The Estrogen Receptor Cofactor SPEN Functions As a Tumor

Published OnlineFirst August 21, 2015; DOI: 10.1158/0008-5472.CAN-14-3475

Cancer Molecular and Cellular Pathobiology Research

The Estrogen Receptor Cofactor SPEN Functions as a Tumor Suppressor and Candidate Biomarker of Drug Responsiveness in Hormone-Dependent Breast Cancers Stephanie Legar e1,2, Luca Cavallone2, Aline Mamo2, Catherine Chabot2, Isabelle Sirois1,2, Anthony Magliocco3, Alexander Klimowicz3, Patricia N. Tonin4,5, Marguerite Buchanan2, Dana Keilty1,2, Saima Hassan1,2, David Laperriere 6, Sylvie Mader6,7, Olga Aleynikova8, and Mark Basik1,2

Abstract

The treatment of breast cancer has benefitted tremendously with microarray-based pathway analyses showed that SPEN from the generation of estrogen receptor-a (ERa)–targeted functions as a tumor suppressor to regulate cell proliferation, therapies, but disease relapse continues to pose a challenge tumor growth, and survival. We also found that SPEN binds due to intrinsic or acquired drug resistance. In an effort to ERa in a ligand-independent manner and negatively regulates delineate potential predictive biomarkers of therapy respon- the transcription of ERa targets. Moreover, we demonstrate that siveness, multiple groups have identified several uncharacter- SPEN overexpression sensitizes hormone receptor–positive ized cofactors and interacting partners of ERa, including Split breast cancer cells to the apoptotic effects of tamoxifen, but Ends (SPEN), a transcriptional corepressor. Here, we demon- has no effect on responsiveness to fulvestrant. Consistent with strate a role for SPEN in ERa-expressing breast cancers. SPEN these findings, two independent datasets revealed that high nonsense mutations were detectable in the ERa-expressing SPEN protein and RNA expression in ERa-positive breast breast cancer cell line T47D and corresponded to undetectable tumors predicted favorable outcome in patients treated with protein levels. Further analysis of 101 primary breast tumors tamoxifen alone. Together, our data suggest that SPEN is revealed that 23% displayed loss of heterozygosity at the SPEN anoveltumor-suppressorgenethatmaybeclinicallyuseful locus and that 3% to 4% harbored somatically acquired muta- as a predictive biomarker of tamoxifen response in ERa-pos- tions. A combination of in vitro and in vivo functional assays itive breast cancers. Cancer Res; 75(20); 4351–63. 2015 AACR.

Introduction growth, proliferation, and survival (1). The ERa is a member of the nuclear hormone receptor family of ligand-dependent tran- Approximately 70% of breast cancers express the estrogen scription factors that regulates gene transcription in the presence receptor-a (ERa) and/or its transcriptional target, the progester- of its ligand by binding cis-regulatory motifs, also known as one receptor (PgR), and are dependent on hormones for their estrogen response elements, lying upstream of target genes or via tethering to DNA by other transcription factors, including AP1 and SP1. DNA-bound ERa recruits the basal transcriptional 1Department of Surgery and Oncology, McGill University, Montreal, machinery and induces the expression of genes implicated in 2 Quebec, Canada. Department of Oncology and Surgery, Lady Davis numerous cancer signaling pathways, a process tightly regulated Institute for Medical Research, Montreal, Quebec, Canada. 3Depart- ment of Pathology, University of Calgary, Calgary, Alberta, Canada. by complex dynamic interactions with coactivators and corepres- 4Department of Human Genetics, McGill University and The Research sors (2, 3). Although the ERa has been shown to be one of the Institute of the McGill University Health Centre, Montreal, Quebec, most important mitogenic drivers in breast cancer in a multitude Canada. 5Department of Medicine, McGill University and The Research Institute of the McGill University Health Centre, Montreal, Quebec, of preclinical and clinical studies, genomic events affecting the Canada. 6Institut de recherche en immunologie et cancerologie, IRIC, ESR1 gene are only observed in metastatic breast cancers (4, 5). Montreal, Quebec, Canada. 7Department de Biochimie, Universitede Interestingly, mutations and chromosomal aberrations appear to 8 Montreal, Montreal, Quebec, Canada. Department of Pathology, Jew- occur at higher rates in coactivators (e.g., AIB1) and corepressors ish General Hospital, Montreal, Quebec, Canada. (e.g., GATA3, NCOR1, and NCOR2) of the ERa, suggesting that Note: Supplementary data for this article are available at Cancer Research their regulation of the receptor's genomic actions may be inti- Online (http://cancerres.aacrjournals.org/). mately linked to the development of hormone-dependent Corresponding Author: Mark Basik, 3755 Chemin de la Cote^ Ste-Catherine, tumors. Room E421, Montreal, QC H3T 1E2, Canada. Phone: 514-340-8222, ext. 4210; Fax: With protumorigenic functions affecting proliferation, 514-340-8716; E-mail: [email protected] growth, and survival, the ERa is the main oncogenic driver in doi: 10.1158/0008-5472.CAN-14-3475 breast cancer and represents an important target for the treat- 2015 American Association for Cancer Research. ment of hormone-responsive tumors. Therapies targeting the

