Dr. Muneesh Tewari Video (Text Version)
Circulating MicroRNAs and Prostate Cancer
Natasha Kyprianou
Last but not least it's my pleasure to introduce Dr. Muneesh Tewari, who will be giving the last presentation of this Game-Changing Research. Dr. Tewari is an Assistant Professor of Medicine at the University of Washington School of Medicine and at the Fred Hutchinson Cancer Research Center in Seattle, Washington.
He's an active oncologist, a clinician-scientist with the expertise in bioinformatics, computational biology, as well as functional genomics. Despite his young age, Dr. Tewari has already made some striking and truly significant contributions to prostate cancer research by identifying micro-RNA regulatory networks in prostate cancer where it has been supported by a New Investigator Award from the DOD Prostate Cancer Research Program in 2007 as well as the Creativity Award from the Prostate Cancer Foundation in 2009. Today, Dr. Tewari will be closing the session by talking about his fascinating work on circulating microRNAs in prostate cancer; Dr. Tewari.
Muneesh Tewari, M.D., Ph.D.; Assistant Professor of Medicine, University of Washington School of Medicine and the Fred Hutchinson Cancer Research Center, Seattle, Washington
Well thank you to Natasha and Wes, of course, for inviting me and giving me the chance to share some of this work with you and I should just-I would like to just mention that the DOD grant, the New Investigator Award that Natasha just mentioned was in fact my first grant, really first major independent grant from-you know starting my own lab about five years ago. So it's really been a pivotal in helping get things launched.
So I'll talk to you today about microRNAs as Natasha mentioned. These are small RNA molecules; they're only 22 nucleotides long. They've been known now for about a decade in-in humans at least and-and maybe an additional number of years in model organisms. And what-what makes micro-RNAs particularly interesting is that they're regulatory molecules, so they physically interact with-with messenger RNAs and one microRNA can interact with multiple messenger RNAs to inhibit their activity. And in fact, when I really started out as an independent investigator I was very interested in these microRNAs because particularly with their potential roles of course in-in cancer, including prostate cancer.
If you look at their roles in normal physiology they've evolved to control really very fundamental processes and development differentiation sort of regulating cell physiology. And it turns out there's at least about 700 of these that are encoded in the human genome. And the other thing that was very attractive starting out was that there's really quite a bit of interest in potentially targeting these essentially as-as therapeutic targets. And so it became very clear even in the first few years that these microRNAs as I mentioned, they're important in-in normal development but in fact they are intimately sort of tied into a number of basic mechanisms of cancer such as evading apoptosis, inducing androgenesis, and so forth. And I've only shown a few examples here.
And so what in parallel to trying to understand what the role of these microRNAs might be in carcinogenesis and in the phenotype you know, we also started wondering whether this could actually be useful-that the fact that these microRNAs are tied to these basic processes means that they capture some information. And could we actually get access to that information from the tumor? And in fact, of course the standard way you know to do this would be to get a piece of the tumor tissue but really almost on a-you know on a-we kind of took a flyer and said well, perhaps these microRNAs might in fact reach the circulation in some way from the tumor tissue. And this was kind of an unorthodox thought at the time because the sense was-and actually it's well known that there is very high activity of ribonucleases in the-in the circulation in plasma or serum and so it was not really expected that-that RNAs would be particularly stable or-or good markers.
Nonetheless we wondered that what-what-or we at least dreamt that if this was actually true there would be very-a number of advantages, and so they're listed here. So one is you know it was already becoming clear that the signatures of these micro-RNAs in tissues themselves were capturing important information and-and yielding useful biomarkers. The number of copies per cell of these molecules is not you know a couple; let's say for a DNA marker these were you know hundreds to thousands and sometimes even tens of thousands. And really I think the most attractive thing was the fact that these were nucleic acids of course. So you know the-the-there was the ability to at least piggyback on all of the great technology that's been developed for-for highly-for highly sensitive detection and measurement of nucleic acids.
And so essentially we've set out to just ask a few relatively seemingly simple questions which were you know can we find cell free micro-RNAs in the bloodstream? And I'll just really summarize quite a bit of data here; and this is just a graph that shows that we measured three different microRNAs represented by-in sort of the three different groups of graphs and three different individuals which are the three different colors and the bottom line is on the-on the Y axis is sort of copies of microRNA per-per micro-liter plasma and this is just in healthy people-that we were seeing amounts really on the order of hundreds of thousands or a million copies. And this was-at least the sort of initial evidence that-that in fact there are microRNAs that are circulating. And there's other experiments that we did to confirm this that we don't have time to get into.
I'd say one of the really key questions we asked is whether or not these were stable-that is if you take a tube of plasma or serum and you just put it at the bench because that's sort of the you know practically what happens; you know frequently in-in blood collection before it can really be frozen away-would these be degraded because this was this question of ribonucleases activity and-and instability of RNA. And in fact, what these graphs show are three different microRNAs over time and actually in two different people. And measuring their levels in the plasma you know of-of plasma incubated up to 24 hours, and you can see they were just essentially rock stable. And then in fact even if you froze-if one freezes and thaws the plasma, there's really no change. So this was I think you know a pretty surprising at least to me but also a very encouraging one with respect to potentially developing these further as diagnostics.
