Genetic mutations can alter how the proteins produced by our genes function and can lead to diseases like cancer. Now researchers have used the gene-editing technology CRISPR/Cas 9 in a less commonly used way, producing liver cancer tumors to better understand the genetic mutations underlying them.
Genes contain the information required to produce proteins. Splicing is the process by which an RNA message copied from information encoded in a gene is edited before it’s used as a blueprint to make a specific protein.
Proteins originating from a single gene that are highly similar in function but with different amino acid sequences are called isoforms. Isoform generation is the body’s way of specializing the properties of a gene or protein. Different isoforms can result in the formation of different types of cancer tumors. These tumor subtypes are hard to produce in a lab, making them difficult to study.
To better understand how isoforms lead to the creation of different types of liver cancer, a new study used the gene-editing tool CRISPR/Cas9 to examine how different isoforms lead to the development of different tumor subtypes.
“Everyone thinks that cancer is just one type,” said Semir Beyaz, a corresponding author of the study. “But with different isoforms, you can end up with cancer subtypes that have different characteristics.”
The researchers targeted a single section of a mouse gene, CTNNB1, using CRISPR/Cas9. The CTNNB1 gene provides instructions for making a protein called beta-catenin which is involved in the regulation and coordination of cell-to-cell adhesion and in gene transcription.
Previous studies have identified beta-catenin as a potent oncogene, a gene that can transform a healthy cell into a tumor cell. Mutations of the CTNNB1 gene have been associated with a wide range of cancers, including liver and colon cancer. Mutations in exon 3 of the CTNNB1 gene – an exon is a section of DNA or RNA that codes for proteins – are key to the transcription of genes involved in tumor formation.
In the current study, researchers wanted to determine how beta-catenin mutations drove the development of liver cancer tumor subtypes, hepatocellular carcinoma (HCC) and hepatoblastoma (HB). HCC is the most common type of adult liver cancer, accounting for around 90% of all liver cancers, while HB is a rare form of liver cancer commonly seen in children.
Usually, CRISPR/Cas9 technology is used to inhibit gene function by removing sections of the DNA sequence (loss-of-function). But here, for the first time, the researchers used it in gain-of-function research to create different cancer-causing mutations in mice.
Using CRISPR/Cas9 in this way stimulated protein activity and, consequently, the growth of tumors. By genetically sequencing the tumor subtypes, HCC and HB, the researchers found that the CRISPR/Cas9-induced beta-catenin isoforms drove the liver tumor subtypes.
“We were able to define those isoforms that [are] associated with different cancer subtypes,” Beyaz said. “That was, for us, a surprising discovery.”
To confirm that these isoforms led to mutation, the researchers tested to see if they could produce the liver cancer subtypes in mice without using CRISPR. They found that they could.
The study highlights the potential for using CRISPR/Cas9 in gain-of-function research and has created a new method of modeling certain liver tumor subtypes. It also further demonstrates the role exon 3 plays in tumor development and the benefits of targeted exon skipping.
Exon skipping is a therapy that uses a mutation-specific antisense oligonucleotide (AON) – a lab-made bit of DNA or RNA that can bind to specific RNA molecules – to induce RNA splicing that causes cells to ‘skip over’ faulty or misaligned exons.
The researchers hope that their findings might guide future research into new therapeutic interventions for cancer.
“Ultimately, what we want to do is find the best models to study the biology of cancer so that we can find a cure,” Beyaz said.
The study was published in The Journal of Pathology.
Source: Cold Spring Harbor Laboratory
