Oncogene (2015) 34, 2297–2308 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc ORIGINAL ARTICLE β-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids S Condello1, CA Morgan2, S Nagdas3, L Cao1, J Turek4, TD Hurley2,5 and D Matei1,2,5,6 Cancer cells form three-dimensional (3D) multicellular aggregates (or spheroids) under non-adherent culture conditions. In ovarian cancer (OC), spheroids serve as a vehicle for cancer cell dissemination in the peritoneal cavity, protecting cells from environmental stress-induced anoikis. To identify new targetable molecules in OC spheroids, we investigated gene expression profiles and networks upregulated in 3D vs traditional monolayer culture conditions. We identified ALDH1A1, a cancer stem cell marker as being overexpressed in OC spheroids and directly connected to key elements of the β-catenin pathway. β-Catenin function and ALDH1A1 expression were increased in OC spheroids vs monolayers and in successive spheroid generations, suggesting that 3D aggregates are enriched in cells with stem cell characteristics. β-Catenin knockdown decreased ALDH1A1 expression levels and β-catenin co-immunoprecipitated with the ALDH1A1 promoter, suggesting that ALDH1A1 is a direct β-catenin target. Both short interfering RNA-mediated β-catenin knockdown and A37 ((ethyl-2-((4-oxo-3-(3-(pryrrolidin-1-yl)propyl)-3,4-dihydrobenzo [4,5]thioeno [3,2-d] pyrimidin-2-yl)thio)acetate)), a novel ALDH1A1 small-molecule enzymatic inhibitor described here for the first time, disrupted OC spheroid formation and cell viability (Po0.001). β-Catenin knockdown blocked tumor growth and peritoneal metastasis in an OC xenograft model. These data strongly support the role of β-catenin-regulated ALDH1A1 in the maintenance of OC spheroids and propose new ALDH1A1 inhibitors targeting this cell population. Oncogene (2015) 34, 2297–2308; doi:10.1038/onc.2014.178; published online 23 June 2014 INTRODUCTION (CSCs),12,13 allowing the development of distant metastases and Epithelial ovarian cancer (OC) is the most lethal of all gynecologic persistence after chemotherapy. malignancies, with the majority of cases being diagnosed at an Recent reports suggest that cells grown as 3D structures behave advanced stage. OC metastasis is the primary cause of clinical differently compared with monolayer cultures and represent a complications and is characterized by several unique features.1 better approximation of tumors developing in vivo. For instance, 14–16 While in other epithelial tumors breakdown of the basement spheroids display distinct genetic expression profiles, 17–20 membrane is required for tumor invasion into lymphatics or specific intercellular signaling and are subjected to different 21–23 vasculature and subsequent dissemination of cancer cells to mechanical forces compared with monolayers. The cellular distant sites,2 hematogenous metastasis is uncommon in OC. dimensionality and the resulting microenvironment exert a critical 24,25 Tumor dissemination occurs directly in the peritoneal cavity, with influence on cell survival, impacting drug sensitivity or 26,27 most sites of secondary implants involving the mesentery, resistance. OC spheroids can be isolated directly from omentum and bowel. This is facilitated by the fact that OC cells malignant ascites or cultured from OC cells by using non- at the primary site are in direct anatomic contact with the adherent conditions or the hanging drop culture method.28 In overlying peritoneal surface and fluid. Their dislodgement from this manuscript, we set out to identify oncogenic pathways the primary tumor on the surface of the ovary or fallopian tube3 regulating the formation of multicellular aggregates with the aim allows cells to float in the peritoneal fluid. Importantly, after of identifying novel targets enriched in 3D culture models. We exfoliation from the primary tumor, OC cells form multicellular hypothesized that inhibition of such targets would disrupt aggregates or spheroids.4 These three-dimensional (3D) cellular spheroid formation and block cancer metastasis. aggregates serve as the vehicle for dissemination in the peritoneal Microarray analysis comparing OC multicellular structures with cavity, protecting cells from anoikis induced by stress in the monolayer cultures identified ALDH1A1, a known CSC marker, extracellular compartment.5,6 upregulated in spheroids. ALDH1A1 was part of a gene network Within spheroid structures, cells adopt mesenchymal features4,7 with β-catenin, and chromatin immunoprecipitation (ChIP) that are regulated by cytokines and growth factors such as demonstrated that ALDH1A1 is a direct β-catenin target. Both estrogen,8 tumor growth factor-β,9 or other proteins secreted in β-catenin knockdown and a novel ALDH1A1 inhibitor (A37; the peritoneal milieu.10,11 The mesenchymal phenotype allows (ethyl-2-((4-oxo-3-(3-(pryrrolidin-1-yl)propyl)-3,4-dihydrobenzo [4,5] cells to invade when they come in contact with the mesothelium,7 thioeno [3,2-d]pyrimidin-2-yl)thio)acetate)) prevented multicellular leading to the establishment of peritoneal implants. We hypothe- aggregation, supporting that inhibition of this pathway effectively sized that cells forming spheres are enriched in cancer stem cells disrupts spheroid formation. 