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324: BRCA1 as a regulator of global cell metabolism – identifying functional targets through integromic analysis
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 cells, whereas integrins in the order solid and alveolar − trabecular structures − discrete groups of tumor cells (p < 0.05). Tubular structures, which are used in histological grading of BC, had the lowest activity of cell adhesion genes. Chromosome aberrations specific for each type of morphological structures have not been found; however, discrete groups of tumor cells demonstrated the lowest number of cytogenetic abnormalities. Solid structures from only one region of breast tumor and lymph node metastases displayed the highest level of chromosome aberrations and two common amplifications of chromosome bands − 17p13.1 (part of TP53 gene) and 12q11q24.33 (including MDM2 gene), which were not been detected in all other structures. Conclusions: Intratumor morphological heterogeneity in BC is not associated with specific chromosome abnormalities and is probably a reflection of different patterns of invasive growth. No conflict of interest. 322 Identification of GREB1 as a potential mediator of estrogen effects on ovarian cancer progression in a mouse model K.M. Hodgkinson1 , L.A. Laviolette1 , B.C. Vanderhyden1 . 1 Ottawa Hospital Research Institute and University of Ottawa, Cellular and Molecular Medicine, Ottawa, Canada Background: Hormone replacement therapy containing estrogen increases the risk of developing ovarian cancer, and 17b-estradiol (E2) promotes growth and survival of ovarian cancer cell lines. We have shown previously that E2 promotes ovarian cancer progression in transgenic and allograft mouse models, and now investigate the mechanism of E2 action on tumour progression. Material and Methods: To identify genes altered by E2 treatment, we used an allograft model in which SCID mice were implanted with E2 pellets and injected with mouse ovarian cancer cells (MAS) derived from the ascites of the tgCAG-LS-TAg transgenic mouse model of ovarian cancer. One E2upregulated gene of particular interest is Gene regulated by estrogen in breast cancer-1 (Greb1), which is an estrogen receptor a (ESR1) target gene which mediates the proliferative actions of hormones in breast and prostate cancer cells. In order to investigate the function of GREB1, we used lentiviral vectors to cause knockdown and overexpression in MAS cells. Results: Survival of mice engrafted with MAS ovarian cancer cells was shortened by E2 treatment and microarray analysis showed upregulation of 197 genes and downregulation of 55 genes in tumours from E2-treated mice. QPCR confirmed upregulation of Greb1 and other genes of interest in tumours from E2-treated mice and cultured MAS cells as well as two human ovarian cancer cell lines. MAS cell proliferation was decreased by GREB1 knockdown and increased by GREB1 overexpression. When injected into SCID mice, MAS cells with GREB1 knocked down resulted in fewer metastases and mice had prolonged survival relative to mice injected with control MAS cells. GREB1 is highly expressed in human ovarian tumours of 4 histological subtypes relative to normal ovarian epithelial cells (on average, 347-fold higher in tumours), and correlates with ESR1 expression, suggesting that GREB1 may be regulated by ESR1 in ovarian cancer (as shown in breast cancer). Conclusions: This study is the first to examine GREB1 action in mouse models and its expression in ovarian cancer cell lines and tumours. Characterization of the function of E2-target genes will elucidate the mechanisms by which E2 increases the risk of ovarian cancer and help clarify the effects of estrogen antagonists on ESR1-positive ovarian cancers. No conflict of interest. 323 Simplifying high throughput 3D tumour spheroid growth and shrinkage assays using live content imaging T. Dale1 , K. Patel1 , B. O’Clair2 , T. O’Callaghan1 , D. Appledorn2 , D. Trezise1 . 1 Essen BioScience, Welwyn Garden City, United Kingdom, 2 Essen BioScience, Ann Arbor, USA Introduction: For several years, 3D tumour spheroid models have been used in the study of cancer biology as these are believed to be more reflective of the in vivo micro-cellular environment than 2D systems. However, the routine application of 3D models within drug discovery has been limited by complexities in methodology and quantification. Our aim was to build relevant, kinetic tumour spheroid growth & shrinkage assays that are as technically straightforward, robust and cost equivalent to 2D assays. Method: In this study, a kinetic, live-cell imaging approach was used to measure the growth or shrinkage of non-adherent tumour spheroids using Ultra Low Attachment (ULA) 96-well and 384-well micro-titre plates (Corning® ). These round-bottomed, hydrogel coated plates facilitate the formation of spheroids by promoting cellular self-adherence as opposed to adherence to the plate surface. Post spheroid formation, quantitative pharmacology on compounds with a range of mechanisms was performed by monitoring spheroid size over 10 days using the IncuCyte™ ZOOM Live Content Imaging system. Spheroid parameters were quantified using fluorescence area and fluorescence intensity metrics via a RFP-labelled non perturbing probe (NucLight-Red™).
