R. Daniel Beauchamp, MD: Developing New Strategies for Colorectal Cancer Prevention and Treatment

Our laboratory has been interested in the problem of colorectal tumorigenesis and how we might take some of the lessons that we learn about this process and try to discover agents, therapeutic agents, that might help us in our fight against colorectal cancer.

[ Slide 01 ]   Model of Tumorigenesis

You're all probably familiar with this model of tumorigenesis that was originally proposed by Vogelstein and his colleagues where you have an initiating event and then you have a sequence of molecular targets or mutations that occur that eventually leads from normal epithelium to an invasive carcinoma. Our laboratory has been interested in the consequences - the functional consequences - of some of these early molecular events, these genetic mutations.

And this is just a list of some of the areas that we've studied and some of the observations that we've made. The alterations in expression of a number of genes that occur early in the process of tumorigenesis in intestinal epithelial cells. Today we'll focus on, really, two areas. One is cyclooxygenase-2 and then what we have discovered to be perhaps an interacting area of TGF-beta and cyclooxygenase-2.

[ Slide 02 ]   Role of COX-1 and COX-2 in Normal and Inflammed Tissue

So just a brief word about cyclooxygenases. As you all know, cyclooxygenases are enzymes that convert - and it's a rate-limiting step - that converts arachadonic acid to prostaglandins. A number of different prostaglandins are produced in this conversion and there are two forms: cyclooxygenase-1 and cyclooxygenase-2. COX-1, if you will, is constitutively expressed in many different tissues and appears to have more of housekeeping function but is necessary for maintaining the mucosal integrity in the gastrointestinal tract and that's why when you take nonsteroidal anti-inflammatory drugs a lot of people get ulceration of the GI tract. Cyclooxygenase-2 is an inducible form of cyclooxygenase and is perhaps more involved in the pathophysiology of a number of different processes and inflammation, including carcinogenesis, as we'll see.

[ Slide 03 ]   COX-2 Expression by Tumor Cells

So what advantage does the expression of COX-2 have in tumor progression to the intestinal epithelial cells, and perhaps in other systems as well? Well, expression of cyclooxygenase-2 appears either in the tumor cell or in the adjacent cells appears to decrease the apoptotic response in those cells. In other words, cells do not like to undergo malignant transformation and they'll frequently undergo apoptosis, a cell death pathway, if they experience a mutation that could lead to a transformation. Cyclooxygenase is a protective or survival factor in this event. Increased cyclooxygenase expression by tumor cells causes increased invasiveness of those cells and at least in in-vitro assays and also expression of cyclooxygenase either by the tumor cell or by surrounding cells leads to an increase in new blood vessel growth.

[ Slide 04 ]   Expression of COX-1 and COX-2 in RIE-Neo, RIE-Raf and RIE-Ras Cells

We made the observation that cells that are transformed, intestinal epithelial cells, in this case RIE cells, Rat Intestinal Epithelial cells, that grow as immortalized cells in culture but they're not tumorigenic. When we transform them with Ras, we note that there's an increase in the expression of cyclooxygenase-2 as compared to the control-transfected cells. And this is the expression of cyclooxygenase-1, which is not induced.

[ Slide 05 ]   LacSwitch System

We've developed a system whereby we could transfect a oncogenic Ras gene into cells using a lac-switch system. In the lac-switch system you transfect in two vectors. One encodes the lac repressor and in the absence of this lactose analog the repressor suppresses expression of this gene that is in the operator vector. When this IPTG or the lactose analog is present, it binds up the lac repressor and allows expression through the lac operon so there we have increased of expression of Ras in the presence of IPTG. So this gives us a nice reversible transformation of these intestinal epithelial cells.

[ Slide 06 ]   Induction of Ras and Morphological Transformation in RIE-iRas Cells

And this is just an example of what happens when we add IPTG to these RIE cells so here you see nice cuboidal flat monolayer epithelial cells. We've added IPTG and look at 24 and 72 hours later. So you see that these cells form foci, they start piling up on top of one another. They lose contact inhibition. Their proliferative rate goes up and these cells are tumorigenic. They're able to form colonies in soft agarose, all characteristics of cell transformation. You take away the IPTG and these cells normalize their morphology. So this is a reversible transformation process. So this just shows you the expression of the Ras protein over time with the addition of IPTG and this is 24, 48 and 72 hours and then this is the lane where we've taken away the IPTG for 72 hours so it's not expressed anymore.

[ Slide 07 ]   The Expression of Ras and COX-2 in RIE-iRas Cells

Now, as we induce Ras with IPTG in these cells, we also see a parallel induction of cyclooxygenase-2 that occurs shortly after the induction of Ras.

[ Slide 08 ]   TGF-Beta

Now the other thing that we have been interested in is TGF-beta. Now TGF-beta ordinarily is a tumor suppressor pathway. The TGF-beta ligand and receptor and signal transduction pathway probably has a normal tumor suppressor role. Unfortunately when cells become transformed, for example by Ras, then this tumor suppressor role is suppressed. However, there are parts of this signaling pathway that are probably preserved that are actually tumor promoting, serve a tumor promoting role. So we also know that Ras transformation can increase the expression of TGF-beta.

