Counterscreen for inhibitors of COUP-TFII (NR2F2): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16)
Name: Counterscreen for inhibitors of COUP-TFII (NR2F2): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16). ..more
BioActive Compound: 1
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Ming-Jer Tsai, Baylor College of Medicine
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01DK45641
Grant Proposal PI: Ming-Jer Tsai, Baylor College of Medicine
External Assay ID: VP16_INH_LUMI_1536_3XIC50 DCSRUN for COUP-TFII (NR2F2)
Name: Counterscreen for inhibitors of COUP-TFII (NR2F2): Luminescence-based cell-based high throughput dose response assay to identify inhibitors of the Herpes Virus Virion Protein 16 (VP16).
Steroid receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) (1), an orphan nuclear receptor and member of the nuclear receptor superfamily, has been shown to be a critical transcriptional regulator in many different cancer types by promoting angiogenesis (2-4), cell proliferation and metastasis (5-11). COUP-TFII has widespread tissue distribution in human; detectable expression has been found in every tissue type examined (12). Currently, the treatment for tumor angiogenesis focuses mainly on blocking VEGFR-2 signaling and has not been effective due to limited efficacy, eventually leading to resistance and/or relapse. COUP-TFII has been shown to promote tumor angiogenesis through modulating multiple angiogenic signals (VEGF/VEGFR-2, Angiopoietin 1/Tie2 and E2F-1) in many different types of cancer (13-14). In addition, COUP-TFII is overexpressed in prostate and several other cancers and is an excellent prognostic marker. By including COUP-TFII data with Cyclin D1, p21, PTEN, and Smad4 data in the prognosis, the prognostic accuracy is improved. The expression level of COUP-TFII and its role in regulating tumor growth and metastasis in prostate cancer has been examined, and these data indicate that COUP-TFII positively promotes prostate tumor growth and metastasis (15). These results provide the rational basis to posit that inhibition of COUP-TFII may offer a novel and broadly efficacious approach for anticancer intervention.
COUP-TFII has also been shown to regulate energy storage and expenditure. We have found that COUP-TFII heterozygous mice have increased mitochondrial biogenesis in white adipose tissue, which results in higher energy expenditure, resulting in resistance to high fat diet-induced obesity and improved glucose homeostasis due to increased insulin sensitivity at peripheral tissues (16). These results indicate that COUP-TFII has an important role in regulating adipocyte differentiation and energy metabolism. Therefore, COUP-TFII inhibitors could potentially serve as agents to improve insulin sensitivity, enhance energy metabolism, and decrease high fat diet-induced obesity.
1. Sagami, I., Tsai, S. Y., Wang, H., Tsai, M. J., and O'Malley, B. W. (1986) Identification of two factors required for transcription of the ovalbumin gene, Mol Cell Biol 6, 4259-4267.
2. Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of cancer: the next generation, Cell 144, 646-674.
3. Lin, F. J., Chen, X., Qin, J., Hong, Y. K., Tsai, M. J., and Tsai, S. Y. (2010) Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development, J Clin Invest 120, 1694-1707.
4. Pereira, F. A., Qiu, Y., Zhou, G., Tsai, M. J., and Tsai, S. Y. (1999) The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development, Genes Dev 13, 1037-1049.
5. Annecke, K., Schmitt, M., Euler, U., Zerm, M., Paepke, D., Paepke, S., von Minckwitz, G., Thomssen, C., and Harbeck, N. (2008) uPA and PAI-1 in breast cancer: review of their clinical utility and current validation in the prospective NNBC-3 trial, Adv Clin Chem 45, 31-45.
6. Harbeck, N., Kates, R. E., Schmitt, M., Gauger, K., Kiechle, M., Janicke, F., Thomassen, C., Look, M. P., and Foekens, J. A. (2004) Urokinase-type plasminogen activator and its inhibitor type 1 predict disease outcome and therapy response in primary breast cancer, Clin Breast Cancer 5, 348-352.
7. Litchfield, L. M., and Klinge, C. M. (2012) Multiple roles of COUP-TFII in cancer initiation and progression, J Mol Endocrinol 49, R135-148.
8. Litchfield, L. M., Riggs, K. A., Hockenberry, A. M., Oliver, L. D., Barnhart, K. G., Cai, J., Pierce, W. M., Jr., Ivanova, M. M., Bates, P. J., Appana, S. N., Datta, S., Kulesza, P., McBryan, J., Young, L. S., and Klinge, C. M. (2012) Identification and characterization of nucleolin as a COUP-TFII coactivator of retinoic acid receptor beta transcription in breast cancer cells, PLoS One 7, e38278.
9. Navab, R., Gonzalez-Santos, J. M., Johnston, M. R., Liu, J., Brodt, P., Tsao, M. S., and Hu, J. (2004) Expression of chicken ovalbumin upstream promoter-transcription factor II enhances invasiveness of human lung carcinoma cells, Cancer Res 64, 5097-5105.
10. Riggs, K. A., Wickramasinghe, N. S., Cochrum, R. K., Watts, M. B., and Klinge, C. M. (2006) Decreased chicken ovalbumin upstream promoter transcription factor II expression in tamoxifen-resistant breast cancer cells, Cancer Res 66, 10188-10198.
11. Shimizu, M., Cohen, B., Goldvasser, P., Berman, H., Virtanen, C., and Reedijk, M. (2011) Plasminogen activator uPA is a direct transcriptional target of the JAG1-Notch receptor signaling pathway in breast cancer, Cancer Res 71, 277-286.
