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Texas A&M s Steve Safe on Using RNAi in His Cancer Research Projects

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At A Glance

Name: Steve Safe

Position: Professor, toxicology, Texas A&M University

Background: Professor, University of Guelph — 1973-1981; Research officer, National Research Council, Canada — 1969-1973; PhD, bioorganic chemistry, Oxford University — 1965; MS, chemistry, Queen’s University — 1963; BS, chemistry, Queen’s University — 1962

Could you talk about what you do in your lab and the kind of research you do?

Almost all of the research we do is cancer research, and we’re doing a couple of different things. We got interested in cancer research because of a receptor called the dioxin receptor, or aryl hydrocarbon receptor. It’s a receptor that binds both toxic ligands and cytochemicals, and activation of this receptor actually down-regulates estrogen activation. In a sense, compounds that bind the Ah receptor are anti-estrogens. So we’ve been looking at exactly the mechanism of that cross-talk between the Ah and estrogen-receptor signaling pathway.

That led us into a number of other things on estrogen-receptor signaling, and I’ve got grants that support studies on a new mechanism of estrogen-receptor signaling [in] both genomic and non-genomic pathways. That’s one area of the research.

The other area is that basically we’ve developed a new class of drugs that work through nuclear receptors that are really interesting and have a good chance of getting into the clinic for treating different types of cancers. Some of the compounds bind PPAR-gamma and inhibit cell and tumor growth. Other compounds bind other nuclear receptors that we’re working on. So it looks fairly exciting, and we have a group of compounds that we hope will get into the clinic.

Where does RNA interference come into your research?

Oh, RNA interference comes into every project. I think the first time we used it was as soon as I saw the first paper in Nature by Tom Tuschl’s group; within a few weeks we were doing studies [applying RNAi].

The one study we wanted to do [stemmed from my lab’s discovery] of a new mechanism for estrogen action in breast cancer. It’s where the estrogen receptor doesn’t bind DNA, but binds another DNA-bound protein called Sp1. Our hypothesis was that a number of genes, important genes in breast cancer, were activated by estrogen-receptor Sp1 complexes. So what we did initially was to knock out Sp1, knock out estrogen receptor, and look at the effects of what hormone-induced gene expression did. Of course, we could knock out hormone-induced gene expression by knocking out Sp1 protein.

That’s when we first started using [RNAi], and then we applied it also to our work with the aryl hydrocarbon- or dioxin-receptor/estrogen-receptor cross-talk, in which we could selectively knock out one or the other and get the appropriate effect. We published a paper in which [we described knocking] out the Ah receptor and the estrogen receptor, and … how it could modulate cross-talk.

Subsequently, we’ve been doing a lot of work on the role of Sp family proteins on the regulation of genes, particularly genes involved in angiogenesis and tumor growth. We’ve been looking at that by, again, knocking out [using RNAi] Sp1, Sp2, Sp3, Sp4, and trying to find out the roles of these individual Sp proteins in gene expression in cancer cells.

As that progresses, is the idea to target these with small molecules?

Well, the initial idea is just mechanistic. [RNAi] is a wonderful tool to show that a particular protein or transcription factor is involved in gene expression, cell proliferation, et cetera, or not. So that’s been the prime intention.

One of the things we’re targeting now is pancreatic cancer. We know that Sp proteins are really important for pancreatic-cancer cell growth and metastases. We’re trying to develop, in collaboration with some other people, siRNA approaches for the in vivo knocking out of Sp proteins, and hopefully knocking out pancreatic-cancer cell growth and angiogenesis.

Who are you working with on this?

We’re collaborating with a company and with the pancreatic cancer group at MD Anderson Cancer Center. I’ve got a lab in Houston in the medical center, and we do a lot of collaboration with MD Anderson scientists.

Is this an RNAi company you’re working with?

Yes.

Which one?

Dharmacon. They’re helping with the in vivo studies because you need a fair bit of siRNA, and they’ve got some new derivatives that appear to be more stable. We haven’t done any of the experiments, but we’re getting ready. We’ve tried a few pilot studies, and we’re going to be looking at some in vivo studies with siRNA approaches in mouse models.

How preliminary is this initial work you’ve done? Have you already gotten a sense of some of the hurdles you’ll need to overcome?

We haven’t gotten anything to work with normal siRNAs, so we’re hoping these derivatives will be useful and a little more stable. But I think probably our problems are more our fault than the reagents’ fault.

Do you see a place down the road where you’ll convert this [RNAi approach] into a therapy, or is that not on the radar?

That is on the radar, and it’s just a matter of showing that it works or doesn’t. My feeling is that it will work.

I think as the technology improves and the reagents improve, we have every reason to assume that some of these techniques are in fact going to work. There’re not going to work for everything, but there’re going to have really important selective applications.

You mentioned a new class of drugs you’re working on. Can you get into that a bit?

They’re interesting — we’re working on them in collaboration with a company called Plantacor. Basically, we got into them because we were interested in plant-derived materials that bind the aryl hydrocarbon receptor; these were indol-dimers called diindolylmethanes, or DIM, which is in fact a metabolite. When you eat cruciferous vegetables you can actually find DIM formed in your gut. It has a lot of really wonderful potential as an anti-carcinogen, and it’s been shown in animal models to be so and is sold as a nutraceutical.

What we’ve done is derivatized and changed the DIM structure. We changed it initially because we thought we would lose activity, but in fact we gained activity in terms of being anti-tumorigenic. What we had was a group of compounds that were pretty active in vivo against tumors, and we didn’t really know how they worked — this was about five or six years ago. What we began to do was screen nuclear receptors.

We know at least one group of these compounds activates PPAR-gamma, which is a really nice and interesting target for cancer therapy, so we’re really pushing that. We have activation of other receptors, but this isn’t published and I can’t really tell you too much about that yet.

Does RNAi come into play in this aspect of your research as well?

Absolutely. In fact, we’re looking at at least three receptors, and to confirm that they’re involved, we knock them out. Sure enough, we show that our compounds don’t act. [RNAi’s] been critical, just absolutely critical to these experiments because some of the results are quite unusual and you really have to prove your case.

You said almost all your work is in cancer research. What about the other part?

Most of my training was in chemistry, and I got into what I’m doing now through toxicology. The main toxicology studies that we’re looking at now are looking at toxi-chemicals that activate the aryl hydrocarbon receptor. We’re also interested in estrogenic endocrine disruptors, and so we do a lot of work on that. And again, when you want to confirm that a gene is involved, or a particular transcription factor is involved, we use RNAi.

Is there any area of [RNAi] that you think needs to be worked on to make it a more efficient tool?

Well, the delivery vehicles need to be improved; the vectors need to be improved. We’re trying to develop a viral vector; there are commercially available ones. We haven’t tested them to see how good they are. We initially tested some of the early plasmids, and we found that we got better results with just the oligonucleotides rather than a plasmid.

So, at least from our point of view, we’d like better expression. You always want better expression, and those are the kinds of things that can always be improved.

You talk to some people, and they’re not as keen on RNAi as we are, but I think in many ways we’re been lucky. We’ve just been able to have some pretty good success — when you get that with a technique, which we often didn’t get trying to knock out particular genes and proteins using other techniques, you become pretty keen on it. I just think [RNAi’s] been a wonderful tool.

[As for RNAi therapeutics,] we dreamt of that right at the beginning, but didn’t have the wherewithal or the expertise to do it. There are still not that many papers out there, but potentially I don’t see why it won’t be used for chemotherapeutics.

You’re working on developing a viral vector?

Nothing special, just trying to get one that works well.

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