SEL1L Expression Decreases Breast Tumor Cell Aggressiveness in Vivo and in Vitro1

[CANCER RESEARCH 62, 567–574, January 15, 2002] SEL1L Expression Decreases Breast Tumor Cell Aggressiveness in Vivo and in Vitro1

Rosaria Orlandi, Monica Cattaneo, Flavia Troglio, Patrizia Casalini, Chiara Ronchini, Sylvie Me´nard,2 and Ida Biunno Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy [R. O., F. T., P. C., C. R., S. M.], and Istitute for Biomedical Technologies–CNR, 20090 Segrate, Milan, Italy [M. C., I. B.]

ABSTRACT SEL1L protein and examined the relationship between SEL1L expression levels and patient survival. We also examined the effect of SEL1L, the human orthologue of the Caenorhabditis elegans sel-1 gene, inducibly up-regulated SEL1L expression on anchorage-dependent is differentially expressed in breast primary tumors and in normal breast growth and the colony formation ability of human breast carcinoma tissues. Analysis of a series of human primary breast carcinomas, using a monoclonal antibody raised against a SEL1L recombinant protein, re- MCF7 cells, which express SEL1L at low levels (16). Our results vealed down-modulation or absence of SEL1L expression in about two- suggest a role for the SEL1L protein in breast tumor growth and thirds of the tumors as compared with normal breast epithelial cells. aggressiveness. Overall survival analysis of breast carcinoma patients indicated a statistically significant correlation between SEL1L down-modulation and poor prognosis. MCF-7, human breast carcinoma cells, were transfected with a MATERIALS AND METHODS construct containing the entire SEL1L cDNA driven by an inducible Patients promoter and showed a dramatic reduction in anchorage-dependent growth and colony formation in soft agar. Growth of the transfected cells A series of 117 cases of primary ductal, lobular, or mixed infiltrating breast in Matrigel, an extracellular matrix rich with laminin, restored colony- cancers with 10-year follow-up from patients surgically treated at Istituto formation ability. These results point to the role for SEL1L in breast Nazionale Tumori (Milan, Italy) was studied. Primary tumor diameter and tumor growth and aggressiveness, possibly involving cell-matrix interac- axillary nodal status were obtained from histopathological reports. Histological tions. grading and biological parameters were determined as described (18).

INTRODUCTION Immunohistochemistry SEL1L (1) is the human orthologue of the sel-1 (suppressor-enhancer- Immunoperoxidase assay was carried out by a sensitive peroxidase-strepta- lin) gene in Caenorhabditis elegans (2) and encodes a predicted vidin method on formalin-fixed, paraffin-embedded sections of breast carci- extracellular protein identified as an extragenic suppressor of lin-12 nomas. Briefly, 1–2 ␮m consecutive sections were cut, deparaffinized, rehy- hypomorphic mutants (3). The lin-12/Notch/glp-1 gene family en- drated, and pretreated using the heat-induced epitope retrieval method (19). Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide in codes proteins with a broad range of action that play an important role methanol for 30 min. After several washes in PBS and treatment with normal in determining developmental choices in several precursor cell types goat serum (1:50) for 30 min at room temperature, sections were incubated (4). Notch signaling is also involved in cell proliferation and in overnight at 4°C with 3 ␮g/ml MSel1 mouse MAb that specifically recognizes apoptosis (5, 6). Two vertebrate lin-12/Notch homologues, murine the SEL1L protein,4 followed by biotinylated antimouse IgG and streptavidin- int-3 and human Tan-1, have been implicated in several cancers (7–9). conjugated horseradish peroxidase (Dako). Peroxidase activity was detected A truncated form of Tan-1 causes human T-cell lymphomas (7) and using 3,3Ј-diaminobenzidine as substrate. Negative controls were incubated can transform rat kidney cells (5). without MAb MSel1. Images were obtained using a Nikon Eclipse 600 SEL1L resides on chromosome 14q24.3–31 near the insulin-depen- equipped with a digital camera. A ϫ60 oil immersion objective was used. dent diabetes mellitus I locus (IDDM11) but unlinked to a locus for Reproducibility of the immunohistochemistry analysis was assessed in the this disease (10–13) or to a locus for autoimmune thyroid diseases preliminary set up of the immunoperoxidase assays with MAb MSel1, which included selection of MAb concentration, scoring system with intra- and residing in the same chromosomal interval (14). SEL1L is primarily interobservers evaluation, and reproducibility on serial slides of the same expressed in embryonic and adult pancreas (15), and is down-regu- cases. lated or undetectable in several primary adenocarcinomas of the pancreas (1) and in gastric cancers (16). Comparative sequence anal- Statistical Analysis ysis of SEL1L in different species has revealed remarkable conser- vation, suggesting an important or even an essential role for this gene Association of SEL1L expression in tumor tissue with breast cancer patient 2 (17). survival was evaluated using the ␹ test. The actuarial probability of mortality We observed previously a differential pattern of SEL1L expression was estimated by Kaplan-Meier analysis, and differences were assessed using the log-rank test. The Cox proportional hazards model was used to identify the in mammary carcinoma cell lines, and in breast cancer and normal clinicopathological variables independently associated with mortality (20). All tissues (16) suggesting a role for SEL1L in breast tumor development. of the statistical tests were two-tailed (significance at P Ͻ 0.05) and performed In the present study we analyzed a large series of human primary using SAS software packages (SAS Institute Inc., Cary, NC). mammary carcinomas using a MAb3 raised against a recombinant Cell Culture Received 7/18/01; accepted 11/6/01. The costs of publication of this article were defrayed in part by the payment of page Human breast adenocarcinoma MCF-7 cells were grown in RPMI (Micro- charges. This article must therefore be hereby marked advertisement in accordance with biological Associates, Walkersville, MD) supplemented with 10% FCS (Hy- 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Associazione Italiana per la Ricerca sul Cancro. M. C. was supported by a fellowship from Fondazione Italiana Ricerca sul Cancro. 3 The abbreviations used are: MAb, monoclonal antibody; DEX, dexamethasone; 2 To whom requests for reprints should be addressed, at Molecular Targeting Unit, RT-PCR, reverse transcription-PCR; SRB, sulforhodamine B; HPRT, hypoxanthine phos- Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1, 20133 phoribosyltransferase. Milan, Italy. Phone: 39-02-23-90-25-71; Fax: 39-23-90-30-73; E-mail: menard@ 4 R. Orlandi, Production of a monoclonal antibody directed against the recombinant istitutotumori.mi.it. SEL1L protein, manuscript in preparation. 567

