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

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

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

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)

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)

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)

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)

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)

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

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)

Tanespimycin reduced GATA3 and SRC-3 DMSO __GW(nM)_ V (uM)___ D T(uM) DMSO 9.0 3.0 1.0 1.0 0.3 0.1 3.0 1 0.3 GATA3 24 hour treatment 0.35 0.54 0.89 1 0.46 0.60 0.72 1 Abbreviation Compound Name V Vorinostat T Tanespimycin P Pioglitazone GW GW8510 B Bexarotene SRC-3 GAPDH DMSO P(uM) DMSO B(uM) ___ DMSO 3.0 1.0 0.3 9.0 3.0 1.0 0.3 DMSO (D) - Vehicle SRC-3 GATA3 GAPDH

Tanespimycin and vorinostat reduced GATA3 and SRC-3 48 hour treatment DMSO V (uM)______ 0.04 0.2 1.0 2.5 DMSO P (uM)_____ DMSO 0.04 0.2 1.0 2.5 Abbreviation Compound Name V Vorinostat T Tanespimycin P Pioglitazone GW GW8510 B Bexarotene GATA3 1 0.75 0.45 0.21 0.24 1 1.04 0.91 0.42 0.38 SRC-3 GAPDH DMSO (D) - Vehicle DMSO B (uM) DMSO 10 2.0 0.4 0.08 T (uM) GW (nM)___ 2.5 1.0 0.2 0.04 10 5 2.5 GATA3 SRC-3 GAPDH

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)

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

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

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