Targeting Foxa1 In Estrogen Receptor-Positive Breast Cancer: Biological Characterization Of Kinase Regulators
CANCER RESEARCH(2016)
Abstract
Estrogen receptor (ER) is the driving and defining transcription factor in 75% of breast cancers and is directly inhibited by targeted therapies such as tamoxifen. DNA interactions and transcriptional potential of ER depend on the pioneer factor, FoxA1, which thereby plays an essential role in determining tumour growth and progression - even when resistance to existing drugs has developed. The aim of this study is to characterize kinase regulators of FoxA1 function in order to identify druggable targets for the indirect inhibition of ER. To identify kinase regulators in ER+ breast cancer, primary screening of multiple cell lines was performed using an siRNA library of the human kinome. After secondary screening of ER+ specific growth-regulatory kinases using a stable luciferase reporter of FoxA1 activation, 50 putative kinase regulators of FoxA1 were identified. These were cross-referenced with kinase inhibitors of FoxA1 activation discovered via a separate cell-based compound screen and competitive-binding kinase displacement assay. This two-pronged screening approach revealed 6 common putative FoxA1-regulatory kinase enzymes. These were characterized by chromatin immunoprecipitation (ChIP) of FoxA1 and ER with quantitative PCR of archetypal ER enhancers, 48 hours after siRNA knockdown of the kinase. This revealed 3 kinases whose inhibition prevented FoxA1 and ER from accessing the chromatin, and 1 kinase that had the opposite effect. Mass spectrometry-based proteomic analysis of endogenous FoxA1-protein interactors using ChIP is underway to assess the change in FoxA1 co-factors following inhibition of these 4 candidate kinases. We used a proteomic purification method called RIME which permits analysis of phosphorylation events on FoxA1. We are currently assessing whether silencing of specific kinases influences phosphorylation of the putative substrate, FoxA1. In summary, the identified kinases demonstrate promising FoxA1 specificity, and may provide druggable targets for the inhibition of ER via FoxA1. This study has potential clinical value for patients with tumours that have become resistant to existing ER-targeted therapies. Citation Format: Simon J. Johnston, Kelly A. Holmes, Jason S. Carroll. Targeting FoxA1 in estrogen receptor-positive breast cancer: biological characterization of kinase regulators. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2906.
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Key words
foxa1,breast cancer,kinase,receptor-positive
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