Research

Oestrogen receptor is the defining feature of luminal breast cancers, where it functions as a transcription factor to induce cell cycle progression. ER is also the target of most endocrine therapies, including tamoxifen and aromatase inhibitors, which are effective treatments. However, some women can develop resistance to these drugs and in many cases, ER simply gets switched back on again, despite the presence of the drug. Therefore, understanding how ER functions is an important issue and one that has not been completely resolved. ER transcriptional activity requires a number of co-factors and co-operating transcription factors that possess enzymatic activity to alter chromatin structure, the outcome of which determines transcriptional activity. It is currently known that a number of ER co-factors can either assist in transcription (including SRC-1 and AIB-1) or are involved in gene repression by tamoxifen (including N-CoR and SMRT).

Recently, using chromatin immunoprecipitation (ChIP) combined with high-throughput sequencing (ChIP-seq), we mapped all ER binding sites in a breast cancer cell model after oestrogen treatment. This unbiased identification of the genomic contact points of ER revealed a number of surprising features about ER biology. These included the observation that ER rarely regulates genes from promoter regions, but instead utilises distal enhancers. We also identified the role of a 'pioneer factor' called FoxA1, which is critical for ER to function. Our lab is interested in extending these findings to fully define the cis- and trans-elements that contribute to ER activity in breast cancer cells, with particular emphasis on the pioneer factors that stabilise ER-DNA interactions.

Characterisation of the role of pioneer factors in ER biology

We are interested in identifying and characterising the role of the pioneer factor FoxA1 in regulating ER activity. We have found that FoxA1 is required for all ER-DNA interactions and for ER to promote cell growth. In the absence of FoxA1, ER function is blocked and cells do not proliferate (Figure 1). We also find that for tamoxifen to successfully block ER function, FoxA1 is also required. This unexpected finding is possibly due to the fact that tamoxifen-ER actively represses gene transcription, which still requires functional ER-chromatin interactions. We find that FoxA1 is essential for cell growth in models of drug resistance, suggesting that FoxA1 may constitute an attractive drug target for endocrine resistant breast cancer. We can show that FoxA1 expression in non-breast cancer cells is sufficient to enable ER to interact with the DNA and to switch on gene transcription. These findings implicate FoxA1 as the sole mediator of ER function and response to hormonal stimuli, in both drug sensitive and drug resistant breast cancer.

Figure 1
ER binding is dependent on the pioneer factor FoxA1. (A) Examples of ER-DNA binding events, determined by ChIP-sequencing experiments in the presence (siControl) or absence (siFoxA1) of FoxA1. (B) A model representing the role of FoxA1 in mediating ER interactions with chromatinised DNA.

Co-operative interactions between ER and RARalpha

We have observed that a number of estrogen-ER regulated genes are subsequently utilise by the ER complex itself in order for effective transcription to take place, resulting in feed-forward loops. We recently found that RARalpha, another nuclear receptor, is an estrogen-ER target gene that can feedback to the ER complex. RARalpha has been shown to be required for oestrogen-mediated gene transcription and proliferation. We have shown, on a genomic scale, that ER and RARalpha co-occupy similar regions of the genome, which occur in a co-operative manner. This was achieved by establishing re-ChIP (double ChIP) sequencing for the first time. RARalpha was subsequently shown to be required for oestrogen-ER to recruit co-factors, providing a bridge between ER and some of the essential co-factors. This investigation links two nuclear receptors together in a co-operative role and may help explain why women with breast cancer can, in some cases, respond to retinoic acids.

Genomic analysis of ER function in primary breast cancer

All ER genomic studies to date have been limited to a single breast cancer cell line, yet they have revealed extraordinary features about ER biology. We have now been able to extend genomic transcription factor mapping experiments into frozen primary breast cancer samples, by performing ER ChIP-sequencing in luminal breast cancer material. The data confirm that ER ChIP-seq can be performed in primary breast cancer samples and that the ER binding events accurately represent the binding sites in the cell lines. However, there are significant numbers of ER binding events that are acquired in tumours with a poor clinical outcome and even more ER binding events acquired in metastatic material that originated from an ER positive breast cancer. These ER binding events, associated with poor prognosis tumours and metastatic samples, occur at genes that predict a poor clinical outcome in patient cohorts. Furthermore, the alterations in ER binding profiles in tumours with poor prognosis and in metastatic samples provide insight into specific genes that may be influencing drug response and risk of metastasis.