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2002 American Association for Cancer Research. SEL1L EXPRESSION DECREASES TUMOR CELL AGGRESSIVENESS clone), and penicillin and streptomycin (100 IU/ml) in a humidified chamber In Vitro Induction of SEL1L Transcription

(95% air and 5% CO2)at37°C. MCF-7 clones containing the SEL1L-pDEX/1 construct in a stable form were treated with 100 nM of DEX for 1–7 days after which the cells were SEL1L-pDEX.1 Construct and Transfection harvested, and the RNA isolated and analyzed by RT-PCR. Full-length SEL1L cDNA (2.4 kb) was obtained by PCR amplification of Immunofluorescence Analysis reverse-transcribed pancreatic mRNA with primers designed to contain a 5Ј KpnI restriction site and cloned into the PCR 2.1 vector (Invitrogen). Plasmid Trypsin-detached MCF-SEL1L or MCF-pDEX/1 cells were incubated with DNA was digested with KpnI and EcoRI, gel-purified (JetSorb; Invitrogen), 3% paraformaldehyde for 10 min at 0°C and permeabilized by a 30-min and recloned into the polylinker of vector pDEX/1. Transcription of SEL1L treatment at 0°C with 0.1% saponin and 0.1% BSA in PBS. Cells were washed was driven by the ␤-globin promoter contained within the plasmid. The and incubated for 30 min at 0°C with 5–10 ␮g of MAb MSel1 in PBS pDEX/1 vector also contained six glucorticoid-sensitive repeats followed by a containing 0.1% BSA and 0.1% saponin. After several washes, cells were TATA box and a G418 resistance gene serving as a selectable marker.5 additionally incubated for 30 min at 0°C with FITC-goat antimouse antibody Cells were stably transfected with the SEL1L-pDEX/1 or control pDEX/1 (Kierkegaard and Perry Labs.) and assessed for fluorescence using a FACS- constructs by electroporation (Bio-Rad gene pulsar) at 960 ␮F and 250 V for calibur (Becton Dickinson) and CellQuest software including the Kolmogorov- three pulses. Exponentially growing cells (107) in RPMI plus 10% FCS were Smirnov statistics. To analyze the pattern of integrin expression, trypsin- incubated with 10 ␮g of XmnI-linearized SEL1L-pDEX/1 or control pDEX/1 detached cells were incubated with mouse MAbs MAR6 (anti-␣6), MAR4 for 20 min at 4°C. G418 (400 ␮g/ml) was added at 24 h after transfection. (anti-␤1), or MLuc5 (anti-67LR) produced in our laboratory, or MAB1964 Individual neomycin-resistant clones were isolated and expanded for addi- (anti-Hu integrin ␤4) obtained by Chemicon and processed as described above. tional analysis. BT-474 cells were seeded on glass coverslips, fixed with acetone, treated with 0.1% saponin and 0.1% BSA in PBS for 20 min, and incubated with 10 ␮g of MAb MSel1 followed by FITC-goat antimouse antibody. Cells were DNA Isolation, Southern Blot, and PCR Analysis then analyzed using a Radiance 2000 (Bio-Rad) confocal microscope using a High molecular weight DNA was extracted from cells using standard FITC filter set. procedures (21). DNA (8 ␮g) was digested with BamHI, fractionated on a 0.7% agarose gel, transferred to Hybond Nϩ filters (Amersham), and hybrid- Western Blotting 32 ized with the [ P]dCTP-labeled SEL1L cDNA probe. The presence of the Cells were lysed using a buffer containing 50 mM Tris-HCl (pH 7.4), 150 ␤ -globin promoter in the transfected clones was confirmed by PCR amplifi- mM NaCl, 10% glycerol, 0.5% NP40, 10 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, Ј cation using a set of primers designed for the promoter and the 3 end of and 1 ␮g/ml phenylmethylsulfonyl fluoride. Protein concentration