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MOLECULAR MEDICINE Breast Cancer Protein Shown to Be Highly Tuned Molecular MachineDefect in Single Part May Steer Cells into Breast or Ovarian CancerEver since it was sequenced in 1994, the BRCA1 gene has held center stage in the quest to understand the causes of hereditary breast and ovarian cancer. And with good reason. Women carrying defects in the gene—and there are several hundred BRCA1 mutations known—are much more likely to develop premature breast or ovarian cancer. Frequency of disease can be as high as 80 percent in some families. Yet while the BRCA1 gene has been laid bare, its nucleotide sequence revealed, the BRCA1 protein is still a shadowy molecular figure. For example, BRCA1 is thought to play a role in repairing damaged DNA, but this hunch has been based on circumstantial evidence—observations of the protein's comings and goings in the cell and the molecular company it keeps. What has been lacking is a direct measure of the protein's activity. A team of Dana–Farber Cancer Institute researchers has recently devised an assay that helps lift the veil on BRCA1, an understanding of which could lead to new therapeutic approaches to the treatment of familial breast and ovarian cancer.
To gain insight into BRCA1's function, Ralph Scully, David Livingston, and their colleagues introduced a healthy BRCA1 gene into human breast cancer cells carrying a mutant BRCA1 gene. Typically, breast cancer cells carrying only the mutant BRCA1 gene are extremely sensitive to gamma irradiation. Their chromosomes break more easily and take a longer time to mend than do those of normal cells. After introduction of the normal BRCA1 gene, cancer cells not only survived at higher rates, their chromosomes were repaired more quickly and were less susceptible to breaks. Earlier studies had shown that knocking out the BRCA1 gene renders cells more susceptible to DNA damage, but this study is the first to show that the DNA-repair function could be restored by a healthy BRCA1 gene. What's more, Scully, an instructor in medicine, Livingston, the Emil Frei professor of medicine and genetics, and their colleagues provide intriguing clues about how BRCA1 goes about repairing DNA damage. All for One and One for AllIn their experiments, reported in the December Molecular Cell, the researchers introduced into the mutant BRCA1-containing cancer cells one of four different mutant BRCA1 genes, each nicked in a different region than the original BRCA1 mutant. The thinking was that by introducing genes mutated in different regions, the cell might piece together a fully functional protein. But no such patchworking occurred, suggesting that the entire BRCA1 protein must be intact to function normally."This is a cooperative machine in that every cog has to be there functioning effectively in order for the machine to operate appropriately," Scully says. "And if that's true then the machine is only as good as its weakest cog—which is why so many mutations appear to have such great harm." What, exactly, the molecular machine does is not clear. Considering its large size, one possibility is that it acts as a scaffold, aligning other repair proteins, making sure they are at the right place at the right time. "Or it may bring proteins in apposition to one another so they can talk to one another in ways they couldn't do otherwise," says Livingston. BRCA1 is known to associate with as many as 15 other proteins. "A defect in its bringing out just one repair factor could make the whole process inefficient," Scully says. Inefficient repair could result in chromosomal breaks and other mutations going uncorrected. Normally, proteins inside the cell sense such uncorrected damage and tell the cell to stop dividing. But if the genes for such tumor suppressor proteins were among those damaged and inefficiently repaired, the cell could keep dividing. In fact, BRCA1 may hasten the process that occurs in non-hereditary, or sporadic, breast cancers, which are often characterized by defects in tumor suppressor genes. "By accelerating the pathway, the natural history of breast cancer may be compressed and concertinaed into a much shorter time period," Scully says. Curiously, very few cases of spontaneously arising breast cancer show evidence of BRCA1 mutations. Why that is true is not known. Nor is it clear why the BRCA1 mutation seems to target only breast and ovarian tissue, though there are hints that BRCA1 and its related proteins may play a role in an estrogen specific pathway. Conspiracy of CluesIt was by piecing together such hints that the researchers arrived at their current model of BRCA1. The first clue came three years ago. Having exposed cells to DNA damaging agents, Scully and his colleagues saw tiny dots of BRCA1 jump from their resting place on chromosomes to sites where DNA was being synthesized. "We were scratching our heads about that," he says. They then observed a curious thing: the moving dots of BRCA1 appeared to be accompanied by another protein. The companion, Rad51, was known to play a vital role in homologous recombination, during which replicating chromosomes, having exchanged pieces, are knit back together.Thinking that BRCA1 might also play a role in chromosomal repair, Scully, Livingston, and their colleagues set out to find a way to directly gauge the protein's activity. Using a retrovirus, they delivered the normal BRCA1 gene—attached to the gene for green fluorescent protein—into the gamma irradiation–sensitive human breast cancer cells. They then exposed the cells to the damaging radiation. Those cells carrying the green fluorescent tag—and by association, the wild type gene—survived gamma irradiation in much higher proportion than did non-glowing cells. It appeared that the cells were capable of more efficient repair and also less likely to break in the face of DNA-damaging agents. "This methodology gives us a chance to begin to explore BRCA1's biological function," Livingston says. The researchers plan to refine the assay and use it to pinpoint BRCA1's role in DNA repair. In the long run, revealing how the BRCA1 mutant disrupts DNA repair could lead to new therapies for breast and ovarian cancer. "The speculation that there might be an Achilles heel for BRCA1 mutant tumors, which singles it out from surrounding tissue, is worth following," Scully says. A possible next step would be to find and deliver a chemotherapeutic agent that is only active in cells with the defect. "My feeling is we've got cogs in a machine," he says. "We've got a machine that's dysfunctional. Can we make use of that dysfunctional machine to kill the cell?" —Misia Landau |
If BRCA1 turns out to play a role in DNA repair, the next step will be to figure out how mutated forms contribute to premature breast and ovarian cancer. "We'd like to fill that knowledge gap," says Ralph Scully (left). David Livingston agrees that this avenue of research is intriguing. "There's a lot to be learned here," he says.