An Expedition of Genetic Proportions Leads Duke Researcher to a New Culprit in Lung Cancer Metastasis
DURHAM, N.C. -- Mapping the human genome isn't his job, but Dr. Gerold Bepler, a researcher at Duke University Medical Center, has tackled part of it anyway. He sought an unknown gene on human chromosome 11 that might be involved in lung cancer, the leading cause of cancer death in both men and women. Locating the gene was the scientific equivalent of finding a house while only knowing the continent where it is located.
Bepler isn't part of the federal Human Genome Project, a 15-year program begun in 1990 to characterize all of the genetic material and identify the more than 80,000 genes that make us human. He doesn't receive funds to map and determine the DNA sequence of large portions of the chromosomes that make us who we are. He doesn't have a dozen DNA sequencers and 30 technicians.
What he did have was a question he wanted to answer, and so he did the mapping anyway. Initial funding from the Jimmy V Foundation got him and two researchers through the first three years, then funds from the National Institutes of Health supported him the rest of the way as his team expanded to five. Using the few DNA sequencers at the University of North Carolina at Chapel Hill and then machines at Duke once they were available, they worked day and night to complete the large project in just under five years.
The end result is a detailed map of about one tenth of chromosome 11. Among the 22 possible genes identified and pinned down in this map, he's identified one that he believes acts unlike other cancer-related genes to make lung cancer spread to other parts of the body, he reports in the January cover story of the journal Genomics. Co-authors are Kathy O'Briant, Young-Chul Kim, Gilbert Schreiber and Diana Pitterle, all of Duke.
Bepler entered the realm of gene mapping after finding that 75 percent of the lung cancer tumors he has studied are missing one copy of a certain region of chromosome 11. Studies in mice linked the chromosome region, called LOH11A, with the ability to slow tumor growth. In LOH11A, he suspected, lay a gene responsible for the aggressive nature of lung cancer, a gene whose remaining copy could no longer prevent the tumors' spread from the lung to other parts of the body in a process called metastasizing. He tracked down the unruly gene by carefully mapping and sequencing a part of chromosome 11 that he knew contained LOH11A.
"We studied the biology of LOH11A," said Bepler, associate professor of hematology and medical oncology. "Then we set out to identify the gene within that region that was responsible for the metastatic spread of lung cancer."
The large map of this part of chromosome 11 solidly places known genes within LOH11A and in its proximity. Bepler said that by knowing which genes are in the same neighborhood, it's easier to determine how certain disease traits occur together, since single genes aren't usually lost but instead a region, such as LOH11A, will be missing.
Finding the house and knocking it down
Bepler's gamble to get into chromosome mapping just to find a gene seems to have paid off. Out of all the genes the researchers found, Bepler thinks a gene called RRM1, known to be involved in DNA production and repair, is also involved in metastatic lung cancer. "I feel pretty confident, with the data that's out there, to say that RRM1 is the gene," he said.
To reach this conclusion, Bepler combined the results of three studies. A report from his lab in 1997 found that adding a second copy of LOH11A to human lung cancer cells slowed or stopped their growth when implanted into mice. A Canadian research group also reported in 1997 that putting the human gene RRM1 into activated mouse cancer cells prevented tumor growth as well. Bepler's current work places the RRM1 gene firmly in the LOH11A region. So RRM1 looks like the culprit.
If further study confirms his work, Bepler's finding could lead to new, desperately needed therapy. Lung cancer claimed the lives of an estimated 160,100 people in 1998, according to the American Cancer Society. One reason for the high mortality rate is that standard treatments such as chemotherapy or radiation aren't as helpful for lung cancer patients as for patients with other kinds of cancer.
Using RRM1 could help improve lung cancer patients' survival rates in several ways, according to Bepler. First, it could serve as a diagnostic tool to indicate severity of the disease: Patients who are missing one copy of the gene could be treated more aggressively since they would be more likely to develop, or already have, metastatic disease. The RRM1 status would probably need to be determined from a tumor biopsy or surgery, since it is unlikely that enough cancer cells would appear in the blood to allow a simple blood test.
While using RRM1 to adjust treatment decisions is in the distant future, Bepler said, a closer goal may prove to be even more important. The gene, he said, could "make a great target for chemotherapy agents."
As it turns out, RRM1 is known to be vital to the life of a cell. Every normal cell (except eggs and sperm) has two copies of the gene, whose job it is to help build an enzyme called ribonucleotide reductase (RR). RR is the only enzyme that changes ribonucleotides to deoxynucleotides, the building blocks of DNA. RRM1 is the only gene for making a certain part of the critical RR enzyme in humans. Without RR or RRM1, cells can't fix their DNA or reproduce.
"Ribonucleotide reductase has been known for 30 years or so and has been studied biochemically," Bepler explained. "It is required for cell viability and cell repair. If you can knock out RRM1, there is no way a cell can overcome the lack of ribonucleotide reductase and survive."
Potential chemotherapy agents could, therefore, either block the enzyme, rendering it ineffective, or could target the remaining copy of the RRM1 gene to prevent it from producing the enzyme. As with all cancer treatments, however, there needs to be selectivity so that cancer cells are killed but normal cells are left alone as much as possible. Normal cells have two copies of RRM1 and should be able to withstand a treatment aimed at the enzyme or gene.
"It should be easier for a therapy agent to completely knock out the enzyme in a cancer cell than in a normal cell, since the cancer cell has only one copy of RRM1," Bepler said. With no ribonucleotide reductase active, the cancer cell will die.
While normal cells may be affected also, they should have enough residual enzyme activity to survive and wouldn't become cancerous just because of diminished RRM1 activity, he said. "The gene doesn't make cancer, it just makes cancer spread."
A new model of tumor suppressors?
What may be most profound about RRM1 is that it could represent a new class of so-called "tumor suppressor genes," Bepler said. Most, if not all, tumor suppressor genes are classified as such because in their normal states they prevent cells from becoming cancerous. Trouble usually arises when one copy of the suppressing gene is deleted from the cell's DNA and the other copy is mutated, becoming inactive.
Surprisingly, Bepler and his team found no mutations in the RRM1 gene in cells from the scores of lung tumors they have examined. Mutations on chromosome 11 near RRM1 may be affecting the gene's performance, but with the data they have now, the researchers believe that the lung cancer cells tend to metastasize simply due to the loss of one copy of RRM1, even though the remaining copy is perfectly normal.
"It's not whether RRM1 is mutated, it's about balance," Bepler explained. "Two copies are good; no copies and the cell dies. One copy seems to make the cell prone to metastasizing."
Although they don't know precisely how RRM1 works to do this, the researchers have a number of ideas. First, metastasis might be induced somehow just because the amount of available ribonucleotide reductase has been reduced -- a hypothesis related to the gene's known function. Alternatively, metastasis could be brought on by an as yet undiscovered job the gene has when only one copy is present. Yet another possibility is that RRM1, officially in a sub-group of tumor suppressors known as metastasis suppressors, may have a sidekick -- another gene in the LOH11A region that helps promote metastasis.
"RRM1 may not be the direct regulator of metastatic spread," Bepler said. "It may be a modulator in a cascade of events that result in metastasis."
As a result of these unanswered questions, Bepler's research team is continuing to study both the LOH11A region as well as the rest of the area they mapped. They have found even more genes in the chromosome 11 segment since the research was submitted for publication. All 22-plus genes are being examined one by one to determine their functions, find their coding sequences (the part of the gene that actually carries the blueprint for a protein), and figure out whether they are mutated in cancerous cells.