Handcuffing Genes
DNA methylation is silencing the very genes that may be needed to stop cells from being cancerous.
At the heart of cancer you’ll find not medicine, but biology: chromosome
rearrangements, genetic defects—and DNA 
methylation.
Researchers who try to explain DNA methylation to a lay person—trying
to express just how critically important it is in cancer research—reach
for a variety of metaphors to describe this essential, yet obscure, biological
process.
“Most genes can be opened or closed,” says Peter Jones, Ph.D., director of the USC/Norris Comprehensive Cancer Center, “but with DNA methylation, the genes become permanently locked.”
And in biological pathways where one gene becomes locked, the entire process
can come to a screeching halt. “It’s like a set of falling dominoes,”
Jones adds. “If you glue one of the dominoes down so that it can’t
tip over, then the ones after it will never fall down either.”
“DNA methylation is like a pair of chemical handcuffs,” says USC/Norris
molecular biologist Peter Laird, Ph.D. “It marks part of the DNA as a
part that should not be used. The gene is intact; it has just been silenced.”
Quiet Destruction
Stripped of metaphor, DNA methylation is simply a process by which a chemical
cluster called a methyl group—which consists of one carbon atom and three
hydrogen atoms—is attached to the surface of a DNA strand by an enzyme
called a methyltransferase. It then provides a literal, physical obstruction
to the normal process of DNA transcription, one of the first steps in creating
proteins and enzymes that are coded by genes.
But, in practice, DNA methylation is so much more than that. For one thing,
it is a process essential to the
normal functioning of a cell—all cells have a full complement of genes,
but the genes a liver cell needs to do its work varies wildly from those a
brain cell needs to work. Methylation enables the cells to switch off the
genes they do not need, so that the ones they do need can work.
It is when methyl groups are appended to genes that are not supposed to be
turned off—when the genes become “hypermethylated”—that
cancer becomes a concern. If a gene that is supposed to prompt a cell to stop
growing becomes locked, the cell may grow continuously and become a cancerous
cell.
Stopping cancer may require an in-depth understanding of the process of methylation—and
a creative vision for how to undo its quiet destruction. It is no exaggeration
at all to say that USC/Norris is uniquely poised to lead that charge, with
a critical mass of talented, energetic researchers tackling the problem from
all angles.
Part of the credit for this unique position goes to Peter Jones. Considered
a pioneer in DNA methylation, he not only helped prove, in 1980, that methylation
can regulate the expression of genes, but made the connection between cancer
and methylation, a connection about which many in the field were originally
skeptical. In recent years, Jones and his lab have been crucial in all aspects
of the field—from understanding the specifics of methylation’s most
basic maneuvers in cells to developing drugs that interfere with or otherwise
counteract these effects.
Last year, the National Institutes of Health (NIH) awarded Jones a five-year,
$6.8 million Program Project Grant, a cluster of at least three separate but
interactive research projects focused on a single subject—in this case,
the molecular mechanisms of human bladder carcinogenesis with a focus on methylation.
The first of the three projects, headed by Jones, focuses on DNA methylation,
its relationship to cigarette
smoking and the use of non-steroidal anti-inflammatory drugs (NSAIDs), and
their effects on cell death.
Part of the grant—a study of two key cellular enzymes (cyclooxygenase-2,
or COX-2, and DNA methyltransferase) and their relationship to smoking and
NSAIDs—is led by Ronald Ross, M.D., the Flora L. Thornton Chair in Preventive
Medicine and the Catherine and Joseph Aresty Chair in Urologic Research, and
Mimi Yu, Ph.D., Keck School of Medicine of USC professor of preventive medicine.
Dynamic Process
But Jones is by no means alone in his expertise or his focus. Ite Laird-Offringa,
Ph.D., assistant professor of surgery and biochemistry at the Keck School,
is looking at ways to use methylation markers to accurately diagnose lung
cancers and mesothelioma (see “Mastering Mesothelioma,” page 4).
She already has shown that it is possible to differentiate between cell lines
of small cell lung cancer and non-small cell lung cancer based on the patterns
of methylation in the tumor cells’ genes.
USC/Norris researcher Chih-Lin Hsieh, Ph.D., associate professor of urology
and biochemistry and molecular biology at the Keck School, is focusing on
the nitty-gritty of methylation—how methyl groups are attached to and
removed from DNA strands. She has shown that methylation is a dynamic process
that continues throughout the life of a cell—and that demethylation is
equally dynamic. And she has provided an in-depth look at the inner workings
of methyltransferases.
“A lot of people are looking at what the methylation changes are in different
types of cancer,” says Hsieh. “I’m more interested in why the
changes occur, and how they occur. What causes them? If we know that, then
there may be things we can do about it.”
And then there is Peter Laird, whose work in the field includes collaborations
with literally dozens of other scientists. It has earned him a patent—awarded
in December 2001—on a technique called MethyLight, which he and his Keck
School of Medicine colleagues created to analyze methylation changes in large
numbers of cells very quickly. And it has attained for him a seat on the board
of directors of the international DNA Methylation Society.
The NIH also supports Laird’s work; last year it approved funding for
four grants on which Laird is the principal investigator, awarding him more
than $6 million in funding over the next five years—all of which is to
be used for DNA methylation research.
