High Throughput Chemical Screening Reveals Multiple Regulatory Proteins on FOXA1 in Breast Cancer Cell Lines
Breast Cancer Research group, Nordic EMBL Partnership, Centre for Molecular Medicine Norway (NCMM), University of Oslo, P.O. 1137 Blindern, 0318 Oslo, Norway
Cell Cycle Research group, Nordic EMBL Partnership, Centre for Molecular Medicine Norway (NCMM), University of Oslo, P.O. 1137 Blindern, 0318 Oslo, Norway
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2018, 19(12), 4123; https://doi.org/10.3390/ijms19124123
Received: 30 October 2018 / Revised: 13 December 2018 / Accepted: 14 December 2018 / Published: 19 December 2018
(This article belongs to the Special Issue Sex Hormone Receptor Signals in Human Malignancies)
Forkhead box A1 (FOXA1) belongs to the forkhead class transcription factor family, playing pioneering function for hormone receptors in breast and prostate cancers, and mediating activation of linage specific enhancers. Interplay between FOXA1 and breast cancer specific signaling pathways has been reported previously, indicating a regulation network on FOXA1 in breast cancer cells. Here in this study, we aimed to identify which are the proteins that could potentially control FOXA1 function in breast cancer cell lines expressing different molecular markers. We first established a luciferase reporter system reflecting FOXA1 binding to DNA. Then, we applied high throughput chemical screening of multiple protein targets and mass spectrometry in breast cancer cell lines expressing different molecular markers: ER positive/HER2 negative (MCF-7), ER positive/HER2 positive (BT474), and ER negative/HER2 positive (MDA-MB-453). Regardless of estrogen receptor status, HER2 (human epidermal growth factor receptor 2) enriched cell lines showed similar response to kinase inhibitors, indicating the control of FOXA1 by cell signaling kinases. Among these kinases, we identified additional receptor tyrosine kinases and cyclin-dependent kinases as regulators of FOXA1. Furthermore, we performed proteomics experiments from FOXA1 inmunoprecipitated protein complex to identify that FOXA1 interacts with several proteins. Among all the targets, we identified cyclin-dependent kinase 1 (CDK1) as a positive factor to interact with FOXA1 in BT474 cell line. In silico analyses confirmed that cyclin-dependent kinases might be the kinases responsible for FOXA1 phosphorylation at the Forkhead domain and the transactivation domain. These results reveal that FOXA1 is potentially regulated by multiple kinases. The cell cycle control kinase CDK1 might control directly FOXA1 by phosphorylation and other kinases indirectly by means of regulating other proteins.