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ERa in breast cancer include antiestrogens, such as tamoxifen Colony formation assay and fulvestrant, and inhibitors of estrogen biosynthesis (aro- Cells were seeded at a density of 5,000 cells per well in 6-well matase inhibitors). Tamoxifen is a selective ER modulator that plates in 1 mL of soft agar (0.3% soft agar in DMEM with 10% competes with estrogen for the ligand-binding domain of the FBS), plated onto 2 mL of solidified agar (0.7% soft agar in DMEM ER and induces an alternative conformation that prevents with 10% FBS) and incubated at 37C. Medium (250 mL) was coactivator binding to the receptor but favors recruitment and supplemented to each well twice weekly for 4 weeks. Colonies interaction with corepressors (6). Although tamoxifen has were scored electronically using an automated cell colony counter been successfully used to treat both early and late hormone (GelCount; Oxford Optronix). receptor–positive breast cancers, more than 50% of patients with advanced hormone-responsive tumors will progress or Plasmids experience disease relapse due to intrinsic and acquired resis- The full-length human SPEN cDNA cloned into the tance to tamoxifen (7). Although many mechanisms of resis- pDream2.1/MCS expression vector was purchased from Gen- tance to tamoxifen have been characterized in vitro,nonehave Script. SPEN-expressing vector and the empty control vector were shown clinical utility as biomarkers guiding treatment with transfected in T47D cells using Attractene (Qiagen) according a tamoxifeninpatientswithER - and/or PgR-positive tumors. to the manufacturer's protocol. After 72 hours, cells were seeded Over the last decades, a number of previously uncharacterized in 6-well plates in DMEM with 10% FBS supplemented with a fi ER partners and cofactors were identi ed, including Split Ends neomycin (Life Technologies) at a concentration of 250 or (SPEN), a protein with essential regulatory roles in transcriptio- 500 mg/mL Clones were isolated and maintained in selection – nal repression (8 12). SPEN, which is also known as SMRT/ media throughout culturing period. HDAC1-associated repressor protein (SHARP), is a large nuclear protein of 402 kDa characterized by four N-terminal RNA-recog- SPEN knockdown by RNA interference nition motifs and a highly conserved C-terminal SPOC (Spen Two SPEN MISSIONshRNA plasmids with the follow- Paralog and Ortholog C-terminal) domain. In various model ingsequences:SPENshRNA1,50-CCGGCCTGTGGTAAAGGT- organisms, SPEN has been shown to be critical during embryo- GGTGTTTCTCGAGAAACACCACCTTTAC-CACAGGTTTTTG-30 genesis and throughout development, in part, due to its regula- (TRCN0000075165) and SPEN shRNA 2, 50-CCGGCGGCTC- tion of the Notch, TCF/LEF, and EGFR signaling pathways CATCATCAATGACATCTCGAGAT-GTCATTGATGAT-GGAGCC- (8, 9, 13). SPEN has also been identified as an estrogen-inducible GTTTTTG-30 (TRCN0000075166) were purchased from Sigma- cofactor able to integrate nuclear hormone receptor activation Aldrich. pLKO.1-puro Non-Target Control and SPEN-specific and repression (10). Despite some evidence for SPEN being shRNA plasmids were transfected in MCF-7 cells using Attrac- implicated in endocrine regulation and development, SPEN func- tene (Qiagen). After 72 hours, cells were seeded in 6-well plates tions have not been investigated in breast cancer. in DMEM plus 10% FBS supplemented with puromycin (Life Using an innovative unbiased integrative genomic approach, Technologies) at a concentration of 0.5, 1.0, or 2.0 mg/m. we identified mutations in the SPEN gene in a breast cancer cell Clones were isolated and maintained in selection media line and four primary breast tumors (14). We also found that throughout the culturing period. SPEN inhibits tumor growth and modulates the transcription of ERa-target genes, including PGR and BCL2.Moreover,we demonstrate that SPEN expression predicts response to tamox- RNA isolation and qRT-PCR fl ifen in vitro and in clinical samples. Together, our findings show Total RNA was isolated from subcon uent cultures using the that SPEN is a novel tumor-suppressor gene and a candidate RNeasy Mini Kit (Qiagen Sciences) and reversed transcribed into predictive biomarker of tamoxifen response in ERa-positive cDNA with the iScript cDNA Synthesis Kit (Bio-Rad). qRT-PCR breast cancers. were performed with TaqMan Gene Expression Assays (Applied Biosystems) and with the following probes: SPEN (Invitrogen, Hs00209232_m1), PGR (Invitrogen, Hs01556702_m1), and18S Materials and Methods (Invitrogen, Hs99999901_s1). Cell lines MCF-7 and T47D cells were obtained from the ATCC and were DNA microarray expression profiling and analysis cultured in DMEM supplemented with 10% fetal bovine serum Expression profiling was conducted according to the manu- (FBS; Wisent). BT20 and MDA-MB-436 were obtained from the facturer's instructions and following the One-Color Microarray- ATCC and cultured in EMEM and RPMI-1640 from ATCC sup- Based Gene Expression Analysis Protocol from Agilent Tech- plemented with 10% FBS, respectively. nologies. Briefly, integrity and concentration of the input RNA was evaluated with the Agilent 2011 Bioanalyzer. One hundred Cell viability assay nanograms of total RNA was reverse transcribed into cRNA, Cells were plated at a density of 2,500 cells per well in 96-well amplified and labeled with Cy3 dye. The resulting labeled cRNA plate for experiments performed in DMEM supplemented with was purified using the RNeasy Mini Kit (Qiagen Sciences) 10% FBS and 5,000 cells per well in 96-well plate for assays carried according to the instructions of the manufacturer and hybrid- under 1% FBS conditions. At 1, 2, 4, and 7 days, cell viability was ized to a Sure Print G3 Human GE 860 K Microarray (Agilent assessed by replacing the medium with a 10% Alamar Blue Technologies) for 17 hours at 65C. The array was then washed solution (Invitrogen) prepared in DMEM. Cells were incubated and scanned on the Agilent DNA Microarray scanner at a with the solution at 37C for 4 hours and fluorescence measured resolution of 3 mmol/L. Images were extracted and normalized with a plate reader, FLUOstar Optima, using 560 nm (Excitation) with Feature Extraction software version 9.5. Expression and 590 nm (Emission) filter settings. values of three biologic RNA replicates for each probe in the

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SPEN Is a Tumor Suppressor in ERa-Positive Breast Cancers

