Selwyn M. Vickers, MD: A New Molecular Pathway in Pancreatic Cancer and Its Potential Application in Gene Therapy

I've been very fortunate at the University of Alabama to the support of Gil Deithelm and we're excited about our new chairman, Dr. Bland, who's coming in the fall.

[ Slide 01 ]   Problem Solving Flowsheet

Much of my entry to the lab is really best described on this next slide. My attitude towards most projects ... was "did it work?" And as I grew in the lab, I sort of ... watching the lives of many of the people here in this audience my attitude changed, not just to be concerned about did it work, but how it worked. So for many of the interns and residents I know this is your attitude on the ward, but in the laboratory it actually needs to be a little bit different, not just does it work, but in fact is there some concern about how it works and why it works. So credit to many of the investigators who have presented who I have had a chance to follow and watch their careers and observe Chuck from a distance and my attitude's changed now. I really want to know how things work and why they work.

[ Slide 02 ]   Surviving Cancer

Secondly, my time at Hopkins certainly gave me an interest in adenocarcinoma of the pancreas. My mentor there, Dr. Cameron, gave me a love for studying and treating this disease. But what I quickly learned since I've been at Alabama, there's been a great progression in how we handle diseases of the major three killers, breast, prostate and colon, but our impact in pancreatic cancer has been minimal.

[ Slide 03 ]   Stage of Presentation

And it will remain minimal if we depend on surgery alone. The major problem with our surgical treatment, although we've made tremendous progress in our perioperative morbidity, only 10 percent of patients present with a surgically curable disease. The rest, as compared to colon, breast and prostate which present with a reasonable amount of local disease that are amenable for surgical cure, only a small amount of pancreatic adenocarcinomas come to us with an ability to be cured. So if we're really going to have an impact here, we've got to look at other ways to try to treat this disease.

[ Slide 04 ]   Molecular Oncogenesis of Pancreatic Cancer

There's been an explosion, as well as our excellence in operative management of understanding the molecular oncogenesis of this disease not only from the understanding of K-ras and the influence, of the latest tumor suppressors, DPC4 from Scott Kern's lab at Hopkins, this has spurred my interest in fibroblast growth factor and the growth factor receptors.

[ Slide 05 ]   FGF in Pancreatic Cancer

In order to look further at how growth factors and these receptors may effect pancreatic cancer growth, we realized that the prototypic growth factors, both acidic and basic, neither of which have a signal peptide are overexpressed in about 60 percent of these tumors. The receptor, which probably plays an even more critical role, is actually overexpressed in about 90 percent of adenocarcinomas.

[ Slide 06 ]   ARIP Cell Line

In order to look at this further, we took probably the closest thing to a normal line, is the ARIP rat cell line. This is an immortalized cell line, but doesn't form tumors. It certainly can be induced through certain means, but in fact its ability to form tumors is severly limited.

[ Slide 07 ]   FGF-1 Retroviral Vectors - 1

In order to study these growth factors further, this simple cartoon shows a paradigm that we used employing a retroviral vector. This module is a chimeric vector containing the human FGF gene. It's powered by the SV40 promoter but it has the signal peptide for FGF4. So it allows us not only to allow the gene and protein to be overproduced, but also to have it secreted which is necessary for acidic fibroblast growth factor to have any effect on the cell.

[ Slide 08 ]   FGF-1 Retroviral Vectors - 2

These cells were then transfected and the housekeeping gene GAPDH was evaluated in the transfected cells. They were both the one that had the betagalactosidase marker and the FGF human gene. You can see the b-Gal in both of those cells but only the transformed cell with the human gene being expressed in this rTPCR.

We looked at protein. We asked the question "Was protein both actually made in the cell cytosol as well as was it being secreted in the conditioned media?" And 3 and 6 are ... is the conditioned media in 3 and the cell lysate in 6. 5 and 2 are from the beta-gal cells which had no FGF present and these two here 7 and 1 are actually markers for human FGF1.

[ Slide 09 ]   Growth Curve

As you know, the cells grow easily in conditioned media but in this case we grew these cells grew in half percent media and they begin to exhibit a transformed phenotype easily growing in an exponential way in reduced serum.

[ Slide 10 ]   Morphological Phenotype

And over time, these cells actually change their morphological phenotype. They grew in an anchorage independent manner and actually changed to a fusiform pattern, as compared to the beta-Gal cells that maintained their simple cobblestone appearance.

[ Slide 11 ]   Oxidant Stress (-ONOO)

Because of a close association with one of our collaborators, Joe Beckman, as well as physical association with our laboratories, we've used peroxynitrite, which you heard addressed by many investigators, as our means of producing apoptosis. So that these cells were evaluated for their sensitivity to peroxynitrite as a means of inducing apoptosis.

