Small, cancer-associated DNA circles “hitchhike” on chromosomes during cell division to spread efficiently to daughter cells by co-opting a process used to maintain cellular identity through generations, Stanford Medicine-led research has found.
These circles, known as extrachromosomal or ecDNA, are major drivers in human cancers. Blocking their ability to associate with chromosomes causes the loss of the circles during cell division and the death of lab-grown cancer cells. Targeting this weak link in the circles’ proliferation could lead to new classes of cancer therapies, the researchers predict.
“Unfortunately, ecDNAs have developed a crafty mechanism that allows them to wreak havoc on human health,” said professor of pathology Paul Mischel, MD. “They are using nature’s own method of gene expression and cell fate to ensure they are safely distributed into the next generation of cells and not lost into the cytoplasm or extracellular space when a cell divides.”
Mischel leads an international team of experts known as eDyNAmiC that was awarded a $25 million grant from the Cancer Grand Challenges initiative in 2022 to learn more about the circles. Cancer Grand Challenges, a research initiative co-founded by Cancer Research UK and the National Cancer Institute in the United States, supports a global community of interdisciplinary, world-class research teams to take on cancer’s toughest challenges.
Mischel, who holds the Fortinet Founders Professorship, and Howard Chang, MD, PhD, professor of dermatology, genetics and pathology who is the Virginia and D.K. Ludwig Professor in Cancer Research, are co-senior authors of the paper, which was published on Nov. 19 in Nature. Graduate student Venkat Sankar and former graduate student King Hung, PhD, are the first authors of the study. Mischel is also deputy director of translational science and an institute scholar at Stanford University’s Sarafan ChEM-H.
The ecDNA origin story
ecDNAs don’t spring into being from nowhere; they are the byproduct of errors in the replication or repair of chromosomal DNA, or spontaneous rearrangements, that release linear portions of the genome into a cell’s nucleus. The ends of these segments can then connect, forming circles. They are likely forming all the time, but only those that confer a survival benefit to the cell and contain what is known as an origin of replication that allows them to be copied have a shot at being inherited when the cell divides.
Frequently, the ecDNA survival benefit is due to the presence of cancer-associated genes called oncogenes. When a cancer cell contains multiple oncogene-encoding ecDNAs, they can supercharge the cell’s growth and allow it to evade internal checkpoints meant to regulate cell division. The ecDNAs also sometimes encode genes for proteins that can tamp down the immune system’s response to a developing cancer — further advantaging tumor growth.
Past research by the eDyNAmiC team has shown that the presence of ecDNA jumpstarts a cancerous transformation in precancerous cells. The researchers found that 17.1% of tumors contained ecDNA, that ecDNA was more prevalent after targeted therapy or cytotoxic treatments like chemotherapy, and that the presence of ecDNA was associated with metastasis and poorer overall survival. But, although the circles have been observed fraternizing with chromosomes as cancer cells divide, the mechanism they relied on to do so has been unclear.
“It’s been a biological mystery,” Chang said. “Chromosomes have regions called centromeres to which cellular machinery attaches to faithfully distribute one copy to each daughter cell during division. But ecDNAs don’t have centromeres. There must be some other specific sequences on the circles that enable them to stick to chromosomes.”
The mechanism is not unheard of. Some viruses, including the Epstein-Barr virus, devote portions of their genomes to regions that stick to human DNA.
To investigate what’s going on with ecDNAs, the researchers developed a screening method called Retain-seq in which the human genome was chopped into short segments and inserted into circular bacterial DNA. These hybrid DNA molecules were then introduced into human cells. The researchers were able to identify sequences, which they term retention elements, that allowed the bacterial DNA to persist in subsequent generations of cells. More than 14,000 possible retention elements were identified; the researchers selected six for further study based on their similarity to sequence of ecDNAs identified in colorectal and brain cancer cells.
“These retention elements are prevalent across the human genome,” Chang said. “They are essential to the survival of the ecDNAs over generations, and often to the survival of the cancer cells that contain them.”
Specifically, many retention elements had characteristics of what are known as promoter and enhancer regions. These regions, which can be far apart on the DNA, are peppered with proteins that can attract one another, resulting in loops that trigger the expression of nearby genes. The retention elements were also lacking in chemical tags called methyl groups associated with the tight bundling of DNA that can make the DNA inaccessible for gene expression.
Most promoters and enhancers, along with the remainder of the genome, are tightly coiled and not active during cell division (a stage called mitosis). But a few key locations remain active — usually around genes that encode proteins specific for cell fate. These regions are called mitotic bookmarks.
“The idea is that these mitotic bookmarks enable the cellular memory that passed from one generation to the next,” Chang said. “The new daughter cells uncoil their chromosomes and see, for example, ‘Oh, I used to be a colon cell. Let’s turn on all the colon genes.’”
Genetic lottery
Chang, Mischel and their colleagues found that retention elements physically interact with mitotic bookmarks in the chromosomes in a way that mimics the interactions of promoters and enhancers in healthy cells. But they do so randomly: There is no process to ensure that equal numbers of each ecDNA in a cell are portioned out to the daughters. This random assortment means that individual cancer cells may inherit all, some or none of the ecDNA from their parent. The genetic lottery allows cancer cells to evolve rapidly, adapting to changing conditions and nimbly developing drug resistance in response to therapy.
“It’s like two buses going to the same destination pull up to the bus stop and passengers grab seats wherever they can,” Mischel said. “Nothing is assigned. If you don’t get on the bus, you’re out of luck. You’re not going anywhere,” meaning the unbound ecDNAs are released into the cytoplasm and essentially disappear.
Finally, the researchers noted that the retention elements were usually lacking in chemical tags called methyl groups that cells use to turn off gene expression. When they triggered the addition of methyl groups on the elements in brain cancer cells grown in the lab, they found that the affected ecDNAs no longer attached to chromosomes and were significantly less likely to be portioned to daughter cells. The switch also substantially reduced the viability and proliferation of the cancer cells — presumably because they had lost the oncogenes encoded on the ecDNA.
“This shows the remarkable specificity of this process,” Mischel said. “It’s not due to random stickiness. The ecDNAs are actively using cellular machinery that evolved for one purpose — maintaining a cell’s identity through generations — to ensure their own survival. This is a remarkable co-opting of nature’s own mechanisms. But it’s also a weakness; if we can interrupt that process, we unlock new therapeutic opportunities for many deadly cancers.”
Researchers from the Charité-Universitätsmedizin Berlin, the Max Delbrück Center for Molecular Medicine and the Berlin Institute of Health contributed to the research.
This work was delivered as part of the eDyNAmiC team supported by the Cancer Grand Challenges partnership funded by Cancer Research UK. It was also supported by the National Institutes of Health (grants OT2CA278688, OT2CA278644, F99CA274692, K00CA274692, and K99CA286968), the National Science Foundation, the Damon Runyon Cancer Research Foundation and the Howard Hughes Medical Institute.
Read more about ecDNA in Stanford Medicine magazine.