was deter- SEL1L (see below). For PCR, 20 pmol of vector-specific sense pDEX/1 primer mined by Comassie Plus Protein Assay (Pierce, Rockford, IL). Total cell and SEL1L-specific antisense (IBD8) primer were added to 300 ng of genomic lysates were resolved on 10% SDS-polyacrylamide gel, and Western blotting DNA; amplification was carried out for 30 cycles at 94°C for 1 min, 60°C for was performed using standard procedures. MAb MSel1 was used at 10 ␮g/ml. 1 min, and 72°C for 3 min in a Perkin-Elmer Corp. thermal cycler. Anchorage-dependent Proliferation Assays RNA Isolation and RT-PCR Analysis SRB. Cells treated with DEX for 1 week were trypsin-detached and seeded ϫ 3 ␮ Total RNA was extracted from cells using the guanidine thiocynate method in 96-well plates (4 10 cells/well; 5 replicates) in 200 l culture medium (1). Total RNA was treated with 1 unit/mg of RNase-free DNase I (Clontech) supplemented with 300 nM DEX and grown for 1, 3, 4, and 7 days. After at 37°C for 15 min. fixation with 10% trichloroacetic acid at 4°C for 1 h and five washes with cDNA Synthesis. Total RNA (1 ␮g) was used in a 20-␮l reaction contain- distilled water, 0.4% SRB (Sigma Chemical Co.) in 1% acetic acid was added ␮ ␮ ϫ and incubation continued for 30 min at room temperature. After several washes ing 5 M of MgCl2,2 l10 reaction buffer [500 mM Tris-HCl (pH 8.8), 80 with 1% acetic acid, SRB bound to cellular proteins was dissolved in 10 mM nM MgCl , 300 mM KCl, 10 mM dithiothreithol], 1 ␮M dNTPs, 50 units of 2 Tris-HCl, pH 10.5. Absorbance at 490 nm, proportional to the number of cells RNase inhibitor (RNAsin), 0.8 ␮g oligo-p(dT) primer, 1.6 ␮g p(dN) random 15 6 attached to the culture plate, was measured by spectrophotometry. primers, and 20 units of avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim). The reaction mixture was incubated for 10 min at Colony Assay 25°C and for 60 min at 42°C. The enzyme was denatured at 99°C for 5 min and chilled on ice. Cells treated with DEX for 1 week or control cells were trypsin-detached, PCR Conditions. The 30-␮l reaction volume contained 2 ␮l of cDNA, 3 seeded in six-well plates at 1000 cells/ml, and cultured for 1 week. Clones ␮ ϫ lof10 buffer [100 mM Tris-HCl (pH 8.8), 15 mM MgCl2, 500 mM KCl, 1% were fixed with methanol and stained with 10% Giemsa. Triton X-100], 0.3 ␮l of each 25 ␮M dNTPs, 0.2 ␮l of vector-specific sense pDEX/1 primers and SEL1L antisense IBD5 primer (100 ␮M), and 1 unit of Anchorage-independent Clonogenicity Assays AmpliTaq DNA polymerase (DYNAZYme). The mixture was heated to 94°C DEX-treated or control cells were seeded in six-well or 24-well plates in for 3 min followed by 30 cycles of amplification using the following condi- semisolid medium containing 0.3% Bacto-Agar (Difco) supplemented with tions: denaturing 94°C for 1 min; annealing at 60°C for 1 min; and extension 30% FCS and 300 ␮g/ml of G418 over a 0.6% agarose layer. Medium at 72°C for 1 min. Amplifications included an initial denaturation at 94°C for containing 30% FCS was added weekly. Colonies were scored after 1–2 week 5 min and final extension for 10 min at 72°C. Reaction products were incubation at 37°Cin5%CO2 in air. For cloning in Matrigel, cells were electrophoresed on a 1% agarose gel and stained with ethidium bromide. The suspended in Matrigel diluted 1:2 in culture medium and seeded in 24-well quality of the neo-synthesized cDNA was verified using HPRT primers. plates at a concentration of 22,000 cells/well.

