc antibody (Fig. 5Citation , line 1) and corresponded to the expected molecular weight of Mr 23,000 for SRBP-2. This labeling was reversed when purified antibodies were preincubated with the corresponding immunogenic peptide (data not shown). This result confirmed that the cDNA sequence encoded the SRBP-2 protein.



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  		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 nitrocellulose membranes, and then analyzed by a Western blot using antibodies targeting: line 1, c-myc; line 2, NH2-terminal SRBP-2; line 3, COOH-terminal 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 Mr ~23,000 for SRBP-2.

 
The subcellular distribution of SRBP-2 was examined in different human cancer cell lines by immunohistochemistry, confocal, and electronic microscopy using the two anti-SRBP-2 antibodies. Both antibodies gave similar results so that representative images obtained only with the antibody targeting the COOH-terminal part of SRBP-2 are shown. First, immunohistochemical analysis was performed on human prostatic tumor PC3 cells. We found that SRBP-2 expression prevailed in the cytoplasmic region. The highest SRBP-2 expression was observed with both antibodies in some cell aggregates, whereas in tumor sections, we noted that a few cells did not express SRBP-2 (Fig. 6A)Citation . Positive SRBP-2 staining was not observed in the absence of primary antibody or with the addition of immunogen peptide (Fig. 6B)Citation . Similar results were obtained by confocal analysis on MDA-MB-231 cells, where strong cytoplasmic labeling was observed, along with slight labeling on the nuclear envelope (Fig. 7A)Citation . When MDA-MB 231 cells were simultaneously labeled with anti-HSI antibody, the merged image demonstrated that both proteins were colocalized in the cytosol and on the nuclear envelope (Fig. 7A)Citation . A similar localization was observed with LNCaP, MCF7, and PC3 (data not shown). The electron microscopic analysis clearly revealed the nuclear localization of SRBP-2 and also that the protein was expressed on the endoplasmic reticulum (Fig. 7B)Citation . Finally, human breast carcinoma tumors were positively stained for SRBP-2 and showed cytoplasmic staining of various intensity very often dramatically increased around the nucleus. Malignant structures as well as normal structures were involved. In the same positive structure, 10–90% of the cells expressed SRBP-2 (Fig. 8)Citation .



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  		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. Magnification: x400.

 


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  		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: x125,000.

 


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  		Fig. 8. Immunostaining of infiltrating breast cancer with anti-SRBP-2 antibody. Magnification: x250 (A) and x400 (B).

 
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)Citation . Every cell line tested expressed SRBP-2. Interestingly, 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.



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  		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 immunofluorescent 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. Discrepancies of expression levels between tumoral and normal cells were statistically tested using the Wilcoxon test and found significant with P = 0.045 < 0.05, for each protein.

 
Pharmacological Characterization of SRBP-2.
The binding properties of the human SRBP-2 protein were characterized in the yeast ERG2 disruptant strain EMY90, which produces SRBP-2. The binding activity of 3H SR31747 was only detected on membrane homogenates and absent in the supernatants, indicating that SRBP-2 is localized in the membranes when expressed in yeast (Fig. 10)Citation . As a control, mock-transformed yeast did not show any binding activity (Fig. 10)Citation . Scatchard plot analysis of those membrane extracts expressing SRBP-2 using 3H-SR31747A revealed a single high affinity site with a Kd of ~10 nM and a Bmax of 208 ± 21 fmoles/mg protein. As a control, no specific binding was observed with a different erg2 gene disruptant (data not shown). Interestingly, in competitive experiments, only Tamoxifen inhibited 3H-SR31747A binding, whereas no {sigma} ligands, such as haloperidol ± pentazocine or DTG, were shown to compete with 3H-SR31747A at SRBP-2 sites (Fig. 11)Citation . Binding experiments were then performed with 3H-DTG, 3H-pentazocine, or 3H-3PPP and confirmed the absence of a binding site for those ligands on the SRBP-2 protein (data not shown). As Tamoxifen has been described to strongly inhibit 3H-SR31747A binding to HSI (20) , we tested the affinity of SRBP-2 for 3H-Tamoxifen, which was found to be 2 nM. Considering their pharmacological profiles, our data indicated that HSI and SRBP-2 have similar binding properties.



