| SLIDES
& TRANSCRIPTS
Saturday,
December 6, 2003
Molecular Therapeutics: Hormone-refractory Prostate Cancer
George Bartsch, M.D. |
| Slide
1: |
 Mr.
Chairman, members and guests. I would like to cite my main co-authors,
Dr. Zoran Culig, Iris Eder and Helmut Klocker. Three of them are
Ph.Ds and are part of our molecular biology lab.
I would like to focus on this so-called common refractive prostate cancer, and show you two applications.
TOP
|
| Slide
2: |
|
 Dr. Huggins refers to apoptosis and the hormone sensitive prostate carcinoma epithelium.
TOP |
| Slide
3: |
|
 About ten years ago, it was dogma that our patients are dying of so-called hormone insensitive tumor cells. A patient is starting with a lot of androgen receptor positive cells, has some androgen receptor negative cells, and then by cloning out them he is going to androgen receptor negative cells.
TOP |
| Slide
4: |
|  At a time where it was not attractive at all, we went into the question of androgen receptor expression, structure and function. I would like to start with expression.
TOP |
| Slide
5: |
|
 It is quite clear that if you go to patients with lymph node metastasis in radical prostatectomy, they express the androgen receptors. But what we found to our surprise is that out of 22 patients, we found the androgen receptor. So the androgen receptor expression in all stages of prostate cancer irrespective of sensitivity of hormone ablation.
TOP |
| Slide
6: |
|  We
looked at a model to look at long term androgen ablation. Therefore
we created the model called the LNCaP lung ablated cells. These
are cells who have grown up in absence of steroids. They are grown
in charcoal stripped serum for several months in about 60 to 80
passages.
What you can
see on the slide is that those cells -- and you see the cells in
the blue line, that they are hyper sensitive to small amounts of
androgens compared to the parental LNCaP cells. What is even more
interesting, those blue line cells, these LNCaP ablated cells, they
carry a three- to fourfold increase of androgen receptors.
TOP |
| Slide
7: |
| 
Then we went
to in vivo studies. You see the parental LNCaP cells and you see
the lung ablated LNCaP cells. If you go to the parental LNCaPcells,
you see this difference in between castration and in between the
control. Also, glutamide is not antagonistic, but it is agonistic.
When you look
at the LNCaP lung ablated cells, what is interesting is that the
most effect on proliferation has castration and pico glutamide,
followed by pico glutamide and then followed by castration.
TOP |
| Slide
8: |
|  Going
now to the structure, this is the androgen receptor with the three
domains, the trans-activation domain, the double-sink finger(?)
and the DNA binding domain and the ligand binding domain. This is
the first mutation we found in '92 in the hormone refractory prostate
carcinoma cell. In the patient with the mutation there was adenine
instead of guanine and there was methionine and valine(?).
We have several tissues of these patients, so we could show that this is not germ line, but this is somatic. If you go to testicular feminization and you have limitation of the androgen receptor, you have a loss of function. But here in prostate carcinoma – and Dr. Chulig has proved this in 15 cell lines all over the world – we get a gain function, and we get activation. We can activate then the androgen receptor by progesterone, by adrogen dione(?) androgen metabolites, even by hydrocyglutamate and even by estradiol. TOP |
| Slide
9: |
|  So the next question we addressed in our group was, do we need at all the androgen receptor in the hormone refractive stage to be activated. We looked at growth factors like IGF, like EGF, like KGF. We looked at cytokines like interleukin-6 and regulators like deactivators like SATK(?) And LHRH.
I want to give you two examples, growth factors IGF-1 and interleukin-6. We also looked to the signaling cascade in the cytokine like injecting state(?).
TOP |
| Slide
10: |
|  I show you this in a supernatant PSA measurement.
This would be
your control by just giving R-18 and 18-1. These are LNCaP cells
that just get IGF-1, so you don't need the R androgens; you can
just activate your androgen receptor. This is wild type, this is
not mutant -- by IGF-1, so there is a cross talk to the androgen
receptor.
TOP |
| Slide
11: |
|  The
same thing happens if you take IL-6. You also get activation of
the androgen receptor. We have found this also in various types
of experiments using the CaP assay. Your question may be, why they
look to IL-6? We found that IL-6 is elevated in serum of patients
with advanced disease, and we found that in the autocrine loop of
those patients there is an expression of IL-6 and the IL-6 receptor.
TOP |
| Slide
12: |
|  The
next thing is, we went to in vivo, and we created the so-called
IL-6 secreting LNCaP cells. That means those cells adapted for a
long time to exposure to IL-6, and then they acquire the ability
to produce IL-6.
What you can see in the xenograph model is that those cells producing IL-6, they have a growth advantage, and in terms of molecular biology, they are going to express the retinal(?) blastoma protein as well as MAP kinases(?). TOP |
| Slide
13: |
|  So the dogma ends like this. At the very beginning, we have apoptosis sensitive prostate epithelium in the untreated patients, but in the hormone refractory prostate cancer patients, we still have the androgen receptor which is hypo reactive. It is hypo reactive because of mutations, because of elevated levels, and because of the cross talk of the ligand independent activations by growth factors and by cytokines.
TOP |
| Slide
14: |
| 
Can you interfere with this? I want to show you two types of experiments we did. First of all, the inhibition of IL-6. This is the work of Dr. Chulig.
TOP |
| Slide
15: |
|  You see this is again going to the xenograph model. This is one medication of an IL-6 antibody to this xenograph model.
TOP |
| Slide
16: |
|
The next thing is, if this is true, why should we not just eliminate the androgen receptor?
TOP |
|
Slide 17: |
|  What we did is, we just constructed an antisense polynucleotide, 15 paces, against the polyglycomine region of the androgen receptor. As you see by the western blot analysis, there is a down regulation of the androgen receptor. You see the untreated control, you see the mis-sense, the mismatch, and you see this after 24 hours.
TOP |
| Slide
18: |
|  We
went into in vitro. You see the control, LNCaP
cells, control, the mis-sense, the mismatch. You see P-colutamide
and you see the oligonucleotide. Then we went to in vivo studies.
This is to LNCaP
xenographs. You see the control, the mis-sense, and again you see
the oligonucleotide. So this is working. At the time we were doing
this, we are bringing this type of oligonucleotide and micro bubbles,
and trying to pop out those micro bubbles using sonography at the
lesions where we think is prostate carcinoma.
TOP |
| Slide
19: |
|
 You see here immunohistochemistry, you see the vesicular(?) control, you see the mis-sense, you see the castration, and you see the antisense oligo.
TOP |
| Slide
20: |
 So in summary, we are saying that the androgen receptor is present and active also in the hormone refractive prostate cancer. At the time being, we have two groups targeting this type of thinking, and this is the inhibition of IL-6 with an IL-6 antibody, and the other thing is the elimination of the androgen receptor using the antisense technique.
Thank you.
TOP
|