Primers Used RESULTS PDEX/1: 5Ј-AAGGCAGGATGATGACCAGG-3Ј IBD8: 5Ј-GCTGGATCCAGTGCCTATTACTGTGG-3Ј Expression of SEL1L Protein in Human Breast Cancer and IBD5: 5Ј-TCTGCTTCCTGCATCTGCCGTCTC-3Ј Correlation with Biopathological Parameters and Prognosis HPRT: 5Ј-AATTATGGACAGGACTGAACGTC-3Ј Ј Ј 5 -CGTGGGGTCCTTTTCACCAGCAAG-3 Expression of SEL1L was evaluated in a series of 117 human primary breast tumors with a 10-year updated follow-up and charac- 5 G. Bunone, personal communication. terized for different pathological, clinical, and biomolecular parame- 568

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2002 American Association for Cancer Research. SEL1L EXPRESSION DECREASES TUMOR CELL AGGRESSIVENESS ters including: nodal involvement, tumor size, grade, menopausal (Fig. 1, a and b). SEL1L protein was detected with a strong staining status, mitosis, necrosis, lymphoid infiltration, HER2 overexpression, in 37% of invasive breast carcinomas analyzed (Fig. 1, c and d) and p53 overexpression, hormone receptor, Bcl2, 67LR laminin receptor, with a weak staining in 35% (Fig. 1, e and f). The remaining 28% and cathepsin D expression. SEL1L protein expression was evaluated scored negative (Fig. 1g). Staining was always cytoplasmic, with a by immunohistochemistry using the mouse MAb MSel1 raised against punctuate pattern suggestive of cytoplasmic vesicles. Normal breast the NH2 terminus of the recombinant human SEL1L protein. MSel1 epithelial tissues adjacent to the tumors were always positive. Two specifically recognizes this recombinant SEL1L protein and a band of MSel1-positive normal mammary ducts are shown in Fig. 1, panels d

Mr 88,000 corresponding to human SEL1L protein in lysates from and e, and MSel1-positive normal breast acini are shown in Fig. 1, MCF7 cells by Western blotting (Fig. 5). BT-474 human breast panel h. MSel1 also reacted strongly with plasma cells (Fig. 1, g and carcinoma cells expressing high level of SEL1L revealed intense h). The “in situ” carcinomas were also positive, often staining more cytoplasmic immunoreactivity with MSel1 in intracellular vesicles intensely stronger than their invasive counterparts (Fig. 1f).

Fig. 1. SEL1L expression in BT474 human breast cancer cells and in normal and neoplastic breast tissues. a, cytoplasmic MSel1 immunoreactivity in intracellular vesicles of BT-474 cells; b, interference contrast image (confocal microscope) of a; c, invasive breast carcinoma cells with strong MSel1 reactivity; d, MSel1-positive normal breast duct and invasive breast carcinoma cells with strong MSel1 reactivity; e, MSel1-positive normal breast duct and invasive breast carcinoma cells with weak MSel1 reactivity; f, in situ carcinoma and invasive breast carcinoma cells with weak MSel1 reactivity; cells adjacent to basal membrane of in situ carcinoma show stronger MSel1 reactivity; g, MSel1- negative invasive breast carcinoma surrounded by several MSel1-positive plasma cells; h, MSel1- positive normal breast acini with some positive plasma cells. Original magnification, ϫ1500 (a and b) and ϫ340 (c–h).

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Table 1 Distribution of clinical, pathological, and biomolecular parameters in three prognosis was associated with patients of which the tumors displayed different subsets divided according to SEL1L expression in primary breast carcinomas SEL1L expression levels similar to those of adjacent normal tissue Number of cases/total (%) in the three subgroupsa according (Fig. 2). to SEL1L expression In univariate analysis, five other clinical and biological parameters SEL1L-neg. SEL1L-weakly pos. SEL1L-strongly pos. including nodal status, grade, HER2, p53, and progesterone receptor b Variables (33 cases) (40 cases) (44 cases) expression were found indicative of prognosis, whereas all of the N-positiveb 22/31 (71) 17/40 (43) 24/43 (56) other variables reported in Table 1 were not. Among the prognostic T2-3 20/32 (62) 21/39 (54) 22/43 (51) GII 13/33 (39) 26/40 (65) 24/44 (55) variables that were statistically significant in univariate analysis, only GIII 12/33 (36) 4/40 (10) 11/44 (25) nodal status maintained its prognostic significance in multivariate Premenopause 13/32 (40) 18/36 (50) 21/44 (48) analysis using proportional hazards model by Cox (Table 2), whereas High mitotic index 15/33 (45) 10/37 (27) 16/42 (38) Necrosis present 17/33 (51) 11/39 (28) 16/44 (36) SEL1L expression was not statistically significant. LPI-present 7/33 (21) 5/39 (13) 11/44 (25) HER2-positive 6/33 (18) 10/40 (25) 7/44 (16) Inducible Expression of SEL1L in MCF7 Cells p53-positive 7/32 (22) 6/40 (15) 4/44 (9 ) PGR-negative 12/33 (36) 13/40 (33) 11/42 (26) MCF-7 cells transfected with the SEL1L-pDEX/1 or pDEX/1 con- Bcl2-negative 10/32 (31) 14/40 (35) 10/44 (23) 67LR-positive 11/33 (33) 6/40 (15) 11/44 (25) trol plasmids were used to investigate whether SEL1L expression Cathepsin D-pos. 18/33 (55) 24/40 (60) 26/44 (59) a In the three subsets information concerning some variables is missing. b N, nodal status; T, tumor size; G, grade; LPI, lymphoplasmacellular infiltrate; PGR, progesterone receptor; 67LR, Mr 67,000 laminin receptor.