expression array were analyzed using Gene Spring GX (Agilent Survival assays Technologies). For experiments with tamoxifen, cells were seeded at a density of 2,500 cells per well in 96-well plates in complete medium and Statistical analysis incubated at 37 C. Twenty-four hours after plating, the medium m Microarray expression data were processed with GeneSpring was replaced with 100 L of hormone-depleted medium [DMEM GX software. Data were normalized to the 75th percentile of all without phenol red (Gibco) supplemented with 10% charcoal- values on the microarrays and to the median expression levels of stripped FBS (Gibco)]. Another 24 hours later, 4-hydroxy-tamox- all samples. The normalized gene expression data were filtered on ifen (Sigma-Aldrich Inc.) or its vehicle was added to each well in m flags and only those classified as detected were allowed to pass the 100 L of hormone-depleted medium. Cell viability was mea- filter and included in the analysis. The expression profiles of genes sured 5 days later with a 10% Alamar Blue solution. differentially expressed by more than 1.5-fold based on three biologic replicates were compared using two-tailed unpaired Immunoprecipitation t tests. Cells were rinsed with ice-cold PBS, harvested, and lysed in lysis buffer (250 mmol/L NaCl, 0.5% NP-40, 5 mmol/L EDTA, 50 mmol/L Tris) freshly supplemented with protease inhibitors Ingenuity pathway analysis [5 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, Microarray data were analyzed using the Ingenuity Pathways 1 mmol/L phenylmethylsulfonylfluoride, 10 mg/mL aprotinin, Analysis (IPA) software available at: http://www.ingenuity. and 10 mg/mL leupeptin]. After Micro-BCA quantification com. Genes up- or downregulated by at least 1.50-fold (P < (Thermo Scientific), at least 250 mg of proteins were incubated 0.05) in Spen 1, SPEN sh1, and SPEN sh3 compared with their overnight with 1 mg of anti-SPEN antibody (Sigma; HPA015825) respective controls were considered for further analyses. Func- or IgG control antibody (Abcam; ab46540-1). Protein A Dyna- tions (P < 0.05) predicted to be increased (z-score > 0.75) in beads beads (Life Technologies) were added for 1.5 hours and SPEN sh1 and SPEN sh3 relative to the nontarget shRNA washed three times with 400 mL of lysis buffer. Precipitated beads transfected MCF-7 clone (NT2) and anticipated to be decreased were then incubated at 95C for 7 minutes in 60 mL of Laemmli (z-score < 0.75) in Spen 1 compared with its empty vector sample buffer followed by a centrifugation at 13,000 rpm for 1 transfected T47D clone (CTL1) were analyzed. Functions con- minute. Proteins were ran in a precast 4% to 20% gel (Bio-Rad) for sistently modulated were considered as regulated by SPEN. 4 hours at 120 V and transferred to a nitrocellulose membrane Similarly, an analysis of the upstream regulators predicted to overnight at 33 V. Membranes were blocked with 5% BSA in TBST be responsible for the observed gene expression changes was and immunoblotted overnight with an anti-SPEN antibody (Sig- conducted. All molecule types were included in the analysis ma; HPA015825, 1:500). except for chemicals and miRNAs. Upstream regulators (P < 0.05) predicted to be activated (z-score > 0.75) in SPEN sh1 and SPEN sh3 relative to the nontarget shRNA-transfected MCF-7 Western blotting clone (NT2) and anticipated to be inhibited (z-score < 0.75) Subconfluent cells were collected by trypsinization, washed in in Spen 1 compared with its empty vector T47D control (CTL1) ice-cold PBS, and lysed in lysis buffer freshly supplemented with were analyzed. Those common to all pairs of cell lines were protease inhibitors. Lysates were then placed on a rocker machine considered as modulated by SPEN. The expression data of for 30 minutes and centrifuged for 5 minutes at 4 C. Supernatants tumors from The Cancer Genome Atlas (TCGA) database were were subjected to Bradford quantification and 50 mg of proteins also analyzed with IPA. For this analysis, a log2 ratio of 2 was were loaded and ran by SDS-PAGE in an 8% gel for 1 hour. used to obtain a list of approximately 3,000 differentially Proteins were transferred to nitrocellulose membranes and incu- expressed genes between the tumor and normal-matched mam- bated with PgR (1:1,000; Cell Signaling Technology) and a-tubu- mary sample. In each case, the predicted score of activation lin (1:10,000; Abcam) antibodies at 4 C overnight. Protein bands (z-score) was computed for the ER and plotted against the log2 were detected using the Amersham ECL Western Blotting Detec- ratio of SPEN RNA expression in tumors compared with nor- tion Reagent. mal-matched breast tissue samples. A log2 ratio of 0.5 was used to stratify patients into high and low SPEN-expressing Clonogenic assays groups. Cells were seeded at a density of 2.5 104 cells per cm2 and grown in DMEM supplemented with 10% FBS for 24 hours. Fluorescence microscopy to quantify cells with chromatin Media was then replaced with 2 mL of hormone-depleted medi- condensation and membrane permeabilization um [DMEM without phenol red (Gibco) supplemented with 10% Cells were seeded at a density of 2.5 105 cells per well in 6- charcoal-stripped FBS (Gibco)]. Another 24 hours later, 5 or m well plates and incubated at 37 C. Seventy-two hours after plat- 10 mol/L of tamoxifen or its vehicle was added to the cells in fi ing, 20 mL of Hoechst 33342 (HO, 100 mg/mL; Sigma) was added hormone-depleted medium. Cells were stained with a xing to the culture medium and plates incubated at 37C for 10 solution containing crystal violet 5 days after the addition of the minutes. Cells were then stained with 2 mL of propidium iodide drug. (PI; 5 mg/mL; Sigma) and analyzed by fluorescence microscopy on an Axiovert 40 CFL (Zeiss) microscope. The percentage of Stimulation with 17b-estradiol normal, apoptotic, and necrotic cells were estimated in six ran- Cells were seeded at a density of 5,000 cells per well in dom fields per condition. Apoptotic cells showed highly pyknotic 96-well plates in complete medium and incubated at 37C. nuclei stained with either HO or PI in the early and late phases of Twenty-four hours after plating, the medium was replaced with apoptosis, respectively. 200 mL of hormone-depleted medium [DMEM without phenol

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red (Gibco) supplemented with 10% charcoal-stripped FBS 1mCi of 35S[dATP], 1 PCR buffer, 200 nm each of dCTP, (Gibco)]. Another 24 hours later, media was replaced with dGTP, and dTTP, 50 pmol of each primer, and 0.5 U of Taq 1nmol/L17b-estradiol in hormone-depleted DMEM plus 10% polymerase. The PCR conditions were as follow: 94Cfor charcoal-stripped FBS for 24 hours. Cell viability was measured 5 minutes (1 cycle), 94C for 30 seconds, 60C for 30 seconds, at day 0, 2, 5, and 7 with a 10% Alamar Blue solution and fold and 72C for 30 seconds (29 cycles) followed by an incubation induction in proliferation by estradiol compared with 100% at 72C for 10 minutes (1 cycle). Products were electrophoresed control was determined by dividing fluorescence values on denaturing gels and autoradiographed at room temperature obtained in the presence of estrogen over that obtained with for 5 days. LOH was scored on the basis of the difference in the the vehicle. relative intensity of signals representing two alleles in tumor DNA samples. All samples positive for LOH or allelic imbal- Patient cohort ance at individual loci were analyzed twice in independent The tamoxifen 50/50 tissue microarray (TMA) cohort is a power assays and Sanger sequenced. series, derived from the Calgary Tamoxifen Breast Cancer Cohort (Cal-TBCC), which includes 50 patients alive 5 years after diag- Mutation analysis of SPEN nosis and 50 patients who died of breast cancer within 5 years of Mutation analyses were performed as previously described diagnosis. The Cal-TBCC is a retrospective database and contains (14). Briefly, the genomic sequence of the protein-encoding demographic data, clinical data, and pathologic data from a region of SPEN was obtained from the UCSC Santa Cruz Genome parent cohort of 819 breast cancer patients diagnosed between Bioinformatics Site available at: http://genome.ucsc.edu. Primer 1985 and 2000 at the Tom Baker Cancer Center in Calgary, pairs for PCR amplification and sequencing of SPEN were gen- Alberta, Canada. Inclusion criteria: confirmed diagnosis of inva- erated using the Primer3 software available at: http://bioinfo.ut. sive breast carcinoma and treatment with primary surgical inter- ee/primer3-0.4.0/ (Supplementary Table S5). PCR products vention with or without postoperative local radiotherapy, fol- derived from breast tumors were purified as per the manufac- lowed by adjuvant tamoxifen endocrine therapy (20 mg orally/ turer's recommendations using the QIAquick PCR Purification Kit day) for 5 years, regardless of ER or PgR status. HER2 status was (Qiagen Sciences) and sequenced at the McGill University (Mon- not systematically performed at the time of diagnosis for the treal, QC, Canada) and Genome Quebec Innovation Center and patients in this cohort. Exclusion criteria: if diagnostic biopsy or the TGEN DNA sequencing facility. Sequence chromatograms primary surgical tissue specimens were unavailable, if patients were aligned and analyzed with the Staden Package (http:// received prior or adjuvant chemotherapy. A total of 534 cases met staden.sourceforge.net/) and the Mutation surveyor software ver- the criteria with a median follow-up time of 82.1 months. The sion 3.24 by using the full-length SPEN reference sequence clinical and initial pathology data were retrieved, data for pro- NM_015001. gression-free survival at 5 years were retrieved, and formalin-fixed fi paraf n-embedded tissue blocks were acquired and replicate Fluorescence-activated cell sorting fi 0.6-mm cores were built into TMAs. All tissues were xed in For cell-cycle analyses, cells were detached with trypsin, fi 10% neutral buffered formalin and embedded in paraf n accord- washed in PBS supplemented with 5 mmol/L EDTA, suspended ing to standard procedures for the time period. Ethical approval in a fixing solution (1 mL of PBS, 5 mmol/L EDTA for 3 mL of for the use of human tissue samples was obtained from the 100% ethanol), and incubated at 20Cforatleast24hours. Conjoint Health Research Ethics Board. Then, cells were washed with PBS/EDTA and resuspended in 2 mL of staining solution (PBS, 50 mg/mL PI, and 20 mg/mL Breast tumors RNAse A). For Annexin V/PI staining experiments, cells were One hundred consecutive breast tumors were collected as detached with trypsin and washed once with PBS. Then, they part of a government-funded (FRSQ) tumor bank at the Centre were resuspended in 200 mL of Annexin V binding solution Hospitalier de l'Universite de Montreal (Montreal, QC, Canada) (1) and stained with 4 mLofPIand4mL of Annexin V. For from 2000 to 2003. Patients had signed informed consent for both assays, cell fluorescence signals were determined imme- breast tumor banking. Tissues sections for each tumor showed diately after staining using a FACScalibur flow cytometer (Bec- >70% tumor cells as determined by an H&E staining for each ton Dickinson). The analysis was performed using the BD sample. DNA was extracted using the Qiagen DNAmp extraction CellQuest (Becton Dickinson), ModFit (Becton Dickinson), kit according to the manufacturer's instructions. and FlowJo software.

