Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of COUP-TFII (NR2F2)
Name: Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of COUP-TFII (NR2F2). ..more
BioActive Compounds: 2602
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: COUPTFII_INH_LUMI_1536_1X%INH PRUN
Name: Luminescence-based cell-based primary high throughput screening assay to identify inhibitors of COUP-TFII (NR2F2).
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.
PRUN, primary, HTS, nuclear hormone receptor, NHR, reporter assay, luciferase, LUMI, COUPTFII, chicken ovalbumin upstream promoter, COUP transcription factor 2, NR2F2, ARP1, COUPTFB, NF-E3, NR2F1, SVP40, TFCOUP2, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this cell-based assay is to identify compounds that can inhibit COUP-TFII transcriptional activity. The expression vectors for COUP-TFII (pcDNA6.2-COUP-TFII) and the luciferase reporter NGFIA-Luc (pXP2-168) are transiently cotransfected into HEK-293T cells.
COUP-TFII has been shown to efficiently activate NGFI-A-Luc expression (17) and the readout can be measured by a luminometer. Small molecules that inhibit COUP-TFII transcriptional activity will decrease the promoter activity that can be detected by luciferase assay. As designed, compounds that inhibit COUP-TFII activity will decrease luciferase activity, resulting in decreased well luminescence.
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 12 ug of the plasmid encoding for COUP-TF2, 8 ug of the pX2-168 luciferase reporter plasmid, and 60 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, COUP-TF2 inhibition was mimicked by transfecting cells in absence of the COUP-TF2 expressing vector (i.e. reporter plasmid only).
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 COUP-TF2 expressed) whereas the rest of the plate was dispensed with COUP-TF2-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_Sample_Field ) / ( Median_High_Control - Median_Sample_Field ) ) * 100
High_Control is defined as wells containing cells transfected with reporter plamid only (pX2-168)
Test_Compound is defined as well containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds
Sample_Field is defined as wells containing cells cotransfected with pcDNA6.2-COUP-TFII and pX2-168 in the presence of test compounds
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells that are within the High and Low Controls (i.e. higher than the average plues three standard deveiations of the Low Control wells and lower than the average minus three stardard deviation of the High Control wells) and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-34, and for inactive compounds 34-0.
List of Reagents:
pX2-168 luciferase reporter plasmid (Assay Provider)
pcDNA6.2-COUP-TFII 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.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)