Researchers have uncovered a new pathway via which cancer cells evade the effects of radiation by deploying self-inflicted – but reversible – DNA breaks to stop the cell cycle and ensure their survival.

The lesions are caused by caspase-activated DNase (CAD), an enzyme involved in DNA fragmentation during cell death. In response to radiation, tumor cells activate CAD, causing genome-wide DNA breaks at sites involved in DNA repair, Claus Sorensen and colleagues at Copenhagen University, Denmark report in an article published in Science on April 29, 2022.

The findings point to a cancer cell-specific mechanism that could be deployed to make tumor cells more vulnerable to the genotoxic effects of radiation.

"Our findings suddenly bring CAD into the spotlight. It is now clear that CAD represents an exciting target for anticancer therapies, which merits dedicated efforts to develop efficient inhibitors," Sorensen said. "This is an exciting area of future research and development, which we also aim to explore," he told BioWorld Science.

The prospect is that CAD inhibitors would be synergistic with radiotherapy, potentially overcoming the acquired resistance that remains a considerable obstacle to complete eradication of tumors.

When healthy cells are irradiated the cell cycle stops at the G1 phase. However, tumor suppressors p53 and pRB, the key factors regulating this checkpoint, are inactivated in many solid cancers. As a result, cancer cells rely on the G2 cell cycle checkpoint to prevent premature entry into mitosis and cell death due to radiation-induced DNA damage.

The researchers screened a library targeting known human nucleases in cancer cells to identify potential nuclease regulators of the G2 cell cycle checkpoint after radiation.

One hit was RBBP8 (retinoblastoma binding protein 8), a known DNA damage repair (DDR) and G2 checkpoint factor.

But unexpectedly, a second hit was CAD, which has not previously been associated with DDR or the control of cell cycle checkpoints.

To investigate the exact role of CAD, the researchers irradiated human wild-type and CAD knockout colorectal cancer cells and measured the extent of DNA damage.

Initially, there were no differences in the number of DNA lesions between wild-type and CAD knockout cells and the progressive reduction in DNA damage burden through active DNA repair was comparable between the two types of cells.

However, 24 hours after irradiation there was a secondary accumulation of DNA lesions in the wild-type cells, which was dependent on the nuclease activity of CAD.

Subsequently, CAD-dependent breaks were observed in a panel of human cancer cell lines, but not in nonmalignant cells.

CAD relies on a chaperone, ICAD (inhibitor of CAD), to be able to fold correctly, and the researchers made similar observations when they irradiated ICAD-deficient cells.

Looking at the extent of single-stranded DNA breaks 24 hours after irradiation, it was seen there were elevated numbers in the control irradiated cells, but not in cells deficient in CAD/ICAD.

Not just irradiation

The same effect has been seen in response to chemotherapy. "We have already tested doxorubicin, which led to similar results [of] a cancer cell protective role of CAD, when they are exposed to the drug," said Sorensen.

CAD-induced DNA damage was in the form of single-strand breaks (SSBs) and the researchers looked to see if these were occurring at specific genomic loci. Initially, the distribution of SSBs was independent of the presence of CAD, but 24 hours after irradiation, a distinct distribution of SSBs was observed in wild-type, but not in CAD-knockout cells.

The researchers mapped these, showing CAD-induced SSBs appeared to accumulate at different hot spots in the genome from the hotspots where SSBs typically occur.

"In summary, genome-wide SSB mapping revealed a characteristic, unusual, CAD-dependent landscape 24 hours after irradiation," the researchers said.

Taken as a whole, the research has uncovered a DDR-mediated G2 checkpoint pathway in which cancer cells exposed to radiation generate reversible CAD-dependent DNA breaks. These stimulate signaling responses that prevent premature entry into mitosis, enhancing cancer cell survival.

As the repair of radiation-induced DNA damage progresses, the number of genotoxic DNA double-strand breaks declines.

At the same time, induction of CAD-dependent DNA breaks amplifies the DDR further, stabilizing the G2 checkpoint and providing more time for repair of more complex and potentially lethal, radiation-induced lesions. The selectivity of the CAD-related stress tolerance pathway likely reflects multiple factors that occur in cancer but not in normal cells, including defects in p53 and pRB pathways.