Environmental Influences
In one of those grants, Laird and Frank Gilliland, M.D., Ph.D., M.P.H., professor
of preventive medicine, received funding from the NIH’s National Institute
of Environmental Health Sciences to create a Center for Environmental Epigenomics,
which will look at the ways in which methylation patterns and other epigenomic
changes are affected by environmental influences.
“DNA methylation is a perfect medium to study the way the environment
influences the human genome,” Laird explains. “Methylation patterns
can be changed throughout our lifetime; they can be easily influenced by our
environment.”
That, says Laird, is why he is particularly excited about the Center for Environmental
Epigenomics. “We’ll be looking at things like how smoking affects
DNA methylation,” Laird says. “We’ll be looking at methylation
and hormone exposure in women, and at nutritional exposures—how diet
affects methylation.”
Other USC/Norris researchers are considering the effects of outside influences
on methylation patterns. Robert Haile, Dr.P.H., professor of preventive medicine
at the Keck School, is primary among them. He is, for instance, currently
investigating the ways in which aspirin and folic acid seem to protect against
colon cancer. “Our preliminary data shows consistent evidence that taking
aspirin decreases the risk of colon cancer by half,” Haile explains,
adding that he has collected similarly promising data on folic acid.
And it seems that these outside influences may be doing their job in part
by protecting key genes from methylation changes that might otherwise kick
cells into cycles of unchecked growth and proliferation. “For example,
folic acid is involved in methylation,” Haile explains, “and so
it may be working to reduce hypermethylation in selected colon cancer-associated
genes.”
Working Results
This intriguing information about the process of DNA methylation is helping
USC/Norris researchers and others to derive drugs that might turn tumor cells
on their ears.
To that end, Jones and his colleagues, including M.D./Ph.D. student Jonathan
C. Cheng, recently published a paper in the Journal of the National Cancer
Institute that showed how oral administration of a methylation inhibitor called
zebularine results in a reduction in the size of malignant tumors in mice.
They found that the drug accomplishes its tumor-whittling by turning on tumor
suppressor genes that have been turned off by methylation.
“This is the first time this type of drug has been able to reactivate
silenced genes through oral administration,” says Jones.
In the study, Cheng, Jones and their colleagues gave relatively high doses
of zebularine to mice engineered to be susceptible to bladder carcinoma. They
administered the drug both intraperitoneally (injecting it into the space
between the abdominal muscles and the abdominal organs) and orally.
The fact that zebularine is still effective when taken orally is no small
deal, says Cheng. Ease of administration can be a major issue in the success
of cancer drugs. And zebularine appears to be both stable in solution and
when exposed to an acidic environment such as the stomach.
Preliminary studies in cultured human cells also seem to indicate that zebularine’s
effects are focused on tumor cells, rather than normal dividing cells. This
may be good news in terms of the drug’s side effects. Still, says Jones,
it may be some time before zebularine is tested in humans.
There is, however, one DNA methylation inhibitor that is already in clinical
trials in humans at the USC/Norris—a drug called decitabine, which is
being used to treat patients with chronic myelogenous leukemia, or CML, who
have failed more conventional treatments.
CML is a disease with three rather distinct phases, says Dan Douer, M.D.,
associate professor of medicine at the Keck School. In its early, or chronic,
phase, patients generally do quite well for a number of years. But eventually,
they move onto a second, accelerated phase, in which their disease begins
to spiral out of control—in about half of CML patients, this happens
within four years of diagnosis. Finally, they enter what is called the blastic
crisis, an acute phase that is invariably fatal, usually within an average
of 12 months after diagnosis.
Until recently, the only hope for CML patients was bone marrow transplant.
Then came the introduction of the anti-cancer drug Gleevec, which inhibits
a key enzyme called tyrosine kinase. Still, says Douer, Gleevec alone can’t
cure all CML patients. “The problem,” he says, “is that some
patients develop resistance to Gleevec. And if they don’t have a transplant
donor lined up, they quickly run out of options.”
The new option is decitabine, which is produced by SuperGen, a Dublin, Calif.-based
biotechnology company. “Our objective with decitabine,” says Douer,
who is the principal investigator of USC/Norris’s arm of this Phase II,
multicenter trial, “is to see if we can get people in the chronic phase
of CML into remission, or to turn people who are in the accelerated or crisis
phases back to the chronic phase.”
Douer and his colleagues are hopeful that demethylation will be a useful tool
in CML, since the same gene that Gleevec targets tends to be hypermethylated.
“We think that if we can affect this gene in a different way than Gleevec
does, then we’ll be able to prolong life in these patients who have failed
on Gleevec—or even get them into remission,” Douer says. “And
the preliminary data back that up, showing that with a relatively low dose
of this drug, patients do indeed respond well.”
Douer notes that this is not only a good example of the sort of translational
medicine USC/Norris is known for—bringing therapies from the laboratory
bench to the patient’s bedside in short order—but that the trial
itself should provide more information for the scientists in the lab. “For
patients in this trial,” he says, “we’ll send samples of their
blood to Dr. Jones, so he can see what is happening in the cells. It’ll
be the first chance we’ll have to really see the in vivo effect of these
sorts of drugs.”
Connecting nuts-and-bolts biology to new, potential drug therapies is really
the driving force behind the Cancer Center’s efforts in the field of
DNA methylation, says Jones. Getting those drugs to the patients, he adds,
is their passion.
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