Estrogen receptor beta is an important modulator of hormone-related carcinogenesis in the human prostate.

Prostate cancer is the most common cancer in men, with 214 new cases diagnosed per 1000 men in Europe. Currently, it is the second most common cause of cancer-related death in men.

The development of prostate cancer is largely dependent on the male hormone, testosterone and so for many patients initial treatment of the disease involves blocking the production and activity of testosterone in order to suppress the disease. Unfortunately, for many men the cancer subsequently becomes resistant to this treatment and continues to grow in the absence of testosterone. Current chemotherapy treatments are not particularly effective against this hormone resistant prostate cancer (HRPC) and patients survive for an average period of 18 months from the time of HRPC diagnosis.

In females, the dominant hormone is estrogen. Estrogen is known to play an important role in the development and growth of breast and ovarian cancers in women. Consequentially, a number of treatments for these diseases are aimed at blocking the production and activity of estrogen in a manner analogous to hormone deprivation treatment of prostate cancer. Estrogen is also present in smaller quantities in men where it regulates a variety of processes in the body. It does this by sending signals to cells using the estrogen receptor. When activated, the estrogen receptor instructs the cell's genetic code (DNA) to create proteins, which in turn influence the behaviour of the cancer cell in a particular way.

Recent scientific research has demonstrated that estrogen and one of its receptors, estrogen receptor beta (ERb) play an important role in controlling some of the genetic mechanisms that can give rise to prostate cancer. However, the mechanisms by which ERb regulates cancer-related genes are poorly characterised. In particular, it is thought that when prostate cancer becomes resistant to testosterone deprivation to become HRPC, estrogen may continue to provide a stimulus for cancer growth acting via the estrogen receptor.

In my research, I aim to increase understanding of how estrogen and ERb can regulate cancer-related genes in the prostate. I will be studying the effects of estrogen and ERb in order to understand how prostate cancer initially develops and how it becomes resistant to testosterone deprivation. Estrogen receptors are dependent upon a protein in the cell called FoxA1, which acts as bridge to bind the receptor to the DNA enabling it to switch genes on and off. Most of our understanding of FoxA1 and its influence on estrogen receptors comes from the study of breast cancer. At present, it has not been determined if FoxA1 has any function with respect to estrogen receptor in the prostate. By increasing or decreasing levels of FoxA1 in prostate cells and tissue I will explore the role that it plays in the prostate and determine whether it could be a potential drug target to control the progression of prostate cancer.

The research will be conducted in the laboratory using cells derived from human prostate cancer tissue as these provide a useful model for studying the disease. I also aim to study prostate tissue obtained from patients undergoing surgery who have given permission for me to do so, in order to see whether some of the findings observed in the cells are also present in 'real-patient' samples.

By increasing the understanding of how estrogen influences the development and progression of prostate cancer I aim to find new ways to treat HRPC.

I aim to increase understanding of the role of ERb in the development and progression of PCa; to identify whether ERb is a potential therapeutic target and to identify a biomarker of ERb activity. I will explore the mechanisms of ERb activity and the role of FoxA1.

1. Mechanistic analysis of ERb activity in cell lines and prostate tumour tissue.
Using PCa cell lines, ERb will be stimulated with known agonists and ERb, AR and FoxA1 mapped by ChIP-seq. In addition, stable inducible short hairpin (sh)ERb expressing cells will be generated to silence ERb expression. I will assess the impact of ERb loss on AR and FoxA1 binding. Under all conditions cell growth will be assessed with or without AR agonists and antagonists. Ex vivo prostate tissue culture will be performed in ERb and AR agonist and antagonist conditions, and transcription factor mapping by ChIP-seq conducted in the tissue to assess ERb mechanistic activity within the context of stromal-epithelial interactions.

2. Identify a protein biomarker demonstrating a functional ERb complex.
Using the cell lines (Aim 1), I will manipulate ERb using specific agonists or inducible shRNA. Total RNA will be collected at multiple time points and RNA seq conducted. Genes differentially regulated under activation and inhibition conditions will be integrated with ERb ChIP-seq data to identify direct ERb target genes. Candidates will be analysed using IHC on a series of PCa TMAs, with the aim of discovering a biomarker representing a functional ERb complex.

3. Determination of the role of FoxA1 and FoxA1 mutations with respect to ERb in PCa.
ERb and FoxA1 interaction assays will be conducted using RIME. I will assess dependence on FoxA1 by silencing it then mapping ERb binding by ChIP-seq. Using validated zinc finger nucleases (ZFN) to target the endogenous FoxA1 genomic locus, stable PCa cell lines with mutant FoxA1 will be generated. The effect of mutant FoxA1 on growth, ERb and AR binding will be determined.

My primary reason for seeking to undertake this research is to benefit patients with prostate cancer and discover new targets for treatment of the disease. Additionally I intend that patients with other estrogen-dependent cancers such as breast and ovarian cancer will similarly benefit. HRPC develops in virtually all men treated with androgen deprivation, with an average median survival from the time of diagnosis of 18 months. Outcomes from current chemotherapeutics are relatively poor. There is therefore an opportunity to greatly impact the lives of affected men and improve clinical outcomes of the disease through research into the responsible underlying molecular mechanisms.

We know that nuclear receptors are key drivers in hormone dependent cancers, yet our understanding of ERb in prostate is rudimentary. There are conflicting data in the literature with respect to the function of ERb in the prostate, which may be related to ERb isoforms. It has been suggested that wild-type ERb is predominantly protective against carcinogenesis, regulating cellular differentiation and apoptosis. However, the splice variant isoforms ERb2 and ERb5 may actually mediate pro-oncogenic changes, contribute to the metastatic potential of PCa and correlate with poor patient prognosis. The mechanisms underlying these observations are presently not well defined. By understanding the molecular and genetic mechanisms involved in ERb-related gene transcription and how they impact carcinogenesis I intend to identify potential therapeutic targets, which could be used in the development and testing of new drugs against prostate cancer. There is the potential for commercial impact via Cambridge Research Technologies (CRT) if viable commercial products result from my research. Development and testing of novel therapeutic agents in the era of molecular medicine is a lengthy process, but based on biological insight, towards which I aim to contribute. ERb specific compounds already exist, but a rational explanation for their use in PCa is currently lacking. This proposal may provide impetus for pursuing the use of existing chemicals for modulation of ERb pathways.

By identifying the genes and proteins associated with ERb transcription I intend to identify a biomarker signature of ERb activity. This will be correlated with patient prognosis by applying it retrospectively to the large cohort of tissue samples in the Addenbrooke's biorepository with long term clinical follow up data. This may be further developed to allow identification of this biomarker in the biopsy samples of men undergoing investigation for prostate cancer, thereby determining the likely influence of ERb-related transcription in their cancer. This opens the possibility of stratifying patients accordingly to determine their suitability for individualised, targeted therapeutics against ERb-dependent pathways. One of the greatest challenges in the management of prostate cancer is in differentiating indolent from clinically significant disease at the time of initial diagnosis and I aim to contribute to improving knowledge in this area.