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Dartmouth s Victor Ambros on microRNAs and Their Possible Role in Cancer

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At A Glance

Name: Victor Ambros

Position: Professor, genetics, Dartmouth Medical School

Background: PhD, biology, Massachusetts Institute of Technology — 1979

BS, biology, Massachusetts Institute of Technology — 1975


Victor Ambros began his post-graduate career in the lab of Robert Horvitz, and didn't travel far for his first academic appointment as an assistant professor at Harvard University. Eventually, he landed a full professorship at Dartmouth Medical School.

Ambros has been at the forefront of microRNA research since the very beginning, co-authoring a paper in Cell in 1997 that described the discovery of the very first of these small non-coding RNAs. Most recently, he collaborated with colleagues at Dartmouth, the Norris Cotton Cancer Center, and Applied Biosystems on experiments examining miRNA expression in human glioblastoma multiforme brain cancer.

Recently, Ambros spoke with RNAi News about his efforts and where the field is heading.

 

You've been involved with microRNAs from the start. Can you give an overview of how it all started?

My lab is studying developmental timing in the nematode C. elegans, and the approach we've been using over the years, starting actually from when I was a postdoc with Bob Horvitz in the early 80s, has been forward genetics. We isolate mutants with interesting developmental defects and try to find the gene that's mutated. We've been studying mutants that are abnormal in their timing of development, and that's how we ended up finding the first microRNA, which is the small RNA product of the lin-4 gene. It was really as simple as that. We didn't know what the lin-4 gene product was going to be, so we just accidentally started working on microRNAs.

It was really as simple as that. We didn't know what the gene product was going to be, so we literally accidentally started working on microRNAs because of that.

When you first identified [the microRNA], was it confusing [in the sense that] here was an RNA that was non-coding? What was the initial reaction to the discovery?

In our lab, the reaction was that we felt we had to be very careful, we had to have it right, and we were not missing anything such as a small open reading frame that could encode for a small protein. So we made sure that we had eliminated all other possibilities. But moreover, the antisense relationship between lin-4 and lin-14 made it easier to accept the idea that that the lin-4 small RNA was the real regulatory gene product that was responsible for repression of lin-14. Prior to actually identifying the product of lin-4, we knew it had to be a repressor of lin-14, so when we found a small RNA that had complementary antisense sequence to lin-14, then we were sure.

The paper was published in Cell, so there was certainly a lot of interest by the editors of Cell; they felt Cell readers would also find the results interesting. So I think there was a considerable amount of interest, but not a lot of confidence on anybody's part, including our own, that this small RNA represented anything other than perhaps a peculiar evolutionary innovation by nematodes.

After publication, what kind of feedback were you getting? Were people saying that this might be going in the wrong direction, or was it more of a wait-and-see attitude?

There was no negative feedback. I think wait-and-see is a good way to capture the sense of others. We knew that it was important for nematodes, and we also knew that the pathway of genes that lin-4 was part of was also important for nematodes. The discovery also permitted us to study an interesting and important process, which is the coordination of cell division and cell differentiation across an animal as development progresses. This is a really important biological problem that we had access to through this genetic pathway in C. elegans. So the study of lin-4 was certainly a valuable effort, even if lin-4 didn't turn out to represent some general class of phenomena.

Can you give an overview of the research you're working on currently?

The main thrust of our lab is still the genetic analysis of C. elegans development. We have also a somewhat smaller project that involves examining the roles of certain microRNA genes in controlling Drosophila development. Finally, we have established some collaborations that involve examining the expression of microRNAs in human tumors. That's particularly in collaboration with Mark Israel, director of the Norris Cotton Cancer Center here at Dartmouth, and Caifu Chen at Applied Biosystems.

Can you give a little more detail on the C. elegans and Drosophila work?

Our central interest in the lab is developmental timing — how do genes control the timing of gene expression and coordinate gene expression throughout the animal; how do the regulatory networks in the cells of a developing animal control sequences of developmental events, which involve dynamic changes in gene expression as development proceeds. That's our basic interest. To that end, we continue to study mutants altered in genes that control developmental timing and try to work out some of the details of the regulatory interactions involved and identify new genes when we can.

The second interest is more generally the role of microRNAs in C. elegans development. For that project, we make mutations in microRNA genes and try to determine what the developmental defects are as a consequence of those mutations. In this case, we're not necessarily expecting those developmental defects to include developmental timing problems because we are looking more broadly at microRNAs in general. Actually we find that, unlike lin-4 and let-7, most of the microRNA genes in C. elegans are not specifically involved in developmental timing.