Fig. 3. Southern blot (left) and genomic PCR analysis (right) of MCF7-SEL1L- transfected clones. For Southern blot, high molecular weight DNA extracted from a mock-transfected MCF-7 clone (Lane 1) and from MCF7-SEL1L-A, -B, and -C clones (Lanes 2–4) was digested with BamHI and hybridized to full-length SEL1L cDNA. A common restriction fragment Ͼ 30 kb is present, as expected, in all of the DNAs tested representing the SEL1L genomic fragment. Clones A, B, and C contain the expected 3.25-kb SEL1L cDNA construct Right panel, integrity of the promoter-cDNA junction was assessed by PCR amplification of DNA extracted from MCF7-SEL1L-A (Lane 1), -B (Lane 2), and -C (Lane 3) clones as well as from a mock-transfected clone (Lane 4). The Fig. 2. Overall survival of breast tumor patients as a function of SEL1L expression in 2.8 kb DNA fragment reflects the entire SEL1L cDNA (2.4 kb) ligated to the pDEX/ F Œ a series of 117 human primary breast tumors: , high SEL1L expressors, , low SEL1L promoter. M, marker. expressors, f SEL1L-negative cases.

Table 2 Univariate and multivariate analysis of prognostic impact of clinical and biological parameters in primary breast carcinomas Univariate Multivariate

Variable pa ␤b pc RRd Ne Ͻ10Ϫ4 0.69 0.01 2.0 Ϫ G Ͻ10 4 0.12 0.55 1.1 Ϫ HER2 1.7 ϫ 10 2 0.50 0.12 1.6 Ϫ p53 4.0 ϫ 10 4 0.14 0.72 1.1 Ϫ PGR 1.5 ϫ 10 2 Ϫ0.37 0.25 0.7 Ϫ SEL1L 5.0 ϫ 10 2 Ϫ0.25 0.15 0.8 a P log-rank. b ␤ parameter estimate. c P from one-degree of freedom Cox test for all variables. d Relative risk. e See Table I for abbreviations.

Fig. 4. Transcription analysis of MCF7-SEL1L clones. Top panel: RT-PCR of DNase No clear correlation was observed between SEL1L expression I-treated RNAs extracted from wild-type MCF-7 before and after DEX addition (Lanes 1 levels, biomolecular/pathological/clinical parameters, and a particular and 2), from mock-transfected clone (Lanes 3 and 4), MCF7-SEL1L-A (Lanes 5 and 6), B(Lanes 7 and 8), and C (Lanes 9 and 10). The expected 760-bp size of SEL1L transcript subset of breast carcinomas (Table 1). The association with the is clearly visible only after induction and is indicated by the arrow. Amplification of the histological subtype was not applicable, because 86% of cases were of neo-synthesized cDNA with primers designed on HPRT is showed below. Bottom panel: ductal or mixed histotype. Interestingly, 4 of 5 pure lobular cases were RT-PCR of retro-transcribed (Lanes 1–6) and total RNA (Lanes 8–13) of MCF7- SEL1L-A as a function of the time exposed to DEX. Appreciable SEL1L levels (indicated SEL1L-negative. by the arrow) were observed on day 5 (Lane 5) and day 7 (Lane 6) of exposure. Lanes 7 SEL1L expression was a good indicator of the overall survival of a and 14 contain negative controls; Lane 15 contains a positive control cDNA amplified ϭ with the same primers. The primers were designed at the 5Ј end of SEL1L (antisense patient (P 0.05). Indeed, SEL1L-negative cases had a poor prog- IBD5) and downstream of the transcription start site on the promoter vector (sense- nosis, and weakly positive cases had intermediate prognosis. The best pDEX). M, marker. 570

