Comprehensive study raises questions about "demethylation" agents

Agents believed to selectively "restart" genes that limit cancer's growth -- a potential treatment option already in early clinical studies -- instead turn off as many genes as they turn on, a team of researchers from the National Cancer Institute and Johns Hopkins has discovered.

"We don't know what effect all these changes might have, but it's clear that when scientists are looking only at the agents' effects on a particular gene or a few particular genes, they aren't seeing the whole picture," says Andrew Feinberg, M.D., M.P.H., King Fahd Professor of Medicine at Johns Hopkins. Their report appears in the October issue of Cancer Cell.

The research team probed the global effects of each of three approaches to unhooking methyl groups from genes' DNA. Cells normally use methyl groups to "mark" certain genes, indicating whether their instructions should or shouldn't be used for making proteins, but the marks are frequently disrupted in cancer cells.

For example, in cancer cells genes that normally stifle cell growth -- so-called tumor suppressor genes -- are shut down because extra methyl groups are hanging on to them. If these extra methyl groups could be removed, the thinking has gone, the gene could be restarted and the cancer slowed or stopped.

But the new work shows that while the agents tested do restart cancer-suppressing genes, they also knock methyl groups off other genes. Moreover, some of the unexpectedly affected genes are turned on, but an equal number -- hundreds -- of other genes are turned off.

The findings don't mean automatic failure for clinical trials of so-called demethylation agents, but they do indicate that careful attention should be paid to results of laboratory experiments and clinical trials that use the agents, since so many genes are affected, says Feinberg.

"It was kind of assumed that removing methyl groups would turn some genes on and others off, but the deactivation side of the coin had been largely ignored as being a minor effect," adds David Gius, M.D., Ph.D., chief of molecular radiation oncology at the NCI. "Now we know for sure that removing methyl groups has both consequences and to equal extents."

In their experiments, the researchers examined the expression of nearly 8,000 genes simultaneously in a colon cancer cell line (called HCT116). By studying the genetic "fingerprint" of a sample before and after demethylation, they could measure how the treatments affected the extent to which the genes' instructions were being used to make proteins.

One chemical agent they tested, 5-aza-2'-deoxycytidine, blocks addition of methyl groups to DNA and is currently in early clinical trials for leukemia. The researchers also tested the effects of knocking out of the cancer cell line either of two genes that encode proteins (DNA methyltransferases) that hook methyl groups onto the DNA, as well as knocking out both genes in the same cell. They compared these methyl-based mechanisms of gene regulation to a chromosome-based one, also in early clinical trials, using a chemical called trichostatin A, or TSA, that gently unravels the chromosomes, exposing genes and allowing their instructions to be read.

Much to the team's surprise, both chemical agents -- one methyl-based and one chromosome-based -- created similar patterns of changes in gene expression in the cell lines, says Hengmi Cui, Ph.D., assistant professor of medicine. However, the genetic knockouts' patterns of genetic changes were not similar to those of chemical demethylation.

While the number of affected genes differed, all of the methods turned off as many genes as they turned on, including entire gene families that might play a role in cancer's development or contribute to its aggressive nature.

The researchers are now investigating further some of the individual genes and gene families affected by the various treatments, and studying the mechanisms by which the chemical agents and the gene knockouts affect methylation and gene expression.

For example, the demethylation chemical was thought to act indirectly -- preventing re-methylation, so to speak -- as proteins were turned over and recycled in the cell. Because the effects of this agent were similar at both one day and five days after treatment, the researchers suggest the agent might have a more direct effect than previously thought.

Methylation of genes is an example of epigenetics, which are inheritable modifications to chromosomes and genes other than changes in the DNA sequence itself.

The research was funded by the National Cancer Institute. Authors on the study are Cui, Feinberg, Sheri Brandenburg and Yali Hu of Johns Hopkins; Gius, Matthew Bradbury, John Cook, DeeDee Smart, Shuping Zhao, Kheem Bisht, Allen Ho, David Mattson, Lunching Sun, Eric Chuang and James Mitchell of the National Cancer Institute; and Lynn Young and Peter Munson of the Center for Information Technology at the National Institutes of Health.

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