Characterisation of deregulated miRNAs during colorectal cancer progression - Interplay between tumour stroma and malignant epithelium

Bowel cancer is the second most common cause of death from cancer in Europe. The vast majority of deaths are not however caused by the cancer itself (which is often treatable with surgery), but by metastasis (cancer spread), to which the most troublesome symptoms can be attributed and to which most patients eventually succumb.
Metastasis is a complex multi-step process about which very little is understood. Unfortunately, this lack of detailed knowledge acts as a barrier to the development of drugs capable of treating or preventing cancer spread. In addition, current treatment methods generally produce disappointing results, highlighting the need for further research in this field.
Some interesting discoveries have recently been made. For example; studies have shown that bowel cancers spread with the help of various cells that exist in the tissues surrounding them. Interaction between these cells appears to influence how aggressively cancer spreads and although many questions remain to be answered, developing a better understanding of this interplay may uncover new opportunities for drug development.
MicroRNAs are a recently discovered form of genetic material. They are expressed in healthy tissue, but corrupted in cancer. They control numerous genes and influence processes involved in metastasis formation and are potential drug targets. However, no previous studies have scrutinised their presence in tissue surrounding bowel cancer, nor do we understand how microRNAs in the surrounding tissue affect cancer spread.
I am a 3rd year surgical trainee with a deep curiosity about the fundamental causes of bowel cancer. I am currently studying for a PhD in cancer science in collaboration with a group of highly experienced, highly reputed surgeons, scientists and pathologists.
The aim of my project is to study the role of microRNAs in bowel cancer spread by examining human bowel cancer specimens and the tissues surrounding them.
I have compared microRNAs in samples from patients who died from cancer spread with those who never developed metastases and have a list of microRNAs that I feel are likely to play an important role. The mechanisms by which abnormal microRNA expression influences bowel cancer spread will be explored using tried and tested laboratory techniques which enable us to quantify cancer invasion and metastasis development. I have also developed a novel artificial cancer model called an organotypic, in which colon cancer cells are grown on a synthetic scaffold containing cells found in the tissue surrounding cancers. This model has already enabled me to demonstrate in a persuasive and visually compelling way, how certain microRNAs enhance invasion and promote colon cancer spread.
As a collaborative group, we offer a unique blend of expertise and provide an excellent platform on which to base this project. A surgical perspective is particularly valuable, as it ensures that our research focus remains concentrated in areas of genuine clinical need and that novel laboratory findings can be rapidly transposed to the clinical setting.

Introduction: Metastases are the principle cause of death in colorectal cancer (CRC). An emerging theme in metastasis development is the role of non-coding microRNAs (miRNAs). An additional area of interest is the role of stromal cells and their interplay with cancer epithelium in dynamically influencing this process. The aim of this project is to establish whether stromal and epithelial miRNA expression patterns have important consequences for the development of metastases in CRC.
Methods: MiRNAs highly differentially expressed in advanced stages compared with early stage disease have been selected as candidates for study. The molecular targets of candidate miRNAs will be identified using Western Blotting and 3'UTR reporter gene assays. Biological significance will be assessed in cultured cell lines in which miRNA expression has been selectively induced or knocked-down. Standard in-vitro functional assays will be used in addition to bespoke organotypic models in which malignant epithelial cells and ex-vivo colonic fibroblasts are juxtaposed in a configuration that closely resembles in-vivo conditions.
Preliminary work: CRC specimens of different, clinically relevant pathological stage have been subjected to Laser Microdissection (LMD) in order to separately profile miRNA expression in stromal and epithelial layers. Stromal expression successfully separated stage-I/II tumours from stage-IV tumours based on a panel of miRNAs, illustrating the potential relevance of stromal candidates. Candidates are currently being tested for functional significance and 3D organotypic models reveal that miR-153 in the epithelium and miR-21 in the stroma are important regulators of CRC invasion.
Applications: Identification of clinically relevant miRNAs in the CRC stroma is novel and may improve staging, guide therapy and reveal new therapeutic targets. Added value is provided as the distinctive biology of CRC stroma has not been reflected in miRNA profiling studies to date.

The focus of my research is the metastatic cascade, a complex and multi-step biological event in which cancer cells acquire the capacity to break free from their sister cells, invade surrounding tissues and establish viable secondary tumour deposits in distant organ systems. This elaborate process is responsible for the vast burden of cancer associated morbidity and mortality and yet relatively little research activity is dedicated to understanding the pathogenesis of metastasis or the molecular mechanisms that underlie cancer progression.
This proposal represents an achievable and innovative research strategy to investigate the role of stromal and epithelial microRNAs (miRNA) in colorectal cancer (CRC) progression and will provide valuable scientific insights in a novel field with significant translational promise. This promise extends to commercial applications in biotechnology and pharmaceutical development and to improvements in health and well being for patients with CRC by informing public health and cancer treatment policy.
In the medium term, the identification of individual miRNAs and miRNA signatures with utility in the management of colorectal cancer will greatly enhance molecular diagnostics and therapeutics. Development of specific miRNA chips with the ability to sub-classify colorectal tumours and improve prediction of disease recurrence or advanced stage would be of great commercial value and could dramatically alter the current management of patients. Thus a molecular pathological approach to disease staging, looking for individual and/or communal metastatic markers and profiles, when combined with modern imaging modalities, may provide a more accurate reflection of stage and better inform the multidisciplinary cancer team about the use of chemoradiotherapy, heralding new opportunities for the delivery of personalised cancer therapy. For example: patients with stage-II CRC are not routinely offered chemotherapy because there is no evidence of benefit in this group as whole, and yet 25% go on to develop metastasis within 5 years. There is strong evidence to support the routine use of chemotherapy in stage-III cancer implying there may be a sub-group of stage-II CRC patients who may benefit from chemotherapy. MiRNA profiles have potentially powerful prognostic and diagnostic applications and our stromal and epithelial profiling studies of patient with stage-II CRC with and without subsequent metastatic progression revealed a panel of differentially expressed miRNAs, which if used alongside other indicators may form the basis of an algorithm of recurrence risk, enabling prognostic stratification and more accurate targeting with chemotherapy.
MiRNAs are potentially targetable by drugs and in the medium and long-term, analysis of these promising individual biomarkers will aid drug discovery programs and ultimately facilitate a shift towards a personalised drug treatment process. Advances in our understanding of the molecular machinery of cancer progression have also been mirrored by technological developments in the field of nano-particle miRNA-based drug delivery systems. Thus, by synthesising two promising fields of research there is potential to create new generations of drugs capable of targeting pathologically relevant molecular pathways exclusively within tumour transformed tissue.
This project has also produced technical innovations in tissue engineering, as we have capitalised on an internal collaboration within the Cancer Sciences Division to produce Organotypic models in which stromal and epithelial candidate miRNA have been synchronously and metachronously over-expressed or knocked down. This powerful experimental model of malignant invasion and tumour/stromal interaction which mimics in-vivo conditions has reduced our dependence on animal experimentation and when used in conjunction with immunohistochemical staining, serves as an intuitive and visually compelling educational tool.