Since the 1950s a chemotherapy drug known as 5-fluorouracil (5-FU) has been used to treat many types of cancer, including blood cancers and cancers of the digestive tract. Doctors believed that the drug works by damaging the building blocks of DNA. However, a new study headed by MIT demonstrates in cancers of the colon and other gastrointestinal (GI) cancers, 5-FU kills cells by interfering with RNA synthesis.
The findings are published in Cell Reports Medicine and could have a significant effect on how doctors treat many cancer patients.
“Our work is the most definitive study to date showing that RNA incorporation of the drug, leading to an RNA damage response, is responsible for how the drug works in GI cancers,” said Michael Yaffe, a David H. Koch Professor of Science at MIT, the director of the MIT Center for Precision Cancer Medicine, and a member of MIT’s Koch Institute for Integrative Cancer Research. “Textbooks implicate the DNA effects of the drug as the mechanism in all cancer types, but our data shows that RNA damage is what’s really important for the types of tumors, like GI cancers, where the drug is used clinically.”
Yaffe hopes to plan clinical trials of 5-fluorouracil with drugs that would enhance its RNA-damaging effects and kill cancer cells more effectively. Yaffe is senior author of the team’s paper, which is published in Cell Reports Medicine and titled “An RNA damage response network mediates the lethality of 5-FU in colorectal cancer.” The paper’s lead authors are Jung-Kuei Chen, PhD a Koch Institute research scientist, and Karl Merrick, PhD, a former MIT postdoc.
Cancer chemotherapy involves the use of drugs that preferentially kill or inhibit the growth of cancer cells, the authors noted. Clinicians use 5-fluorouracil (5-FU) as a first-line drug for colon, rectal, and pancreatic cancers. The drug is usually given in combination with oxaliplatin or irinotecan, which damage DNA in cancer cells. “In modern clinical practice, 5-FU is usually administered in combination with platinum-based DNA-crosslinking agents or DNA-damaging topoisomerase inhibitors,” they further explained. The combination was thought to be effective because 5-FU can disrupt the synthesis of DNA nucleotides. Without those building blocks, cells with damaged DNA wouldn’t be able to efficiently repair the damage and would undergo cell death.
Yaffe’s lab, which studies cell signaling pathways, wanted to further explore the underlying mechanisms of how these drug combinations preferentially kill cancer cells. For their reported study the researchers began by testing 5-FU in combination with oxaliplatin or irinotecan in colon cancer cells grown in the lab. To their surprise, they found that not only were the drugs not synergistic, in many cases they were less effective at killing cancer cells than what one would expect by simply adding together the effects of 5-FU or the DNA-damaging drug given alone. “Surprisingly, combining 5-FU with oxaliplatin or irinotecan resulted in sub-additive loss of viability in nearly all cell lines examined,” they wrote.
“One would have expected that these combinations to cause synergistic cancer cell death because you are targeting two different aspects of a shared process: breaking DNA, and making nucleotides,” Yaffe said. “Karl looked at a dozen colon cancer cell lines, and not only were the drugs not synergistic, in most cases they were antagonistic. One drug seemed to be undoing what the other drug was doing.”
Yaffe’s lab then teamed up with Adam Palmer, an assistant professor of pharmacology at the University of North Carolina School of Medicine, who specializes in analyzing data from clinical trials. Palmer’s research group examined data from colon cancer patients who had been on one or more of these drugs and showed that the drugs did not show synergistic effects on survival in most patients. “Analysis of this clinical trial data revealed, at best, an additive but non-synergistic effect for the combination of oxaliplatin with 5-FU, and a sub-additive effect for the combination of 5-FU and irinotecan,” the investigators wrote. “Taken together, these results indicate that the cytotoxic activities of 5-FU minimally enhanced the DNA damage-induced cytotoxicity conferred by these agents and are instead consistent with the beneficial effects of combination chemotherapy arising from patient-to-patient variability rather than drug additivity or synergy …”.
