Published OnlineFirst June 25, 2019; DOI: 10.1158/0008-5472.CAN-18-3264 Cancer Tumor Biology and Immunology Research EN1 Is a Transcriptional Dependency in Triple- Negative Breast Cancer Associated with Brain Metastasis Guillermo Peluffo1,2, Ashim Subedee1,3, Nicholas W. Harper1, Natalie Kingston1, Bojana Jovanovic1,2, Felipe Flores1,4, Laura E. Stevens1,2, Francisco Beca5,6, Anne Trinh1,2, Chandra Sekhar Reddy Chilamakuri7, Evangelia K. Papachristou7, Katherine Murphy1, Ying Su1,2, Andriy Marusyk1,2, Clive S. D'Santos7, Oscar M. Rueda7, Andrew H. Beck5,6, Carlos Caldas7, Jason S. Carroll7, and Kornelia Polyak1,2,3 Abstract To define transcriptional dependencies of triple-negative titative proteomic analyses of EN1-bound chromatin com- breast cancer (TNBC), we identified transcription factors high- plexes revealed association with transcriptional repressors and ly and specifically expressed in primary TNBCs and tested their coactivators including TLE3, TRIM24, TRIM28, and TRIM33. requirement for cell growth in a panel of breast cancer cell High expression of EN1 correlated with short overall survival lines. We found that EN1 (engrailed 1) is overexpressed in and increased risk of developing brain metastases in patients TNBCs and its downregulation preferentially and significantly with TNBC. Thus, EN1 is a prognostic marker and a potential reduced viability and tumorigenicity in TNBC cell lines. By therapeutic target in TNBC. integrating gene expression changes after EN1 downregulation with EN1 chromatin binding patterns, we identified genes Significance: These findings show that the EN1 transcrip- involved in WNT and Hedgehog signaling, neurogenesis, and tion factor regulates neurogenesis-related genes and is associ- axonal guidance as direct EN1 transcriptional targets. Quan- ated with brain metastasis in triple-negative breast cancer. groups (1). Knowledge of the molecular properties of luminal Introduction þ þ ER and HER2 subtypes has led to the development of endocrine Breast cancer is a heterogeneous group of diseases with different and HER2-targeted therapies. However, currently there is no biological and clinical characteristics. On the basis of the presence effective targeted therapy for triple-negative breast tumors. of estrogen and progesterone receptors (ER and PR), and þ þ À À À Triple-negative breast cancer (TNBC) constitutes 10%–20% of HER2, tumors are classified into ER , HER2 , and ER PR HER2 breast cancer cases in the United States and more commonly (triple-negative) subtypes, whereas gene expression and epige- affects younger and African-American women (2, 3). TNBCs have netic profiles divide breast tumors into luminal and basal higher risk of developing distant metastases and in general have poor clinical outcome. TNBCs are also heterogeneous and have been grouped into luminal, basal, and mesenchymal subtypes 1Department of Medical Oncology, Dana-Farber Cancer Institute Boston, based on gene expression patterns (2–4). However, these sub- Massachusetts. 2Department of Medicine, Harvard Medical School, Boston, types, besides luminal androgen receptor (AR)-positive tumors, Massachusetts. 3BBS Program, Harvard Medical School, Boston, Massachusetts. have not impacted the clinical management of patients with 4Harvard University, Cambridge, Massachusetts. 5Department of Pathology, 6 TNBC (3) highlighting the need for additional molecular markers Harvard Medical School, Boston, Massachusetts. Department of Pathology, to guide treatment decisions. TNBC genome sequencing studies Beth Israel Deaconess Medical Center, Boston, Massachusetts. 7Cambridge Research Institute, University of Cambridge, Cambridge, United Kingdom. have so far failed to identify novel recurrent mutations besides TP53, PIK3CA, and PTEN (2, 3), suggesting that TNBC phenotypes Note: Supplementary data for this article are available at Cancer Research may be driven by nongenetic alterations such as perturbed epi- Online (http://cancerres.aacrjournals.org/). genetic and transcriptional programs. G. Peluffo and A. Subedee contributed equally to this article. The luminal phenotype is defined by a set of lineage-specific Current address for A. Subedee: NIH, Rockville, Maryland; current address for transcription factors including ESR1, FOXA1, GATA3, and SPDEF Y. Su: Deciphera Pharmaceuticals, Waltham, Massachusetts; current address for that also represent transcriptional dependencies in luminal breast F. Beca: Stanford University, Stanford, California; current address for A. Marusyk: fi tumors (2, 3). We hypothesized that transcription factors (TF) Mof tt Cancer Center, Tampa, Florida; and current address for A. H. Beck: PathAI, fi Cambridge, Massachusetts. speci cally expressed in TNBCs may also exemplify