1Department of Medicine, Indianapolis, IN, USA; 2Department of Biochemistry and Molecular Biology, Indianapolis, IN, USA; 3University of Virginia Medical School, Indianapolis, IN, USA; 4College of Veterinary Medicine Purdue University, Indianapolis, IN, USA; 5Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, USA and 6VA Roudebush Hospital, Indiana University School of Medicine, Indianapolis, IN, USA. Correspondence: Dr TD Hurley, Department of Biochemistry and Molecular Biology, MS-4019, Indianapolis, IN, USA or Dr Professor D Matei, Indiana University Melvin and Simon Cancer Center, 535 Barnhill Drive, RT 473, Indianapolis, IN 46202, USA. E-mail: [email protected] or [email protected] Received 20 December 2013; revised 10 May 2014; accepted 12 May 2014; published online 23 June 2014 ALDH1 in ovarian cancer S Condello et al 2298 RESULTS validating the β-catenin–ALDH1A1 interaction in the generation of Gene expression profiles of OC spheroids OC spheroids. OC cell lines (IGROV1, SKOV3 and A2780) and primary OC cells derived from malignant ascites were grown as monolayers, β-Catenin signaling regulates spheroid formation and self-renewal spheroids or transitioned back from spheroid-to-monolayer Semiquantitative RT–PCR validated upregulation of β-catenin and cultures. When grown under non-adherent conditions, OC cells of its target genes c-myc and cyclin D1 in IGROV1 and SKOV3 formed 3D aggregates with distinct features. For instance, SKOV3 spheroids compared with monolayers (Figure 2c). An additional adenocarcinoma cells formed glandular structures with prominent exploratory analysis used quantitative RT–PCR for the human Wnt extracellular matrix secretion; IGROV1 endometrioid cells formed signaling pathway and demonstrated an overall upregulation of branching spheroids, whereas A2780 and OVCA primary cells this pathway in spheroids compared with monolayers cultures. formed compact round multicellular aggregates. Spheroids Specifically, 23 Wnt-related transcripts were upregulated 42-fold derived from human primary cells displayed calcifications, similar in spheroids vs monolayers, with 17 genes being known positive to psammoma bodies formed in human tumors (Figure 1a). regulators of the pathway (Figure 2d and Supplementary Tables 4 To identify genes and pathways upregulated in spheroids, we and 5). Notably, Frizzled receptors 1, 4 and 7 were upregulated compared expression profiles of monolayers, spheroids or 44-fold in spheroids cultures, supporting enrichment in Wnt spheroid-to-monolayer IGROV1 cultures using Affymetrix micro- signals leading to increased β-catenin activity under 3D arrays. Unsupervised hierarchical clustering demonstrated distinct conditions. profiles of 3D vs 2D cultures, with reversal of the spheroid To measure β-catenin function in spheroids vs monolayers, the genotype when cells were transitioned back to monolayers T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay (Figure 1b) and analysis of variance-based statistical analysis was used. A 43-fold increase in TCF/LEF activity was noted in identified 473 transcripts differentially expressed in spheroids SKOV3 spheroids compared with monolayers, and a lesser compared with monolayers (Po0.01 and false discovery rate magnitude, but still significant increase was recorded in o0.01). Of those, 15 transcripts were upregulated 44-fold and 25 IGROV1 cells grown as multicellular aggregates (Figure 3a). The transcripts were downregulated 44-fold in spheroids vs mono- results indicate that the increase in spheroids proliferation was layers (Tables 1 and 2). Validation of top differentially expressed accompanied by an active β-catenin/TCF transcriptional activity. genes using semiquantitative reverse transcriptase (RT)–PCR To further assess the role of β-catenin signaling in spheroid confirmed upregulation of aldehyde dehydrogenase 1 A1 (ALDH1), formation, SKOV3 cells were transiently transfected with short angiopoietin-like 2 (ANGPTL2), thrombospondin type I (THSD) and interfering RNA (siRNA) targeting β-catenin or with scrambled neurotensin (NTS) and downregulation of family 25, member B siRNA before plating in ultra-low adherent plates to allow sphere (FAM25B) and v-ets erythroblasosis virus homolog (ETS) in spheroids