aCGH of breast tumors Intersection probabilities DNA quality was assessed using a 2100 bioanalyzer using a To determine the statistical signiﬁcance of intersection between DNA 12000 Lab ChIP Kit (Agilent Technologies). Array compar- two lists of genes, we assessed the probability of this intersection ative genomic hybridization (aCGH) was performed as previous- to occur by performing 10,000 independent simulations with ly reported (15). randomly selected lists of genes of the same size. P values were calculated using a hypergeometric test. Loss-of-heterozygosity assessment using polymorphic microsatellite repeat markers Chromatin immunoprecipitation Loss-of-heterozygosity (LOH) analysis for the region sur- Cells were plated (1.2 106 cells in 150-mm culture dishes) rounding 1p36.1 was performed using the AFM217zc3a poly- and grown for 72 hours in DMEM containing 10% FBS. Cells were morphic marker (Supplementary Table S4). PCR was per- ﬁxed at room temperature for 30 minutes in 2 mmol/L Ethylene formed in 12.5-mL volume containing 100 ng of genomic DNA, glycol bis(succinimidyl succinate) (16) followed with 10 minutes

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SPEN Is a Tumor Suppressor in ERa-Positive Breast Cancers

in 1% formaldehyde. Glycine was added (125 mmol/L final tions of SPEN revealed that 17.4% (4 of 23) of tumors with LOH concentration) and left at room temperature for 5 minutes to harbored a somatic mutation within the gene. Two missense quench formaldehyde. Cells were rinsed twice and scraped in ice- mutations (P2158A, A3327T) were detected in two different cold PBS. Collected cells were then centrifuged at 1,400 rpm for 5 samples, whereas the same nonsense mutation (Q3141X) was minutes and lysed into lysis buffer (50 mmol/L HEPES-KOH pH found in two other tumors (Fig. 1B and C). 7.5, 140 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100, 0.1% To corroborate the prevalence of genomic and genetic altera- sodium deoxycholate, 0.1% SDS) freshly supplemented with tions affecting SPEN, we then surveyed data from the TCGA and protease inhibitors. Samples were incubated on ice for 10 minutes the catalogue of somatic mutations in cancer (COSMIC). We and sonicated to obtain fragments of 300 to 1,000 bp in size found that SPEN RNA expression is downregulated in invasive (setting: 25%, 10 times 15 seconds). After centrifugation (15 breast carcinoma compared with normal breast tissue (P ¼ minutes 13 000 rpm, 4C), the sheared chromatin was diluted 1.43E07) and that SPEN is homo- or heterozygously deleted in dilution buffer (1% Triton X-100, 2 mmol/L EDTA pH 8, 20 in 27.2% of breast cancer samples (Supplementary Fig. S2A and mmol/L Tris–HCl pH 8, 150 mmol/L) freshly supplemented with S2B; refs. 19–22), similar to the prevalence of LOH in our protease inhibitors. Samples were then incubated with 1.5 mgof own tumor set. Data analysis also revealed that tumors har- antibody (SPEN: HPA015825, Sigma ERa: sc-543, Santa Cruz boring nonsense mutations in SPEN express low to very low Biotechnology, IgG: ab46540-1, Abcam) at 4C with rotation SPEN mRNA levels, even in the absence of copy number loss overnight in a final volume of 1 mL. DNA–protein interactions and that breast cancer samples with LOH at the 1p36 locus were washed three times for 10 minutes with wash buffer (0.1%, express significantly lower SPEN transcript levels compared 1% Triton X-100, 2 mmol/L EDTA pH 8, 20 mmol/L Tris–HCl pH with tumors without copy number alterations (Supplementary 8, 150 mmol/L NaCl) and once for 10 minutes with final wash Fig. S2B; refs. 20, 22). In both databases, somatic mutations in buffer (0.1%, 1% Triton X-100, 2 mmol/L EDTA pH 8, 20 mmol/L SPEN were reported in 2% to 3% of breast cancers as well as in Tris–HCl pH 8, 500 mmol/L NaCl). Chromatin was eluted with many other cancer types, with a prevalence reaching 14.3% and 350 mL of elution buffer (1% SDS, 100 mmol/L NaHCO3)by 11% in cervical and endometrial cancers, respectively (Supple- rotation for 15 minutes and reverse cross-linked with 4 mLof mentaryFig.S2C;ref.23). proteinase K solution at 55C for 90 minutes followed by an overnight incubation at 65C. DNA was purified using the SPEN acts as a tumor-suppressor gene in ER-positive breast DNeasy Mini Spin columns (Qiagen) and following the manu- cancer cells facturer's instructions. PCR amplification of a DNA fragment, 311 Given the suggested role of SPEN in endocrine regulation kb upstream of the PGR transcription start site was performed and our identification of an insertion–truncation mutation in using the following primers: forward 50-CCA CTT TGC CAC ATG SPEN in the ERa-positive T47D breast cancer cell line, we ACA TC REV-30 REV 50-AAC TCC CAA GGG ACC ATT TC-30. attempted to investigate the functions of SPEN in the context of ERa-positive breast cancers. We first transfected a SPEN- expressing vector in T47D cells and isolated two stable clones Results (T47D-Spen 1–2; Fig. 2A and B), with varying degrees of SPEN is mutated and underexpressed in breast tumors restored SPEN expression. T47D-SPEN cells displayed a marked Using an integrative genomic approach, we previously identi- reduction of proliferation in both normal and low serum fied a number of genes containing nonsense mutations and conditions (Fig. 2C and Supplementary Fig. S3A) and exhibited mapping to regions exhibiting LOH in breast cancer cell lines increased sensitivity to apoptosis under serum-starved growth (14, 17). Briefly, this approach involved the profiling of cell lines conditions (Supplementary Fig. S3B). Reintroduction of SPEN treated with emetine, an inhibitor of the nonsense-mediated RNA into T47D cells did not lead to their accumulation in G1 or G2 decay (NMD) pathway (17). Genes whose transcript levels were either in 10% or 1% FBS conditions, suggesting that the increased by emetine treatment potentially contained a nonsense observed effects are not due to cell-cycle arrest (Supplementary mutation and were prioritized on the basis of those that aligned to Fig. S3C and S3D). A soft agar assay revealed that restoration of regions of LOH or deletion as assayed by genotyping with single- SPEN levels in T47D cells abrogates anchorage-independent nucleotide polymorphisms (SNP) arrays. Using this strategy, we growth (Fig. 2D). To further establish SPEN as a tumor-sup- identified and further established the AT-rich interactive domain pressor gene in breast cancer, in vivo xenografts studies were 1a (ARID1A) as a candidate tumor-suppressor gene in breast performed with BALB-c nude mice implanted with 60 days slow cancer, a finding that has since been confirmed by the discovery release 17b-estradiol pellets and injected with T47D control of ARID1A mutations in breast tumors by several groups (16, 18). cells in one mammary fat pad and T47D-SPEN cells injected Similarly, we identified an insertion/truncation mutation at contralaterally. Of a total of 8 mice that developed palpable nucleotide 1184 in the SPEN gene and LOH at the SPEN locus tumors, 7 developed tumors from control cells without tumors (chromosome 1p36), resulting in undetectable SPEN protein arising from contralateral SPEN-overexpressing clones (Fig. 2E levels in the ERa-positive, T47D breast cancer cell line (Supple- and Supplementary Fig. S3E and S3F). Notably, only one tumor mentary Fig. S1A–S1C). derived from T47D-SPEN cells was collected after completion To assess the prevalence of somatically acquired genomic of the study and its volume was much smaller than control aberrations and mutations affecting SPEN in breast cancer, we tumors. Together, these in vitro and in vivo results support the performed aCGH or microsatellite PCR at the AFM217zc3a poly- tumor-suppressive functions of SPEN in breast cancer. morphic marker on a cohort of 101 primary breast tumors (Fig. 1A Then, to further characterize SPEN functions in ERa-positive and Supplementary Table S1). We found that 22.8% (23 of 101) breast cancer cells, we silenced its expression in another hor- of tumors had LOH or a copy number loss at the SPEN locus. mone-dependent breast cancer cell line, MCF-7, which express Sequencing of the protein-encoding exons and splice-site junc- high endogenous levels of the protein (Supplementary Fig. S1B

