1. Identifying compounds that inhibit the transcription factor GATA3 in MCF-7 breast cancer cells
Vignesh Ramachandran
SMART Summer Research Program
Department of Molecular and Cellular Biology
Baylor College of Medicine

2. Breast cancer facts
Breast cancer is the 2nd leading cause of cancer death in women, exceeded only by lung cancer1
An estimated 1.7 million new breast cancer cases occurred worldwide among women in 20122
Breast cancer accounted for 521,000 deaths worldwide in 20123
American Cancer Society
Susan G. Komen
World Health Organization

3. ER+ breast cancers are the most common types
Subtype 
These tumors tend to be  
Prevalence (approximate) 
Luminal A
ER+ and/or PR+, HER2-, low Ki67
40%
Luminal B
ER+ and/or PR+, HER2+ (or HER2- with high Ki67)
20%
Triple negative/basal-like
ER-, PR-, HER2-
15-20%
HER2 type
ER-, PR-, HER2+
10-15%
ER+ breast cancers are defined by being positive for and driven by ERα
provides a means to find targeted treatments, which typically have less harmful side effects than traditional chemotherapy
ERα interacts with the transcription factor GATA3
Table obtained from: Susan G. Komen

4. GATA3 mRNA levels correlate with that of ERα
GATA-3 binds to two cis-regulatory elements located within the ERα gene, and this is required for RNA polymerase II recruitment to ERα promoters. Reciprocally, ERα directly stimulates the transcription of the GATA-3 gene, indicating that these two factors are involved in a positive cross-regulatory loop
GATA3 participates with ERα (ESR1) in a complex, positive cross-regulatory loop that drives tumor growth4
Table constructed in: cBioPortal for Cancer Genomics Dataset: Breast Invasive Carcinoma (TCGA, Nature 2012)
4. Brown et al. (2007)

5. GATA3 mutations and mRNA levels correlate with ERα mRNA levels
GATA3 mutated
ESR1 mutated
Both mutated
Neither mutated
ESR1, mRNA expression (microarray)
Graph constructed in: cBioPortal for Cancer Genomics Dataset: Breast Invasive Carcinoma (TCGA, Nature 2012)
GATA3, mRNA expression (microarray)

6. GATA3 mutations are driver mutations in breast cancers
Cross-cancer alteration summary for GATA3 (69 studies / 1 genes)
Red – Amplification
Green – Mutation
Blue – Deletion
Grey – Multiple alterations
Graph obtained from: cBioPortal for Cancer Genomics Dataset: Breast Invasive Carcinoma (TCGA, Nature 2012)

7. GATA3 mutations are heavily concentrated at the C-terminus
Second zinc finger determines DNA binding affinity5
Heterozygosity for the truncating mutation (D336fs) in MCF-7 stabilizes and reduces turnover rate of mutant GATA3 through an undefined mechanism5
Figure obtained from: cBioPortal for Cancer Genomics,
5. Wade et al. (2014)

8. Hypotheses
Tanespimycin, vorinostat, pioglitazone, GW8510, and bexarotene will inhibit GATA3 in MCF-7 cells
Compounds that inhibit GATA3 will more effectively reduce cell viability if used in combination with compounds that hinder other cellular proteins implicated in breast cancer (ie. SRC-3 and ERα)

9. Use of UCSC Cancer Genome Browser
GATA3
ESR1
FOXA1
SRC-3
MCF-7
Compounds
RXR agonist - RXR act as ligand-dependent transcription factor that functions as heterodimer with retinoid acid receprtor (RAR) to modulate the transcriptional activity of retinoid receptor target genes associated with cell growth and differentiation
Compound
Function
Vorinostat
Histone deacetylase (HDAC) inhibitor
Tanespimycin
Heat shock protein 90 (Hsp90) inhibitor
Pioglitazone
Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist
GW8510
Cyclin-dependent kinase (CDK) inhibitor
Bexarotene
Retinoid X receptor (RXR) agonist
UCSC Cancer Genome Browser

10. Cell viability assays GW8510 Vorinostat Pioglitazone Tanespimycin
Viable Cells (% of Control)
Viable Cells (% of Control)
nM (log 10)
nM (log 10)
Pioglitazone
Tanespimycin
Viable Cells (% of Control)
Viable Cells (% of Control)
nM (log 10)
nM (log 10)
Bexarotene
Viable Cells (% of Control)
nM (log 10)

11. Tanespimycin reduced GATA3 and SRC-3
DMSO __GW(nM)_ V (uM)___ D T(uM) DMSO
GATA3
24 hour treatment
Abbreviation
Compound Name V
Vorinostat T
Tanespimycin P
Pioglitazone GW
GW8510 B
Bexarotene
SRC-3
GAPDH
DMSO P(uM) DMSO B(uM) ___ DMSO
DMSO (D) - Vehicle
SRC-3
GATA3
GAPDH

12. Tanespimycin and vorinostat reduced GATA3 and SRC-3
48 hour treatment
DMSO V (uM)______
DMSO P (uM)_____ DMSO
Abbreviation
Compound Name V
Vorinostat T
Tanespimycin P
Pioglitazone GW
GW8510 B
Bexarotene
GATA3
SRC-3
GAPDH
DMSO (D) - Vehicle
DMSO B (uM) DMSO
T (uM) GW (nM)___
GATA3
SRC-3
GAPDH

13. Combinational Treatment with Tamoxifen (4HT) and Bufalin
Tamoxifen is an antagonist of the estrogen receptor via its active metabolite, 4-hydroxytamoxifen; shuts down ERα pathway
Bufalin is a potent small-molecule inhibitor of SRC-3, which is implicated in cancer cell proliferation, invasion, and metastasis6 ; inhibit the potential SRC-3 alternative mechanism when the ERα pathway is cut off
6. O’Malley BW, et al. (2014)

14. Cell viability assays: T and V with Tamoxifen (4HT) or bufalin
Viable Cells (% of Control)
Combinational treatment with Tamoxifen or bufalin seem to demonstrate additive effects
Synergistic effects do not seem evident
Concentration of Tanespimycin
Viable Cells (% of Control)
Concentration of Vorinostat

15. Go back to hypothesis; restate + answer Future work + implications
Summary
Hypothesis: Tanespimycin, vorinostat, pioglitazone, GW8510, and bexarotene will inhibit GATA3
Tanespimycin and vorinostat inhibited GATA3
Hypothesis: Compounds that inhibit GATA3 more effective in combinational treatments
Combinational treatment with Tamoxifen or bufalin were seemingly not synergistic (an additive effect was observed)
Go back to hypothesis; restate + answer
Future work + implications
Slide thanking peeps
Slide per point

16. Future Work and Implications
Investigation of synergistic, combinational compound treatments
Use of mouse models to study effectiveness of tanespimycin and vorinostat in vivo
Tanespimycin and vorinostat are FDA-approved drugs; clinical trials can be conducted on patients with GATA3 mutations similar to that found in the MCF-7 cell line