[ Slide 12 ]   -ONOO Treatment of Pancreatic Cell Lines

And when we evaluated the transformed cell lines, with the beta-Gal cell line, they were quite sensitive to 150-micromolar peroxynitrite, on the order of 90 percent of the cells were killed. These were the reverse order addition controls and no treatment vehicle alone.

But as to no surprise, those cells that now had the overexpression of the acidic fibroblast growth factor and its secretion, were quite resistant to peroxynitrite even at higher levels when ... used to treat them. The cells were stained with pyridium iodide and fluorescein diacetate to look for dead and alive cells as well as this process was also reversed by ATA Aurintricarboxylic acid which blocks the DNA disruption as well as by TUNEL-staining.

[ Slide 13 ]   FGF-1 Protects Against ONOO Induced Death

To really ask the question "Did FGF play a role?" We then added exogenous FGF1, 20 nanograms, to the cells that were nontransformed. And we were able to mimic the protection when we added peroxynitrite so at zero hours there was similar death, at two hours there was similar death, but at eight hours, near the time of cell cycle, we could induce the same protection we saw in the transformed cells with the retroviral vector by adding exogenous FGF, which extended out as we continued the addition of this FGF.

[ Slide 14 ]   Tumor Study

These cells also then were put in as xenografts and in general the ARIP line will not form tumors easily either in their syngeneic host or in the xenograft model. But when we placed these cells at 2 times 10 to the 7th in nude mouse, we were able to get ... 5 out of 6 of the mice to grow tumors. When we harvested these tumors we obviously stained them for FGF, which was positive as seen here, but we also stained for nitrotyrosine and nitric oxide synthase II. And both of these tumors stained ... all of those tumors stained positive for nitrotyrosine and for nitric oxide Type 2.

We subsequently actually went on to look at a battery of human tumors which we recently published, showing the association of acidic fibroblast growth factor and positive staining for nitric oxide Type 2 and nitrotyrosine.

[ Slide 15 ]   NO + O2 = ONOO

Around this time... this slide simply shows what we thought may be a process, and as mentioned by a number of investigators, peroxynitrite is sort of the product of nitric oxide and superoxide. And under pathologic conditions, NO is the one compound that has the ability to out-compete superoxide dismutase for this compound. Obviously tyrosine can be modified and plays a critical role in cellular growth through phosphorylation but in this scenario we believe that nitration at the meta position plays a role maybe in inactivation of proteins in most settings.

[ Slide 16 ]   Have An Array of Proteins Been Nitrated?

We then took some of these cells, these tumors that we had grown and actually looked to see if there was an array of proteins that may have been nitrated. And we took the cells that had been transformed and asked the question by probing on a Western blot with a polyclonal anti-nitrotyrosine antibody, there are multiple proteins that were nitrated, but particularly at 60 kilo Daltons. We could increase this nitration of tyrosine by adding peroxynitrite or treating. When we asked the same question of the nontransformed cells without treatment with peroxynitrite, we had very little, if any, nitration of proteins. But we were intrigued by these proteins that we saw consistently at this level.

[ Slide 17 ]   Overexpression and Activation of the Tryosine Kinase SRC in Human Pancreatic Carcinoma

And about the time we did this, here's a paper published from France that described an overexpression and activation of a tyrosine Src kinase in human pancreatic adenocarcinoma. And in fact, in all the tumors they looked at, Src kinase was overexpressed. They didn't go into understanding why it was overexpressed or why its activity was increased, but we had some ideas why that might be the case.

[ Slide 18 ]   Phosphorylation and Nitration of Src

Subsequently, we took those same protein lysates you saw, IP'd them with either anti-phosphotyrosine or ... polyclonal anti-nitrotyrosine and probed with a Src II antibody that binds only the activated Src. And these are two separate lanes, really duplicates both, in the FGF-1 transformed cells with the FGF. And we found that Src both was nitrated and was phosphorylated in the cells, which is a bit of a sort of conundrum, because in most cases nitration tends to inactivate proteins, but in this scenario there was phosphorylation of Src and nitration of it.

[ Slide 19 ]   Phosphotyrosine IP/Probe with Anti-Src

We then actually looked at some human cancers and found that looking at normal tissues and this being the obviously the heavy and light chains of the antibodies from the immunoprecipitate but in chronic pancreatitis and in cancer, and then this is the antibody alone, we found only actually in the cancers, and a number of human cancers, that Src was actually phosphorylated...

[ Slide 20 ]   Nitrotyrosine IP/Probe with Anti-Src

...and in fact Src was also nitrated only in the human cancers as compared to normal pancreas ... from a transplant... from resections of normal pancreas and tumors as well as in chronic pancreatitis.

[ Slide 21 ]   Pancreatic Cancer

So this interest in the Src kinase, there are nine family members, these four are probably actually are found in tumors. What's interesting is that, as you know, Src kinases have an affinity for tyrosine activated receptors, particularly FGFR1 and probably the major role is the Src SH2 domain which interacts with a number of effector proteins, particularly ras, cortactin, PI3 kinase, which all probably play a role through the MAP kinase pathway. We believe that in fact critical to this and nitration relates to this SH2 domain.

