More on FOX News: FOXA1 on the horizon of estrogen receptor function and endocrine response

Estrogen receptor α (ER) is a major driver of breast cancer and the target of endocrine therapy. Full disclosure of the cofactors regulating ER interactions with chromatin and its transcriptional regulatory activity is still elusive. Novel genome-wide profiling tools have mapped ER binding events in breast cancer cells and delineated cofactors important in ER activity. Among these, the Forkhead protein FOXA1 is emerging as a key factor dictating global chromatin structure and the transcriptional function of ER in breast and non-breast cancer cells. The significance of FOXA1 in the chromatin interactions and transcriptional regulation of both estrogen- and tamoxifen-bound ER, and in supporting tamoxifen-resistant cell growth, may impact current endocrine therapies.


Background
Th e estrogen receptor α (ER) protein is present in over two-thirds of breast cancers, where it functions in the nucleus as a ligand-dependent transcription factor to drive cell proliferation, survival, and invasiveness. Endocrine therapies to block ER activity are the most important systemic treatments for ER-positive breast cancers, though resistance is prevalent [1]. We need to understand the molecular determinants regulating ER DNA binding and activity to elucidate the mechanisms underlying this resistance.
Th e advancement of chromatin immunoprecipitation (ChIP)-based technologies, which combine ChIP with microarrays or high throughput sequencing (ChIP-seq), has helped to identify a network of co-regulators and their genome-wide DNA binding sites (known as their cistrome) that cooperate to regulate ER DNA binding and transcriptional activity. Th ese technologies have revealed that, in breast cancer cells, ER mostly binds to distal enhancers that are also enriched for Forkhead motifs [2][3][4]. Furthermore, the Forkhead protein FOXA1, a favorable prognostic factor that correlates with the luminal A breast cancer subtype and hormonal sensitivity [5], has been shown to act as a pioneer factor, opening chromatin regions for the recruitment of ER to these DNA binding sites [6]. However, how global the importance of FOXA1 is in mediating ER function in breast cancer, as well as in other target tissues and under diff erent ligand conditions, and what are the underlying factors that determine FOXA1 specifi city remain open questions.

The article
To more broadly investigate the genome-wide relationship of ER and FOXA1 DNA-binding sites, Hurtado and colleagues [7] fi rst performed ChIP-seq of ER and FOXA1 in three diff erent breast cancer cell lines. FOXA1 binding events were found to be dynamic and cell-linespecifi c, a phenomenon potentially related to the insulator protein CTCF. Within each cell line, a signifi cant overlap of over 50% was found between ER and FOXA1 sites. FOXA1 was also found to mediate ER function in non-breast cancer cells and to act upstream of ERchromatin interactions, enabling ER binding at more con densed chromatin regions. Additionally, FOXA1 was required to globally facilitate ER-mediated transcription, since downregulation of FOXA1 aff ected the transcription of more than 95% of estrogen-regulated genes. Finally, FOXA1 knockdown resulted in signifi cant growth inhibition of MCF7 cells, substantiating the key role of FOXA1 in the estrogen response of breast cancer cells.
To study the ER cistromic profi le and the role of FOXA1 in mediating tamoxifen inhibition, estrogendeprived MCF-7 cells treated with estrogen or tamoxifen were subjected to ER ChIP-seq and gene expression micro array analyses. In contrast to a previous report [8],

