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Thu Nguyen Poster 2014 MIdyear
1
Role of Casein Kinase 2 Inhibi1on in Estrogen Receptor Posi1ve Breast Cancer Thu Nguyen 1 , Marlon Williams 1 , Jamal Pratt 1 , Patrick Carriere 1 , Shawn Llopis 1 , Syreeta L. Tilghman 1 , Christopher Williams 1 Division of Basic Pharmaceutical Sciences, College of Pharmacy 1 , Xavier University of Louisiana, New Orleans, LA 70125 Hypothesis Methodology Protein kinase CK2 is involved in ER-dependent cell cycle progression of breast cancer cell proliferation. Abstract Background: De novo or acquired resistance to anti-estrogens in estrogen receptor (ER)+ breast cancer has been attributed to ligand independent activation of the ER. Cross-talk with kinase signaling pathways may mediate ligand-independent activation of the ER, resulting in tumor progression despite anti-estrogen therapy. Protein kinase CK2, a ubiquitously expressed serine threonine kinase, has been shown to impact the transcriptional activity of nuclear receptors. In this study, we sought to ascertain the impact of CK2 inhibition on gene expression and impact of CK2 inhibition on ER dependent breast cancer cell proliferation. Methods: In order to ascertain the impact of CK2 on estrogen dependent gene expression, T47D breast cancer cells were cultured in the presence of tetrabromocinnamic acid (TBCA), a selective inhibitor of CK2, and ERE-driven luciferase activity and estrogen responsive gene expression (PR, cMYC, CCND1). Following exposure to TBCA in the presence or absence of estradiol, cell cycle progression and stress-associated cellular senescence were assayed by flow cytometry. Results: TBCA treatment resulted in decreased estradiol-induced ERE-luciferase activity. Interestingly, TBCA had gene specific effects on estrogen responsive genes, potentiating estradiol induced activation of cMYC and PGR, while inhibiting induction of CCND1 expression. Conclusions: These studies show, for the first time, that CK2 modulates ER transcriptional activity in a gene specific manner, and that CK2 is involved in ER-dependent cell cycle progression by supporting estrogen induced CCND1 expression. These studies suggest a role for CK2 as nuclear receptor co-activator, and that CK2 inhibition may be a viable adjunct to anti-estrogen therapy in the treatment of breast cancer. Discussion Acknowledgments Research Centers for Minority Institutions (RCMI) Louisiana Cancer Research Consortium Xavier University of Louisiana RCMI Grant 3G12MD007595-04 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 DMSO 800nM CK2 Inh 400nM CK2 Inh 200nM CK2 Inh 1nM E2 100nM Tamoxifen 100nM Tam + 400nM CK2 Inh 1nM E2 + 400nM CK2 Inh % Control (DMSO) Treatments CK2 Inhibi:on Decreases ERE Transcrip:onal Ac:vity Results Figure 3. ERE-driven Luciferase activity of T47D breast cancer cells T47D breast cancer cells were treated with varying concentrations of a selective CK2 inhibitor (TBCA), alone or in combination with Tamoxifen or estradiol (E2) and the resultant activity is shown as percent control of activity. Cells treated with TBCA showed a decrease in estradiol-induced ERE-luciferase activity. 0 1 2 3 4 5 6 7 8 Con E2 Fold Expression PGR (TCBA) (+TBCA) 0 5 10 15 20 25 30 Con E2 Fold Expression CMYC (TCBA) (+TBCA) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Con E2 Fold Expression CCND1 (TCBA) (+TBCA) Figure 4. Gene expression of TBCA-treated T47D breast cancer cells Estrogen responsive genes were analyzed in cells treated with CK2 inhibitor, TBCA. (Top panel) Cells treated with TBCA showed an increase in progesterone receptor (PCR) independent of estradiol. (Middle panel) Oncogene CMYC, which is typically known as a cell cycle promoter, shows increased expression with TBCA, agreeing with its paradoxical function as a cell cycle arrest promoter. (Bottom panel) TBCA reduces the expression of cyclin- D1 (CCND1). Figure 1. Luciferase Assay Protocol to measure ERE-activity Figure 2. Chemical Structure of CK2 inhibitor TBCA (E)3(2,3,4,5tetrabromophenyl)acrylic acid • CK2 modulates ER transcriptional activity in a gene specific manner • CK2 is involved in ER-dependent cell cycle progression • CK2 has a possible role as a nuclear receptor co-activator
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Transcript of Thu Nguyen Poster 2014 MIdyear
Role of Casein Kinase 2 Inhibi1on in Estrogen Receptor Posi1ve Breast Cancer
Thu Nguyen1, Marlon Williams1, Jamal Pratt1, Patrick Carriere1, Shawn Llopis1, Syreeta L. Tilghman1, Christopher Williams1 Division of Basic Pharmaceutical Sciences, College of Pharmacy1, Xavier University of Louisiana, New Orleans, LA 70125
Hypothesis
Methodology
Protein kinase CK2 is involved in ER-dependent cell cycle progression of breast cancer cell proliferation.