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  		Fig. 10. Assessment of SR31747A binding in SRBP-2 expressing yeast. Membrane homogenates and supernatants from yeast ERG2 disruptant strain EMY90 transformed 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 SRBP-2.

 


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  		Fig. 11. Competitive binding experiments on binding of 3H SR31747A to SRBP-2 expressing yeast membrane. Membranes were incubated with 3H SR31747A in the presence of various concentrations of unlabelled SR31747A ({bullet}), Tamoxifen ({circ}), (+) pentazocine ({blacktriangledown}), (-) pentazocine ({triangledown}), 3PPP ({blacksquare}), DTG ({square}), or haloperidol ({diamondsuit}). Membranes were filtered, and radioactivity was measured by scintillation counting. Data points represent mean of the percentage of 3H SR31747A bound using triplicate measurements, bars ± SE.

 
Sterol Isomerase Activity.
Sterol isomerase activity was assayed using cholest-8-en-3ß-ol and microsomes from {Delta}erg2 and {Delta}erg2- SRBP-2-transformed cells. Despite the overall homology with HSI and the fact that some important residues required for sterol isomerase activity are conserved between the two sequences, SRBP-2 did not exhibit any sterol isomerase activity (Table 3)Citation , which was also confirmed by the absence of complementation in ERG-2-disrupted yeast (data not shown; see protocol in Ref. 12 ).


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  		Table 3 Measurement of {Delta}8-{Delta}7 sterol isomerase activitya

 
Associations among SRBP-1, SRBP-2, and HSI Expression with Clinicopathological Variables and Prognostic Relevance.
When analyzing the potential of SRBP-1 and HSI as prognostic markers in a population of 95 patients with operable primary breast cancer, we showed previously that the presence of HSI and absence of SRBP-1 expression were associated with poorer DFS (P = 0.007; Ref. 20 ). This analysis was extended to SRBP-2 using the COOH terminus-targeting antibody. No correlation was shown between SRBP-2 labeling and age, tumor grade, and tumor size. In addition, there was no correlation between the SRBP-2 immunoreactivity and PR or estrogen receptor status. Although a slight correlation was noted between SRBP-1 and SRBP-2 (P = 0.073), as well as between nodal status and SRBP-2 (P = 0.09), the correlation was stronger between HSI and SRBP-2 (P = 0.008). There was also a significant correlation between HER2 and SRBP-2 (P = 0.042). As far as the prognosis of SRBP-1, HIS, and SRBP-2 on DFS is concerned, we noted that SRBP-2 had a significant effect on DFS when considering the subgroup of patients with SBR 1 and 2 [4.34 (95% confidence interval: 0.79–23.84), P = 0.067, stratified on nodal status], whereas SRBP-1 (P = 0.38) and HSI (P = 0.56) were not significant in this subgroup of patients. This effect was essentially seen in node-positive SBR 1 or 2 patients where the relative risk of failure was equal to 8.7 (95% confidence interval: 0.9–85.1) for SRBP-2-positive patients. Although this latter subgroup included a few patients, there were 13 versus 75% failures for SRBP-2-negative and -positive patients, respectively.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Cloning of {sigma}2 would be warranted on the basis of the {sigma}2 expression profile in human tumors, its potential use as a marker of proliferation, and by the fact that the antiproliferative effect of SR31747A is not fully understood in mammals. With the completion of the human genome sequencing project, sequence databases provide a wealth of information that should be investigated. Our strategy was thus to explore these sequence databases to identify the {sigma}2 protein sequence. Homology searches indicated that SRBP-1 did not exhibit significant sequence homology with any known human proteins. However, it showed 35% identity with ERG2, the yeast sterol isomerase that is also known to bind SR31747A with nanomolar affinity. Despite its sequence homology with ERG2, SRBP-1 did not exhibit detectable sterol isomerase activity. HSI is the human counterpart to the yeast sterol isomerase. This Mr 25,000 protein bound {sigma} ligands and especially SR31747A with nanomolar affinity. No significant sequence homology was noted when comparing human SRBP-1 and HSI. To identify additional novel proteins that could bind SR31747A, we used 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 screenings led to the identification of SRBP-2, an original Mr 23,000 protein that exhibited 41% homology with HSI at the protein level. In our database search, we also found the mouse homologue of human SRBP-2. The human SRBP-2 gene was localized on chromosome 13q14.3-q21.1. The other corresponding 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 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 regulate the SR31747A effect. Additional studies are required to test this hypothesis.