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Figure 1. SPEN genomic alterations and mutations in breast cancer. A, representative image depicting copy number anomalies of chromosome 1 in a breast tumor sample measured using the 244 K aCGH microarray. Individual probe values are represented as normalized log2 ratios. Red dots represent probes with increased copy number values (log2 ratio > 1) and green dots represent probes with decreased copy number values (log2 ratio < 1). DNA copy number losses are apparent at the telomeric end of the 1p arm, including the genomic locus containing SPEN. B, sequencing chromatograms of SPEN at the site of mutation in the four primary tumor samples (bottom) and their corresponding normal tissues (top). Arrows, mutation sites in tumor samples. C, schematic diagram of SPEN protein domains (RRM, RNA recognition motif; NLS, nuclear localization sequence; RID, receptor interaction domain; SPOC, SPEN paralog and ortholog C-terminal domain), and locations of somatically acquired mutations resulting in the amino acid alterations, P2158A, Q3141X, and A3327T found in the sequence analysis of breast tumors.

and S1C). Using two different short-hairpin RNAs targeting between MCF-7-shRNA-SPEN clones and their control (data SPEN,fourMCF-7-shRNA-SPEN clones showing decreased not shown). The results of the functional assays performed with SPEN expression were generated (Fig. 3A and B and Supple- MCF-7 cells are complementary to those obtained with T47D mentary Fig. S3G–S3I). shRNA-mediated knockdown of cells and demonstrate that SPEN regulates proliferation, tumor SPEN slightly increased proliferation (Supplementary Fig. growth, and survival in ERa-positive breast cancer cells. Inter- S3J) but markedly increased colony formation in soft agar estingly, using a cohort of 1,784 breast cancer patients with assays (Fig. 3C and Supplementary Fig. S3K) and reduced the luminal A tumors, we assessed the clinical significance of SPEN rate at which MCF-7 cells undergo apoptosis under low serum expression and found that high SPEN RNA levels were signi- conditions (Supplementary Fig. S3L). As in T47D-SPEN clones, ficantly associated with good survival (HR, 0.78; P ¼ 0.005) no significant difference in cell-cycle distribution was observed over 20 years (Fig. 3D; ref. 24).

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Figure 2. SPEN regulates cell growth and survival in T47D cells. A, representative blot showing immunoprecipitated SPEN protein levels in SPEN-overexpressing clones (Spen 1 and 2) and the control clone (CTL1). Immunoprecipitation with nonspeciﬁc rabbit IgG in Spen 1 cells was done as a negative control. B, qRT- PCR of SPEN expression in SPEN- overexpressing T47D cells (Spen 1-2) relative to control clones (CTL1-2). Represented is the mean (SEM) expression value of SPEN in four biologic replicates, normalized to the control clone (CTL1; , P < 0.005; , P < 0.01; , P < 0.05). C, growth curves of SPEN-overexpressing T47D clones (Spen 1-2) and control clones (CTL1-2) grown in DMEM plus 1% FBS. Data points represent mean ﬂuorescence values (SEM) of four experiments performed in quadruplicates. D, representative images of soft agar colony assays performed with T47D Spen 1 and Spen 2 clones and the control clones. Bar graphs represent the mean (SEM) number of colonies formed per well in four experiments performed in triplicates. E, tumor volume in fat pads of nude mice injected bilaterally with CTL1 and SPEN-overexpressing T47D clones. Data points represent the mean tumor volume (SEM) of 7 CTL1 and 1 Spen 1 primary tumors.

Then, to evaluate whether the tumor-suppressive function of SPEN regulates the expression of genes related to cell death SPEN extends to ER-negative breast cancer cells, we silenced its To delineate the transcriptional program regulated by SPEN expression using siRNAs in BT20 and MDA-MB-436, two triple- in ERa-positive breast cancers, gene expression profiling using negative breast cancer cell lines expressing high RNA and protein DNA microarrays was conducted on RNA from one of the two levels of SPEN (Supplementary Fig. S4A and S4B), and measured clones generated with the SPEN expression vector (Spen 1, the SPEN effects on proliferation. Contrary to our expectations, we highest expressor), as well as one of the two clones generated found that SPEN knockdown limited the proliferation of both cell with each of the shRNA hairpin vectors, SPEN shRNA1 (SPEN lines in proliferation assays, suggesting that SPEN may have sh1) and SPEN shRNA2 (SPEN sh3), along with their respec- proproliferative functions in ERa-negative breast cancer cells tive controls. Microarray results confirmed the knockdown or (Supplementary Fig. S4C–S4F). Consistent with these findings overexpression of SPEN in each stably transfected clone (Sup- is the observation that SPEN RNA expression levels are predictive plementary Table S2). IPA performed with genes significantly of poor prognosis (HR, 1.49; P ¼ 0.016) in a cohort of 581 (P < 0.05) altered (1.5-fold) in each clone compared with patients with basal breast tumors (Supplementary Fig. S4G; its control revealed that the largest proportion of genes ref. 24). Although further experiments are required to define the (32%–37%) in our dataset are associated with "cell death and roles of SPEN in basal breast cancers, our results suggest that SPEN survival" (Supplementary Fig. S5A). Using the DeathBase has opposing functions in ERa-positive and ERa-negative cancer database, we found that 31% of genes reported to be involved cells while its expression may serve as a useful marker for patient in cell death (27 of 86) are differentially expressed in SPEN prognostication and stratification in both subtypes. clones compared with their controls (Supplementary Fig. S5B–

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Figure 3. SPEN regulates cell growth and survival in MCF-7 cells. A, representative blot showing immunoprecipitated SPEN protein levels in MCF-7 (SPEN sh1-sh4) clones and their control (NT, Non- Target shRNA-transfected control). Immunoprecipitation of SPEN in T47D cells was done as a negative control. B, qRT-PCR of SPEN expression in SPEN- silenced MCF-7 clones (SPEN sh1-sh4) relative to their control. Represented is the mean (SEM) expression value of SPEN in four biologic replicates, normalized to the control (NT2). C, representative images of soft agar colony assays performed with MCF-7 clones and their control (NT1). Bar graphs represent the mean (SEM) number of colonies formed per well in ﬁve experiments performed in triplicates (, P < 0.005; , P < 0.01; , P < 0.05). D, Kaplan–Meier graphs depicting progression-free survival of 1,784 patients with luminal A breast tumors, stratiﬁed according to high and moderate/weak (black) expression of SPEN.