And then the ultimate question really was whether or not cancer-derived microRNAs could get into the blood because this was all done-the work I've shown so far with just plasma from healthy individuals and again I don't have time to go into all the details, but we did two experiments. So one was in a mouse model where we knew exactly where the microRNA was coming from because it was a prostate cancer xenograft model and in fact you know in a dozen mice we could easily distinguish the mice that had the xenograft from the ones that didn't specifically by-by measuring microRNA.
But the other experiment was in fact then really a measurement of a specific candidate micro-RNA that we chose because of its expression in prostate cancer in the serum from 25 men with advanced prostate cancer compared to 25 controls. And I'll show you know this-this graph really is sort of the kind of proof of principle of all of this; it's again you know these are men with advanced metastatic prostate cancer. The-the Y axis here in fact is a-is a log scale. So as you can see that you know there's one-there's a pretty big difference in groups; the left side is the normal and the right side is the patients. But in addition you can see that in some of these men there's extremely high levels of this particular marker which happened to be miR-141.
And so we-we published this a couple of years ago, actually maybe now almost 3 years ago in PNAS. And I was asked actually by you know the organizers and the organizing committee to have one slide which-which actually says well what's the impact of this has been? So it's not been that long, I mean relatively speaking I guess in-in research time, but I would say that you know I think the-the biggest impact of this work really has been that it's introduced or helped introduce and others have been working in this field as well, a new approach for-for these blood-based biomarkers. And I guess you know the-the long-term vision at least we have you know based upon some of those advantages I mentioned earlier is that the use of circulating microRNAs as markers potentially at various stages in the disease spectrum really has the potential at least if the right markers can be found for developing inexpensive and highly sensitive tests, again because they're-they're based upon nucleic acid technology and potentially I guess our long-term vision would be that these would in fact be point of care and-and-and very simple and not even require a clinical lab. That's of course far into the future.
And I think you know as part of this impact of course has been that this-this area has really burgeoned as an area of research and I've just shown at the bottom here, there's a few-now other investigators that have made some really pretty interesting findings with respect to circulating microRNAs in prostate cancer and some of those papers are shown here. But in fact this is also now you know being taken out to a variety of other cancer types and even all kinds of diseases. So this-that's been actually pretty gratifying. And I guess you know the-the potential impact here I think in the future at least of what my lab is interested in certainly is in addition to just detecting and potentially monitoring cancer but really to learn something about tumor physiology and potentially dynamically from-from blood-based measurements. And again you know I think there's-there's quite a bit of work now ongoing in this area which makes it a-an early but-but very exciting field.
So that I think you know more or less summarizes you know most of the work to date. I'm going to just take one slide really to just update you-maybe a couple slides actually about where we're going next and what at least--what--what we're working on. So in addition of course you know looking for new markers for various clinical indications which I think is ongoing work, to really move this forward I think to clinical useful application, I think it's going to require a number of different things and a few of them are listed here. And these are essentially the ones that at least in my lab we've--we've been working on.
So one is the mechanism of the stability, I think will be important; the other is understanding all the different variables that can sort of affect these measurements-relatively mundane but very important question to answer; and the third of course is--is trying to really understand what the microRNAs are telling us. And I will just have time today to tell you about this idea of the mechanisms of microRNA stability since it's a question that frequently comes up. And just in this one slide what I'll just summarize to you is that essentially it looks like there are at least two mechanisms of microRNA stability. So these microRNAs don't circulate just sort of naked. And one of these is in encapsulation in vesicles, which are exosomes or--or one kind of vesicle but the other mechanism appears to be in fact protection by a protein complex. And in fact we just published this; it just came out and this--this figure is relatively busy but basically what we did was fractionated plasma by size exclusion so we could separate the vesicles from the protein complexes.
And if you look from sort of top to bottom and then left to right, the--the stuff in the sort of far up--or lefthand corner are microRNAs that are particularly in these vesicles. And the ones on the right side are the ones that are in the protein complexes. So why is this important? I think the you know the reason this is important is as we are moving forward in this field it shows us that there are specific sub-populations of these microRNAs and now you know again it's a very early phase in this field but I think there's a lot to learn and-and this may in fact carry more unique information if we look at these microRNAs not just in whole plasma but in-but in specific sub-populations that for instance might derive from tumor cells. And of course, this also opens up strategies for purification; the exosome approach has already been--sort of is already under way by many different labs but I think now using protein handle for the immuno-purification may also help in developing tests.
So I guess I would just really summarize by-by saying that this is a really I think new area; we have a lot to learn. But I think it-in the long-term vision of-of ultimately learning more from patients and-and learning more about tumor dynamics you know and real people I hope that this is-this will ultimately lead to-to real tests and real contributions. And just to acknowledge since I'm over time, I will just-you know I think a number of the funding sources have already been acknowledged. And I'd just like to acknowledge the people in my lab, in particular I'd like to mention John Chevillet since he has a poster here-here at the meeting, as well. Thank you very much.