[ Slide 09 ]   Ras Induces the Expression of TGF-beta1 in RIE Cells

And we know that many tumors over-express TGF-beta. So this just an example in the RIE cells when you turn on Ras with IPTG, then you get an induction of TGF-beta I expression in these cells. So this is a northern blot and this is RNA.

[ Slide 10 ]   TGF-beta1 Induction of COX-2 Protein

And then this is a Western blot and this shows protein. We know that in the cells, the RIE cells that are not transformed by Ras that there is a transient increase in cyclooxygenase expression when they are exposed to TGF-beta. So these are nontransformed cells and when we treat with TGF-beta, we see this transient induction of cyclooxygenase-2.

[ Slide 11 ]   Both TGF-beta1 and COX are Overexpressed in AOM Adenomas

We also know that or we have observed that there's a correlation, appears to be a correlation of expression of TGF-beta I and cyclooxygenase-2 in tumors. And in this case these are azoxymethane induced adenomas and in some cases, invasive carcinomas from rats where we see increases in TGF-beta. T stands for tumor; M stands for normal mucosa and so we see matched pairs here. And so in the tumors there is increased expression of TGF-beta and there's also increased expression of cyclooxygenase-2. Cyclooxygenase-1 on the other hand remains fairly stable.

[ Slide 12 ]   Activating Ras Mutation Decreases Sensitivity to TGF-beta Growth Inhibition

So we've observed a potential correlation of TGF-beta and cyclooxygenase-2. We know that when you have transformation with Ras, that the cell's response to TGF-beta is altered. And this is just an example of this, this is a slide from John Barnard, one of our collaborators, that shows what happens when you treat normal RIE cells with TGF-beta. You have a profound inhibition of growth of those cells. In contrast, when you transform these cells with Ras, they are resistant, completely resistant to growth inhibitory effects of TGF-beta. These are control untreated cells and this is Ras cells treated with TGF-beta. They are refractory.

[ Slide 13 ]   Expression of Ras Reduces the Levels of TRI and TRII

So one of the reasons that, or at least one of the reasons we think that these cells become resistant to the growth inhibitory effects of TGF-beta is that induction of Ras down regulates the expression of the TGF-beta receptors. In this case this TR-1 is a Type 1 receptor and Type 2 receptor takes both of these receptors to cause signal transduction after ligand binding. And in this case we see that with IPTG treatment, by 24 hours there's a profound decrease in the expression of both of these receptors. And we also know from work in other laboratories that this is not the only thing that happens, that Ras interferes with the growth inhibitory signaling pathway of TGF-beta that Tien Ko is going to talk about some later.

[ Slide 14 ]   TGF-beta1 Exnhances Ras-induced Expression of COX-2

Despite the fact that there's inhibition of the signaling through the TGF-beta pathway and a down regulation of the receptors, when you add TGF-beta to cells that are transformed with IPTG, we see a super-induction of the cyclooxygenase-2. So here is TGF-beta alone without IPTG in these cells. You see a transient induction of cyclooxygenase-2. This is RNA here. With IPTG alone we see a progressive increase in cyclooxygenase-2. It's modest. With the combination, we see a profound increase in the message that is synergistic.

[ Slide 15 ]   TGF-beta Neutralizing Antibody Inhibits the Expression of COX-2 in RIE-iRas Cells

We've also asked the question whether the induction of TGF-beta by Ras has an autocrine effect and whether this autocrine TGF-beta feeds back on those cells and has a role in increasing the expression of cyclooxygenase-2 with just Ras transformation alone. And in this experiment we've used a TGF-beta neutralizing antibody and added it in combination with IPTG and this is over time. And we see that with IPTG alone there's the expected induction of, in this case, the cyclooxygenase-2 protein and with the TGF-beta neutralizing antibody present, this was inhibited. So this indicates to us that, yes, the Ras induction in those cells is turning on TGF-beta and then TGF-beta is having an autocrine effect.

[ Slide 16 ]   TGF-beta1 Stabilizes COX-2 mRNA

Now, we've gone on to look at the mechanism by which this induction of cyclooxygenase-2 and the profound increase of the message occurs in response to the combination of Ras and TGF-beta. We've looked at transcriptional regulation and the regulation does not appear to be primarily transcriptional. It does, however, appear to be stabilization of the message RNA for COX-2. In this case we've blocked transcription using a polymerase-2 inhibitor, DRB, and then we've followed decay of the messenger RNA. And what we see with no treatment it's a very short-lived message with a half-life between 10 and 20 minutes. In the presence of IPTG or Ras transformation there's some stabilization. TGF-beta alone causes a similar degree of stabilization of the message. The combination causes a profound stabilization of the message with a half-life of more than an hour.

[ Slide 17 ]   Schematic Representation of COX-2 Transcript Isoforms

And this has led us to do some experiments in looking at this region, this is the 3' untranslated region of the cyclooxygenase-2 message RNA. And we think that this is going to be the target of this regulation or whatever proteins are being regulated by the combination of Ras and TGF-beta that would bind to this region of the message RNA which is highly conserved among species. And that this provides us with the potential to identify these proteins that are regulated specifically in the process of transformation that might be new therapeutic targets.

That's it. Thank you.