retained the entire SEL1L-cDNA construct in-frame with the promoter (2.8 kb). DNA from a mock-transfected clone was used as a negative control (Fig. 3, Lane 4). Transcription Analysis of MCF7-SEL1L Clones as a Function of DEX Induction. Transcription of the inserted SEL1L-cDNA into the selected clones was analyzed by RT-PCR using DNase I-treated RNAs and a primer set designed on the 5Ј end of the SEL1L-cDNA (antisense IBD5), and on the promoter vector downstream of its transcription start site (sense pDEX/1). As expected, control RNAs from wild-type MCF-7 (Fig. 4, Lanes 1 and 2) and a mock-transfected clone (Fig. 4, Lanes 3 and 4) showed no SEL1L signal before or after DEX induction, whereas clone A (Fig. 4, Lanes 5 and 6), B (Fig. 4, Fig. 5. Flow cytometry analysis of noninduced (open curve) and DEX-induced (filled curves) MCF7-pDEX/1 or MCF7-SEL1L clones. Mean fluorescence and Kolmogorov- Smirnov statistics [D/s (n)] results are reported. Cells were treated with MSel1 MAb followed by FITC-conjugated goat antimouse IgG and analyzed with a FACScalibur (Becton Dickinson) using CellQuest software. Mean fluorescence of cells treated with unrelated mouse MAbs did not exceed 30. In the panel is shown a Western blotting analysis of lysates from noninduced and DEX-induced clone A. affects the growth of breast cancer cells. Eleven of 30 G418-resistant clones were analyzed for the presence and integrity of the construct and for RNA expression. Clones were designated as MCF7-SEL1L and MCF7-pDEX/1 mock-transfected. Southern Blot and Genomic PCR Analysis of MCF7-SEL1L and MCF-pDEX/1 Clones. High molecular weight DNA was digested with BamHI, transferred to nitrocellulose filters, and hybridized with radiolabeled SEL1L cDNA insert (“Materials and Meth- ods”). BamHI was used because it cuts the entire cDNA ligated to the promoter, releasing a 3.25-Kb fragment. Fig. 3 shows a Southern blot of three representative clones and from a mock-transfected clone. Clones A (Fig. 3, Lane 2), B (Fig. 3, Lane 3), and C (Fig. 3, Lane 4) but not the control (Fig. 3, Lane 1) showed the expected DNA fragment. The constitutive SEL1L genomic fragment was present, as expected, in all of the DNAs. The 8-kb fragment present in clone C (Fig. 3, Lane 4) may indicate construct chromosomal rearrangements. To determine the status of the promoter-cDNA junction within the Fig. 7. Anchorage-independent clonogenicity in soft agar. MCF7, MCF7-pDEX/1, and genome of the selected clones, PCR amplification was carried out MCF7-SEL1L-A, -B, and -C cells were pretreated with 100 nM of DEX for 7 days to using a primer set designed on the 3Ј-end of the cDNA (antisense induce transcription of exogenous SEL1L (f) or left without inducer (Ⅺ), and trypsinized, counted, and seeded in soft agar in the absence of DEX. Colonies were scored after 10 IBD8) and on the pDEX/1 promoter (sense pDEX/1). As shown in days. Five fields of each duplicate determination were examined under the microscope and Fig. 3, clones A, B, and C (Fig. 3, Lanes 1, 2, and 3, respectively) colonies counted. Data are given as mean; bars, Ϯ SD.

Fig. 6. Proliferation assay of adherent MCF7-pDEX/1 control cells and clones MCF7-SEL1L-A, -B, and -C. Cells were pretreated with 100 nM of DEX for 7 days (f) or not pretreated (Ⅺ) and trypsinized, counted, seeded in 96-well plates, and grown for 1, 3, 4, and 7 days. Proliferation was assessed spectrophotometrically based on absorbance at 490 nm of SRB bound to cellular protein. Data are given as mean of quintupli- cate determinations; bars, Ϯ SD.

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Fig. 8. Anchorage-independent clonogenicity in the laminin- rich extracellular matrix Matrigel and in soft agar. MCF7- pDEX/1 and MCF7-SEL1L-A clones were pretreated with 100 nM of DEX for 7 days (f) or grown uninduced in culture medium (Ⅺ), then trypsinized, counted, and seeded in Matrigel or soft agar in the absence of DEX. Matrigel colonies were scored after 7 days and soft agar colonies after 12 days. Data are given as mean; bars, Ϯ SD.

