New research suggests the non-coding BRAF pseudogene can produce a lymphoma phenotype in mice.
Pseudogenes, a sub-class of long non-coding RNA that developed from the genome’s protein-coding genes but lost the ability to produce proteins, have long been considered genomic “junk.”
Yet the retention of pseudogenes during evolution has suggested they may have biological functions and contribute to disease development.
Now, researchers have provided evidence that one of these pseudogenes does have a role in causing cancer, and they described the discovery in Cell.
The investigators found that, independent of any other mutations, abnormal amounts of the BRAF pseudogene led to the development of an aggressive, lymphoma-like disease in mice.
The team said this finding suggests pseudogenes may play a primary role in a variety of diseases, and the functional genome could be much larger than we thought.
“Our mouse model of the BRAF pseudogene developed cancer as rapidly and aggressively as it would if you were to express the protein-coding BRAF oncogene,” said study author Pier Paolo Pandolfi, MD, PhD, of Harvard Medical School in Boston, Massachusetts.
“It’s remarkable that this very aggressive phenotype, resembling human diffuse large B-cell lymphoma, was driven by a piece of so-called ‘junk RNA.’”
The researchers’ discovery hinges on the concept of competing endogenous RNAs (ceRNA), a functional capability for pseudogenes Dr Pandolfi and his colleagues described almost 5 years ago. The team discovered that pseudogenes and other noncoding RNAs could act as “decoys” to divert and sequester microRNAs away from their protein-coding counterparts to regulate gene expression.
“Our discovery of these ‘decoys’ revealed a new role for messenger RNA, demonstrating that, beyond serving as a genetic intermediary in the protein-making process, messenger RNAs could actually regulate expression of one another through this sophisticated new ceRNA ‘language,’” Dr Pandolfi said.
He and his colleagues showed that, when these decoys prevented microRNAs from fulfilling their regulatory function, there could be severe consequences, including making cancer cells more aggressive.
With their new research, the investigators wanted to determine if this same ceRNA cross-talk took place in a living organism and if it would result in similar consequences.
“We conducted a proof-of-principle experiment using the BRAF pseudogene,” explained Florian Karreth, PhD, a fellow in the Pandolfi lab. “We investigated whether this pseudogene exerts critical functions in the context of a whole organism and whether its disruption contributes to the development of disease.”
The researchers focused on the BRAF pseudogene because of its potential ability to regulate levels of the BRAF protein, a well-known proto-oncogene linked to numerous cancer types. In addition, the BRAF pseudogene is known to exist in both humans and mice.
The team began by testing the BRAF pseudogene in tissue culture. Their findings showed that, when overexpressed, the pseudogene operated as a microRNA decoy that increased amounts of the BRAF protein.
This, in turn, stimulated the MAPK signaling cascade, a pathway through which the BRAF protein controls cell proliferation, differentiation, and survival and which is commonly found to be hyperactive in cancer.
The investigators went on to create a mouse model in which the BRAF pseudogene was overexpressed. And they found these mice developed an aggressive, lymphoma-like cancer.
“This cancer of B-lymphocytes manifested primarily in the spleens of the animals but also infiltrated other organs, including the kidneys and liver,” Dr Karreth said. “We were particularly surprised by the development of such a dramatic phenotype in response to BRAF pseudogene overexpression alone since the development of full-blown cancer usually requires two or more mutational events.”
Mice overexpressing the BRAF pseudogene displayed higher levels of the BRAF protein and hyperactivation of the MAPK pathway, which suggests this axis is critical to cancer development.
The researchers confirmed this by inhibiting the MAPK pathway with GSK1120212, a MEK inhibitor that dramatically reduced the cancer cells’ ability to infiltrate the liver in transplantation experiments.
The investigators further validated the microRNA decoy function of the BRAF pseudogene by creating two additional transgenic mice, one overexpressing the front half of the BRAF pseudogene, and the other overexpressing the back half.
Both of these mouse models developed the same lymphoma phenotype as the mice overexpressing the full-length pseudogene, a result the researchers described as “astonishing.”
“We never expected that portions of the BRAF pseudogene could elicit a phenotype,” Dr Karreth said. “[W]hen both front and back halves induced lymphomas, we were certain the BRAF pseudogene was functioning as a microRNA decoy.”
The investigators also found the BRAF pseudogene is overexpressed in human B-cell lymphomas, and the genomic region containing the BRAF pseudogene is commonly amplified in a variety of human cancers. This suggests the group’s murine findings are of relevance to human cancer development.
Moreover, the researchers found that silencing the BRAF pseudogene in human cancer cell lines that expressed higher levels led to reduced cell proliferation. This reinforces the importance of the pseudogene and suggests a therapy that reduces BRAF pseudogene levels may be beneficial to certain cancer patients.
“While we have been busy focusing on the genome’s 20,000 coding genes, we have neglected perhaps as many as 100,000 noncoding genetic units,” Dr Pandolfi noted. “Our new findings not only tell us that we need to characterize the role of all of these non-coding pseudogenes in cancer, but, more urgently, suggest that we need to increase our understanding of the non-coding ‘junk’ of the genome and incorporate this information into our personalized medicine assays.”
“The game has to start now. We have to sequence and analyze the genome and the RNA transcripts from the non-coding space.”
