Two-stage genome-wide 5-hydroxymethylcytosine (5hmC) reprogramming during colorectal carcinogenesis and liver metastasis

This research is designed to investigate the role 5hmC plays in colon cancer. We already know that levels of 5hmC are much higher in normal colon cells than colon cancer cells, and previous research into other cancers has shown that stimulating cells to produce more 5hmC leads to slower tumour growth. 5hmC is therefore seen as a potential diagnostic biomarker for many cancers. However, not all cancers behave identically and there is still very little understanding of how the loss of 5hmC actually contributes to colon cancer. Our goal is to determine whether targeting 5hmC is a viable therapeutic option for treating colon cancer specifically.

We have two research objectives:
1. To discover what role 5hmC plays during colon cancer progression
2. To discover how changes in 5hmC levels affect metastasis

For the first objective we will investigate two specific models for 5hmC function. The first addresses whether reduced levels of 5hmC affect the way RNA is created ("spliced"). There is evidence that when present near the splice sites, 5hmC facilitates successful splicing and it is therefore logical to hypothesise that when 5hmC is removed splicing may occur aberrantly. Several cancers have a population of aberrantly spliced RNA variants that can drive cancer cells and enable them to divide continually and become invasive.
The second model is based on findings that have shown that the production of 5hmC is necessary for normal colon cell development. The colon is lined with a series of finger-like projections called villi, at the base of which there is a pool of colon stem cells capable of developing into mature colon cells. These stem cells have very little 5hmC and acquire 5hmC when they develop into the cells that form the mature villi. Colon cancer cells have many similar features to colon stem cells including a lack of 5hmC. Therefore we are testing whether the lack of 5hmC helps cancer cells to remain stem-like and continually divide.

For the second objective we have evidence that 5hmC levels might actually increase when colon cancer cells spread ("metastasise") to the liver. During metastasis cells change their shape in order to migrate to different sites. This shape-changing process is the same process that occurs during embryonic development when cells migrate to different regions in order to form various organs. The enzymes involved in producing 5hmC play a prominent role during this developmental process. We will therefore test whether tumour cells that have been treated to produce more 5hmC have an increased or decreased ability to form metastatic tumours. We will also do the complementary analysis to see whether cancer cells with an inability to produce 5hmC can become more or less invasive.
The outcome of this research will help us understand how this epigenetic mark works and also tell us whether targeting 5hmC levels in colon cancer is a safe therapeutic option from which patients would be likely to benefit.

The purpose of this programme is to discover the function of 5-hydroxymethylcytosine (5hmC) during colorectal cancer (CRC) and liver metastasis.

5hmC is produced as an intermediate DNA demethylation product when 5-methylcytosine is oxidised by the TETs (Ten-Eleven-Translocation proteins). In normal tissue 5hmC is a stable epigenetic mark associated with active gene expression. Recently 'Loss of 5hmC' has emerged as a hallmark of many primary cancers, an early detection and potential cancer
staging tool. The TETs are exceptionally responsive to metabolic stimuli and this opens up several avenues for therapeutic intervention. However, the therapeutic potential seems to be cancer-specific, since unexpectedly, in some cancers, (e.g. prostate cancer), metastasis and recurrence are associated with increased levels of 5hmC and poor survival. We have preliminary data that suggest that this may also be the case for CRC.

In this project we are using a combination of bioinformatics, mouse models and a unique series of CRC model cell lines in an organoid culture system to discover the causal relationship between 5hmC levels, the tumour phenotype and metastatic transformation in CRC. We propose to test two models for 5hmC function. The first model proposes that changes in 5hmC mediate oncogenic splice switching to drive cancer progression. The second model proposes that 5hmC levels regulate the stem-like phenotype of cancer cells. These models are not mutually exclusive.
The outcome of this research will inform how 5hmC contributes to the cancer phenotype and whether it is a viable therapeutic target. Although the focus is on the role of 5hmC, an additional outcome of this project will be to further elucidate the catalytic function of TET enzymes in cancer progression.

Colorectal cancer (CRC) is a major public health problem, being the third most commonly diagnosed cancer and the fourth largest cause of cancer death worldwide. Several studies have identified changes in DNA methylation associated with disease states including colon cancer. There are also chemotherapy drugs that target DNA methylation. However, these drugs are not in use for CRC. There is still a gap in our knowledge with regard to the causal relationship of DNA methylation changes, and how this relates to cell biology and ultimately the tumour phenotype. Our research into 5-hydroxymethylcytosine (5hmC) looks at the mechanistic links between epigenetics and cancer. We anticipate that academics interested in epigenetics, DNA methylation, 5hmC, gene regulation, alternative splicing, cancer, metastasis, stem cells and regenerative medicine will be interested in this research and that they will be able to build upon our findings and methodologies to answer related questions in their research fields.

In the longer term clinical and pharmaceutical stakeholders will be beneficiaries of this research, which will ultimately be translated for patient benefit. Our research will produce information on 5hmC that clinicians treating CRC patients may use for diagnostic and prognostic purposes. They will benefit from our research showing the effects on gene expression and the relevance to tumour progression and metastasis. Our data will specifically indicate whether 5hmC levels need to be therapeutically upregulated during early stages of colon cancer and possibly inhibited at later stages to prevent metastasis.

Our methodology and cellular models will be of interest to pharma as well as researchers interested in using these for high throughput screening and as alternatives to animal experimentation.

Finally, our research provides high learning and development potential for researchers working on the programme who will get the opportunity to learn state-of-the-art mass spectrometry techniques in epigenetics, quantitative cell biology imaging, organoid culture, all applied to hypothesis-driven research.

Overall, this research builds upon the UK's strength in epigenetic research and aligns with the MRC's strategic plan to understand the regulatory mechanisms that underpin disease.