Defining the Rho-kinase PKN2 as a 1p22 encoded tumour suppressor in colorectal cancer.

Bowel cancer is the 4th most common cancer in the UK and the second largest cause of cancer deaths. Preventing and treating these diseases represents a huge unmet clinical need. The bowel is one of the most challenged organs in our body, through exposure to physical stresses, ingested toxins, gut bacteria and digestive processes, all of which can induce tissue damage and inflammation.

In order to remain healthy and replace damaged cells and tissue, the bowel undergoes constant cell growth, and this presents an ideal environment for cancers to develop. To limit cancer development, cell growth in the bowel is under very tight control. Only a small group of cells, called stem cells, have the ability to replace damaged tissues, and these cells are the most likely to change into cancer cells.

Stem cells are partly controlled through genes called tumour suppressors. Normally, these genes stop stem cells growing unless they are provided with very specific instructions to allow the bowel to repair. In cancers these tumour suppressor genes are mutated or lost, leading to uncontrolled growth, resulting in bowel cancer.

Using bioinformatic data from human cancers and a mouse model of colon cancer, we have identified an entirely new tumour suppressor gene, called protein kinase N2 (PKN2). In human cancers, PKN2 is commonly suppressed through a variety of mechanisms. As an example, a region of chromosome 1 called 1p22, where the gene for PKN2 sits, is lost in 30% of aggressive colon cancers.

In addition, there are genetic mutations in PKN2 in some colon cancers, which can indicate a role in cancer development. Further, we show that PKN2 is reduced in samples from cancer that has spread from the colon to other organs in the body. Using a genetic mouse model, we have found that specifically removing the PKN2 gene results in mice becoming highly susceptible to colon cancer.

This provides strong and direct evidence that PKN2 is a tumour suppressor, which can prevent colon cancer from developing.

In this grant we wish to explore how PKN2 stops bowel cancers from developing. Preliminary experiments suggest that PKN2 may prevent tissue damage and inflammation, which are known to play important roles in cancer development in the colon. Our data suggest that PKN2 is important for strengthening the connections between the cells lining the intestine, which could explain why loss of PKN2 is associated with a greater susceptibility to intestinal injury and cancer development.

We will use our PKN2 mouse model to show how genetic deletion of PKN2 enhances tissue damage and inflammation in the bowel. We will also use laboratory cancer cell lines and 'mini-gut' models called organoids to examine the molecular machinery regulated by PKN2 in normal intestinal cells. Next, we wish to examine colon cancer in a genetic mouse model.

Human colon cancers are almost always initiated by mutation of an important tumour suppressor gene called adenomatous polyposis coli (APC). We will breed our PKN2 genetic mouse model with a mouse in which the APC gene is mutated. These mice develop spontaneous bowel tumours and we expect that deleting PKN2 will make tumours develop more rapidly and in a more aggressive way.

This will help us understand how PKN2 contributes to cancer progression driven by the most common mutation found in human colon cancer. Finally, we will examine the role of PKN2 in mini-gut cultures developed from human colon cancer patients. We will delete PKN2 using CRISPR-Cas9 technology and see how this changes the growth, and invasiveness of a panel of cancer-patient derived mini-guts.

This will provide crucial information to help translate our findings to clinical benefit. Together, this research will help define an exciting new tumour suppressor pathway, which can help prevent colon cancer, and potentially other cancers, from developing.