[ Slide 22 ]   Src Activity Increase in Cancers

And what's most important, although this picture is sort of faded and it's a poor reproduction, when we asked in fact was Src kinase activity increased, when we used a 32p enolase substrate, we found that actually only in the cancers was there an increase in Src activity and this also reproduce this when we added peroxynitrite to human cells.

[ Slide 23 ]   ONOO - Upregulates Src Activity

You can see again with this substrate when we looked at the enolase, and this is better reproduced, that when we asked was Src kinase increased, in fact it was in the cancers as well as when we added peroxynitrite. We also asked, "Was this Src associated with any other proteins?" And we immuno preciptated with the anti-Src antibody and probed then with cortactin, this Src activated and these tumors actually coprecipitated with cortactin showing that in this tumor at least, when activated, it co-localizes with cortactin, maybe indicating one of the paths which it possibly uses to promote oncogenesis.

[ Slide 24 ]   Src Inactivity

What we actually [think] may take place is that Src, in its inactive form, the terminal phosphatase binds to the SH2 domain and this keeps it inactive. We believe however that phosphatases obviously take this phosphate off, but that probably nitration could mimic actually phosphorylation but if this does occur it prevents it from binding to its SH2 domain. As far as we know this process of nitration is sort of a permanent effect and is not easily removed and this could constitutively activate Src. Once Src is activated, the reason that you see phosphorylation is that it undergoes autophosphorylation at multiple sites in the SH3 and SH2 domains so that you possibly would get nitration and evidence of phosphorylation as it interacts with other effector proteins. Obviously in this form when it stays phosphorylated it's active, but nitration may also provide a means of why it gets constitutively activate in this setting of a pancreatic cancer.

[ Slide 25 ]   Big Picture

This is what we think possibly is the big picture. We've seen certainly evidence that nitric oxide Type 2 is overexpressed in the human pancreatic adenocarcinomas and in our cell model. We've also demonstrated in our lab and recently published over the last two years, that peroxynitrite induces release of FGF1 from these cells. There are studies that I won't talk about that show that FGFR1 beta is critical for the signal and FGFR1 alpha probably localizes in a perinuclear form but bypasses all the signal transduction pathways in this tumor, so that FGFR1 beta which when phosphorylated through the binding of either FGF1 or FGF2 binds with cSrc, which then activates it to allow it to interact with either through focal adhesion kinase or through the multiple other effector proteins ending up with a cellular proliferation. Peroxynitrite may play a significant role in that we see nitration of Src in all of these cells which could constitutively activate Src, allowing it to go on to this malignant transformation.

[ Slide 26 ]   Experimental Concept

My title involved gene therapy and the applications from what we've learned is in a simple manner to apply some of our understanding of FGF biology to gene therapy principles. And this is some of the work we've carried out with our gene therapy center at UAB. Our paradigm has been fairly straightforward [in] that we've used an ad-CMV cytosine deaminase prodrug therapy using 5-FC as the prodrug. The reason we like this for pancreatic cancer is because it uses and benefits from the radiation sensitizing effect of 5-FU. So that in general we've used this as a means to treat both in vivo and in vitro setting for our proof of principal.

[ Slide 27 ]   Redirected CD Enzymatic Activity in Pancreatic Carcinoma Cells

Where we've applied some of our basic science understanding is that we've actually, with the collaboration with the company called SelectiveGenetics, we have used the FGF ligand and an antibody to the adenoviral knob, to redirect the adenoviral cytosine deaminase gene to pancreatic cancer cells. And in that that redirection we go through the ... FGF receptor rather than the CAR receptor and in doing that we've seen a significant increase in our ability to transfect and transduce these cells. And we've done this at a much lower MOI. At MOIs of 1 when we redirect we get a significant conversion of 5-FC to 5-FU as compared to when we use the non-redirected adenovirus. So starting at lower MOIs of 1 and 2 we get almost a 3 to 5 fold increase in transfection by going through the FGF receptor versus going through the CAR receptor.

[ Slide 28 ]   In Vivo Efficacy for Fab-FGF Redirection of AdCMVCD with XRT

That alone has even shown us that in an in vivo model when we use the standard AdCD versus the Ad FGF, we can get a significant improvement in tumor kill at a much lower MOI, which may predict a better means for trying to do our clinical trial because we can use a much smaller MOI. And that probably is the best way I can show that we've applied some of our basic science understanding to gene therapy.

[ Slide 29 ]   Acknowledgments

There are number of people who I want to thank, particularly Dr. Deithelm who is now retiring, but a number of collaborators in the lab particularly Tony Thompson and LeAnn McMillan Crow.

[ Slide 30 ]   The California Coast

And this slide is sort of to remind Dr. Brunicardi of where he ... at least to make him homesick ... California coast.

Thank you.