S5D; ref. 25). Notably, most of these differentially expressed the organization of transcriptional programs. In addition, we genes implicated in cell death and survival (e.g., BCL2, BMF, found that the ER is the only upstream regulator whose pre- and BIK) are localized at the mitochondrial membrane and dicted activation is inversely correlated with SPEN expression participate in apoptosis induced by intracellular signals. We (Fig. 4A). To strengthen this relationship between SPEN and the also found that the expression of BCL2, an estrogen-target gene ER, we then cross-referenced our microarray data with two that has strong antiapoptotic functions, is significantly down- publicly available lists of 7,095 and 5,342 genes bound by regulated in both SPEN-overexpressing T47D clones (Supple- ERa and ERb, respectively, within 25 kb of their transcription mentary Fig. S5F), a finding that may explain, at least in part, start site as assayed by ChIP-seq experiments in MCF-7 cells the increased susceptibility of T47D-Spen 1 and T47D-Spen 2 treated with 17b-estradiol (26, 27). In both cases, almost half to apoptosis. Then, to better define the tumor-suppressive the number of genes significantly upregulated in MCF-7- activity of SPEN in breast cancer, we focused on functions shRNA-SPEN cells compared with their control were included predicted to be increased in MCF-7-SPEN-sh1 and MCF-7- in these lists (P ¼ 0.00003; Supplementary Fig. S6B–S6E; SPEN-sh3 silenced cells and decreased in T47D-Spen 1. With ref. 28). Interestingly, no such enrichment was observed with these criteria, only the biologic function of "cell survival" was the lists of downregulated genes in MCF-7-shRNA-SPEN cells. identified as consistently repressed by SPEN expression (Sup- Then, because ERa and ERb often dimerize with one another, plementary Fig. S5E). The data obtained from transcriptome we next examined whether the observed enrichments were analyses thus point to a role for SPEN in the regulation of cell specific for genes bound by both receptors and/or uniquely viability and these results are consistent with our findings of bound by ERa or ERb. Interestingly, we found a significant cells grown under low serum and anchorage-independent overlap between our microarray data and genes bound by both conditions. receptors or reported to be uniquely bound by ERa upon stimulation with estrogen. No significant overlap with genes Transcriptional regulation of the ERa targets by SPEN solely bound by ERb was observed, suggesting that our data are Further analyses with IPA revealed that most upstream reg- mainly enriched for ERa but not ERb target genes. In line ulators affected by the modulation of SPEN expression are with these results, we also observed that restoration of SPEN involved in the regulation of transcription (Supplementary Fig. levels significantly dampens estradiol-induced proliferation S6A), providing additional evidence that SPEN participates in in T47D cells (Fig. 4B; P ¼ 0.002). Next, to evaluate whether

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SPEN Is a Tumor Suppressor in ERa-Positive Breast Cancers

Figure 4. SPEN represses ERa transcriptional activity. A, IPA showing that the ER is the only upstream regulator whose activation is inversely correlated with SPEN expression in the SPEN-overexpressing clone, Spen 1, and SPEN-silenced clones, SPEN sh1 and SPEN sh3. B, Alamar Blue growth assay with the parental T47D cells, SPEN-overexpressing clones (Spen 1-2) or the control clone (CTL1) treated with vehicle (ethanol) or 1 nmol/L 17b-estradiol for 7 days. Cell proliferation is expressed as fold induction in proliferation (SEM) relative to vehicle-treated cells in three experiments performed in quadruplicates. C, coimmunoprecipitation of ERa with SPEN in MCF-7 cells grown in DMEM plus 10% FBS or phenol- and hormone-free DMEM plus 10% charcoal-stripped FBS (HD), supplemented or not with 1 nmol/L 17b-estradiol (E2), or 10 mmol/L tamoxifen (Tam) for 24 hours. Immunoprecipitation with nonspeciﬁc rabbit IgG was done as a negative control. D and E, Western blot analyses showing baseline expression of PgR in Spen 1–2andSPENsh1–sh4 clones compared with their respective controls (NT, Non-Target shRNA-transfected clone). F and G, qRT-PCR of PGR gene expression in T47D (Spen 1–2) and MCF-7 (SPEN sh1–sh4) clones compared with their respective controls. Represented is the mean (SEM) expression value of PGR in four (F) and three (G) biologic replicates, normalized to the control (, P < 0.005; , P < 0.01; , P < 0.05). H, chromatin immunoprecipitation studies of SPEN and ERa binding to DNA, 311 kB upstream of the PGR transcription start site, in MCF-7 and T47D cells, indicating that SPEN interacts with the PGR promoter in MCF-7 but not in T47D cells. Bar graphs represent the fold signal enrichment (SEM) of SPEN and ERa relative to IgG in three independent experiments. I, box plot depicting the relationship between SPEN expression in 60 hormone-sensitive tumors and ER activation score, as predicted by IPA analysis of each patient samples transcriptional proﬁle.

SPEN mediates its effects on the ERa throughaninteraction stimulation with 17b-estradiol. Our data showed that SPEN with the receptor complex, coimmunoprecipitation studies interacts with both liganded and unliganded ERa (Fig. 4C). were conducted under conditions of hormone depletion and However, a larger fraction of ERa coimmunoprecipitated with