In the fly project, we have focused on a set of microRNAs that are very well evolutionarily conserved in their sequence. We feel, then, that the fly might be a good model for trying to figure out what the roles of those microRNAs are in the development of an animal that is bigger than a worm — and aspects of the development of the fly more closely resemble the sorts of processes that occur in a mammal where large fields of cells need to organize themselves and differentiate into patterns that are regulated by cell-cell signaling across longer distances than occur in nematodes.

There was recently research [in which you were involved] regarding microRNAs and brain cancer presented [at the European Society of Human Genetics annual meeting]. I understand they're preliminary, but can you give an overview of that and the findings?

Yes, these findings are quite preliminary, but we feel that what we've been able to see so far in our data suggests that this is a useful question to pursue — that is, what are the microRNAs that are expressed in tumor cells and whose levels may change, either increase or decrease, in tumors of various types in relation to normal tissue?

Our hope is that by doing a really careful and through quantitative study of the levels of all the known mammalian microRNAs in these tumors we'll be able to make some contribution that might lead to some deeper insights into the nature of these tumors.

The reason why microRNAs are important molecules to study in a disease context such as cancer is that they are regulatory molecules — they regulate other genes, and it's believed that any one microRNA can regulate dozens or perhaps hundreds of other genes. So each microRNA has potentially a great impact on the physiology of a cell. Therefore, they are particularly important molecules for us to know about.

How were these experiments conducted? Can you give a description of what you did?

We've just begun to examine a whole series of tumor cell lines and tumor samples from patients. What we've done to begin with is profile all the known human microRNAs in about a half dozen cell lines and brain tumor samples to determine whether or not we see any consistent differences in the patterns of the microRNAs amongst those tumors and cell lines. If we did see a difference, we would then scale up the project.

So that's the state we're at. We've done a pilot study of a small set of representative cell lines and tumors, and we see very interesting differences in the levels of certain microRNAs. This could mean that microRNA levels will be useful indicators of significant characteristics of various tumors. Now, we're going to scale up the study and get the data we now need to test the hypothesis that there will be consistent patterns of microRNA expression changes in brain tumors that will be useful and have predictive value for the behavior of the tumors and their treatment, eventually.

All this is being done with an assay from Applied Biosystems?

Right.

Can you touch on the difference between the assay and previous approaches?

The assay is very sensitive, so that allows us to use small samples, and sample size is limited when one is using biopsies from humans. Secondly, it is very quantitative and reproducible, so the data is going to be really robust — we feel we'll be generating a data set that will have a long legacy in the scientific community [and] will be useful for a long time. So we feel this is an important study to invest time and resources in because the data will be so good.

As you scale up and start getting more and more data, what's your sense or hope for where this will lead? Do you see microRNAs as just a way to diagnose [disease], or do you see potential along the same lines as what's being done with siRNAs?

I'm not very good at predicting the future, because I always seem to be off the mark. But to be honest, I see it leading, as you suggested — in a direction where people will identify microRNAs that seem to have important roles in disease physiology, whether that be cancer … or other human diseases such as autoimmune diseases and neurodegenerative diseases.

I think profiling microRNA levels in various human disease conditions is going to be a growing field, for sure. Whether or not they'll be used as therapeutics like siRNAs, I sort of doubt it because siRNAs are so much more effective at silencing genes than are microRNAs. However, I think that we will be able to inhibit the expression of a chosen microRNA gene. So, if a microRNA is increased in level and thereby causes a disease condition, then we can potentially use siRNA-like technology to knock down the microRNA and intervene in that fashion.

Those are the two areas that I say are going to be pretty exciting: profiling microRNAs, and thereby finding microRNAs that are important in cancer and other human diseases, then perhaps intervening with RNAi therapeutics or modified RNA oligonucleotides to knock down the microRNAs.

Are you doing any work in other cancers or other diseases at this point?

In collaboration with Mark Israel, of the Dartmouth Norris Cotton Cander Center, and Caifu Chen, at Applied Biosystems, we are profiling microRNA expression in a set of 60 tumor cell lines from a variety of human tumors. This should be a valuable data set, because these particular cell lines have been extensivly studied by many investigators. This background of molecular and physiological knowledge of these cancer cell lines can, in principle, be integrated with our data on microRNA expression to hopefully yield new insights about regulatory pathways contibuting to important properties of the tumors.

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