Scientists suggest that blocking the cell cycle protein cyclin D1 in tumor cells may provide a new approach to increasing the sensitivity of a wide range of cancers to radiotherapy. A team led by Siwanon Jirawatnotai, Ph.D., at the Dana-Farber Cancer Institute, has demonstrated that ionizing radiation causes cyclin D1 to directly interact with a protein known as RAD51 that is involved in the homologous recombination process, and that this cyclin D1-RAD51 interaction leads to the repair of DNA damaged by radiotherapy.
Reducing levels of cyclin D1 in different types of cancer cells effectively impaired this homologous recombination-mediated DNA repair, and increased the sensitivity of cancer cells to radiation both in vitro and in experimental mice. The team’s studies are reported in Nature, in a paper titled “A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers.”
Cyclin D1 is a component of the core cell cycle machinery, and is found at abnormally high levels in many human cancer types, Dr. Jirawatnotai and colleagues report. To elucidate the molecular functions of cyclin D1 in human cancer cells, the researchers analyzed the proteins with which cyclin D1 interacts in different cyclin D1-overexpressing tumor lines. They found that along with its expected interaction with cell cycle control proteins, cyclin D1 appeared to connect to proteins involved in the cells’ DNA repair processes, and particularly with RAD51, a key DNA recombinase that mediates DNA repair via homologous recombination. “These observations suggested that cyclin D1 may play a role in DNA damage repair,” the authors write.
When cyclin D1 was subsequently silenced in different types of cancer cells using using a short hairpin RNA (shRNA), the cells became more sensitive to ionizing radiation. This effect of cyclin D1 silencing was confirmed in retinoblastoma protein-negative cell lines, which don’t require D-cyclins for proliferation, and in mouse embryonic fibroblasts that lack D-cyclins but have normal cell cycle progression. Cyclin D1 activates the CDK4 and CDK6 kinases, but treating cancer cells with a specific inhibitor of these kinases had no effect on cell sensitivity to ionizing radiation, suggesting the kinases were not involved in the cyclin D1-related DNA repair mechanism.
Interestingly, subjecting cyclin D1-depleted and non-depleted cancer cells to radiation induced comparable initial levels of DNA damage. Sixteen hours later, however, there was more unrepaired DNA in the cyclin D1-depleted cells than in the controls. This was linked with significantly reduced rates of homologous recombination, which again pointed to a potential role for cyclin D1-RAD51 interaction. Adding cyclin D1 to the initially depleted cells reinstated homologous recombination rates after ionizing radiation, indicating that cyclin D1 was required for an efficient homologous recombination DNA repair process, the authors write.
Further analyses confirmed that cyclin D1 binds directly to RAD51 in several human cancer cell lines. This interaction was induced by radiation, and intensified with increasing doses of radiotherapy. Both cyclin D1 and RAD51 were both found to be recruited directly to the sites of DNA damage, and colonized at the sizes of double-stranded DNA breaks. Ionizing radiation did actually lead to reduced total levels of cyclin D1 (but not RAD51), but the researchers suggest the strong induction of cyclin D1-RAD51 interaction after radiation, even in the face of reduced total cyclin D1 levels, just prompts more of the remaining cyclin D1 molecules to be recruited to form complexes with RAD51 to facilitate DNA repair.
The recruitment of cyclin D1 to DNA damage sites was found to rely on its interaction with BRCA2, another homologous recombination protein which is recruited to DNA damage sites prior to RAD51. Knocking down cyclin D1 had no effect on the recruitment of BRCA2, supporting the notion that it acts upstream of cyclin D1.
In a final set of in vivo studies, the researchers generated retinoblastoma-negative, cyclin D1 knockout cancer cells and injected them into nude mice. Cyclin D1 silencing per se had no effect on the initial growth of tumors in the experimental animals. However, treating the animals to ionizing radiation slowed the growth of these tumors to a greater extent than cyclin D1-overexpressing tumors, indicating increased sensitivity to the radiotherapy.
“Collectively, these results are consistent with the model that cyclin D1 is recruited to DNA damage sites through BRCA2; cyclin D1 then helps to recruit RAD51 through a direct cyclin D1-RAD51 interaction,” the authors conclude. “Our observations suggest that depletion of cyclin D1 could sensitize human cancers to radiation by limiting DNA repair.”
“This is the first time a cell cycle protein has been shown to be directly involved in DNA repair,” Dr. Jirawatnotai states. “Our results potentially change the way we think about cyclin D1 and cancer and may encourage targeting cyclin D1 in a very large pool of human cancers which do not need cyclin D1 for proliferation, but may still depend on cyclin D1 for DNA repair.”