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Results: In a monoculture model, NucLight-red labelled A549 lung epithelial carcinoma cells developed into a single spheroid in each well of 96- and 384well plates within 48−96 hours in culture. In both plate types, the resulting spheroid size, ranging from 330 mm to 600 mm, was proportional to the number of cells seeded (1000–5000 cells per well). Data was obtained for a set of compounds yielding a range of pIC50 values. For example, the clinically used cytostatic agent cycloheximide inhibited spheroid growth (pIC50 5.8) with no substantial effect on spheroid shrinkage. In contrast, the chemo-therapeutic paclitaxel (Taxol® ) both inhibited spheroid growth (pIC50 7.9) and at high concentrations (0.1 mM) induced spheroid shrinkage. Conclusion: The data shown here describes validation of a technically straightforward, robust and economical kinetic, live-cell approach to measuring spheroid size. Such facile 3D assays allow for adoption into early phase drug discovery projects. No conflict of interest. 324 BRCA1 as a regulator of global cell metabolism − identifying functional targets through integromic analysis A. Powell1 , K.I. Savage1 , D.P. Harkin1 . 1 Queen’s University Belfast, CCRCB, Belfast, United Kingdom Introduction: Mutations within the BRCA1 tumour suppressor gene predisposes carriers to a high risk of breast and ovarian cancers. BRCA1 primarily functions to maintain genomic stability through a role in multiple cell processes including DNA repair, cell cycle arrest and transcriptional regulation. Recent studies suggest a role for BRCA1 in regulating fundamental metabolic processes. We performed a discovery metabolomic screen to explore the role of BRCA1 in regulating cellular metabolism and its relevance to tumour progression. These data were integrated with existing BRCA1 genetic and transcriptional data to identify metabolically relevant BRCA1 transcriptional targets. Materials and Methods: BRCA1-deficient HCC1937 cells were virally transduced with wild type BRCA1 or GFP and matched cell and medium samples were analysed by Metabolon Inc. Metabolite fold-change data were then functionally annotated. This dataset was analysed in parallel with BRCA1 gene expression microarray and ChIP-chip data using the IMPaLA integromic analysis tool in order to identify metabolically relevant targets of BRCA1 transcriptional regulation. Results: Of 347 unique metabolites in the screen, 50 were significantly associated with BRCA1 status. Loss of BRCA1 results in a trend of general upregulation in diverse classes of molecules. Highly desaturated and long chain fatty acids were strongly represented in the dataset; loss of BRCA1 results in upregulated levels of C18 and C20 desaturates including arachidonic acid (AA). Conversely, levels of antioxidant a-Tocopherol were found to be reduced, suggesting a complex involvement of fatty acid metabolism and oxidative balance. Pathway-driven integromic co-analysis of BRCA1 metabolite and transcription data identified 13 pathways among which lipid and lipoprotein metabolism, signal transduction and small molecule transporters were enriched for BRCA1 regulated transcripts. The potential role of BRCA1 in regulating these transcripts was cross-validated in publically available datasets in order to produce a high-confidence list of functionally important BRCA1-regulated transcripts that are likely to have phenotypic relevance. Conclusion: Our data suggest that BRCA1 has a much more diverse role in regulating metabolism than was previously appreciated. Through an integromic approach we have identified BRCA1-regulated genes that may represent targets with relevance to the phenotype of BRCA1 deficient cancers. In order to assess the functional relevance of this data, secondary screening techniques will be employed: a real-time based screen of genetic targets in our primary model and a metabolic dependency screen, consisting of a panel of small molecule inhibitors of key enzymes in identified pathways. No conflict of interest. 326 Expression analyses of long non-coding RNAs in breast cancer E. Knutsen1 , T. Fiskaa1 , S.L. Figenschau1 , E.S. Brun1 , S. Fismen1 , O.M. Seternes2 , E.S. Mortensen1 , S.D. Johansen1 , M. Perander1 . 1 UiT The Arctic University of Norway, Department of Medical Biology, Tromsø, Norway, 2 UiT The Arctic University of Norway, Department of Pharmacy, Tromsø, Norway Background: Long non-coding RNAs (lncRNAs) are regulatory transcripts longer than 200 nucleotides, the majority being transcribed from RNA pol II promoters and processed by 5 capping, polyadenylation, and splicing. They have recently attained much attention as important regulators of gene expression at different levels. The lncRNAs exert their function by mediating interaction with DNA elements, other RNA molecules, or proteins either by complementary base pairing or by adapting specific structures. Many of them show tissue-specific expression and are expressed at specific time points during embryonic development, indicating an important role in turning on and off genes at specific circumstances. Deregulated expression of many lncRNAs
