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. 2015 Apr 30;34(18):2297-308.
doi: 10.1038/onc.2014.178. Epub 2014 Jun 23.

β-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids

S Condello  1 , C A Morgan  2 , S Nagdas  3 , L Cao  1 , J Turek  4 , T D Hurley  5 , D Matei  6
Affiliations

β-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids

S Condello et al. Oncogene. .

Abstract

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 (P<0.001). 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.

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Figures

Figure 1
Figure 1. Gene Expression Analysis of OC spheroids and monolayers
A, Morphology of OC cells grown as spheroids and stained with methylene blue and fuchsin (400X magnification). Shown are spheroids derived from SKOV3, IGROV1, A2780 and primary human cells derived from OC ascites. Arrows point to extracellular matrix deposited by SKOV3 cells and calcifications (psammoma bodies) formed in the ascites derived spheroids. B, Hierarchical clustering displays differential expression profiles for IGROV1 cells grown as monolayer, spheroid, or spheroid to monolayer cultures (n=3 replicates). Rows represent individual samples and columns represent genes. Each cell corresponds to the level of expression of a particular gene in a given sample. A visual dual color code is utilized with red and blue indicating relatively high and low expression levels, respectively. The scale of color saturation, which reflects the gene expression levels, is included. C, Differentially expressed genes between spheroids and monolayer were validated by semi-quantitative RT-PCR. Densitometry shows relative gene expression normalized for GAPDH. D, Cell morphology of SKOV3, IGROV1, and OC primary cells grown as monolayers (m) and spheroids (s), 100X magnification (left panels). Semiquantitative RT-PCR assessed ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH3A1 mRNA expression levels in SKOV3, IGROV1, and primary OC cells cultured as monolayers (m) compared with spheroids (s, right panels). E, Flow cytometry measures Aldefluor positive cells in SKOV3 and IGROV1 cells grown as spheroids compared to monolayers. DEAB-treated cells serve as negative controls. Measurements were performed in three replicates.
Figure 2
Figure 2. Gene networks in OC monolayers versus spheroids
A, Gene networks generated using the IPA bioinformatics tool were ranked by log p-values and compared spheroid versus monolayer cultures. Networks with larger log p-values are more significant. B, Analysis within the top ranked networks (log p value > 25) displays interconnected genes as nodes. Genes are colored according to expression level values; red symbols correspond to up-regulated genes, while green symbols indicate down-regulation. Dashed lines between nodes show indirect interactions, while continuous lines indicate direct interactions. C, Semiquantitative RT-PCR assessed mRNA expression levels for β-catenin and its targets (c-myc and cyclin D1) in SKOV3 and IGROV1 cells monolayers compared with spheroids. D, Stacked diagram representing number of genes upregulated >2 fold or downregulated >2-fold in IGROV1 spheroids vs. monolayers. Positive and negative Wnt pathway regulators are included (full list of genes is shown in Supplementary Tables 4 and 5). The RT2 Profiler PCR Array for human Wnt signaling pathway was used to compare expression profiles in spheroids vs. monolayer cultures.
Figure 3
Figure 3. β-catenin regulates OC spheroid and tumor formation
A, SKOV3 and IGROV1 cells grown as monolayers were co-transfected with TCF/LEF1 luciferase reporter and Renilla control plasmid, prior to plating as monolayers or spheroids. Luciferase activity relative to renilla activity compared monolayers and spheroids at 24 and 48 hours and is expressed as fold increase. Data are shown as means of duplicate measurements +/− SD. Experiments were repeated at least three times. Significant differences are marked. B, SKOV3 cells were transfected with scrambled or β-catenin targeting siRNA prior to plating in ultra-low attachment plates. Sphere counts are shown as means +/− SD of quadruplicate measurements. C, Semiquantitative RT-PCR measures β-catenin and c-Myc expression levels in SKOV3 spheroid cells transfected with scrambled or β-catenin targeting siRNA. Densitometry shows relative gene expression normalized for GAPDH. D, Real-time PCR measures the expression levels of β-catenin target genes c-Myc and cyclin D1 in SKOV3 cells transfected with scrambled or β-catenin targeting siRNA. Data are shown as means +/− SD of 3 replicate measurements. E, Western blotting shows β-catenin expression levels in SKOV3 cells stably transduced with control- and β-catenin targeting shRNA and used for ip inoculation of nude mice. F, Tumor weights, volumes, and numbers of peritoneal metastases derived from SKOV3 cells stably transduced with control- and β-catenin targeting shRNA and injected ip in nude mice (n = 5 and 7, respectively). Data are shown as means +/− SEM. Significant differences are marked.
