Investigating the role of the Notch atypical ligand delta-like homologue 1 (DLK1) in adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is a rare, aggressive tumour occurring in the adrenal cortex, the steroid-producing outer layer of the adrenal gland. Early diagnosis and surgical resection is crucial to long-term survival. In many cases, the cancer has spread by the time of diagnosis and the prognosis is bleak, with five-year survival rate in advanced disease under 15%. The only approved medical treatment is the drug mitotane but it is poorly tolerated and often fails to prevent disease progression.

It is important to correctly identify ACC to ensure its prompt management. Current practice revolves around prediction of malignancy based upon clinical, biochemical and radiological features. Once a mass has been removed, histological diagnosis is based upon properties of the tumour and a further prediction of malignant potential. This is often complex especially in borderline cases. Therefore it is important to identify new biological markers that can aid in the diagnosis, prognosis and monitoring of disease.

It is not known which cells are involved in the development of ACC, nor is it known which cells within an established tumour confer resistance to chemotherapeutic drugs and may be responsible for tumour relapse. We have recently identified a novel population of cells in the adrenal gland, which we believe might be the precursor of adrenal cancer. These cells express a protein called delta-like homologue 1 (DLK1). DLK1 is not normally found in most healthy human tissues. However, expression has been reported in other malignancies and in some of these increased DLK1 expression is associated with a worse prognosis. This makes it an attractive option for further investigation not only as a novel biomarker in ACC but also as a player in the development of this malignancy and as such a potential target for novel therapeutic intervention.

In this project I will be investigating the role of DLK1 in adrenocortical carcinoma on two fronts. First, I will be studying its role as a potential novel biomarker in this condition. I will be doing this by looking at expression of DLK1 in collected tumour samples and in blood and seeing if there is a relationship with survival rates as well as clinical and biochemical presentation of disease. In addition, I will also be looking at DLK1+ adrenocortical carcinoma cells and characterising how they differ from other cells in the cancer.

To carry out this project, I have developed collaborations with one of the largest groups investigating this condition from Germany (Prof. Fassnacht, University of Würzburg) and also my local pathology department to gain access to over 100 tissue sections of ACC along with the clinical records to assess details of presentation and long-term outcomes.

I am collecting ACC tumour samples from four surgical centres across London. From our local hospital I am also collecting normal adrenal tissue from patients having surgery for other non-cancerous pathologies. In addition, I will be performing experiments using two new patient derived adrenocortical carcinoma cells lines through a collaboration developed with University of Colorado Denver in addition the commercially available, H295R cell line.

This project combines cellular biological investigation with a clinical focus. It has the promise to identify and characterise a new molecular biomarker for diagnosis and prognosis of ACC, to deliver new biology defining a cell population in this malignancy and to identify targets which may be amenable to novel therapeutic intervention.

Adrenocortical carcinoma (ACC) is a rare malignancy with a devastating prognosis. Treatment options are limited. Early surgical resection offers the only chance of cure but frequently disease presents late and in the advanced stage five-year survival is <15%. Histopathological diagnosis of ACC is complex, based on a prediction of malignancy, and there are few widely used biomarkers in clinical practice as yet.

We have identified a novel cell population in the adrenal cortex that express the protein delta-like homologue 1 (DLK1). DLK1 expression is low in most healthy tissues; however, expression is reported in numerous malignancies and is associated with a worse prognosis. We have identified that DLK1 is overexpressed in ACC compared to normal adrenals and non-malignant adrenal pathology. In preliminary experiments using the ACC cell line H295R, we have shown that DLK1+ cells have increased colony forming ability, chemoresistance and features of adrenocortical progenitor/stem cells compared to DLK1- cells.

Aims:
1) To investigate the role of DLK1 as a novel biomarker in adrenocortical carcinoma
2) To characterise the features of DLK1+ adrenocortical carcinoma cells in vitro and ex vivo

Methods:
- Correlation of immunohistochemical expression of DLK1 with survival rates and details of clinical and biochemical disease presentation
- Assessment of serum DLK1 levels in patients with ACC vs control population of non-malignant adrenal pathology before and after surgical resection
- Assessment of characteristics of DLK1+ ACC cells (3 cell lines and primary tumours): spheroid generation, chemo-resistance, colony formation, RNA extraction and quantitative PCR, tumour-initiating ability

Impact:
This project promises to identify and characterise a new molecular biomarker for diagnosis and prognosis of ACC, to deliver new biology defining a cell population in this malignancy and to identify targets which may be amenable to novel therapeutic intervention.

ACC is a rare malignancy with a bleak prognosis. Medical treatment options are limited, are often poorly tolerated and regularly do not prevent disease recurrence or progression. Any increase in the knowledge of the cells and pathways involved in the development and chemo-resistance of this malignancy is going to benefit the research community studying this condition. Identification of potential biomarkers for this disease will aid in the diagnosis and prognosis of ACC. It is likely that this will account for the first clinical benefit from this project. Ultimately, this project aims to identify novel therapeutic targets and provide substrate for the development of novel medical options to improve the prognosis of patients with this condition. This will likely be of interest to the pharmaceutical industry and has the potential for commercial enterprise. Better prognostication and more tailored treatment strategies with novel agents will lead to an improvement in disease outcome, reduction of disability and improvement of quality of life. This will be of broad economic benefit to the NHS and other health services worldwide.

We have identified that a novel cell population in this malignancy that show increased resistance to common therapeutics in vitro, marked by the expression of the transmembrane protein DLK1. This project will study further the phenotype of these cells, how they differ from other cells of the adrenal cortex and from other populations of cells involved in ACC and if they have tumour-initiating ability. The adrenal cortex is a good model to study pathogenesis of malignancy as the cells and pathways involved in its embryological development in humans are relatively well understood compared to other organs. An increase in knowledge about the behaviour of defined cell groups within a tumour, and how they relate to the corruption of the normal developmental and self-renewal processes of organs, will likely be informative to the wider community studying other malignancies and those studying inducible pluripotent stem cells. Better understanding in this area will lead to more effective and better tailored treatment in cancer and other conditions with improved outcomes and beneficiaries in healthcare systems, associated charities and patient groups.

The potential outcomes of this project in science and translational medicine will promote world-leading research at Queen Mary, University of London and will contribute to the breadth and quality of academic research in the UK. It will enable all members of our group to develop further skills in research and translational medicine. From a personal point of view, it will allow me to develop skills required for a career as a clinical academic and form a solid foundation for attainment of this goal.