Era of Hope 2011 Investigator Vignette

Title: Modeling Impact of BRCA1 and BRCA2 Mutations in Mammary Epithelial Cells

Investigator: Meggan E. Keith, PhD, State University of New York at Albany

I have a postdoctoral grant from the BCRP, and the focus of it is to create a model system of cell lines from women who are heterozygous for BRCA-1 or BRCA-2 mutations and to use these cell lines to investigate the initiation and progression of breast cancers in women with this hereditary predisposition.

Many of these women choose to have prophylactic mastectomy and so my goal is to collect the tissue from these women at the time of prophylactic mastectomy meaning that it will be normal tissue instead of cancerous. And so I’m going to use this tissue to make cell lines which will be normal mammary cells but contain the BRCA mutation that the woman had.

It became kind of obvious to me that there was a gap in what we had in research. We had normal cells and we had BRCA completely null cancer cells but that middle step, cells along the progression pathway there aren't very many of, and so that became my goal was to actually start making some of those cell lines. It’s both a research goal and kind of a personal goal for me because I carry a BRCA-2 mutation and that’s what got me started on this whole train of thought was I realized that you know this was--I was kind of in the unique spot because I’ve been on both sides of the table. And so it became very obvious to me that knowing the sorts of things that women that have these mutations need and want to know and knowing what I know about science, I could figure out how to most quickly get at a way to answer those questions. And so that’s what I’m trying to do with my work.

We’re collaborating with a surgeon who does about 100 of these prophylactic mastectomies a year. He’s actually the surgeon who did my surgery so that’s how I met him and that was the other way we got started on this.

So far I’ve been doing the background work with the existing normal and cancerous cell lines. Basically I’ve been trying to set up assay systems so that once I get the tissue from the women and make the cell lines, I can very quickly progress through a standard set of experiments so that I’ll very quickly be able to generate a basal set of data characterizing these cell lines. I can get that out, and then hopefully other people from the research community can start using these lines to answer their own questions.

Women with BRCA-1 and BRCA-2 mutations undergo a lot of mammography which is also ionizing radiation. Ionizing radiation is a DNA damaging agent; it’s very commonly used in studies that look at BRCA-1 and BRCA-2 function. I think that’s going to be very important when we have the cell lines that I make because we’ll be able to see what does this increased mammography do over time, you know if—in trying to prevent cancer from occurring in these women if we’re actually making it more possible for it to occur because we’re constantly eradiating these cells with low doses? So I think that could be very—potentially very interesting later.

But for right now, sort of the take-home message from this overall is that all of the cells with BRCA-1 and BRCA-2 mutations are very sensitive to radiation and what we hope to get at with this when we have the cell lines that I’ll make is whether having only one working copy of BRCA-1 or BRCA-2 makes you more like this or more like this and what does that tell us about potential outcomes to these women.

This graph represents about a 6 to 8 months worth of—of work so I’m very proud of it. This is all five of the cell lines. I show two different time points—6 hours and 48 hours post treatment. And these are flow cytometry images; the green is the G1 phase of the cell cycle, the cells that are not dividing; the little brownish area is S phase, the cells that are starting to replicate their DNA; and the blue is G2M which are the cells that are starting to undergo mitosis and actually divide. And it’s been shown before that when you irradiate cells they will arrest and so we wanted to see whether that was occurring which is why we chose the 6-hour time point.

The other issue that we wanted to look at was how quickly could these cells resolve the damage which is why chose the 48-hour time point. So in the normal cells here, irradiating them does—cause a slight increase in the amount of blue which indicates that they have a G2M arrest. But after 48 hours, this arrest has been resolved. And all of the other BRCA-mutated cell lines at 48-hours after irradiation, the majority of these cells are still stuck in this arrested phase. And that’s because they can't repair the damage to their DNA so they can't finish that cycle and go on.

So again the question is the cells that have a half set of the BRCA-1 or BRCA-2 will they look more like this or will they look more like this, and then we can start to make some projections about what that means for women that have these mutations.