12. Suzuki, T., Moriya, T., Darnel, A. D., Takeyama, J., and Sasano, H. (2000) Immunohistochemical distribution of chicken ovalbumin upstream promoter transcription factor II in human tissues, Mol Cell Endocrinol 164, 69-75.
13. Qin, J., Chen, X., Xie, X., Tsai, M. J., and Tsai, S. Y. (2010) COUP-TFII regulates tumor growth and metastasis by modulating tumor angiogenesis, Proc Natl Acad Sci U S A 107, 3687-3692.
14. Qin, J., Chen, X., Yu-Lee, L. Y., Tsai, M. J., and Tsai, S. Y. (2010) Nuclear receptor COUP-TFII controls pancreatic islet tumor angiogenesis by regulating vascular endothelial growth factor/vascular endothelial growth factor receptor-2 signaling, Cancer Res 70, 8812-8821.
15. Qin, J., Wu, S. P., Creighton, C. J., Dai, F., Xie, X., Cheng, C. M., Frolov, A., Ayala, G., Lin, X., Feng, X. H., Ittmann, M. M., Tsai, S. J., Tsai, M. J., and Tsai, S. Y. (2012) COUP-TFII inhibits TGF-beta-induced growth barrier to promote prostate tumorigenesis, Nature.
16. Li, L., Xie, X., Qin, J., Jeha, G. S., Saha, P. K., Yan, J., Haueter, C. M., Chan, L., Tsai, S. Y., and Tsai, M. J. (2009) The nuclear orphan receptor COUP-TFII plays an essential role in adipogenesis, glucose homeostasis, and energy metabolism, Cell Metab 9, 77-87.
17. Pipaon, C., Tsai, S. Y., and Tsai, M. J. (1999) COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site, Mol Cell Biol 19, 2734-2745.
DCSRUN, counterscreen, secondary, triplicate, dose, dose response, titration, dilutionVirion Protein 16, VP16, herpes simplex virus, VP-16, lumi, luminescence, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to determine whether the hits identified as active in a set of experiments entitled, Luminescence-based cell-based primary high throughput screening assay to identify antagonists of COUP-TFII (NR2F2) (AID 686940), are assay artifacts affecting luciferase activity, cell viability or the transcription/translation machinery in a non-selective manner.
HEK293 cells were routinely cultured in T-175 flasks containing 25 mL of DMEM media supplemented with 10% v/v fetal bovine serum and 1% v/v antibiotic-antimycotic mix at 37 C, 5% CO2 and 95% relative humidity (RH). The day prior to run the assay, the HEK293 cells were harvested using 5 mL of TrypLE reagents and seeded in fresh media at a density of 10 million cells per T175 flask. The following day, cells were transfected with 1 mL of serum-free OptiMEM containing 9.9 ug of the plasmid encoding VP16 (pAB-Gal4-VP16), 9.9 ug of the p17 mer x4-TK-Luc luciferase reporter plasmid, and 65 uL of X-tremeGene 9 transfection reagent. Twenty four hours post transfection, cells were harvested using 5 mL of preheated TrypLE and resuspended at a concentration of 800,000 cells per mL in phenol-red free DMEM supplemented as above. In the absence of a pharmacological positive control, VP16 inhibition was mimicked by transfecting cells in absence of the VP16 expressing vector (i.e. reporter plasmid only). Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 85.4 uM.
The assay was started by dispensing 5 uL of cell suspension into each well of white, sterile, solid-bottom 1536-well plates. The first three columns received control cells (no VP16 expressed) whereas the rest of the plate was dispensed with VP16-transfected cells. After addition of cells the plates were spun down. The plates were incubated for 4 hours and then treated with 43 nL/well of test compounds or DMSO (final concentration 0.65%) on COUP-TF2 cells and Control cells. Plates were incubated for eighteen hours at 37 C, 5% CO2 and 95% RH. Plates were then removed from the incubator and equilibrated to room temperature for 10 minutes. Luciferase activity was detected by addition of 5 uL of One-Glo reagent to each well. After a 10 minute incubation time, light emission was measured with the ViewLux reader (PerkinElmer).
The percent inhibition for each compound was calculated as follows:
%_Inhibition = ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Control_Field ) ) * 100
Low_Control is defined as wells
High_Control is defined as wells containing cells transfected with reporter plamid only (p17 mer x4-TK-Luc)
Test_Compound is defined as well containing cells cotransfected with pAB-Gal4-VP16 and p17 mer x4-TK-Luc in the presence of test compounds
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 100-0.
List of Reagents:
p17 mer x4-TK-Luc luciferase reporter plasmid (Assay Provider)
pAB-Gal4-VP16 plasmid (Assay Provider)
HEK293 cells (ATCC, part CRL-1573)
DMEM (Invitrogen, part 21063)
FBS (Hyclone, part SH30088.03)
Antibiotic-Antimycotic 100X Liquid Solution (Gibco, part 15240)
X-tremeGENE 9 DNA Transfection Reagent (Roche, part 06365809001)
OptiMEM (Invitrogen, part 31985)
TrypLE Trypsin Replacement Enzyme (Invitrogen, part 12604)
OneGlo (Promega, part E6130)
1536-well plates (Corning, part 7298)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Assay: Dictionary: Version: 0.1
Assay: CurveFit : Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit : Mask: Excluded Points
* Activity Concentration. ** Test Concentration.
Data Table (Concise)