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Maria Kavallaris On Using Proteomics to Study Mechanisms of Cancer Drug Resistance

Maria Kavallaris
Group leader, Pharmacoproteomics, Experimental Therapeutics Program
Children's Cancer Institute of Australia for Medical Research

At A Glance

Name: Maria Kavallaris

Position: Group leader, Pharmacoproteomics, Experimental Therapeutics Program, Children's Cancer Institute of Australia for Medical Research, since 2001. Project leader, CCIA, 1998-2002.

Background: Postdoc, Albert Einstein College of Medicine, 1996-1997

PhD in biochemistry, University of New South Wales, Australia, 1994.

Last week, Maria Kavallaris gave a talk on mechanisms of resistance to anti-leukemia drugs at Select BioScience's OncoProteomics conference in South San Francisco. ProteoMonitor spoke with Kavallaris after her talk to find out more about her research and how she got into the field.

How did you get into using proteomics to study mechanisms of drug resistance?

I did my PhD in children's cancer research in drug resistance. I was mainly doing a lot of cell biology, and some biochemistry as part of that. I started investigating drug transport and drug-targeted interactions. That led to doing my postdoctoral studies at the Albert Einstein College of Medicine in New York City. When I went to work there, I started working with Susan Horowitz who had discovered how Taxol works. As part of my studies there, I got involved with trying to understand how the drug interacts with its target, and how changes to the target affected the interaction of the drugs. And that was a pretty exciting time.

These were all very new things for me. I guess near the end of my postdoctoral studies there, I started getting really interested more in the protein side of things, because I did a lot of work with gene regulation and changes in gene expression in drug-resistant cell lines and clinical samples. Then I started thinking the target of these things is the proteins. So when I went back to Australia in 1998, I started thinking that I really want to get involved in better understanding what's going on with the proteins.

At that time, I thought this is where cancer research is going to go, and I thought that we might make better breakthroughs if we started investigating the proteins further. So I went and enrolled in a 2D gel electrophoresis course in the Australian Proteome Analysis Facility. I did some training there, and took it back and I was investigating some very specific questions about drug resistance.

Very early on, I started finding things that ended up being really interesting and quite valuable in advancing our research. I took the initiative then to really expand the proteomics, and also to build proteomics up where I was, because I went back to the Children's Cancer Institute of Australia, and they didn't really have any sort of proteomics set up there at all — they weren't doing a lot of protein work.

I spoke to the director, and said, 'I really want to establish proteomics within the institute.' She was very supportive and helped get funding for some of the equipment that I needed. Because we're an independent research institute and we rely on donations as well as government funding through grants, I maintained a very strong collaboration with the Australian Proteome Analysis Facility in Sydney. And that was fantastic. By that collaboration, we really got the research moving quite rapidly. We made some key discoveries with protein pathways involved in the mechanisms of resistance to anti-cancer drugs.

We took the paradigm of the leukemia model that we had. We had very nice drug-resistance lines, as well as animal models that we could use. But I see these as a stepping stone to expand into other tumor types, as well as to work with other drugs as well. So that's sort of historically how I got into it.

Did you continue with 2D gel electrophoresis, or did you expand to use other proteomic technologies as well?

We started off with the traditional 2D gels and Coomassie staining and silver staining-type gels, and then about three years ago, we adopted the 2D DIGE technology, and the fluorescence-based 2D gels. We found that very useful because it's easier for the quantitation and the reproducibility of the samples. And you get statistical analysis done on your protein spots, and it often follows what we've gotten by Western blotting as well.

So we've gone with that approach, but we're also aware that we have limitations in using 2D gels. We're now starting to look at some of the less abundant proteins, and also some of the high molecular weight proteins. So we'll probably start moving soon, for some of our applications, to LC-MS. But I think the way we'll go is probably by enriching, and then going to that system.

How did you get from doing 2D gels to actually honing in on the mechanisms of drug resistance?

We started off looking at drug-resistant cell lines. Traditionally, for drug resistance, often people have developed drug-resistant cell lines and then tried to find differences between their cell lines and the original parent cells. And we thought, 'Well, we're going to see a lot of changes.' Drug resistance is multi-factorial, and you don't expect that just one change is going to answer all your questions. And what we thought is, 'Well, if we go there and we look at drug resistance, we're going to find a lot of changes. But then how do we know which changes to pursue?' Because some changes will be directly related to the resistance mechanism, and some of the changes will be indirectly related.

The way we decided to approach it was to look at what happens when the drug-sensitive cells actually respond to the drugs. In other words, treat the drug-sensitive cells with increasing doses of drug, and look for changes that either go up or down.

We focused on the proteins that were common to both drug resistance and drug response. And by doing that, we were able to really then focus our interest on 10 key proteins. And when we looked through PubMed to find out what was known about those proteins. All of them had somehow been related to cytoskeletal changes. So it's probably regulating the target of the drugs.

I think with a lot of these target studies, it's rarely one thing, but rather the combination of factors — changes in the cellular target and changes in the proteins regulating the stability of the cellular target — are all contributing to the resistance phenotype that we were seeing.

Were you surprised that all the proteins you found were related to the cytoskeleton?

There were a few that weren't, but the majority were. So over 50 percent were related to the cytoskeleton. At first, I thought, 'Maybe they're just compensating.' The original drugs targeted the microtubules, so that's a component of the cytoskeleton. And we know that if you manipulate the expression of the microtubule cytoskeleton, you'll affect the stability of the actin cytoskeleton, and vice versa.

But what we're talking about here are permanent changes, because these cells are already resistant, and these were now stable changes in the cell. So the question is, 'Do these changes occur in concert when you're selecting out resistant cells, or does one come before the other?' We're sort of investigating what comes first.

I must admit I was skeptical when we first saw [the cytoskeletal protein changes] in our resistant cell lines, but when we then went into the animal model of intrinsic and acquired resistance, then I started to think, 'Maybe there's something really in this.'

We validated the key proteins that we thought were involved, and now we're slowly going through and checking functionally which ones are directly involved in the action of the drugs.

How will this research have applications in cancer therapy?

We're hoping in the future that it might help identify which patients are resistant to certain types of chemotherapy, and ultimately we want to find ways to reverse the resistance phenotype.

Now that we've identified some potential new targets, the future lies in how to reverse resistance.

Is any of this work being patented?

It is. One aspect of the discovery, using differential proteomics, is.

Do you think that these proteins are applicable, in general, to all sorts of drug resistance, or are they only applicable, in particular, to leukemia drug resistance?

At this point, we don't know if they are applicable to other cancers yet. That's going to be something we have to investigate, and that's part of our future plans.

Do you have other projects planned for the future?

Yes. A lot of our work is looking at aspects of the cytoskeleton in cancer cells, and manipulating different aspects to see what can either increase or decrease sensitivity to anti-microtuble drugs. So we've been looking at the cellular targets, like the tubulins in drug resistance, and looking at how we can manipulate those. So there's certain forms of the tubulin that, if we switch them off, results in an increase in sensitivity to certain drugs, and other ones that, if you manipulate them, result in a decrease in sensitivity. By slowly dissecting the pathway of how these drugs work, we can then start better targeting, and even developing new therapeutics. That's what I'm very interested in getting into.

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