Inactivation of the PRH/HHEX tumour suppressor and its effects on inflammatory signalling in prostate cancer.

Prostate cancer is the most common cancer in men in the UK and this disease has a particularly high incidence in men of African origin. Around 50,000 new cases are diagnosed each year and this is increasing as men live longer. Most men diagnosed with prostate cancer do not die from this disease. However, for patients with disease that has spread to other sites in the body, surgical castration is the recommended treatment. For patients with less advanced disease radiotherapy and/or drug treatments are effective. However, these treatments can have serious side effects including osteoporosis and bone fracture. In many less advanced cases treatment is probably unnecessary as the cancer is very unlikely to progress and spread. Better ways of identifying patients who need treatment and better treatments would be of immense value to patients and result in major cost savings for the NHS. The incidence of prostate cancer in Kenya and other low- and middle-income countries (LMIC) is also rising and in these countries the cost of treatment and the cost of lives lost to this disease are major barriers to economic development. The aims of this project are to improve our understanding of the causes of prostate cancer progression and to create an archive of prostate cancer samples that will be of value to this study and to future work in this area.
Inflammation in the prostate is common and this is known to be an important factor in prostate cancer. Inflammation in the prostate in LMIC and UK patients can have many causes including bacterial and viral infections and autoimmune responses. Cells from the immune system are found in the prostate and in prostate cancers they release signals that increase the replication of cancer cells and increase their ability to spread.
We have discovered that a protein called PRH stops prostate cells from replicating and stops them from invading other tissues. We found that as prostate cancer becomes more advanced the activity of PRH is decreased in three ways: first, the PRH protein is inactivated by a modification known as phosphorylation; second, the gene that normally produces PRH is stopped from making PRH; and third, this gene is deleted altogether in some cancer cells. Our work showed that the PRH protein works by controlling the activity of many genes important in allowing prostate cells to respond to signals from immune cells and in particular we showed that PRH controls the response to a molecule called Transforming Growth Factor beta (TGFb) released from some types of immune cell. In addition we have shown that TGFb itself controls PRH activity by decreasing how much PRH is produced by the PRH gene and by increasing phosphorylation of PRH.
We will investigate the importance of immune cells and immune signals in prostate cancer in Kenya and in the UK and find out how changes in the levels and activity of PRH alter the response of prostate cells to TGFb and other immune signals. To achieve this we will collect blood, urine, and cancer samples from Kenyan patients and document the clinical history associated with prostate cancer in these patients using questionnaires. We will then determine the levels of PRH and phosphorylated PRH in Kenyan and UK prostate cancer samples using a technique called immunohistochemistry (IHC) that allows us to visualise proteins in cancer cells. We will also measure the levels of inflammatory signals and immune cells in the Kenyan blood samples. Finally we will determine how changes in PRH levels and activity influence how prostate cells respond to signals from immune cells and how these signals control PRH levels. These studies will advance our understanding of how immune signals are involved in prostate cancer. They will also tell us whether measuring the levels of immune signals in blood and PRH or phosphorylated PRH in prostate cancer might be a good ways to predict which patients require treatment.

Transforming Growth Factor beta (TGFb) and other cytokines are secreted by tumour associated macrophages and these cells are important in prostate cancer progression, epithelial-mesenchymal transition (EMT), and metastasis. We have shown that PRH/HHEX inhibits prostate cell proliferation, EMT, and cell migration and invasion in part by activating the transcription of the gene encoding Endoglin, a TGFb co-receptor. Moreover, TGFb down-regulates PRH mRNA and protein levels in prostate cells. This is important because PRH is inactivated by multiple mechanisms in benign prostatic hyperplasia (BPH) and prostate cancer including the accumulation of phosphorylated and transcriptionally inactive pPRH. We hypothesise that PRH activity in prostate cells shapes their response to signals from immune cells and that the inactivation of PRH as a result of signals from immune cells, results in increased cell proliferation and cell migration/invasion and thereby contributes to cancer progression. Our aim is to test this hypothesis and in doing so investigate the importance of immune signals in cancer progression. To achieve this aim we will: 1. Establish a Kenyan archive of blood and urine samples from patients with prostate disorders and an archive of prostate tumour sections to include BPH, prostate carcinomas, and castration resistant carcinomas and document the epidemiologic risk factors and clinical histories associated with these samples. 2. Determine the levels of PRH and pPRH in Kenyan and UK prostate cancer samples using immunocytochemistry (IHC). 3. Determine the inflammatory signals (cytokines and immune cells) that are relevant in the Kenyan population using flow cytometry and IHC. 4. Determine how changes in PRH levels alter the response of prostate cells to signals from immune system cells and how signalling from immune system cells down-regulate PRH activity.

Prostate cancer represents a major barrier to economic development in Kenya and other LMIC as there is significant mortality associated with this disease and the existing health systems are ill-equipped to deal with complex and expensive cancer treatments. The UK faces similar problems in that there is a high incidence of prostate cancer (50,000 cases per year). However, over-treatment is also an important issue in the UK resulting in unnecessary treatment and major cost implications. In large part this is due to the fact that Prostate-Specific Antigen (PSA) testing has a high false-positive rate and a high false-negative rate and there are limited validated biomarkers for general clinical use in conjunction with PSA to report on prognosis. There is therefore an unmet clinical need for new prostate cancer biomarkers that distinguish benign tumours that will proliferate locally but not become invasive from those that will become androgen-insensitive metastatic carcinomas. Although several novel biomarkers are in development, none of these are in widespread clinical use. Ideally any new biomarkers that are developed should be suitable for use in the UK and in LMIC. This favours low cost approaches based on simple blood tests. In this project we will investigate the role that inflammation plays in prostate cancer. A better understanding of the role of inflammation in this disease could result in improved methods for diagnosis and prognosis, as well as new and effective treatments. This would have an important impact on human health in the UK and mitigate a major barrier to development in Kenya and other LMIC.
In this project we will establish a Kenyan archive of blood and urine samples from patients with prostate disorders and an archive of prostate tumour sections and we will catalogue the epidemiologic risk factors associated with these samples. This will create a significant resource for future genetic and biomarker studies and increase research capacity in Kenya. Using these samples we will identify inflammatory signals that are relevant in this cohort. This will help us to understand the underlying causes of inflammation and we expect that this knowledge will have an impact on health care. In addition we will compare immune cell infiltration in prostate cancer sample from Kenya and from the UK. This will also produce data that will be of relevance to human health. We will go on to determine whether pPRH and/or PRH can act as biomarkers to identify patients with a poor prognosis most likely when used in combination with other biomarkers. pPRH and/or PRH may identify patients likely to respond well to personalised cancer treatment options involving kinase inhibitors. The results of this work could therefore influence the design of clinical trials involving direct and indirect CK2 inhibitors. This is a route that is expected to have a significant impact on clinical practice.
This project will improve our understanding of this complex disease with potential identification of key markers for the management of prostate cancer while creating a Kenyan biobank that will be of great value to future studies. We will hold joint meetings in the UK and Kenya to increase the skills and knowledge base in both countries. This project will therefore promote economic growth and welfare as well as increasing research capacity and contributing to staff training and development. The outcomes of previous work from our laboratories are expected to have a major impact in the development of new biomarkers and new combinatorial cancer treatments. Insights from this project will also be communicated to the medical and scientific community, patient groups, and the wider public in the UK and in Kenya to ensure maximum impact of this work. We will communicate the results of these studies through publications, workshops in both countries, and through scientific meetings.