Defining Mechanisms of Hormone Therapy Breast Cancer Risk Reduction and Biomarkers of Response

When breast tissue undergoes x-ray (i.e. a mammogram), the fatty component appears black whilst the areas containing the glandular tissue appear white. Mammograms with a large proportion of white are said to have high mammographic density (high MD). This is largely due to large amounts of supporting material in the breast called stroma.
Over recent years it has become apparent that having high MD confers a significant increased risk for the development of breast cancer, although the mechanism which confers this increased risk is not understood.
High MD is a common finding in the mammograms of women in the UK and globally. Therefore, interventions targeted towards reducing MD have the potential to benefit large numbers of women.
Recently it has been shown that the anti-oestrogen drug tamoxifen can reduce the risk of developing breast cancer when given to women with a strong family history. This reduction in risk corresponds to a reduction in MD. In addition, those women who show no reduction in breast density with tamoxifen treatment show no associated reduction in breast cancer risk, suggesting that the protective effect of this drug is mediated through reduction of MD.
This project aims to build on these key observations to investigate how MD can be modulated to establish markers that may help us predict which women will be protected or benefit from anti-oestrogen treatments. This project will look at the effect of tamoxifen and aromatase inhibitors on human breast tissues.
At Barts Cancer Institute we routinely consent patients undergoing surgery for donation of breast tissue not needed for diagnosis to be used in research. We will use these samples in our research to investigate the mechanisms by which anti-oestrogens exert their protective effect. Stromal cells will be treated with tamoxifen and an aromatase inhibitor and the effect on a series of molecules potentially involved will be assessed. We will also validate the results on samples taken prospectively.
The importance of this project is that by understanding the pathways contributing to high MD we will better understand the factors leading to development of breast cancer and this will help devise new ways of preventing it. Therefore we anticipate the results of this research will particularly benefit women who are at high risk of developing breast cancer.
The results from this research may also assist doctors in predicting which of these high risk women will respond well to treatments such as tamoxifen and aromatase inhibitors, that are aimed at reducing MD, and critically, identify those women who will not respond. This will mean that they will only be prescribed to women who are likely to have a good response to the treatment. Those that are unlikely to respond well will be spared some of the treatment associated side effects.

Aim: To elucidate how anti-oestrogens modulate MD in order to establish biomarkers to predict which women will be protected by these treatments, and to ultimately identify potentially targetable, alternative pathways that could be modified to reduce risk.
Objectives:
I) To use primary human in-vitro and ex-vivo model systems to establish genes and pathways modulated by anti-oestrogenic agents.
II) To establish the functional significance of pathways identified in (i) using co-culture systems.
iii) To investigate the clinical utility of key functional markers in predicting response to anti-oestrogens in a prospectively recruited patient cohort.
Methodology: Fibroblasts from patient samples and pieces of breast tissue maintained in culture will be used as model systems. Cultures will be treated with Tamoxifen (+/- estradiol) Aromatase Inhibitor (Letrozole) or vehicle control. Proliferation and fibrogenic signalling will be analysed using MTS assays, Ki67 analysis, western blotting, RT-PCR, ELISA and immunohistochemistry. RNA-Seq will be used to assess genome-wide changes. Targets will be validated by qRT-PCR or western blotting. The impact of pathways identified on epithelial cell proliferation and invasion will be assessed in 2D and 3D co-cultures, using siRNA knock-down, antibody inhibition or gene over-expression approaches. Clinical utility will be assessed in patients offered preventive tamoxifen through Family History clinics, and those receiving adjuvant AIs through the breast clinic. Biopsy and/or blood samples will be taken at baseline and 1 year and markers measured (by ELISA, IHC, RT-PCR), and compared to changes in MD.
Scientific and Medical Opportunities: These novel model systems are highly physiologically relevant, applicable to many other areas of research. Medically, this project aims to deliver precision medicine to the clinic.

The findings of this research have the potential to make a major impact on the management of individuals at high risk of developing breast cancer.
Gaining an improved understanding of the biological pathways which contribute to density-associated risk will allow researchers in the field of breast cancer to make significant progress in understanding MD. Establishing the functional significance or utility of genes and pathways modulated by anti-oestrogenic agents provides potential targets for new therapies and markers to predict response. Drug companies in the commercial sector may also seek to exploit these findings to develop novel treatments, which can reduce MD via similar mechanisms but potentially have a better side effect profile than tamoxifen or AIs, which although considered well-tolerated by the medical profession actually cause significant loss of quality of life, particularly when taken over an extended period, as these agents are.
This research will identify markers that can be used to identify women who are likely to respond to risk reducing anti-oestrogens. Importantly, it should also illustrate groups who will not benefit from these treatments, signifying an economic saving and avoiding drug side effects for some women. This takes forward the concept of precision medicine into the preventive setting.
Despite the recent recommendations from NICE for the use of tamoxifen in the preventive setting, there remains some reluctance amongst the medical profession to prescribe the drug in this setting, largely related to side effects. By providing a set of markers that can indicate whether a woman is likely to be responding to anti-oestrogen therapy, clinicians can be better guided in their management, and women can make a more informed choice. Although AIs are not yet recommended in the preventive setting, their superior protective effects make it likely that this will happen. The parallel analysis of AI in this study will provide a timely evidence base. Thus, this research will impact on those individuals responsible for managing high risk individuals and GP practices and breast cancer charities may also be indirectly affected, as they will have an important role in engaging women and counselling them about MD measurement and anti-oestrogen use. Through our Communications Plan, we also aim to inform women of the concepts of screening for protective benefit and hope therefore to communicate the concept of better informed choices for their treatment, particularly important in the preventive setting.