Too Much FGFR2 Can Increase Risk of Breast Cancer

Increased Breast Cancer Risk Because of a New Runx2 Site in the FGFR2 Gene

by Dr. Barry Starr, Stanford University

May 14, 2008

Lots of studies are done these days to find DNAs that can predict someone's chances for getting a disease. Most of them don't explain why a DNA difference can lead to a disease though. A new study is different.

This study shows why a DNA difference in the FGFR2 gene increases a woman's wirk for breast cancer. This is significant because knowing why a DNA change leads to increased risk means that scientists can work on finding ways to reverse the effect. In other words, this knowledge can help researchers find a treatment or maybe even a cure.

In this case, the DNA difference leads to an increased risk because it causes too much FGFR2 protein to be made. Remember, a gene is really just the recipe for a certain protein. In this case, the FGFR2 gene has the instructions for making the FGFR2 protein. So the DNA difference causes the FGFR2 gene to make too much FGFR2 protein.

It isn't surprising that having too much FGFR2 might lead to cancer. This protein is involved in breast cancers in both mice and humans. It has also been shown to be important in starting a cell on its way to being a tumor.

Knowing some people make too much FGFR2 means scientists can work on ways of lowering FGFR2 levels to decrease these people's cancer risk They also figured specifically why one of the DNA differences caused too much protein to be made. It was because of something called a transcription factor (TF).

Transcription Factors Determine How Much Protein Gets Made from a Gene

Transcription factors stick
to the DNA near a gene.
There they affect how much
protein gets made.

Cells "know" how much protein a gene should make because of something called transcription factors (TFs). Remember, genes are made of DNA. And DNA is made up of four different "letters"—A, G, C, and T.

The instructions for how a protein is made are written in this simple language. How much protein gets made is determined by these letters too.

But the letters don't directly determine how much protein gets made. Instead, it is the TFs that determine this by sticking to specific DNA sequences near the protein instructions.

For example, there is a TF called Runx2 that binds the sequence ACCGCA best. This is the TF that the researchers propose is at least partly responsible for why certain people make more FGFR2 protein. And are therefore at a greater risk for getting breast cancer.

Making a New Runx2 Site

Most people have the sequence ACTGCA in the DNA near their FGFR2 gene. Certain people with an increased risk of breast cancer have the sequence ACCGCA. The second sequence is much closer to a Runx2 site.

The researchers went on to provide evidence that Runx2 probably does bind this DNA site in these people. And that by binding there, Runx2 causes the gene to make more FGFR2 protein. Perhaps one day scientists can directly target Runx2 in these people and decrease their risk of breast cancer.

The researchers showed Runx2 was involved by doing three basic kinds of experiments that scientists do to try to show that a certain TF binds a certain piece of DNA. These experiments are called gel shift, ChIP, and reporter assays.

More Information

Assays to Look for DNA Binding by a TF

Gel shift

A gel shift looks at whether there is something from a cell that can stick to a certain piece of DNA. This is a lot harder than it sounds because the DNA and protein are both too small to see. So scientists need to make one (or both) visible.

Usually this is done by "labeling" a piece of DNA with something radioactive. Scientists can the see the radioactivity either with a piece of film or with imaging equipment.

The radioactive DNA is then added to a nuclear extract to see what sticks. A nuclear extract is basically a tube full of many of the proteins from a certain cell's nucleus. (The nucleus is the part of the cell where the DNA and TFs are.)

After awhile (the time varies), the mix of radioactive DNA and proteins is loaded onto a gel. A gel can separate things based on how big they are. (The gel has lots of little holes in it so that big things travel more slowly.)

The radioactive DNA will move through the gel at a certain speed. If a TF sticks to the DNA, then it will be slowed down.

Think about the DNA as a dad running in a race. He will definitely be slowed down if his 7 year old is on his back. So too will the DNA when a TF sticks to it.

The researchers found that the piece of DNA from people who were at a higher risk for breast cancer had an extra protein:DNA complex. So something binds the FGFR2 gene in these people that does not bind in other people. Now the researchers had to figure out what the protein in that DNA/protein complex might be.

