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GENETICS Genes of Rare Disorder May Shed Light on Breast Cancer Growth Study something rare and you may learn about something common. For 10 years Alan D'Andrea, HMS professor of pediatrics at the Dana–Farber Cancer Institute, has studied the very rare autosomal recessive disease Fanconi anemia (FA) with just that hope. "We have always thought that if we could understand the genes responsible for this disease we would have something general to say about cancer in the whole population," said D'Andrea. Fanconi Anemia Proteins Respond to DNA Damage A newly identified protein (D) involved in Fanconi anemia (FA) is the missing link between the previously identified FA protein complex and DNA-repair machinery. In response to DNA damage (bellow) the FA complex (made up of Fanconi anemia proteins A, C, E, F, and G) allows one ubiquitin (Ub) to be added to D. Ubiquitinated D then moves to nuclear foci that contain BRCA1, a protein that is defective in the majority of inherited breast cancers and is thought to play a role in DNA repair. Fluorescence microscopy shows that D, usually diffusely located throughout the nucleus (top left), concentrates into nuclear foci after DNA damage (top right). Adapted from originals from the Alan D'Andrea laboratory Children with FA suffer from a variety of congenital abnormalities and by age 8 develop complete bone marrow failure. If they survive bone marrow failure, an alarmingly large percentage of patients go on to develop a wide range of cancers early in life. Recent work by D'Andrea, two postdoctoral fellows in his lab, Irene Garcia-Higuera and Toshiyasu Taniguchi, and their collaborators at the Oregon Health Sciences University has contrib-uted greatly to the understanding of the molecular basis of FA. In the February Molecular Cell they describe the isolation of an FA gene, FANCD2, and link the FANCD2 protein to DNA repair. Their research also specifically connects FA with BRCA1, a gene associated with breast cancer. Fragile DNA At the cellular level, FA appears to be due to genomic instability. Chromosomes from patients break easily and are extremely sensitive to DNA-damaging agents. The vulnerability of their genome to damage translates into cancer. Although FA patients appear similar from a clinical standpoint, FA can be caused by mutations in any one of eight genes. The striking similarity among FA patients, in spite of their genetic heterogeneity, has led researchers to believe that the FA genes act together in a common pathway. Until recently only five of the FA genes had been isolated. Each of these is an orphan gene, one that has nothing in common with any other known gene in any other organism. Their sequence therefore reveals nothing about their function nor does it suggest a model organism to facilitate study of the genes. Not surprisingly, the orphan status of the FA genes has frustrated researchers' attempts to understand their function. D'Andrea and colleagues were not surprised to discover that cells from patients with mutations in FANCD2 had little or no FANCD2 protein. They were intrigued, however, by their finding that although two forms of FANCD2 exist in normal cells, only one is present in cells from patients with mutations in the five previously identified FA genes. This observation placed FANCD2 downstream of the other FA proteins and indicated that the form of FANCD2 that was lacking in FA cells may be important for normal cellular function. Irene Garcia-Higuera, Toshiyasu Taniguchi, Alan D'Andrea (l to r), and colleagues have found a connection between the rare disease Fanconi anemia and DNA repair. Their research suggests a new pathway is involved in breast cancer. Photo by Steve Gilbert In what D'Andrea describes as a "complete coincidence" and a "stroke of luck," the form of FANCD2 lacking in FA cells is different from the other in that it is ubiquitinated, a process that D'Andrea had been studying for years independently of his work on FA. The other FA proteins were necessary for the addition of ubiquitin to FANCD2, supporting the idea that they lie upstream of FANCD2 in the FA pathway. Unlike most ubiquitinations, which consist of multiple ubiquitin molecules, the ubiquitination of FANCD2 consists of only one molecule. Since very few other proteins are known to be monoubiquitinated, little is known about this modification. The addition of a single ubiquitin, however, is thought to have a different effect from the degradation-targeting effect of polyubiquitination. Repair Crew In its unmodified form, FANCD2 is diffusely located throughout the nucleus. When ubiquitinated, FANCD2 forms dots, or foci, in the nucleus. The re-searchers discovered that this process—the ubiquitination of FANCD2 and subsequent formation of nuclear foci—occurs in response to DNA damage. The finding provided the first definitive connection between FA proteins and a response to damaged DNA. The connection between FAN-CD2 and DNA repair grew stronger when the researchers recognized that the dotted pattern formed by FANCD2 was similar to the pattern formed by the breast cancer protein BRCA1. Mutations in BRCA1 are responsible for the majority of inherited breast cancers, and there is strong evidence that BRCA1 plays a role in DNA repair. Indeed, the FANCD2 foci also contained BRCA1. When looking at the localization of FANCD2 in closer detail, the researchers saw that the protein associated with broken chromosomes. BRCA1 also associated with the same parts of the broken chromosomes. Localizing FANCD2 to the chromosome with BRCA1 puts FANCD2 in a prime site to play a role in the maintenance of chromosomal integrity. Mutations in the FA pathway may be involved in the development of breast cancers in which BRCA1 mutations are not present. If this is the case, testing the integrity of the FA pathway in breast cancer cells will be an important diagnostic tool. By testing breast cancer cells for the presence of the monoubiquitinated form of FANCD2, the entire FA pathway has effectively been screened, said D'Andrea. "What might be the biggest contribution of this work is that it clearly defines monoubiquitination as a regulatory switch," D'Andrea said. Future goals include determining the steps involved with the turning-on and turning-off of this switch. He said that further work may uncover a novel class of enzymes, monoubiquitin ligases, or a novel deubiquitinating enzyme. The researchers are now primed to determine the role of FA proteins in maintaining genomic integrity. FANCD2 has homologues in Drosophila and C. elegans, making FANCD2 the first nonorphan gene in the FA pathway. "This sets the stage for the genetic dissection of the FA pathway," D'Andrea said, "and should provide further insight into the normal mechanisms regulating chromosome stability." —Heather Ettinger