Understanding how an MLH1 promoter polymorphism predisposes to colorectal cancer

Colorectal cancer affects 1 in 20 people in the western world. Mutations that cause colorectal cancer can be inherited or, more often, acquired during a patient's lifetime. There are also some common genetic variants (small differences found in the DNA of many people) that can increase their risk of cancer by a small percentage. These variants usually do not have a big effect on the gene, but may influence how active the gene is in particular cells. One such colorectal cancer risk variant is found near the MLH1 gene. MLH1 is disrupted in about 15% of colorectal cancers, but instead of mutations in its DNA, it is repressed by acquired chemical marks on the DNA (called DNA methylation or epimutation). The common variant near MLH1 is strongly linked to increased DNA methylation and MLH1 gene repression, but we do not yet understand exactly how and why this leads to a higher risk of this specific type of colorectal cancer. The main aim of this project is to answer these questions.

We will work out how the common genetic variant near MLH1 causes different risks of MLH1 repression. We think that 1) the high risk variant binds to proteins that cause DNA methylation and that this represses MLH1 activity, or 2) the high risk variant disrupts the binding of proteins necessary to keep MLH1 active, reducing MLH1 expression and allowing methylation to spread across the DNA causing further reduction in expression, or 3) the high risk variant just affects the binding of activating proteins and that the less active gene is more easily silenced by DNA methylation than occurs by chance.

We will test these alternatives by
1) studying the genetic variant and MLH1 methylation and repression in a large number of patients with colorectal tumours
2) using cell lines to look for proteins which bind the DNA preferentially on one of the variants, and to study how MLH1 methylation and repression occur over time
3) finding out how the genetic variant influences cancer growth in mouse models of the human disease

The knowledge from these studies will help us to understand the effects of MLH1 disruption on tumours and ultimately provide information to help with patient diagnosis, prognosis and therapy. New treatments which remove methylation in tumours are being developed, and our findings, and the cell and mice models we create, should help to determine if and when these therapies could be used to treat colorectal cancer patients with MLH1 repression.

Microsatellite-unstable (MSI+) tumours are commonly found in the colon where they comprise ~15% of all cancers. The underlying cause is mismatch repair (MMR) deficiency which in the majority of cases is caused by MutL homologue 1 (MLH1) promoter methylation. Why MLH1 is inactivated rather than other MMR genes and why methylation occurs rather than gene mutations are unknown, although there may be a causal link to the putative CpG Island Methylator Phenotype (CIMP). Intriguingly, a polymorphism, rs1800734, lies within the MLH1 promoter upstream of the translational start site of MLH1, and this SNP shows a strong association with the risk of MSI+ colorectal cancer (CRC), but not with MSI-negative cancers.

We shall
1)expand the analysis of the association between rs1800734 and CRC risk, and mine existing data for rare germline genetic variants near rs1800734
2)determine associations between rs1800734 genotype, MLH1 methylation, MLH1 expression and CIMP in human normal, benign and malignant colorectal samples and find the statistically most likely causal links
3)construct isogenic CRC cell lines with all three rs1800734 genotypes
4)investigate chromatin structure and transcription factor binding to identify molecules and processes differentially associated with rs1800734 alleles
5)modulate DNA methylation and follow subsequent methylation (re-)acquisition and consequences according to rs1800734 genotype.
6)repress MLH1 mRNA transcription and determine whether promoter methylation occurs as a consequence
7)set up a mouse model of rs1800734 and determine effects of each SNP genotype on MLH1 methylation and intestinal tumours in a Braf-mutant model of MSI+ carcinogenesis
8)if time permits, develop a mouse with a general tendency to MLH1 methylation and determine the influence of rs1800734 therein
These experiments will identify many of the mechanisms underlying the association of rs1800734 with MSI+ CRC and provide broader insights into MMR-deficient canc

ACADEMIC IMPACTS
1) RESEARCH DELIVERABLES
(Please refer to section on Academic Beneficiaries for full list of research users). The deliverables will relate to the MLH1 gene and the SNP rs1800734 including its methylation, gene expression, role in tumorigenesis, association with colorectal cancer risk, mutation selection, cancer evolution, general epigenetic and gene regulation mechanisms and may provide information useful for medical genetics, screening, diagnostics and therapeutics. The proposal will directly relate to the field of colorectal cancer biology and more broadly the cancer, epigenetic, gene regulatory research fields and cancer medicine.
2) TRAINING THROUGH RESEARCH
The research assistant will be given full laboratory training in technical skills, project design, data analysis, manuscript preparation and critical literature review. Internal and external training courses and opportunites to work with collaborators will be encouraged whenever appropriate, equipping the post holder for future higher degrees or independent research.
3) BIOLOGICAL AND METHODOLOGICAL RESOURCES
The isogenic cell lines and transgenic mice created to study rs1800734, MLH1 and cancer predisposition, and any additional biological resources created during the project, will be an asset to those in the colorectal cancer field and will be made available to the research community after publication. New or improved methodology including developments of the CRISPR/Cas9 system may be useful to the wider community as described above and will be shared and published.
SOCIO-ECONOMIC IMPACTS
4) HEALTH AND MEDICINE
rs1800734 is known to increase colorectal cancer risk and an increase in understanding may help to decide its importance in future screening, diagnostic or treatment programmes. In addition, the importance of and ability to manipulate methylation at the MLH1 locus could have long-term prospects for therapeutics.
5) ENHANCING SKILLS OF MEDICAL AND SCIENCE STUDENTS
The University of Oxford expects medical and basic science students to undertake research projects as part of their course. This proposal would allow the PI to host and train undergraduate and masters students from Oxford and other leading universities.
6) PUBLIC ENGAGEMENT WITH RESEARCH
Cancer research is able to attract the attention of the general public. We will take available opportunities to explain the importance of basic research in the advancement of cancer screening, diagnosis and treatment.