Dysregulation of tissue-specific bone morphogenetic protein signaling initiates and promotes colorectal carcinogenesis

The human gut is made up of units called crypts, in which all cell types within it are derived from the stem cells housed at the crypt base. Control of these stem cells is thought to occur from the action of chemical messages secreted by cells that surround the stem cell. Derangement of these messages can lead to abnormal stem cell growth leading to colorectal cancer formation which affects 39,000 people a year in the UK and is responsible for more than 16,000 deaths annually.

Much of our understanding of the development of bowel cancer comes from the study of rare diseases in affected families who pass down (inherit) an increased risk of developing cancer. Study of some of these rare familial syndromes - Juvenile Polyposis (JP) and Hereditary mixed polyposis syndrome (HMPS), together with larger case-control genetic studies of sporadic colorectal cancer in the general UK population has suggested that derangement of one of the chemical messenger pathways called the Bone Morphogenetic Protein (BMP) pathway may be important in the initiation of the human colon cancers.

Many of the components of the BMP pathway are normally expressed in the cells that surround and support the intestinal crpyt (the mesenchymal cells) rather than the cells lining the crypt itself (the epithelium). We have shown that the familial syndrome HMPS results from the aberrant 'switching on' of Gremlin1, a mesenchymal BMP inhibitor in epithelial cells. We have also demonstrated disruption of the normal expression pattern of other BMP signaling pathway constituents in colorectal polyp and tumour cells and propose that this disruption affects normal mesenchymal-epithelial cross talk and can promote tumour formation.

A team of clinicians and scientists intend to examine the genetic mechanisms that regulate the switching on and off of Gremlin 1 in normal and cancer tissues. We will also investigate at what stage of tumour development the pathway disruption occurs and generate cell culture models to examine the effects of derangement of BMP messaging. We believe that this work will lead to a better understanding of tumour development and growth and highlight potential target areas for anti-cancer treatment

Objectives: Overall - To establish constituents of the BMP pathway's involvement in the initation and progression of human colorectal cancer
Specific 1. To identify tissue-specific regulatory regions upstream of Grem1
2. Explore the timing and effect of dysregulation of the BMP pathway in human tissue and colorectal cancer cell lines
3. Assess the contributions of canonical and non-canonical second messenger systems in BMP signal mediation

Experimental design
1. Regulatory regions - We will use Formaldehyde assisted identification of regulatory elements (FAIRE) Chromatin immunoprecipitation (CHiP) to identify transcription factor occupancy profiles and chromosome conformation capture (3C, 4C) together with luciferase reporter assays in cell lines to assess the enhancer structure of this key genes upstream region
2. Up and down regulation - Clonal ordering studies, in situ hybridisation and immuno will be used on archival or fresh human tissue to assess the site and timing of BMP constituent dysrgulation. Lentiviral stable transduction of cell lines with doxycycline responsive elements or shRNA will be used to up and down regulate BMP2, 4 and Grem1 to allow assessment of effects on cell morphology, proliferation, clonogenicity.
3. Second messenger systems - We will use immuno and qRT-PCR to assess target gene activity followed by specific receptor and pathway inhibitors to determine the contribution of canonical (SMAD4 dependent) signaling and non-canonical (SMAD4 independent) pathways on BMP signaling mediation.

Exploitation: We hope to highlight the role of the mesenchyme in control of epithelial stem cells and establish constituents of the BMP pathway as colorectal cancer initiating and promoting genes in familial and sporadic colorectal cancer. This will help to facilitate the development of novel biomarkers and therapeutic agents

The likely beneficaries from this research include:

The research team. The projects are designed to make use of established and state-of-the art molecular biological techniques. This will allow the researchers to develop skills in a broad range of techniques, enabling some members to establish future independent research groups in this field.

The scientific community (as discussed under academic beneficiaries). If our hypothesis is correct then we will identify the BMP pathway as capable of initiating and promoting CRC, with derangement of tissue compartment specific signalling as a novel carcinogenic mechansism.

Patients and the health service. Colorectal cancer (CRC) remains a major health burden and is the second commonest cause of cancer death in the UK. Much of what we understand of intestinal stem cell homeostasis and CRC genetics we have learnt from the study of rare familial polyposis syndromes and our investigation of rare hamartomatous polypoisis syndromes and genome wide association studies of sporadic CRC has identified components of the BMP pathway's central involvement in CRC pathogenesis. Confirming our findings and the mechanism of action of these genes in CRC aetiology will help in the development of new biomarkers and drug targets. Additionally we hope to be able to show that dysregulation of compartmentalised signaling is sufficient to initiate carcinogenesis which would be a novel tumourigenic mechanism in CRC. There is growing interest in heterotypic cell interaction and the role of the stroma in tumour initiation, as therapeutic stromal manipulation of epithelial cancer stem cells is a tantalising prospect in solid tumour malignancies.

Commercial beneficiaries. This research is of an exploratory nature aiming to assess the homeostatic and carcinogenic role of key intestinal signaling pathways. No commercial opportunities are expected to arise directly from this work although it may pave the way for the future development of biomarkers or chemotherapies. Any commercially viable finding will be discussed with Isis Innovations, a nationally renowned technology transfer company.