Yaffe added, “This confirmed that when you give these combinations to people, it’s not generally true that the drugs are actually working together in a beneficial way within an individual patient. Instead, it appears that one drug in the combination works well for some patients while another drug in the combination works well in other patients. We just cannot yet predict which drug by itself is best for which patient, so everyone gets the combination.”
These results led the researchers to wonder just how 5-FU was working, if not by disrupting DNA repair. Studies in yeast and mammalian cells had shown that the drug also gets incorporated into RNA nucleotides, but there has been dispute over how much this RNA damage contributes to the drug’s toxic effects on cancer cells.
Inside cells, 5-FU is broken down into two different metabolites. One of these gets incorporated into DNA nucleotides, and other into RNA nucleotides. In studies of colon cancer cells, the researchers found that the metabolite that interferes with RNA was much more effective at killing colon cancer cells than the one that disrupts DNA.
That RNA damage appears to primarily affect ribosomal RNA, a molecule that forms part of the ribosome—a cell organelle responsible for assembling new proteins. If cells can’t form new ribosomes, they can’t produce enough proteins to function. Additionally, the lack of undamaged ribosomal RNA causes cells to destroy a large set of proteins that normally bind up the RNA to make new functional ribosomes. The authors stated, “Motivated by the unexpected finding of a lack of synergistic or even additive anti-tumor responses to clinically used 5-FU-based combination chemotherapy in a systematic study of human clinical trial data and a panel of CRC cell lines, we found using systems pharmacology, phospho- and ubiquitin proteomics, cell biology, and biochemical validation, that apoptotic cell death induced by 5-FU in CRC cells is primarily mediated by its effects on ribosomal RNA leading to impaired ribosome biosynthesis, rather than through its effects on DNA replication or direct DNA damage.”
They are now exploring how this ribosomal RNA damage leads cells to under programmed cell death, or apoptosis. They hypothesize that sensing of the damaged RNAs within cell structures called lysosomes somehow triggers an apoptotic signal. “My lab is very interested in trying to understand the signaling events during disruption of ribosome biogenesis, particularly in GI cancers and even some ovarian cancers, that cause the cells to die. Somehow, they must be monitoring the quality control of new ribosome synthesis, which somehow is connected to the death pathway machinery,” Yaffe commented.
The findings suggest that drugs that stimulate ribosome production could work together with 5-FU to make a highly synergistic combination. In their study, the researchers showed that a molecule that inhibits KDM2A, a suppressor of ribosome production, helped to boost the rate of cell death in colon cancer cells treated with 5-FU. “… we showed that activation of rRNA transcription by inhibiting KDM2A significantly enhanced the sensitivity of CRC to the RNA damage effects of 5-FU,” they wrote. “… we show that treatments upregulating ribosome biogenesis, including KDM2A inhibition, promote RNA-dependent cell killing by 5-FU, demonstrating the potential for combinatorial targeting of this ribosomal RNA damage response for improved cancer therapy.”
The findings also suggest a possible explanation for why combining 5-FU with a DNA-damaging drug often makes both drugs less effective. Some DNA damaging drugs send a signal to the cell to stop making new ribosomes, which would negate 5-FU’s effect on RNA. A better approach may be to give each drug a few days apart, which would give patients the potential benefits of each drug, without having them cancel each other out.
“Importantly, our data doesn’t say that these combination therapies are wrong. We know they’re effective clinically. It just says that if you adjust how you give these drugs, you could potentially make those therapies even better, with relatively minor changes in the timing of when the drugs are given,” Yaffe says. Noting limitations of their reported study, the authors concluded, “Nonetheless, this study sheds insights into better mechanisms for clinical utilization of 5-FU, both as a single agent and in combination with DNA-damaging chemotherapies.”
The researchers are now hoping to work with collaborators at other institutions to run a Phase II or III clinical trial in which patients receive the drugs on an altered schedule.