By looking at the letters in the DNA, researchers can make educated guesses about what protein might stick. They can then confirm their guess by using antibodies.

Antibodies bind certain proteins very specifically. What this means is that an antibody to Runx2 will only recognize Runx2. And when an antibody sticks to a protein/DNA complex, that complex will get bigger.

Again think about the dad with the 7 year old on his back. Now imagine the dad's 4 year old son piles on too. Dad will now run even slower. And so will a DNA:protein:antibody complex.

The researchers found that when they added a Runx2 antibody to their gel shift assay, their protein/DNA complex got bigger. So Runx2 is definitely sticking to this piece of DNA.

Unfortunately, gel shift assays can give results that have little to do with what happens in the cell. This is because the experiments are done in a tube with only bits and pieces from the cell.

There might not be the same proteins that are found in a cell—some might be missing or there might be more or less of others. Also the experiment uses a small, naked piece of DNA.

DNA in a living cell is connected to lots of other DNA and there are all sorts of proteins stuck to the DNA. For example, TFs can help each other bind or keep other TFs away. Proteins like histones can hide DNA from TFs. And there are lots of other reasons too. This is why scientists invented the ChIP assay.

ChIP assay

A ChIP assay looks
at DNA as it is found
in a cell.

A ChIP assay looks at what proteins are actually bound to a certain piece of DNA in a cell. The reason scientists don't do this first is a researcher needs to know in advance what he or she is looking for. And a ChIP assay is really time consuming to do.

From the gel shift assay, the researchers knew they should look for Runx2 in their ChIP assay. And that is what they did.

The first step in a ChIP assay is to freeze the proteins where they are on the DNA. A cellular environment is a dynamic one—proteins are coming off and on the DNA all the time.

Think about it like ants on a log. Let's say someone wanted to know which ants were where on a long branch. It would be very hard to see them all at once. But if you could spray the tree with something sticky, then the ants would be frozen in their tracks. Now a scientist could look at the branch at his or her leisure.

The sticky thing scientists use in a ChIP assay is formaldehyde. Formaldehyde sticks proteins to DNA.

After the proteins are stuck, the scientists need to focus on just the protein they're interested in. In this case, that protein is Runx2.

Remember, scientists can't see the protein and separate them out that way. Instead they have to do some tricky things with antibodies.

Before they can do that, though, they need to break the DNA up into smaller chunks. Often they use sound waves.

The next step involves pulling out only the smaller pieces of DNA that have the Runx2 protein attached. For this scientists use two different kinds of antibodies.

The first antibody is one to Runx2. The second antibody is called Protein A or Protein G.

Both of these antibodies stick to other antibodies. So what we have now is Runx2 stuck to a piece of DNA. We have an antibody stuck to Runx2 and an antibody stuck to the antibody to Runx2. How does having such a complicate mess help?

Because the Protein A (or G) is stuck to a bead. This bead is like the snow in a snow globe. After someone shakes a snow globe, the snow settles.

Here the beads are spun down and collected. Now the scientist has lots of pieces of DNA that Runx2 sticks to. The next step is just to look at the FGFR2 gene.

A scientist accomplishes this with PCR. (Click here to see how this process works.) After all of this, the researchers were able to show that people with the DNA difference had twice as much Runx2 at their FGFR2 gene.

The problem with both of these assays is researchers can't tell if the extra TF has any effect on the gene. To test this, researchers use a reporter assay.

Reporter Assay

In a reporter assay, a scientist directly tests whether or not a DNA sequence can turn on a gene. The researcher takes the DNA sequence he or she is interested in and puts it in front of a gene that is easy to read.

In this case, the researchers used the luciferase gene. This is the gene that makes fireflies light up. So basically the researcher determines which DNA sequence makes more light in a cell. When they did this, they found that the piece of DNA with the difference that leads to an increased risk of breast cancer made more light.

More Information

Content provided by the Department of Genetics, Stanford University.