Lanes 7 and 8), and C (Fig. 4, Lanes 9 and 10) clearly showed a form adherent colonies was also impaired. In a representative exper- marked increase of the 760-bp transcript only after induction. The iment, clone A seeded at 1000 cell/ml formed 24 colonies when presence of a faint band in these uninduced samples suggests pro- DEX-induced but formed 173 colonies when uninduced. A mock- moter leakage resulting in a low DEX-independent expression of the transfected clone formed 172 and 176 colonies with and without SEL1L-transfected gene. As control, amplification of retrotranscribed induction, respectively. In a series of three colony assay experiments, RNAs was performed with HPRT-specific primers. A 200-bp band the number of colonies from DEX-treated clone A was significantly was present in all of the RNAs, with slight variation in intensity. lower than from the untreated clone A or from the DEX-treated or The kinetics of glucorticoid-mediated SEL1L induction was studied untreated mock-transfected clone (P Ͻ 0.0001; Student’s t test). using clone A. Cells were treated with 100 nM of DEX from 1 to 7 days, and total RNA was isolated and analyzed by RT-PCR (Fig. 4, Effect of SEL1L Levels on Anchorage-independent Lanes 1–6). Appreciable SEL1L levels were seen beginning at day 5 Clonogenicity of MCF7-SEL1L Clones (Fig. 4, Lane 5) and were sustained by day 7 (Fig. 4, Lane 6). Soft agar colony assay was carried out on MCF7-mock and MCF- Amplification of total RNA was used as control for DNA contami- 7-SEL1 clones treated and untreated for 1 week with DEX. The nation (Fig. 4, Lanes 8–13). Fig. 4, Lanes 7 and 14 reflect loading of colony formation efficiency was dramatically reduced in DEX- negative controls, whereas Fig. 4, Lane 15 shows the positive control. induced MCF7-SEL1L cells as compared with the uninduced cells, Flow Cytometry Analysis of SEL1L Protein Levels in MCF-7- whereas the control clone formed a comparable number of colonies in SEL1L Clones. Induction of exogenous SEL1L protein in the SEL1L the presence or absence of DEX (Fig. 7). Even wild-type MCF7 cells, clones was measured by indirect immunofluorescence using MAb grown with or without DEX and subjected to soft agar assay, showed MSel1. Fig. 5 shows SEL1L protein levels expressed by clones A and no significant difference in cloning efficiency (Fig. 7, top). Similar B before and after DEX addition. Despite basal fluorescence attrib- results were obtained when the induction time was increased from 3 utable to endogenous SEL1L, a significant mean increase in fluores- to 7 days and when the number of cells seeded was increased from cence was observed in both clones after DEX induction, as determined 5 ϫ 104 to 1 ϫ 105. The percentage of colonies formed in DEX- using Kolmogorov-Smirnov statistical analysis. Mock-transfected induced in regard to the uninduced cells ranged from 3% to 30% controls (only one clone is shown) showed no significant increase. (mean ϭ 12%) for MCF7-SEL1L cells and from 83% to 105% Fig. 5 panel shows a Western blot containing lysates from uninduced (mean ϭ 92%) for control cells. and DEX-induced clone A confirming the increase of SEL1L levels The colony-forming ability of DEX-treated MCF7-SEL1L clones after DEX treatment. was restored in the presence of Matrigel, an extracellular matrix rich Effect of SEL1L Expression Levels on Anchorage-dependent in laminin. Fig. 8 shows a representative experiment with clone A Growth of MCF7-SEL1L Clones indicating that the same number of colonies were obtained in Matrigel independent of SEL1 induction, whereas the same DEX-induced cells MCF7-SEL1L clones A, B, and C, and a mock-transfected control were highly impaired in regard to colony formation in soft agar. The clone were grown in the absence or presence of DEX for 1 week to control clone grew in Matrigel as well as in soft agar. Immunofluo- induce the transcription of exogenous SEL1L and to increase SEL1L rescence analysis of expression of laminin-binding integrins (␣6 and cellular levels. Cells were then grown in adherence for 1, 3, 4, and 7 ␤1) and the 67LR laminin receptor revealed no differences between days, fixed, and assessed for protein content as described in “Mate- clone A, induced or noninduced, and a mock-transfected clone (Table rials and Methods.” DEX-induced SEL1L expression leads to 50%, 3). The only appreciable difference (ϩ56%) was the level of SEL1L 58%, and 60% inhibition of growth rate at 7 days in clones A, B, and protein, detected with MAb MSel1, in induced MCF7-SEL1L-A cells, C, respectively (Fig. 6). DEX did not significantly affect growth of the as expected. Similar results were obtained with clones B and C. two MCF7-pDEX/1 mock-transfected clones (shown in Fig. 6 for one clone). Comparable results were obtained using MCF7-SEL1L clones DISCUSSION pretreated with DEX for 2 weeks. Chronic DEX treatment caused growth arrest and cell detachment of MCF7-SEL1 clones after 3–4 This report aims to show that the protein encoded by the gene weeks treatment, with no affect on the growth of control MCF7- SEL1L plays a role in the progression of human breast carcinomas. pDEX/1 cells. Terminal deoxynucleotidyl transferase (Tdt) -mediated SEL1L is a novel human gene of which the function is still unknown, nick end labeling analysis of adherent cells treated with DEX for 1 or but its homology to the C. elegans gene sel-1 (2) and to the yeast 2 weeks revealed no increase in the number of apoptotic cells (ϳ5%) HRD3 genes (22) suggests that it may be involved in a yet-to-be- in MCF7-SEL1 clones as compared with uninduced cells or mock- defined secretory/endocytic pathway. SEL1L is differentially ex- transfected DEX-treated cells. The ability of MCF7-SEL1L clones to pressed in neoplastic cells of different origin (1, 16), raising the 572