Abstract
Estrogen receptor α (ER) is a major driver of breast cancer and the target of endocrine therapy. Full disclosure of the cofactors regulating ER interactions with chromatin and its transcriptional regulatory activity is still elusive. Novel genome-wide profi ling tools have mapped ER binding events in breast cancer cells and delineated cofactors important in ER activity. Among these, the Forkhead protein FOXA1 is emerging as a key factor dictating global chromatin structure and the transcriptional function of ER in breast and non-breast cancer cells. The signifi cance of FOXA1 in the chromatin interactions and transcriptional regulation of both estrogen-and tamoxifen-bound ER, and in supporting tamoxifen-resistant cell growth, may impact current endocrine therapies. the results demonstrated that tamoxifen induced ER binding events similar to those induced by estrogen. Additionally, estrogen and tamoxifen were found to regulate common genes. FOXA1 knockdown showed that tamoxifen-ER employs similar FOXA1-dependent mechanisms as estrogen to interact with chromatin. However, the experimental setting prevented direct assess ment of whether FOXA1 is required for the tamoxifen antiproliferative eff ects in breast cancer cells. Of note, in tamoxifen-resistant derivatives of MCF-7 cells, chromatin binding profi les of both ER and FOXA1 dramatically diff er from those of the wild-type cell line, and the binding occurred independently of tamoxifen treatment. However, ER and FOXA1 binding regions still signifi cantly overlapped and, most importantly, ER chromatin binding and cell proliferation in the tamoxifenresistant line still required FOXA1.

The viewpoint
Unbiased, genome-wide mapping and profi ling of ER interaction with chromatin and its transcriptional regulation activity in breast cancer have recently been estab lished by leading groups in this fi eld [2-4,9,10] and have created a valuable resource to increase our basic under standing of estrogen/ER action and to improve thera peutic strategies. Th ese studies have collectively demon strated the fundamental role of FOXA1 in guiding and regulating ER chromatin binding events and gene transcription. While the recent report of Hurtado and colleagues [7] strongly substantiates these previous data and notions, its novel insights into the role of FOXA1 in both tamoxifen action and resistance, and into mechanistic aspects of FOXA1 action, are of clinical and biological signifi cance.
Th e pivotal role of FOXA1 in ER-DNA interaction and transcriptional activity, as well as in the growth of wild type MCF7 cells, points to FOXA1 as a potential therapeutic target for opposing ER activity and tumor growth, either alone or in combination with additional endocrine targets. Conversely, since the tamoxifen-ER interaction with DNA, which is a key component of tamoxifen's inhibitory action on ER-dependent gene expression, was also found to depend tightly on FOXA1, one could argue that co-targeting of FOXA1 might antagonize, instead of enhance, the inhibitory capabilities of anti-estrogens such as tamoxifen. Notably, however, in tamoxifen-resistant cells, the extensively altered pattern of ER binding and resistant cell growth phenotype both required FOXA1, an observation pointing again to FOXA1 as a therapeutic candidate. Interestingly, Lupien and colleagues [11] recently showed that epidermal growth factor stimulation of MCF7 cells induces a ligand-independent distinct profi le of ER binding sites that is still enriched for Forkhead binding sites. Th is growth factor (GF)-specifi c ER cistrome dictates a unique transcriptional program that correlates with gene expression signatures found in poor-outcome as well as HER2-positive breast cancers [11]. Similarly, a unique ER cistrome and ER trans criptional activation were also documented upon AKT activation [12], and others have shown that blocking this pathway with a PI3K inhibitor can restore the 'classic' pattern of E2-induced gene expression [13]. Acquired tamoxifen resis tance of MCF7 cells and xenografts is also associated with increased GF/HER1/2 signaling and a distinct expression profi le that correlates with growth factor gene expression signatures [14,15]. As aberrant expression/activation of GF pathways is commonly associated with endocrine resistance [1,15], the data cumulatively imply that the diff erential ER chromatin binding and subse quently altered transcriptional program that derive from hyperactive GF signaling are fundamental underlying mechanisms of endocrine resistance. Th e relative contri butions to this global molecular switch of ER-chromatin interactions and gene expression provided by (1) non-genomic ER function, (2) additional Forkhead family members, (3) other chromatin remodeling factors and/or ER coregulators, (4) posttranslational modifi cation of these key factors, and (5) additional epigenetic modifi ca tions are still open questions. However, the critical roles of FOXA1 and ER in this process, as shown by Hurtado and colleagues [7], indeed suggest that co-targeting of FOXA1 together with more potent ER inhibitors or degraders [11] might represent an improved strategy to circumvent endocrine resistance in breast cancer.

Competing interests
The authors declare that they have no competing interests.