Elucidating the molecular and epigenetic basis of cancer initiation

Changes in gene function, which are not caused by alterations within the gene sequence, are referred to as "modulation of gene function by epigenetic mechanisms". Epigenetic mechanisms involve addition and removal of tiny chemical groups attached to DNA and proteins. These epigenetic modifications play a key role in helping cells within the body perform the correct function. For example, brain cells and skin cells have essentially the same genes and genetic sequence, but they are fundamentally different both in structure and function, and their distinct identities need to be maintained throughout their lifetime. During development, the fertilised egg gives rise to over 200 different types of cells in the body, which accumulate epigenetic modifications and together confer an identity or signature to each cell type - these alterations are defined as their epigenetic information.

Cancer cells have in essence "forgotten" their identity, and epigenetic mechanisms are thought to be involved in erasing this memory. Generally, cancer cells evolve from a so-called "cancer stem cell" into more mature cancer cells, accumulating both genetic and epigenetic mutations, which eventually confer a survival advantage to the cancer cells in response to the body's normal defences. The fundamental question is how such cancer stem cells with this "forgotten identity" arise in the first place, and to what extent epigenetic modifications contribute to this process. Recently scientists have demonstrated that tumours can be induced in the absence of genetic changes, confirming that at least in some instances only epigenetic changes, such as "forgetting identity", are sufficient to initiate cancer.

We aim to identify the aberrant epigenetic changes, which can trigger a normal cell to become cancerous. Technological advances in the last couple of years have offered unprecedented potential in addressing this question. Scientists have developed a novel tool called "CRISPR/Cas9 based gene editing". With this tool we can induce a cut in the cell's DNA in essentially any region with the help of a "guide" RNA. This guide finds the sequence of interest, and recruits the enzyme Cas9 to this site to induce a cut in the DNA and generate a genetic modification. Our unique approach in inducing epigenetic mutations is that we use an inactive Cas9 enzyme which is fused to a DNA methylating (gene silencing) enzyme. In this way, wherever the guide attracts the inactive Cas9 to a DNA site, it will hypermethylate (silence) that specific DNA sequence, inducing an epigenetic change rather than a cut. Following this, we can assess the cancerous potential of cells harbouring such epigenetic changes.

Cancer is associated not only with aberrant hypermethylation and silencing of some genes (as described above) but also with general loss of methylation in the cell's DNA, therefore loss of epigenetic information. Recently, I have discovered a link between signalling pathways and widespread loss of DNA methylation in mouse embryonic stem cells (which often have common features with cancer cells). In this proposal, I aim to find proteins/enzymes involved in this process. Using an embryonic stem cell model system for DNA demethylation will allow identification of factors that might play a role in general loss of methylation in cancer. As a result, we hope to find new targets which can be used in developing novel cancer drugs.

Ultimately, by understanding the epigenetic basis of cancer stem-cell production, our aim is to stop cancer before it starts.

In Objective 1 of this proposed research we will use state-of-the-art technologies to induce epigenetic modifications (or epimutation) and ask what functional consequence they have on cell function. These powerful tools are currently being developed and involve the CRISPR/Cas9 system with slight modifications whereby the Cas9 is inactive and fused to DNA methyltransferases or 5-methylcytosine TET oxidases. We are implementing this system in collaboration with the Jurkowski lab (Stuttgart University) to understand epigenetic processes that occur during cancer initiation.

Selection of the genomic regions targeted for epimutations is based on the knowledge accumulated so far in cancer epigenetics research. Numerous promoters are hypermethylated in cancer but key is the identification of combinations of genes that have the potential to be drivers in the process of cancer initiation when no genetic mutation is required.

The cell types to test these epimutations on were chosen based on the hypothesis that cancer stem cells can arise from adult stem cell compartments or through dedifferentiation/reprogramming of progenitors or differentiated cells. This is compatible with the knowledge that cancer stem cells harbour stem cell-like properties. Therefore we will start by using mesenchymal stem cells (MSC), neural stem cells (NS), mammary epithelial and myoepithelial cells.

Cells harbouring the induced epimutations will be analysed for tumourigenic properties in vitro and injected in immune-compromised mice in order to measure tumour growth in vivo. Subsequently these tumours will be utilised to unravel the molecular analysis of cellular transformation.

In Objective 2, we will use the embryonic stem cell model system where transition between two states (primed to ground) is accompanied by widespread DNA demethylation. The methodology involves use of shRNA libraries (whole genome and sub-libraries) to find proteins involved in the DNA demethylation pathway.

The proposed research uses state-of-the-art technology called Epigenetic Editing based on the CRISPR/Cas9 gene editing system as detailed in the summary and case for support sections. This is a highly promising technology, which will have impact in many areas of research and future therapies. Since we are at the forefront of academic research using this technology, the proposed work may be used as a reference protocol by other researchers planning to use this technology in cancer research, ageing and regenerative medicine fields. The academic community will therefore benefit by us implementing, using, improving and functionally exploring this technology.

The overall impact of this research (both Objectives 1 and 2) can be divided in three categories: Academic Research, Medical/Therapeutic and Commercial/Societal.

As described in the Academic Beneficiaries section, the proposed research will advance the cancer epigenetic field by solving a key problem, which is to identify driver epimutations in cancer and separate them from passenger epimutations. Technologically, the academic community will benefit from us analysing the advantages and limitations of the Epigenetic Editing system, assessing its versatility and functional relevance in basic research. It is highly likely that combinations of epimutations will induce a transformed cell phenotype therefore this can be used as a model system for epigenetically induced cancer, which does not exist at present.
Objective 2 is a novel discovery project using embryonic stem cells as a model system for global demethylation. I am very familiar with this system, and it will reveal proteins involved in the demethylation pathway, a topic of high interest in the epigenetic research and reprogramming community. This has potential to open up novel avenues in DNA methylation research and improve iPS reprogramming protocols.

Impact in Medical Research and Therapy: our ultimate goal is to further develop these tools into targeted epigenetic therapeutics whereby, in combination with in vivo delivery methods (such as virus like particles, etc.), specific cells will be targeted to induce epimutations. This could lead to a change in the cell's identity with the end result the induction of cell death or transdifferentiation. In fact, we are currently developing such tools in collaboration with Dr. Jens Gruber at the Goettingen Primate Centre in Germany.
In addition, this research will contribute to understanding and potentially identifying the elusive cancer stem cells and where/how they originate. Ultimately, finding cancer inducing combinatorial epimutations could lead to new diagnostic tools and hopefully a novel approach to detecting rare malignant cells in tissues.
With regards to Objective 2, defining the demethylation pathways and the factors involved could lead to identification of new molecules that contribute to the genomic hypomethylation observed in cancer. This could contribute to new cancer therapies by inhibiting processes that disrupt the correct maintenance of genomic DNA methylation.

Commercial and Societal benefits: discoveries resulting from this work and implementing targeted epigenetic therapeutics could lead to potential business partnerships with biotechnology companies. This method will allow in vivo delivery of a targeted epimutation system in the cell type of choice and can therefore be custom designed provided unique cell surface molecules are identified. This work will contribute to maintaining the UK at the forefront of novel concepts in epigenetic therapies and ultimately introduce new therapies with a positive impact on public health and well-being. If epigenetic mechanisms are involved in cancer initiation, this work has the potential to result in cancer biomarkers, which could be used in prevention screening strategies to ultimately stop cancer development.