Research into how gross chromosomal rearrangements (GCRs) occur may have major implications for the discovery and development of new cancer therapies, according to a Japanese study reported in the July 22, 2021, edition of PLoS Genetics.
GCRs, in which portions of the genome become moved, deleted or inverted, can lead to cell death and the development of various types of cancer, including breast and ovarian cancer, but how GCRs occur has remained unclear.
"We had previously published two related papers suggesting that targeting the mechanism underlying GCR formation might be a potential new approach to cancer treatment," principal investigator Takuro Nakagawa told BioWorld Science.
The associate professor in the Department of Biological Sciences of the Graduate School of Science at Osaka University said his group's new study had demonstrated the central involvement of the protein Rad8 in this process.
Rad8 is an ubiquitin ligase, the enzyme responsible for ubiquitination, or the attachment of a ubiquitin tag to a particular place on another protein.
The Osaka University research team demonstrated that Rad8 attaches a ubiquitin molecule to proliferating cell nuclear antigen (PCNA).
During DNA replication or self-repair, three copies of PCNA bind together and form a ring-like structure around the DNA strand, along which the ring structure can move.
RAD8 attaches a ubiquitin molecule at lysine 107, which is the interface between the different PCNA molecules. Ubiquitination at lysine 107 weakens interactions between the PCNA molecules and changes the structure of the protein ring, which alters how PCNA functions and leads to GCR formation, rather than accurate DNA repair.
"Rad8 and PCNA K107R mutations reduced GCR rates, which were not further reduced by... double mutations, showing Rad8 and PCNA K107 ubiquitination act via the same GCR pathway," said Nakagawa.
"A previous study using budding yeast cells has shown that the Rad8 homologue is required for PCNA lysine 107 ubiquitination." Moreover, introduction of an interface mutation D150E, which interferes with PCNA-PCNA interaction, increased GCR rates in the PCNA K107R or rad8 mutant strains, suggesting PCNA ubiquitination at lysine 107 interferes with the PCNA-PCNA interaction," said Nakagawa.
The team also identified another enzyme, Mms2-Ubc4, that participated in the process.
"Mutations in Mms2 and Ubc4 reduced GCR rates and that these were not further reduced by Rad8-Ubc4 double mutations, again showing that Rad8 and Mms2-Ubc4 act via the same GCR pathway," Nakagawa said.
The combined action of Rad8 and Mms2-Ubc4 is therefore responsible for causing GCRs, but how these findings in a single-celled organism such as yeast might translate to cancer in complex organisms such as humans is less clear.
In particular, little is known about helicase-like transcription factor (HLTF), the human equivalent of the Rad8 found in yeast, although HLTF has been observed to be activated and upregulated in human cancers.
"HLTF gene amplification has been reported in human breast, ovarian, lung and head and neck cancers, suggesting that HLTF is positively involved in or required for tumorigenesis," Nakagawa said.
Collectively, these study findings indicate that the inhibition of HLTF, or inhibiting ubiquitin attachment to PCNA at the human equivalent of lysine 107, could be a new strategy for cancer therapies.
Regarding drug discovery and development, "we have shown that Rad8/HLTF interacts with Mms2-Ubc4 via the Rad8/HLTF RING domain and ubiquitinates PCNA at lysine 107," said Nakagawa.
Therefore, "any molecule that binds with the HLTF RING domain or Mms2-Ubc4 and inhibits their interaction might also inhibit this ubiquitination pathway and cancer," he said, noting, "such molecules might be identified using a yeast two-hybrid assay."
However, "first it would be necessary to confirm that HLTF-dependent ubiquitination of PCNA at the specific site are actually involved in tumorigenesis," said Nakagawa.
Furthermore, "because HLTF and ubiquitination have many different roles, it is critical to identify a molecule that specifically interferes with HLTF-dependent ubiquitination at the specific site of PCNA," he said.
Meanwhile, "the exact molecular mechanisms by which GCR occurs remain unclear, so our group is trying to identify all the factors that play essential roles in GCR in order to fully elucidate that mechanism."