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ESI Special Topic of:
"Gene Silencing," Published December 2003

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Gene Silencing

An INTERVIEW with Dr. Stephen Baylin

ESI Special Topics, January 2004
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According to our analysis of gene silencing research over the past decade, Stephen Baylin of Johns Hopkins University ranks at #2 among researchers publishing in this field, with 11 papers cited a total of 1,554 times. He is also a co-author of the paper ranked at #1 in our survey: "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands," (PNAS 93[18]: 9821-6, 3 Sept. 1996). In the Essential Science Indicators Web product, Dr. Baylin has 86 papers cited a total of 10,295 times to date in the field of Clinical Medicine and 31 papers cited a total of 1,911 times to date in the field of Molecular Biology & Genetics. At the Johns Hopkins University School of Medicine, Dr. Baylor is the Virginia & D.K. Ludwig Professor in Cancer Research, the Associate Director for Laboratory Research at the Sidney Kimmel Comprehensive Cancer Center, the Director of the Cancer Biology Program, and the Chief of the Tumor Biology Laboratory. Below, Special Topics correspondent Gary Taubes talks with Dr. Baylin about his work in gene silencing.

ST:  Your laboratory was partly responsible for launching this whole field of DNA methylation and gene silencing—how did you first come upon this phenomenon?

My entry into the DNA methylation story is somewhat serendipitous. I had a group interested in the molecular reasons why certain human cancers express endocrine features that are important to growth, and the way that these genes behave. We were trying to understand the transcriptional pathways that allow this behavior. I started looking at DNA methylation on the promoter of a target gene. In the 1980s, we thought DNA methylation might be one way to silence genes. In looking for that, we discovered an area of the promoter of this endocrine gene that putatively should not be methylated, but was methylated in the cells we were studying. That was in our experimental system and those systems were cancer cells. It became apparent that this might be an alternative way for tumor-suppressor genes to be silenced, an alternative to mutations. We followed that line of thinking and that’s how we got into this. At the time we found this, Frommer, Bird, and others defined what they called CpG islands. Evolution has progressively depleted the CpG dinucleotide in the mammalian genome, so most of the genome is CpG poor. But CPG dinucleotides tend to be clustered in these small stretches of DNA, the CpG islands. In normal cells, these CpG islands are maintained free of methylation, especially in gene promoter regions. We were seeing methylation of the gene we were studying in a promoter region CpG island, so we thought this abnormality might be associated with neoplasia.

ST:  Take us from that point in a short arc to the 2001 Cancer Research paper (M. Esteller, P.G. Corn, S.B. Baylin, J.G. Herman, "A gene hypermethylation profile of human cancer," Cancer Research 61: 3225-9, 2001).

…the idea is that this panel of markers might be utilized in screening for early detection of cancer.”

Well, by the early 1990s we began to associate loci that seemed to have hypermethylation in cancer to regions of loss of heterozyosity, which are known as LOH. Most tumor-suppressor genes are mutated on one copy of the gene and the other copy is often lost to chromosomal deletion. So LOH occurs when you lose a portion of the chromosome for one of two alleles of the genes. And that is Knudson’s hypothesis: you have to lose function of two copies of the tumor-suppressor gene to achieve a tumor progression event. We therefore started associating hypermethylation of loci to regions of LOH. We then began to look for specific candidate tumor-suppressor genes in which this might be a player. In a paper we published in PNAS about 1994, Jim Herman and I reported that we had found such a gene in the Von-Hippel Lindau tumor-suppressor gene—most people for short call it the VHL gene. This is a gene that can be mutated in the germ line of families who get inherited types of renal cancer. What we found was that in the non-inherited form of renal cancer, in about 20% of tumors, you had promoter hypermethylation and no mutations. In about 60% tumors, you have mutations in VHL. We did this in collaboration with a group at the National Cancer Institute. We also demonstrated that the gene could be reactivated with a compound called 5-azacytidine, which is an inhibitor of DNA methyltransferases and is known to demethylate regions. Then in collaboration with David Sidransky’s lab in 1995, we showed that a very common change in human cancers was for p16 gene to show hypermethylation. Those were the first two genes—VHL and p16. Since then a whole host of researchers, in our lab and elsewhere, showed that about half the tumor-suppressor genes have this change. There’s now a growing list of these genes, all leading up to that paper in 2001. This started to be something lots of people were looking for.

ST:  So is the 2001 Cancer Research paper mostly a review of all this work?

It was a review but with a lot of primary data in it as well. In particular, by the late 1990s, we thought that this hypermethylation might be a molecular marker of cancer you could use to look at the DNA—from tumor, or urine, or sputum, or whatever—with diagnostic purposes in mind. This became apparent not only from a basic understanding of how tumor-suppressor genes work and how genes get silenced, but through the constant position of this hypermethylation in the promoter region of these genes, and the ability of investigators to develop very sensitive techniques to detect it. So the idea was that these specific hypermethylated promoter regions could be used as a molecular marker of tumor suppressors, and by putting together tumor panels of four or so hypermethylated genes, we might be able to detect lung cancer, colon cancer, breast cancer, and leukemias. We showed we could get up to 80% coverage of the genome with such panels. In other words, one or more of the markers are likely to be methylated in at least 80% of tumors. Now people are getting closer to 100%. So the idea is that this panel of markers might be utilized in screening for early detection of cancer.

ST:  How exactly would you use such a panel of markers?

