High Throughput Chemical Screening Reveals Multiple Regulatory Proteins on FOXA1 in Breast Cancer Cell Lines.

Shixiong Wang,Sachin Singh,Madhumohan Katika,Antoni Hurtado,Sandra Lopez-Aviles

doi:10.3390/ijms19124123

Shixiong Wang, Sachin Singh + Show 3 more

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https://doi.org/10.3390/ijms19124123

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Abstract

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.

Highlights

Forkhead Box A1 (FOXA1), known as hepatocyte nuclear factor 3 alpha, belongs to the forkhead family of transcription factors and plays pivotal roles in the development of prostate and mammary gland [1,2]
The findings from the drug screening indicate that additional kinases have a positive control in HER2 positive cells compared to MCF-7 cells
These results suggest the hypothesis that the binding of FOXA1 to the chromatin in HER2 enriched cells is

Summary

Introduction

Forkhead Box A1 (FOXA1), known as hepatocyte nuclear factor 3 alpha, belongs to the forkhead family of transcription factors and plays pivotal roles in the development of prostate and mammary gland [1,2]. FOXA1 is a pioneer transcription factor with a DNA binding domain that resembles the structure of linker histones [3,4]. FOXA1 is able to control the expression of TET1 and interact with it to regulate local DNA methylation and the activity of corresponding enhancer activity [9]. These properties confer the ability of FOXA1 to act as a pioneer factor for both estrogen receptor α [10,11] and androgen receptor [12], controlling their binding, location, and function in breast and prostate cancer respectively

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