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SPEN under hormone-depleted conditions, a finding that is The clinical significance of SPEN expression levels on tamox- consistent with corepressors interacting more strongly with un- ifen response was determined by assessing its nuclear staining bound nuclear receptors. Interestingly, no interaction between in TMAs containing triplicate core biopsies from 100 early-stage SPEN and ERb was detected under these conditions, suggesting breast cancer patients treated exclusively with adjuvant tamox- that SPEN preferentially interacts with ERa over ERb,consistent ifen. This cohort was composed of 50 patients who had disease with our aforementioned transcriptomic analysis. Then, to recurrence and died from breast cancer and 50 patients without further support a role for SPEN in the repression of ERa-depen- recurrence or death from the disease. Tissues were scored dent transcription, the expression of the PgR, a primary ERa according to SPEN nuclear staining intensity and percentage target gene, was measured in our SPEN transfectants (29). We of SPEN-positive tumor cells. Kaplan–Meier survival analyses þ þ observed an inverse relationship between SPEN and PgR of the 65 hormone-responsive (ERa PgR HER2 )breast expression, at both mRNA and protein levels (Fig. 4D–Gand tumors revealed that high SPEN protein expression was signif- Supplementary Table S2). In addition, chromatin immunopre- icantly predictive of favorable clinical outcome (P ¼ 0.029; cipitation experiments performed with MCF-7 and T47D cells Fig. 5E). Moreover, high SPEN RNA expression was strongly revealed that SPEN modestly but consistently interacts with correlated with good relapse-free survival (HR, 0.55; P ¼ DNA upstream of the PgR gene at a site also bound by the ERa 0.0055) in an independent cohort of 424 luminal A ER-positive in MCF-7 cells but not in T47D cells (Fig. 4H and Supplemen- patients treated with tamoxifen alone (Fig. 5F; refs. 24, 30). tary Fig. S6G). Interestingly, SPEN interaction with DNA This prognostic effect was not observed in patients with ERa- at this genomic site was restored by its reexpression in T47D negative tumors (Supplementary Fig. S7I), suggesting that cells (Supplementary Fig. S6H). Together, these results provide SPEN expression levels may serve as a predictive biomarker of evidence for SPEN as a negative regulator of the in ERa-depen- tamoxifen response in hormone-sensitive breast cancers. dent transcriptional program in ERa-positive breast cancer cells. Discussion SPEN genomic content and expression levels predict ER One of the hallmarks of cancer is the accumulation of activation genetic mutations in tumor cell DNA, resulting in the activa- To begin to evaluate the clinical significance of our findings tion of oncogenes and the loss of tumor-suppressor genes that suggest a tumor-suppressive role for SPEN in ERa-positive (31). Although their expression is usually lost in many tumors breast cancers due to its regulation of ERa-dependent tran- compared with normal tissues due to genetic and epigenetic scription, the expression profiles of 60 luminal A breast tumors events, the limited expression of tumor-suppressor genes may and their normal-matched counterpart were extracted from the nevertheless be of clinical relevance. Our identification of TCGA database and subjected to pathway analysis using IPA. SPEN as a tumor-suppressor gene and candidate predictive ApairwisecomparisonofSPEN RNA expression in this subset marker of tamoxifen response, due to its repressive activity of of 60 tumors showed that low SPEN expression predicts higher ERa-dependent transcription in breast cancer, demonstrates ER activation (Fig. 4I). As anticipated, no such correlation was that the study of tumor-suppressor genes might uncover novel observed in a subset of 40 basal breast tumors (data not mechanisms of drug resistance and biomarkers of drug shown). Taken together, our data suggest that SPEN represses response. ER-driven transcription in ERa positive but not hormone Using a genomic approach, we discovered LOH at the SPEN receptor–negative breast tumors and that ERa activity is strong- locus in 23% of breast cancers. Sequencing of SPEN in tumors ly enhanced in breast cancer cells expressing low SPEN mRNA with LOH at the 1p36 locus identified four nonsynonymous levels. mutations, including a nonsense mutation recurrent in two independent tumors, adding to the 29 mutations in breast SPEN sensitizes ERa-positive breast cancer cells to tamoxifen tumors reported in the COSMIC database (23). This finding, Having shown that SPEN represses the transcription of genes in addition to our observation that samples from TCGA with downstream of the ERa, we hypothesized that SPEN may affect nonsense mutations in SPEN express low SPEN mRNA levels, cellular responses to the antiestrogen, tamoxifen. Using phar- suggest that SPEN gene expression might be regulated by the macologically relevant tamoxifen concentrations, we found NMD pathway in breast cancer cells, a mechanism that we have that SPEN silencing confers resistance to the drug in cell shown to be responsible for undetectable SPEN protein levels viability and clonogenic assays while its overexpression sub- in T47D cells. stantially increases sensitivity to tamoxifen (Fig. 5A and B; To establish SPEN as a tumor-suppressor gene and define its Supplementary Table S3; Supplementary Fig. S6A–S6E). Inter- functions in breast cancer, we used an in vitro model with MCF-7 estingly, induction of apoptosis following tamoxifen treatment and T47D cells, two ERa-positive breast cancer cell lines expres- was considerably reduced in MCF-7-shRNA clones and 2- to sing high and very low SPEN protein levels, respectively. The 3-fold higher in T47D-SPEN cells than in their respective forced expression of SPEN in T47D cells and its knockdown in controls (Fig. 5C and D; Supplementary Fig. S6F–S6H), indi- MCF-7 cells unmasked an inhibitory effect on cell proliferation, cating that SPEN sensitizes cells to tamoxifen-induced apo- growth, and survival. In addition, gene expression analyses of ptosis. Notably, modulation of SPEN expression did not affect MCF-7 and T47D clones identified "cell survival" as the major cells sensitivity to ICI 182780 (fulvestrant; Supplementary biologic function affected by the modulation of SPEN expres- Fig. S8A–S8D), a pure ERa antagonist that induces ERa deg- sion. The enrichment of our SPEN-regulated gene expression radation, suggesting that SPEN's interaction with an intact profiles for pro- and antiapoptotic genes provides a molecular ERa may be critical to predispose cells to apoptosis in response mechanism for the altered response of SPEN-silenced MCF-7 to tamoxifen treatment. and SPEN-overexpressing T47D cells to apoptotic stimuli, such

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Figure 5. SPEN regulates ERa-positive breast cancer cells response to tamoxifen. A and B, Alamar Blue survival assay performed with T47D (Spen 1–2) and MCF-7 (SPEN sh1–sh4) clones treated with tamoxifen (T47D: 8 mmol/L, MCF-7: 6 mmol/L) or its vehicle for 5 days. Bar graphs represent the mean percentage of survival (SEM) of tamoxifen-treated cells relative to vehicle-treated cells in three (T47D) and at least four (MCF-7) experiments performed in quadruplicates. C and D, Annexin-V/PI flow cytometric analyses performed with T47D (Spen 1–2) and MCF-7 (SPEN sh1–sh4) clones treated with tamoxifen or its vehicle for 5 days. Bar graphs represent the percentage of PI-positive þ (PI ) cells (SEM) collected after treatment with tamoxifen or its vehicle in five experiments (, P < 0.005; , P < 0.01; , P < 0.05). E and F, Kaplan– Meier graphs depicting progression-free þ þ survival of ERa /PgR /HER2 patients treated with tamoxifen alone, stratified according to high and moderate/weak (blue–black) expression of SPEN.

as suspension in soft agar and growth under serum-deprived two ERa corepressors. Our observation that SPEN represses the conditions. expression of ERa target genes, including the transcription of Further analysis of our microarray data revealed that SPEN the PGR, and that it coimmunoprecipitates with the ERa regulates the expression of a number of genes downstream of complex in the presence of estradiol, demonstrates that endog- the ER, conﬁrming its role as a repressor of the ERa-dependent enous SPEN can bind and repress ligand-bound ERa.Although signaling pathway in breast cancer cells. These results extend most corepressors bind unliganded receptors, we have demon- those of Shi and colleagues (10), who demonstrated that SPEN strated that SPEN acts on both estradiol-bound and unbound inhibits estradiol-induced ERa-dependent transcription in a receptors, a property unique to few ERa coregulators. This luciferase assay. Although not derived from experiments con- function may be particularly important in limiting the genomic ducted with breast cancer cells, their results were highly sug- effects of 17b-estradiol and preventing uncontrolled cell pro- gestive of SPEN having repressive effects on ERa transcription. liferation and survival between successive hormonal cycles in In a yeast-two hybrid assay, they also showed that SPEN the normal breast. Indeed, our ﬁnding that SPEN knockdown interacts with other ERa cofactor, including SRA, an RNA potentiates the proliferation of the normal-like epithelial cell molecule with coactivating functions, and SMRT and NCoR, line, MCF10A, exposes a possible a role for SPEN in preventing