Abstract Background: De novo or acquired resistance to anti-estrogens in estrogen receptor (ER)+ breast cancer has been attributed to ligand independent activation of the ER. Cross-talk with kinase signaling pathways may mediate ligand-independent activation of the ER, resulting in tumor progression despite anti-estrogen therapy. Protein kinase CK2, a ubiquitously expressed serine threonine kinase, has been shown to impact the transcriptional activity of nuclear receptors. In this study, we sought to ascertain the impact of CK2 inhibition on gene expression and impact of CK2 inhibition on ER dependent breast cancer cell proliferation. Methods: In order to ascertain the impact of CK2 on estrogen dependent gene expression, T47D breast cancer cells were cultured in the presence of tetrabromocinnamic acid (TBCA), a selective inhibitor of CK2, and ERE-driven luciferase activity and estrogen responsive gene expression (PR, cMYC, CCND1). Following exposure to TBCA in the presence or absence of estradiol, cell cycle progression and stress-associated cellular senescence were assayed by flow cytometry. Results: TBCA treatment resulted in decreased estradiol-induced ERE-luciferase activity. Interestingly, TBCA had gene specific effects on estrogen responsive genes, potentiating estradiol induced activation of cMYC and PGR, while inhibiting induction of CCND1 expression. Conclusions: These studies show, for the first time, that CK2 modulates ER transcriptional activity in a gene specific manner, and that CK2 is involved in ER-dependent cell cycle progression by supporting estrogen induced CCND1 expression. These studies suggest a role for CK2 as nuclear receptor co-activator, and that CK2 inhibition may be a viable adjunct to anti-estrogen therapy in the treatment of breast cancer.
Discussion Acknowledgments Research Centers for Minority Institutions (RCMI) Louisiana Cancer Research Consortium Xavier University of Louisiana RCMI Grant 3G12MD007595-04
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
DMSO 800nM CK2 Inh
400nM CK2 Inh
200nM CK2 Inh
1nM E2 100nM Tamoxifen
100nM Tam + 400nM CK2
Inh
1nM E2 + 400nM CK2
Inh
% Con
trol (D
MSO
)
Treatments
CK2 Inhibi:on Decreases ERE Transcrip:onal Ac:vity
Results
Figure 3. ERE-driven Luciferase activity of T47D breast cancer cells T47D breast cancer cells were treated with varying concentrations of a selective CK2 inhibitor (TBCA), alone or in combination with Tamoxifen or estradiol (E2) and the resultant activity is shown as percent control of activity. Cells treated with TBCA showed a decrease in estradiol-induced ERE-luciferase activity.
0 1 2 3 4 5 6 7 8
Con E2
Fold Expression
PGR
(-‐TCBA)
(+TBCA)
0
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Con E2
Fold Expression
CMYC
(-‐TCBA)
(+TBCA)
0 0.2 0.4 0.6 0.8 1
1.2 1.4 1.6
Con E2
Fold Expression
CCND1
(-‐TCBA)
(+TBCA)
Figure 4. Gene expression of TBCA-treated T47D breast cancer cells Estrogen responsive genes were analyzed in cells treated with CK2 inhibitor, TBCA. (Top panel) Cells treated with TBCA showed an increase in progesterone receptor (PCR) independent of estradiol. (Middle panel) Oncogene CMYC, which is typically known as a cell cycle promoter, shows increased expression with TBCA, agreeing with its paradoxical function as a cell cycle arrest promoter. (Bottom panel) TBCA reduces the expression of cyclin- D1 (CCND1).
Figure 1. Luciferase Assay Protocol to measure ERE-activity
Figure 2. Chemical Structure of CK2 inhibitor TBCA (E)-‐3-‐(2,3,4,5-‐tetrabromophenyl)acrylic acid
• CK2 modulates ER transcriptional activity in a gene specific manner
• CK2 is involved in ER-dependent cell cycle progression • CK2 has a possible role as a nuclear receptor co-activator
