Report
Teaser, summary, work performed and final results
Periodic Reporting for period 3 - ER_disease (Defining hormonal cross-talk and the role of mutations in estrogen receptor positive breast cancer)
Teaser
The proposal revolves around understanding and characterising hormone dependent breast cancer. There are 55,000 new cases of breast cancer very year in the UK alone, a full 75% of these are driven by the estrogen receptor (ER) pathway. Drugs that block ER activity have had a...
Summary
The proposal revolves around understanding and characterising hormone dependent breast cancer. There are 55,000 new cases of breast cancer very year in the UK alone, a full 75% of these are driven by the estrogen receptor (ER) pathway. Drugs that block ER activity have had a dramatic impact on survival rates, but a third (~12,000) of patients will relapse and die of metastatic breast cancer. We and others have recently shown that the three key factors that make an ER pathway function are ER, FoxA1 and GATA3. Importantly, these factors are mutated in cancer, but we don’t know what these mutations do. In addition, recent evidence has suggested that parallel hormonal pathways, particularly progesterone receptor (PR) and androgen receptor (AR) can impact ER activity, potentially by competing with ER for DNA access.
The key goals of the proposal are to understand how the hormonal pathways influence ER activity and to exploit these findings therapeutically. We have safe, off patent drugs that both activate and inhibit PR and AR, but we are unclear about the biological interplay between these pathways and ultimately about what patients to use them in. The goals of the proposal are to characterise these hormonal cross-talk mechanisms.
In addition, a key goal is to identify what the mutations do to ER, GATA3 and FoxA1 and to identify mechanisms that regulate GATA3 and FoxA1. The overall objectives are to delineate mechanisms of estrogen receptor activity in breast cancer, with a focus on identifying and exploiting the changes in protein fidelity and the potential for transcription factor cross-talk.
Work performed
Defining the cross-talk between steroid nuclear receptors in breast cancer
We have invested considerable time in identifying the best cancer models that have comparable expression levels of ER, PR and AR. This has revealed an excellent cell line model (ZR751) cells that have comparable levels of these three key nuclear receptors. We have assessed the impact of progesterone (P4) or androgen (DHT) treatment on this cell line and have found both drugs to be antiproliferative, as we had hypothesised in the proposal. We have now conducted a series of ER, PR and AR gemome-wide mapping experiments (ChIP-seq) under the different hormonal conditions and the data is currently being analysed. Preliminary analysis suggested that P4 reprograms ER binding (validating our previous published work) and that the ER reprogramming mediated by P4 is substantial. Unexpectedly, we did not see any ER or PR reprogramming following DHT treatment, suggesting that the activity of DHT is not via molecular inhibition or modulation of ER or the repressive mechanisms elicited by P4-PR on ER.
In parallel, we have set up the large scale proteomic pulldown (RIME) experiments, as suggested in the proposal. These are currently underway, but we have validated the reagents and the cell line model to undertake these experiments.
We have been exploring the role of progesterone (P4) in contexts were ER (ESR1) is mutated and our data suggest that the ESR1 mutation associated with a third of distant metastases, retains sensitivity to P4. This work provides a therapeutic alternative for treatment of women with this common mutation and we are currently exploring the functional and mechanistic basis of this.
In addition, we have attempted to tackle a couple of the key controversies and complexities in the field. We know that Medroxyprogesterone acetate (MPA) when used in Hormone Replacement Therapy (HRT) is associated with a small but significant increase risk of ER+ breast cancer. These findings have dramatically decreased HRT use globally and unfortunately progesterone and all progestins have been considered dangerous. We have recently explored whether MPA behaves the same as P4/progestins and have some data to suggest that they are not the same. MPA appears to be able to induce changes in ER binding, independent of PR, which is the first molecular evidence that MPA is not the same as other progestins or P4.
Similarly, there is controversy in the field because it is know that antiprogestins (i.e. PR antagonists) can inhibit growth of ER+ breast cancer cell growth. The conclusion was that if antiprogestins can inhibit growth then PR agonists should do the same and promote growth. In some circles of the community, there is only interest in pursuing antiprogestins as therapies, yet these either lack efficacy in patient trials or have unacceptable toxicities. In addition, it is well established that PR agonists (i.e. progestins such as Megace) have good antitumour activity. We have conducted a series of experiments to compare, for the first time, progestins and antiprogestins side by side. The results have been suprising and suggest that both agents can reprogram ER and block growth. As such, a paradox exists whereby both PR agonists and antagonists can have the same effect on cancer cell growth, since they both negatively impact ER activity.
Characterising the impact of ER, FoxA1 and GATA3 mutations and FoxA1 PTMs on ER signalling and the consequences on steroid nuclear receptor cross-talk.
ER (ESR1) is known to be mutated in metastases. We had originally planned on engineering in ESR1 mutations into ER+ cancer cell lines, to investigate the role of these mutational changes. However, following the funding of this proposal we discovered that a colleague in London (Prof Simak Ali, Imperial College) has already engineered the two common mutations into MCF-7 breast cancer cells. As such, we have collaborated with Prof Ali to assist in the characterisation of these cell
Final results
A number of unexpected observations have come from the work to date.
- MPA appears to behave differently than other progestins, suggesting that the progestin used in HRT that is linked with increased breast cancer risk, can behave differently to progesterone and other progestins.
- The observation that both progestins and antiprogestins can do the same thing to breast cancer cell growth, is a paradox, but explains the complexities and conflicting data in the field.
- The observation that mutant GATA3 is a protective event was very unexpected and implies that a common breast cancer mutation is a protective event that provides a better clinical outcome. To our knowledge this is the first time that a mutation that is this common has been linked with a better prognosis.
- The functional discovery of a link between GATA3 and TET2 is unexpected and suggests that there is a functional connection between enhancer transcription factors and DNA methylation.