Researchers have developed a CRISPR/Cas9 method that can create myriad mutations and combinations of mutations in mice. A researcher says that the method makes studying how individual mutations affect the development of cancer much easier.
Mutations can cause cancer, but cancer often results not from one mutation but from a combination of mutations.
In cancer research, however, studying the combined effects of mutations can be difficult because focusing on one mutation is the most widely used approach.
Studying several mutations at the same time requires crossing the mice. This might be possible for two mutations but is much more difficult for 11 mutations.
Conversely, discovering the effect of one mutation can be difficult when it is present in cancer along with 10 other mutations.
To overcome this problem, researchers have now developed a CRISPR/Cas9 method that can create single mutations or combinations of mutations in mice they want to investigate.
“When sequencing a tumour to look for mutations, we often find them in many exotic genes, but we cannot say whether the mutations affect cancer development or not. This is especially true if several mutations are present, some of which may be passenger mutations that appear alongside cancer-causing mutations but have no effect. Our CRISPR/Cas9 method enables us to create the mutations in mice that we want to investigate and to study the relevance of the mutations individually,” explains a researcher involved in developing the method, Martin K. Thomsen, Associate Professor, Department of Biomedicine, Aarhus University, Denmark.
The research has been published in Nature Communications.
Induced mutations in mice
The method uses mice that have Cas9 expressed in the organs in which the researchers want to investigate cancer – in this case, the prostate.
Cas9 is the molecular scissors in CRISPR/Cas9, and the CRISPR guide identifies the location in the gene in which the scissors will create a mutation.
When the researchers want to create a very specific mutation in the prostate of living mice, they need to add a targeted CRISPR guide, and Cas9 in the mice will then make the required mutation.
For example, this mutation could be found in a few or many people with prostate cancer, but the importance of the mutation is unknown because it often occurs together with other mutations.
The researchers have also shown that they can create one, three, five, six or even eight mutations in the prostate cells in the same mouse.
“Then, by combining mutations, we can determine what each mutation does and whether it accelerates or delays the development of cancer? We can use this method to answer these questions,” says Martin K. Thomsen.
Mutations increase the risk of metastasis
The researchers used the method to investigate the function of the KMT2C gene in prostate cancer.
KMT2C is relatively frequently mutated in prostate cancer, but its specific role has been a mystery until now. Both cell lines and mice with just this mutation do not develop cancer.
The researchers therefore investigated what happens when this mutation is added to other mutations in prostate cancer.
This part of the research showed that if a tumour is already present and KMT2C is mutated, the cancer metastasises. This indicated that KMT2C affects the ability of tumours to metastasise.
Effect of mutations
Having identified that KMT2C affects metastasis, the researchers investigated the region in which it exerts its effect.
The study showed that mutations in KMT2C upregulate a region within one chromosome with five to 10 genes: the genes in this region of the chromosome are overexpressed.
The researchers investigated the specific effects of the Odam and Cabs1 gene, whose function has not previously been described.
Using CRISPR/Cas9 to mutate the genes, the researchers then investigated whether the mice still metastasised even though KMT2C had mutated, and they did not.
“We identified some drivers that improve understanding of how mutations affect prostate cancer. When KMT2C is mutated, this upregulates the Odam and Cabs1 gene clusters, each of which are essential genes in metastasis. We do not yet know what these genes specifically do, but further research may reveal their function,” explains Martin K. Thomsen.
He also says that after the researchers identified the role of KMT2C in developing metastasis in prostate cancer, some of his colleagues have also become more aware of this among their patients.
“Suddenly they are noticing how often KMT2C is mutated among people with metastasis in prostate cancer – which they would not normally observe when investigating the effects of many mutations. Our discovery may therefore be relevant for human cancer – knowledge that could be worth its weight in gold. The next step will be to discover how this could become clinically relevant,” adds Martin K. Thomsen.
Also initiates other types of cancer
Martin K. Thomsen says that the method can make researchers more aware of the effects of various mutations not only in prostate cancer but also in several other types of cancer.
The researchers have used the method to create specific mutations and combinations of mutations and thereby give mice brain cancer, lung cancer and pancreatic cancer.
Nevertheless, the researchers are struggling to make the method work for colon cancer.
“We are proficient at understanding the mutations in which a gene loses its function, but we struggle a bit with the mutations in which a gene is overactivated. Naturally, we are working on being able to solve this in the future,” concludes Martin K. Thomsen.