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cellular transformation. Genetic aberrations affecting SPEN in in each of our SPEN clones are also downstream of the ER (P < normal and premalignant breast epithelial cells could therefore 0.0001; Supplementary Fig. S6F) suggests that the antiapopto- potentiate ERa transcriptional activity, especially during the tic transcriptional program regulated by the ERa is likely follicular and estrogen-driven phase of the menstrual cycle, and controlled by SPEN (28). lead to the expression of estrogen-responsive genes, including Generally, our findings suggest that the inactivation of SPEN the PgR as well as antiapoptosis-related genes, all of which may by deletion and/or intragenic mutation may contribute to contribute to cell survival and cancer development. breast tumor formation and progression. As SPEN mutations Given our results showing that SPEN expression establishes a were reported in other cancer types, we can speculate that SPEN proclivity to apoptosis in ERa-positive breast cancer cells, and tumor-suppressive functions extend to many other tissues. that it represses the transcriptional activity of the ERa,we Future studies should therefore seek to evaluate the functions evaluated the sensitivity of SPEN-silenced MCF-7 and SPEN- of SPEN in other cancers, such as cervical and endometrial overexpressing T47D cells to the antiestrogen, tamoxifen, a cancer, in which the occurrence of SPEN mutations is very high drug that antagonizes the ERa. Whereas several mechanisms (14% and 11%, respectively). In conclusion, our results estab- of resistance to tamoxifen are known, such as ERa phosphor- lish SPEN as a regulator of ERa-dependent transcription ylation at S167, HER2 overexpression, and hyperactivation of of apoptosis-related genes in breast cancer and provide func- the PI3K pathway, no predictive biomarker for tamoxifen tional and clinical evidence for SPEN as a tumor-suppressor response is in current clinical use other than the ERa and the gene and a candidate predictive biomarker of tamoxifen PgR (21, 32–35). Using an in vitro breast cancer model with response in ERa-positive breast cancers. MCF-7 and T47D cells, we showed that SPEN affects tamoxifen but not fulvestrant sensitivity in ERa-positive breast cancer fl cells. SPEN protein and RNA expression in two patient cohorts Disclosure of Potential Con icts of Interest fl that had received tamoxifen therapy alone in the adjuvant No potential con icts of interest were disclosed. setting support our findings, with significantly better outcomes for patients with breast tumors showing high SPEN expression Authors' Contributions than for those with low/moderate SPEN expression. Although Conception and design: S. Legare, L. Cavallone, P.N. Tonin, M. Basik further clinical validation is needed, our findings suggest that a Development of methodology: S. Legare, A. Mamo, C. Chabot, I. Sirois, subpopulation of ERa-positive early-stage breast cancer A. Magliocco, A. Klimowicz, P.N. Tonin, M. Buchanan, D. Keilty, M. Basik patients may not benefit from adjuvant tamoxifen. In addition, Acquisition of data (provided animals, acquired and managed patients, our observation that SPEN expression does not affect cells provided facilities, etc.): S. Legare, A. Magliocco, A. Klimowicz, P.N. Tonin, a M. Buchanan, D. Keilty, M. Basik sensitivity to ICI182780, a drug that induces ER degradation, Analysis and interpretation of data (e.g., statistical analysis, biostatistics, suggests that SPEN interaction with the ERa may be critical to computational analysis): S. Legare, L. Cavallone, I. Sirois, A. Magliocco, establish a proclivity for cell death in response to tamoxifen A. Klimowicz, S. Hassan, D. Laperriere, S. Mader, O. Aleynikova, M. Basik treatment. It will thus be important in future studies to also Writing, review, and/or revision of the manuscript: S. Legare, C. Chabot, evaluate the effect of SPEN expression on sensitivity to other I. Sirois, A. Magliocco, P.N. Tonin, S. Mader, M. Basik Administrative, technical, or material support (i.e., reporting or organizing endocrine therapies, such as aromatase inhibitors, which do a data, constructing databases): S. Legare, I. Sirois, A. Magliocco, A. Klimowicz, not alter intracellular ER levels. In addition to having poten- M. Buchanan, M. Basik tial clinical applications, such studies may provide new insights Study supervision: C. Chabot, P.N. Tonin, M. Basik into SPEN functions in the context of antiestrogen drug response. Taken together, our data indicate that breast cancers expressing SPEN may be more sensitive to tamoxifen-induced Acknowledgments apoptosis and that SPEN expression could serve as a marker of The authors thank past and current members of the M. Basik laboratory, a especially Banujan Balachandran and Elaheh Ahmadzadeh, for technical assis- tamoxifen response in ER -positive breast cancers. tance, as well as Isabelle Royal, Vincent Giguere, and Volker Blank for assistance It is noteworthy that modulation of SPEN expression con- and discussion. The authors also acknowledge the help of Kathy Ann Forner and sistently affected serum deprivation and tamoxifen-induced Christian Young from the animal care and flow cytometry facility, respectively, apoptosis in both T47D and MCF-7 breast cancer cell lines, of the Lady Davis Institute for Medical Research. given that they have different defects in apoptotic mechanisms: T47D cells contain a p53 mutation and MCF-7 cells do not Grant Support express the apoptotic mediator caspase-3 (36, 37). With geno- This study was supported by a McGill Integrated Cancer Research mic and nongenomic actions affecting proliferation, migration, Training Program studentship (FRN53888) and a doctoral award from the — and apoptosis, the ERa has a central role in the biology of Fonds de recherche du Quebec Sante(S.Legare); the Eileen Iwanicki postdoctoral fellowship in Breast Cancer Research from the Canadian breast cancer. Indeed, prior studies have demonstrated that the a Institutes of Health Research in partnership with the Breast Cancer Society ER can protect breast cancer cells from program-induced cell of Canada (I. Sirois); and by grants from the Cancer Research Society and death, in part, by modulating the expression of apoptosis- the FRQS Reseau de Cancer Axe Cancer du Sein et Ovaire as well as a grant related genes, such as BCL2, BIK,andBMF (16, 38, 39). Where- from the Quebec Breast Cancer Foundation (M. Basik, P.N. Tonin, and as very few of the well-established ERa-target genes (i.e., S. Mader). GREB1, TFF1, CCND1, etc.) were differentially expressed in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked our microarray data besides PGR, the modulation of SPEN advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate expression in MCF-7 and T47D cells affected the expression this fact. of a number of apoptosis-related genes, including BCL2, BIK, and BMF.Ourfinding that approximately 35% of genes Received November 25, 2014; revised May 21, 2015; accepted July 10, 2015; involved in "cell death and survival" and regulated by SPEN published OnlineFirst August 21, 2015.

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www.aacrjournals.org Cancer Res; 75(20) October 15, 2015 4363

The Estrogen Receptor Cofactor SPEN Functions as a Tumor Suppressor and Candidate Biomarker of Drug Responsiveness in Hormone-Dependent Breast Cancers

Stéphanie Légaré, Luca Cavallone, Aline Mamo, et al.

Cancer Res 2015;75:4351-4363. Published OnlineFirst August 21, 2015.

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