By ecancer reporter Clare Sansom

Breast cancers that over-express the oestrogen receptor (generally termed ER⁺, reflecting the American spelling “estrogen”) have a good prognosis, largely because they can be treated with hormone therapies such as tamoxifen.

Oestrogen receptor positive tumours that also express the progesterone receptor but not the herceptin receptor (ER⁺ PR⁺ HER2^- tumours) generally respond to treatment better than those that express the herceptin receptor or neither (respectively, ER⁺ PR^- HER2⁺ and ER⁺ PR^- HER2^-). There are two forms of oestrogen receptor, alpha and beta. Oestrogen receptor alpha (ER-a) is the one expressed in breast tissue; it is known to control gene expression through binding to chromatin at many different points on the genome, most of which are cis-regulatory elements.

Jason Carroll and Carlos Caldas of Cancer Research UK’s Cambridge Research Institute, and the University of Cambridge, UK and their co-workers have now mapped the binding of this receptor to chromatin in breast tumours. The researchers used chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to analyse the ER binding in eight primary ER⁺ PR⁺ HER2^- tumours, representative of those likely to respond best to hormone therapy, and seven with a receptor status that predicted a worse outcome.

The clinical outcome of each case was known, with all patients with cancers predicted to have a poor outcome having died of their disease. Three samples of distant metastases taken from women with oestrogen receptor positive breast cancer and two oestrogen receptor negative “control” tumours expressiong high levels of ER-b were also analysed.

The researchers identified a core set of 484 DNA regions where oestrogen receptor binding occurred in at least 75% of the complete sample of ER⁺ tumours but in neither of the ER^- tumours, implying that drug resistance in the poor prognosis tumours is not due to a loss of oestrogen receptor binding to DNA. The overall global strength of receptor binding, however, was found to be highest in the metastases and lowest in primary tumours with good response.

Next, they used differential binding analysis to identify DNA-binding regions associated with differential response to treatment. After normalisation for overall binding strength, binding to 599 separate DNA regions was found to be enhanced in the tumours with good prognosis, whereas binding to 1,192 regions was enhanced in the poor prognosis tumours.

DNA sequence motifs associated with the binding of the forkhead protein FOXA1, known to be a “pioneer factor” for interactions between the oestrogen receptor and chromatin, were found principally in the binding regions associated with poor response.

The researchers then identified genes located within 20kB of each of these regions, generating “gene lists” associated with both good and poor prognosis. The genes in each list were found to be preferentially upregulated in the tumours in the respective group.

They then tested these gene sets as outcome predictors in a large, independent group of oestrogen receptor breast cancers and found the expression of both gene sets to be predictive of distance metastasis free survival (with p=3x10^-5 for the good outcome genes and p=3x10^-8 for the poor outcome genes).

There was no correlation between the expression profile of either gene set and outcome in four cohorts of oestrogen negative breast cancers, implying that oestrogen receptor binding to regulatory elements close to these genes is biologically important in breast cancer.

To validate these findings, the researchers then mapped oestrogen receptor binding in a number of tamoxifen-sensitive and tamoxifen-resistant cell lines. Interestingly, almost 80% of the ER-binding regions differentially associated with poor response were found to bind the receptor in the tamoxifen-sensitive cell line MCF-7, indicating that the oestrogen receptor-binding profile of this cell line is intermediate between those of the good-response and poor-response tumours.

Next, oestrogen binding receptor binding in this cell line was tested after treatment with mitogens and found to enhance binding particularly at the FOXA1 motifs that were associated with poor response. Co-expression of the protein FOXA1 with the oestrogen receptor was found to occur in distant metastases, supporting the theory that this protein mediates changes in oestrogen receptor binding associated with drug resistance and metastasis.

Reference

Ross-Innes, C.S., Stark, R., Teschendorff, A.E. and 12 others (2012). Differential oestrogen receptor binding is associated with clinical outcome in breast cancer. Nature, published online ahead of print 4 January 2012. doi:10.1038/nature10730