Figure 4
Figure 4. ALDH1A1 and β-catenin expression in OCspheroids
A, Cell morphology of SKOV3 and IGROV1 cells grown as monolayers (m) and three spheroid generations (s1-s3, left panel). Semiquantitative RT-PCR for β-catenin and ALDH1A1 mRNA expression levels comparing monolayers and the three generation of spheroids (right panel). B, Western blotting measures expression levels of β-catenin and cyclin D1 in monolayer cultures and three spheroid generations. Densitometry quantifies β-catenin, ALDH1A1, and cyclin D1 expression levels normalized for GAPDH. C, IF staining for β-catenin (Cy5, red) and ALDH1A1 (AlexaFluor488, green) in SKOV3 cells grown as monolayers or spheroids (200X magnification). Nuclear β-catenin localization is identified by emergence of purple spectra on merged images.
Figure 5
Figure 5. ALDH1A1 is a β-catenin target in OC cells
A, Morphology of primary cells derived from OC malignant ascites grown as monolayers (m), three spheroid generations (s1-s3), or spheroid to monolayer culture (s to m). B, Semiquantitative RT-PCR for β-catenin and ALDH1A1 mRNA expression levels comparing monolayers, three generation of spheroids, and spheroid to monolayer cultures. Densitometry quantified β-catenin and ALDH1A1 normalized with the house-keeping gene 18S. C, Flow cytometry quantifies Aldefluor positive cells derived from OC ascites and grown as spheroids compared with monolayers. DEAB-treated cells serve as negative controls. Measurements were performed in duplicates. D, Semiquantitative RT-PCR for ALDH1A1 expression levels in SKOV3 cells transfected with scrambled or β-catenin targeting siRNA. E, Scheme representing the TCF/LEF1 binding sequences within the ALDH1A1 promoter relative to the designed primers (top panel). ChIP assay used chromatin from IGROV1 cells immunoprecipitated with β-catenin or IgG (control). Results of PCR amplification are as follows: DNA ladder; chromatin from IGROV1 cells not subjected to IP (input) and amplified with 2 sets of primers corresponding to the two predicted TCF/LEF1 binding sequences on the ALDH1A1 promoter (lanes 1-2,f1/r1 and f2/r2) or with primers corresponding to the TCF/LEF binding site on the c-myc promoter (lane 3, positive control); chromatin immunoprecipitated with β-catenin antibody and amplified with ALDH1A1 promoter specific primers (lanes 4-5, f1/r1 and f2/r2); ALDH1A1 promoter nonspecific primers (lane 6, up f/r, negative control), or c-myc promoter specific primers (lane 7, positive control); or chromatin immunoprecipitated with IgG and amplified with ALDH1A1 and c-myc specific primers (lanes 8-10, negative controls).
Figure 6
Figure 6. Structure, properties, and effects of A37 in OC cells
A, Structure of 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)}. B, Normalized residual activity of selected ALDH isoenzymes in the presence of 20 μM A37 in the presence of saturating concentrations of aldehyde substrate. C, Representative Lineweaver-Burk plot for the non-linear fit to the competitive inhibition equation for A37 inhibition of ALDH1A1 versus varied acetaldehydes. D, Aldefluor activity measured by flow cytometry in IGROV1 cells treated with control (DMSO and DEAB) and A37 (1-50μM) for 3 days. E-G, Morphology (E), number of spheres (F), and percentage of viable cells measured by the CCK-8 assay (G) after treatment with DMSO (control) and A37 (1, 5, 10, 25, and 50μM) for 3 days. Data are shown as means of triplicate measurements +/− SD. Significant differences are marked. H-I, Effects of cisplatin (25nM-5μM) and A37 (1μM) in OC cells grown as spheroids. Dose-response curves representing sphere numbers (H) and percentage of surviving cells (I) were plotted using GraphPad Prism against the logarithmic concentrations of cisplatin used during a 72h treatment period.
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