You would take your PCR procedure, such as the one that we reported in a 1996 PNAS paper. You would isolate DNA, say, from the sputum. That procedure is extremely sensitive. If there is one hypermethylated tumor cell in there—one in 1,000 or 2,000 or maybe even 10,000—we’d be able to detect it and that would be the marker. So you look at the hypermethylation of these promoter regions of specific genes. And you look in a specific repertoire of genes for any given tumor type.

ST:  How far do you see this going as a clinical tool?

There are lots of small observations that have built up over the last several years indicating the tremendous promise of this. It’s going to take bigger studies, though, exploring hypermethylation in large numbers of patients in given situations—such as screening sputum for lung cancer, or screening urine for prostate cancer. All these things are under active exploration from lots of groups. It’s going to be the data from these kinds of studies that will tell us how far things will go.

ST:  What have you found to be the most challenging aspect of this research?

I think the challenge and excitement of this research is that it spans everything from basic science all the way to clinical possibilities. The basic science dovetails into the exploration of how chromatin, the proteins that interact with DNA, dictates the gene expression pattern that emerges from our genome. This has also been a very exciting, explosive area over the past few years; how the DNA methylation associated with gene silencing is intimately linked to understanding how chromatin dictates gene expression status. So why I am mentioning this? The biggest challenges in the field at the moment are understanding two things: how does DNA methylation participate in gene silencing (and that includes what molecules target the DNA methylation to these promoter regions), and why is the cancer cell doing this? At the molecular level, why is this taking place? What’s happening during tumor progression? Understanding these two processes constitutes the biggest fundamental challenge in the field. That’s what my lab works on a lot.

ST:  What do you see as the therapeutic implications?

They’re pretty obvious. For one thing, unlike mutations, which are permanent changes in genes that lead to improper protein readout and function, this is a reversible change. Certainly, experimentally, you can make this gene expression come back on. You can strip away the DNA methylation, and change chromatin from the inactive to the active pattern around these promoters. There’s an entire enterprise growing up around this idea. As with all things related to cancer, however, it’s not so simple. What drugs can specifically do this to a sufficient extent to get these genes back on and actually have a good therapeutic effect or actually work to prevent cancer is an open question. This hypermethylation is a very early change in cancer cells, though, so we’re optimistic that this might be utilized.

ST:  If you have four hypermethylated marker genes for these various cancers, does that tell you anything about the cancer process itself?

Sometimes it does. Definitely in some genes, it’s given us exciting leads to understand the progression of cancer cells. Here’s an example: about 15% of all colon cancers have a phenotype in which you have multiple regions of mutation in and outside of the genes in the genome of those tumors—it’s called microsatellite instability. Researchers first discovered, in families who inherit this form of tumor, that they inherit it because they have mutations in the enzymes that repair mutations that arise during DNA replication. If these repair enzymes are defective, then they build up other mutations. The leading way this happens in the non-familial form of the cancer, in the 15% of these sporadic cancers, is hypermethylation in a promoter of a gene called MLH1, which is a mismatch repair gene. So 15% of the colon cancers from patients have this microsatellite instability, and about 70% of that 15%—the majority—have hypermethylation of MLH1. So here’s an epigenetic event that would set up a genetic series of changes that people think are fundamental to why that type of colon cancer gets started. That’s one example. VHL is another. The hypermethylation and mutations in VHL are very specific, mostly for renal cancer. They clearly play a role. Since families inherit mutations in VHL, we know that methylation and mutations are equally causative in the progression of the cancer. The functional significance of many of these genes is now becoming apparent.

Now that people are devising ways to screen cancer genomes for these hypermethylated genes, we then have to say "OK, if we find them, are they important to function?" One way we can do that is to knock that gene out in a mouse model. We have done that for one gene we found in this kind of screen, and showed that it was important to embryonic development. When one copy of the gene is knocked out in mice, the mice go on to develop tumors later in life as the other copy of the gene becomes hypermethylated.

ST:  Can you give us a five-year prediction of where the research in DNA methylation and gene silencing is likely to go?

I think I’ll say what we are likely to know in five years. We will certainly be a lot farther along in understanding the promise of this marker technology. Some people see this promise as very high right now, but we have to test it. In five years we should either really be using these markers and knowing which direction to go with them, or we will have obtained data for whatever reasons that tell us the promise is not as high as we thought. So we will have a much better understanding of the clinical promise of markers. By the same token, we will have drugs that can block DNA methylation, such as 5-azacytidine, which will probably be approved by the FDA for at least one condition, a pre-leukemic syndrome known as myelodysplastic syndrome, or MDS. Patients with MDS have anemia and deficiency of other blood elements, and they have a high incidence of developing leukemia. They don’t have proper function of the bone marrow, and we have known for some time now that they respond to this drug with long-term improvement and normalization of marrow function. The FDA probably will approve 5-azacytidine for use in this syndrome. The challenge will be to find out whether it works through reactivation of genes. If so, it will further open up the question of how efficacious reversal of gene silencing may be for cancer in general. I think in the next few years we will get farther along in understanding that possibility.

That’s what I’m excited about. The research is real and the clinical possibilities are real. And we’re making progress. Over the next five years we will see how far it is likely to go.End

Stephen Baylin, M.D.
Johns Hopkins University
Baltimore, MD, USA

Read comments from Dr. Stephen Baylin; discussing his Fast Breaking Paper in the field of molecular biology & genetics titled "The fundamental role of epigenetic events in cancer" from August 2003.

Read comments from Dr. Stephen Baylin and Dr. Manel Esteller; discussing their Fast Breaking Paper in the field of clinical medicine titled "A gene hypermethylation profile of human cancer" from June 2002.

ESI Special Topics, January 2004
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ESI Special Topic of:
"Gene Silencing," Published December 2003

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