To a functional point of view, although numerous critical residues for sterol isomerase activity in HSI are conserved in SRBP-2, we did not evidence any significant sterol isomerase activity for SRBP-2. This may suggest either that some residues are more important than expected for sterol isomerase activity or that SRBP-2 may interact with an additional partner that is lacking in yeast and would contribute to the sterol isomerase activity of this protein. Additional studies are warranted to address this issue.

The pharmacological profile of SRBP-2 was assessed to further characterize this protein. We found that SR31747A bound to SRBP-2 with a 10 nM affinity. Although this binding potency was lower compared with HSI, both SRBP-2 and HSI proteins exhibited similar binding properties when considering other {sigma} ligands. Neither haloperidol, DTG, nor pentazocine competed with SR31747A binding. Tamoxifen was the only ligand that displaced SR31747A with high potency (Kd = 3 nM). Altogether, these pharmacological data (and specifically the absence of binding of DTG on SRBP-2) indicate that SRBP-2 and {sigma}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 respective 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 function of these binding sites.

Recently, SRBP-1 and HSI expression were immunocytochemically investigated relative to a series of clinicopathological and immunocytochemical prognostic factors in a trial involving 95 patients with operable primary breast cancers. The combined analysis of their expression revealed that the presence of HSI and absence of SRBP-1 receptor expression were associated with poorer DFS (19) . As we found that SRBP-1, HIS, and SRBP-2 were expressed in various human tumor cell lines, especially in breast cancer cells, the above-mentioned study has been pursued with SRBP-2 to assess the potential use of this newly described protein as a prognostic marker in human breast cancer. Despite the absence of any correlation between SRBP-2 and the receptor status (PR or estrogen receptor), SRBP-2 expression seems to correlate with poorly differentiated and highly aggressive tumors, reporting to the high correlation of SRBP-2 and HER2 expression and to the tendency for highly expressing SRBP-2 tumors to have involved positive lymph nodes. Our results showed that, as compared with HSI, SRBP-2 expression had a greater effect on DFS when considering node-positive patients with SBR = 1 or 2. Although additional studies, including a greater number of patients, need to be done, our results suggest that the expression of SRBP-2 might be a potential breast cancer marker.

In conclusion, we have identified and characterized an additional binding protein for SR31747A. The family of human proteins that binds SR31747A now comprises four members. Among these, three are molecularly characterized. They share some structural features and pharmacological activities. Their expression in human tumors together with their potential usefulness as discriminative markers for the immunohistochemical assessment of tumors further support the interest of SR31747A in oncology.


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at Sanofi-Synthelabo, 371 rue du Professeur Joseph Blayac, F-34184 Montpellier cedex 04, France. Phone: (33) 4 67 10 62 90; Fax: (33) 4 67 10 60 00; E-mail: pierre.casellas{at}sanofi-synthelabo.com Back

2 The abbreviations used are: DTG, 1,3-Di-(o) tolylguanidine; 3PPP, HOPh-Pip-Pr; DFS, disease-free survival; DAPI, 4',6-diamidino-2-phenylindole; PR, progesterone receptor; SBR, Bloom and Richardson grading system; FISH, fluorescence in situ hybridization. Back

Received 11/ 1/02. Revised 4/16/03. Accepted 6/13/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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