The p53 gene plays an important role in cell biology as it regulates the cell cycle and halts the formation of tumors. While the gene was discovered more than four decades ago, researchers are still determining exactly how it works to improve cancer therapy. Now, researchers at the Wistar Institute have uncovered a key mechanism as to how p53 suppresses tumors.
The findings are published in Cancer Discovery in an article titled, “An African-specific variant of TP53 reveals PADI4 as a regulator of p53-mediated tumor suppression.”
“TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified,” wrote the researchers. “Here, we characterize a rare, African-specific, germline variant of TP53 in the DNA binding domain, Tyr107His (Y107H). NMR and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the non-natural amino acid citrulline.”
By using a genetic variant of p53 and comparing what that variant failed to accomplish with what the healthy “wild type” p53 gene could do, the researchers discovered the mechanism by which p53 triggers immune function that, in turn, kills the tumor.
“The paradigm shift is that instead of asking ‘What does p53 do’ we were able to use a lesser-functioning but cancer-predisposing genetic variant in African Americans to tell us ‘What does p53 not do when it doesn’t suppress cancer?’” said Maureen E. Murphy, PhD, senior author on the paper and deputy director of the Ellen and Ronald Caplan Cancer Center and the Ira Brind professor and program leader of the molecular & cellular oncogenesis program at the Wistar Institute.
The researchers sought to determine how p53 suppresses tumors by exploring how one particular hypomorph fails to suppress them. The researchers chose an African-specific variant called Y107H due to the fact that African Americans have the largest cancer burden of any ethnic group in the world. Their first hypothesis was that they could use the hypomorph to find which “downstream” genes—which p53 would ordinarily turn on—are critical for suppressing tumors. Their second hypothesis was that they could then screen for drugs that would kill the hypomorph tumors: Murphy’s group accomplished both goals.
The researchers began by using CRISPR engineering to make a mouse model of their African-specific hypomorph Y107H. Next, the researchers created tumor cell lines with their Y107H hypomorph, as well as cell lines with a G334R. They then compared which genes were turned on by normal, or wild type, p53 (to suppress the tumor) but not turned on by the two hypomorphs (which failed to suppress the tumor). The gene that met these conditions was PADI4.
“It’s as though this was the key p53 target gene that, every time you have a genetic variant that predisposes you to cancer, it cannot turn on this gene,” said Murphy. She added that it makes sense that PADI4 would be implicated because this gene helps the immune system recognize tumors. It does this by modifying components of tumor proteins so that they become citrulline, which is a non-natural amino acid. When the immune system recognizes citrulline as a foreign body, it attacks.
“Essentially, when a tumor cell goes from one cell to two and it’s not supposed to, p53 is alarmed, it turns on PADI4, and PADI4 says, ‘Immune system, you better come get me,’” said Murphy.
The researchers then turned to machine learning to help them to predict which patients would respond to immunotherapy and which would not.
“Enter Noam Auslander, PhD, who is a brilliant machine-learning artificial intelligence person here at Wistar,” said Murphy. “She said, ‘Let me find the genes that p53 and PADI4 control together using bioinformatic approaches and create a gene signature.’”
Auslander analyzed 60,000 tumors in the TCGA database and identified five genes that were coregulated together by wild type p53 and PADI4 and that the Y107H hypomorph couldn’t turn on. She also found that this five-gene signature predicted cancer survival, immune infiltration into the tumor, and who would respond to immunotherapy.