Table 3 Cell surface expression of integrin and 67LR in MCF7-pDEX/1 and MCF7- they showed a marked decrease in cell growth ability. We believe that SEL1-A clones grown in the presence or absence of DEX the increased level of SEL1L in DEX-induced MCF7-SEL1L cells is Variation of mean fluorescence as compared to a re-expression of the SEL1L molecule at physiological level that a noninduced cells causes a decrease in tumor aggressiveness. Induced-MCF7-pDEX/1 Induced-MCF7-SEL1-A The extracellular matrix profoundly influences the growth and SEL1L Ϫ5% ϩ56% differentiation of breast epithelial cells both in culture and in vivo ␣6 ϩ8% Ϫ9% (23), and regulates apoptosis and cell cycle progression through an ␤1 Ϫ14% Ϫ30% 67LR Ϫ30% Ϫ25% integrin-dependent signaling (24). Interestingly, colony-forming abil- a Immunofluorescence analysis was performed as described in “Materials and Meth- ity of MCF7-SEL1L clones was restored when cells were grown in ods.” ␤4 expression was tested but was nearly undetectable in all samples. anchorage-independent conditions in the presence of Matrigel. It is possible that SEL1L influences the interaction of the cells with the extracellular matrix, for example, by modulating membrane adhesion- possibility of its involvement in some aspects of neoplasia. Our receptors. The increased level of SEL1L in the stained cells adjacent present data obtained in a series of human primary breast tumors to the basal membrane of the in situ carcinoma (Fig. 1f) is consistent reveal a correlation between SEL1L expression and tumor progres- with this possibility. The finding that SEL1L expression did not sion. Down-regulation of SEL1L expression, compared with normal modulate the expression of two different laminin receptor expression breast epithelial cells, strongly correlates with aggressiveness of the does not rule out this hypothesis considering the large repertoire of disease. Although the poor prognosis associated with SEL1L nega- molecules involved in cell/matrix interaction. Alternatively, SEL1L tivity might reflect resistance to chemotherapy, because node-positive expression may inhibit endogenous synthesis of extracellular matrix cases of this series were treated with adjuvant chemotherapy based on by the tumor cells. Indeed, if laminin is not produced by the SEL1L- CMF, the in vitro data strongly suggest a direct involvement of expressing cells, impairment of anchorage-independent growth and SEL1L in the control of cell proliferation. The finding that the also of adherence would be expected, whereas a substrate rich in prognostic value of SEL1L expression lost statistical significance in laminin such as Matrigel could still favor the growth of SEL1L- multivariate analysis suggests an interaction with one of the other expressing cells. This is in keeping with a possible role of SEL1L in factors associated with prognosis in this series such as nodal status, a secretory pathway as also suggested by the localization of SEL1L to although bias because of the low number of cases analyzed cannot be intracellular vesicles. Gene expression profiling by microarray anal- excluded. ysis and two-dimensional gel electrophoresis followed by mass spec- Immunohistochemical analysis of mammary breast normal and troscopy of our biological system may shed light on this issue. tumor tissues, and confocal microscopy analysis of breast cancer cell In conclusion, our data suggest that SEL1L is a cytoplasmic protein lines clearly indicated the cytoplasmic localization of SEL1L in involved in cell growth control of breast cancer cells, and its down- vacuoles consistent with the compartmentalization of transfected modulation in breast carcinomas is associated with tumor aggressive- SEL1L/green fluorescent protein recombinant gene expression in cy- ness. The mechanism involved is still unknown. Recent studies sug- toplasmic vesicles (3). This localization is also in line with a possible gest that activin A induces SEL1L expression during morphogenesis role for SEL1L in protein trafficking. of the salivary gland (25) and leads to a dramatic decrease in growth To investigate the involvement of SEL1L in breast carcinoma, we of T47D human breast cancer cells (26). On the basis of these findings transfected the MCF-7 human breast carcinoma cells, found previ- and our data, we speculate that SEL1L may be regulated by the ously to constitutively express low levels of SEL1L, with a construct transforming growth factor-␤ family of growth factors and receptors, containing the SEL1L entire open reading frame under an inducible and may exert its function by influencing cell-matrix interactions promoter. 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