such depen- dencies that can be exploited therapeutically and may divide Corresponding Author: Kornelia Polyak, Dana-Farber Cancer Institute, 450 TNBC into clinically relevant subsets. To test this hypothesis, first Brookline Ave, Boston, MA 02215. Phone: 617-632-2106, Fax: 617-582-8490, fi E-mail: [email protected] we selected TFs that are highly and speci cally expressed in primary TNBCs followed by a targeted cellular viability screen Cancer Res 2019;79:4173–83 for these TFs in a panel of breast cancer cell lines of different doi: 10.1158/0008-5472.CAN-18-3264 subtypes. Using this approach, we have identified several TFs Ó2019 American Association for Cancer Research. specifically required for the survival of TNBCs and among these www.aacrjournals.org 4173 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst June 25, 2019; DOI: 10.1158/0008-5472.CAN-18-3264 Peluffo et al. further characterized EN1, a TF with known roles in brain (5, 6) PBS, cells were plated in fresh medium into collagen-coated and dermomyotome (7) development. plates. Cells were collected at 0, 3, 9, 12, and 24-hour timepoints for FACS and immunoblot analyses. For cell-cycle analysis, cells were harvested, washed in PBS, and fixed in ice-cold 70% ethanol Materials and Methods at À20C overnight. Fixed cells were resuspended in a solution Cell lines containing 100 mg/mL RNase and incubated for 30 minutes at Breast and colon cancer cell lines were obtained from ATCC or 37C with agitation. The cells were then resuspended in a solution generously provided by Steve Ethier (SUM149 and SUM159 cell containing 40 mg/mL propidium iodide (Sigma), and the analysis lines, University of Michigan, Ann Arbor, MI). Cells were cultured was performed on a FACS Aria II Cytometer (BD Biosciences). in media recommended by the provider, except SUM149 and SUM159 cells were cultured in DMEM/F12 supplemented METABRIC expression and survival analysis with 5% FBS, 10 mmol/L HEPES pH7.4, 1 mg/mL hydrocortisone, Expression analysis was performed on METABRIC dataset. 5 mg/mL insulin, 50 U/mL penicillin, and 50 mg/mL streptomycin. Using the EN1 mRNA expression distribution we separated EN1 MCF7 cells were cultured in 4.5mg/L glucose DMEM supplemen- expression into four clusters using a univariate Gaussian mixture ted with 10% FBS, 10 mg/mL insulin, 50 U/mL penicillin, and model–based clustering (mclust version 5.4.2 package for R; 50 mg/mL streptomycin. Cells were cultured at 37 C with 5% CO2. refs. 8–10). Using the EN1-defined clusters, Kaplan–Meier sur- The identities of the cell lines were confirmed by short tandem vival curves were plotted and a log-rank P value was computed repeat analysis; and they were regularly tested for Mycoplasma. using the function km.coxph.plot in the R package survcomp. Next, the survival in the C4 cluster was compared with the other Generation of cell line derivates clusters. A univariable Cox proportional hazards analysis was SUM149 and SUM159 cells expressing TET-inducible short performed using the coxph function in R to assess the association hairpin RNAs (shRNA) targeting EN1, CTNNB1,orNLGN4X in of EN1 mRNA expression with overall survival. A multivariable pLKO lentiviral vector were generated by selecting with 5 mg/mL Cox proportional hazard analysis was performed using the coxph puromycin for 5 days after lentiviral infection. Entry cDNA open function in R, and the age, the Nottingham Prognostic Index, and reading frame (ORF) for EN1 in pENTR221 was obtained from continuous EN1 expression were considered. The EN1 expression human ORFeome collection v5.1. pCDNA3-CTNNB1 and z-scores of 243 triple-negative tumors from the METABRIC cohort pCDNA3-CTNNB1S33Y were obtained from Addgene. Lentiviral were computed and the mean levels in the primary tumor for expression constructs were generated by Gateway swap into patients that had a brain metastasis (n ¼ 17) and patients that did pLenti6.3/V5-Dest Vector (Life Technologies) and sequence ver- not (n ¼ 226) were compared using a two-sided t test. Differential ified. MCF7-lacZ and MCF7-EN1 cells were selected with 5 mg/mL gene expression was performed using two-class unpaired signif- blasticidin. icance of microarray analysis (SAM 2.0 package, R 3.2.2) in basal carcinomas in the C1, C2, and C3 cluster and the C4 cluster. Next, Colony growth assays we performed a preranked GSEA (gene set enrichment analysis) A total of 500–1,500 cells expressing TET-inducible EN1- or using software provided by the Broad Institute (http://www.broad CTNNB1-targeting shRNAs were plated into each well of a 6-well institute.org/gsea/msigdb/annotate.jsp) on a ranked gene list plate. Next day, regular media (no doxycycline control) or ranked (after exclusion of EN1 gene) based on