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Results: In a monoculture model, NucLight-red labelled A549 lung epithelial carcinoma cells developed into a single spheroid in each well of 96- and 384well plates within 48−96 hours in culture. In both plate types, the resulting spheroid size, ranging from 330 mm to 600 mm, was proportional to the number of cells seeded (1000–5000 cells per well). Data was obtained for a set of compounds yielding a range of pIC50 values. For example, the clinically used cytostatic agent cycloheximide inhibited spheroid growth (pIC50 5.8) with no substantial effect on spheroid shrinkage. In contrast, the chemo-therapeutic paclitaxel (Taxol® ) both inhibited spheroid growth (pIC50 7.9) and at high concentrations (0.1 mM) induced spheroid shrinkage. Conclusion: The data shown here describes validation of a technically straightforward, robust and economical kinetic, live-cell approach to measuring spheroid size. Such facile 3D assays allow for adoption into early phase drug discovery projects. No conflict of interest. 324 BRCA1 as a regulator of global cell metabolism − identifying functional targets through integromic analysis A. Powell1 , K.I. Savage1 , D.P. Harkin1 . 1 Queen’s University Belfast, CCRCB, Belfast, United Kingdom Introduction: Mutations within the BRCA1 tumour suppressor gene predisposes carriers to a high risk of breast and ovarian cancers. BRCA1 primarily functions to maintain genomic stability through a role in multiple cell processes including DNA repair, cell cycle arrest and transcriptional regulation. Recent studies suggest a role for BRCA1 in regulating fundamental metabolic processes. We performed a discovery metabolomic screen to explore the role of BRCA1 in regulating cellular metabolism and its relevance to tumour progression. These data were integrated with existing BRCA1 genetic and transcriptional data to identify metabolically relevant BRCA1 transcriptional targets. Materials and Methods: BRCA1-deficient HCC1937 cells were virally transduced with wild type BRCA1 or GFP and matched cell and medium samples were analysed by Metabolon Inc. Metabolite fold-change data were then functionally annotated. This dataset was analysed in parallel with BRCA1 gene expression microarray and ChIP-chip data using the IMPaLA integromic analysis tool in order to identify metabolically relevant targets of BRCA1 transcriptional regulation. Results: Of 347 unique metabolites in the screen, 50 were significantly associated with BRCA1 status. Loss of BRCA1 results in a trend of general upregulation in diverse classes of molecules. Highly desaturated and long chain fatty acids were strongly represented in the dataset; loss of BRCA1 results in upregulated levels of C18 and C20 desaturates including arachidonic acid (AA). Conversely, levels of antioxidant a-Tocopherol were found to be reduced, suggesting a complex involvement of fatty acid metabolism and oxidative balance. Pathway-driven integromic co-analysis of BRCA1 metabolite and transcription data identified 13 pathways among which lipid and lipoprotein metabolism, signal transduction and small molecule transporters were enriched for BRCA1 regulated transcripts. The potential role of BRCA1 in regulating these transcripts was cross-validated in publically available datasets in order to produce a high-confidence list of functionally important BRCA1-regulated transcripts that are likely to have phenotypic relevance. Conclusion: Our data suggest that BRCA1 has a much more diverse role in regulating metabolism than was previously appreciated. Through an integromic approach we have identified BRCA1-regulated genes that may represent targets with relevance to the phenotype of BRCA1 deficient cancers. In order to assess the functional relevance of this data, secondary screening techniques will be employed: a real-time based screen of genetic targets in our primary model and a metabolic dependency screen, consisting of a panel of small molecule inhibitors of key enzymes in identified pathways. No conflict of interest. 326 Expression analyses of long non-coding RNAs in breast cancer E. Knutsen1 , T. Fiskaa1 , S.L. Figenschau1 , E.S. Brun1 , S. Fismen1 , O.M. Seternes2 , E.S. Mortensen1 , S.D. Johansen1 , M. Perander1 . 1 UiT The Arctic University of Norway, Department of Medical Biology, Tromsø, Norway, 2 UiT The Arctic University of Norway, Department of Pharmacy, Tromsø, Norway Background: Long non-coding RNAs (lncRNAs) are regulatory transcripts longer than 200 nucleotides, the majority being transcribed from RNA pol II promoters and processed by 5 capping, polyadenylation, and splicing. They have recently attained much attention as important regulators of gene expression at different levels. The lncRNAs exert their function by mediating interaction with DNA elements, other RNA molecules, or proteins either by complementary base pairing or by adapting specific structures. Many of them show tissue-specific expression and are expressed at specific time points during embryonic development, indicating an important role in turning on and off genes at specific circumstances. Deregulated expression of many lncRNAs