Dr. Donald J. Tindall Video (Text Version)
Androgen Receptor Basic Science
Dr. Howard Soule
So on the—for every great medical breakthrough there is a great senior scientist upon whose shoulders we all stand. And our next person—speaker is such a person. Don Tindall from the Mayo Clinic who is a Professor of Biochemistry and—and Urology is really a treasure of a scientist who has led to essentially every major discovery around the androgen axis over very many years. I’m really pleased to have been able to contribute to the PCRP Integration Panel with Dr. Tindall, who was our—our last Chair of—of that organization making great contributions to prostate cancer research. So Don, we look forward to your scientific doctorate.
Donald J. Tindall, Ph.D.; Professor of Biochemistry and Urology, Mayo Clinic
You embarrass me Howard. I—I thought I was the warm-up act for Howard Scher. But what I would like to do is just give an overview of what we think we know at this point about the role of the androgen receptor in prostate cancer and—and hopefully lead into some thoughts about how we might target this for treatment. So this is what we think we know about the mechanism of androgen action in prostate cells in general and that is that testosterone enters the cell by passive diffusion, very important step then occurs, a conversion to more active androgen, dihydrotestosterone, by 5-alpha reductase enzymes; this steroid—both testosterone and dihydrotestosterone binds with the androgen receptor but DHT binds much tighter and therefore is more active.
Once the steroid binds to the receptor it causes conformational changes in the protein allowing release of chaperoned proteins that have stabilized the receptor. These are heat shock proteins. There’s dimerization of the receptor which can enter the nucleus and bind to elements on DNA called androgen response elements. And then there’s recruitment of co-activator proteins, other transcriptional machinery, which allows the—the DNA to loosen up and can now transcribe genes such as PSA and other genes that are responsible for growth differentiation of the cell.
So this shows the—the—the androgen receptor protein at the bottom left. At the top left is the chromosome X where the gene resides. There’s—there’s transcripts and then ultimately the protein, and we heard about the structure of the protein from Scott Dehm this morning. The ligand binding domain is on the right. There are two zinc fingers that allow the protein to bind to the DNA and then this transcription regulation domain on the left.
Now regarding the progression of prostate cancer, we can think in terms of it schematically as being a continuum from an androgen-dependent state to a castration recurrent prostate cancer or castration-resistant prostate cancer. And if we think in terms of tumor burden, in the presence of androgen there’s increased tumor burden. Following androgen ablation this burden is reduced, but with time the tumor will eventually recur. And it has this castration-resistant phenotype that we want to understand in order to develop therapeutics. So the—these cells are very smart. They’ve been able to hijack a number of mechanisms in order to survive in this castration environment. And what I’d like to do this morning is simply go through this list and give you some examples of the data that supports these conclusions.
And one is most of our observations have depended on models and we have some very nice animal models which can recapitulate this clinical condition. And one of these models was developed in Leland Chung’s lab when he was at MD Anderson and what they did was inject these prostate cancer cells into mice, allow them to grow for a period of time, and then castrate the mice. And the tumors underwent this regression that I just showed you but eventually relapsed into tumors that were castration resistant. They were able to grow a cell line out of these tumors called the C4 cells and then repeat the process to get a more aggressive cell line called the C42 cell line. These exhibit androgen-independent growth in culture but very importantly they still require the androgen receptor for their maintenance. And they express androgen-dependent genes such as PSA.
Now I think one of the best examples I know of is with showing or demonstrating this ability of the—these cells to respond in an androgen-independent manner is an experiment shown here where—sorry—where these androgen-sensitive cells are compared to the castration-resistant cells. And you can see here in the presence of androgen, PSA message is highly stimulated in the androgen-sensitive cells. But in the castration-resistant cells there’s a constitutive level of PSA message even in the total absence of androgens in the media. Nonetheless, these cells can respond to androgens when androgens are added as shown here where PSA message is highly increased.
And I’d like to highlight conceptually some work that Scott Dehm did when he was in my lab where Scott dissected the androgen receptor and asked which portion of the receptor was critical for this androgen-dependent activation and which portion of the protein was critical for this castration-resistant phenotype. And the conclusion from Scott’s data is that this AF2 function which is the—this transcription activating domain on the right hand side of the protein is very important for the androgen-dependent activity. But it becomes less and less important as you go to this castration-resistant phenotype. On the other hand, this Tau5 region shown in red is very—plays little role in androgen-dependent activation but becomes more and more important for this castration-resistant phenotype. And so, the take-home message that I’m sending to the pharmaceutical industry is that we need to start focusing on this N-terminal domain for more effective drugs.
You saw this slide this morning. I think Jim Mohler’s observation that there is synthesis of androgens by the tumors themselves in my opinion was a paradigm shift in our thinking. Obviously Jack Geller had shown this a number of years before, but to be honest with you it was buried in a paper and I’m not sure even Jack fully recognized this at the time. But I know—I’ve known Jim for a long time and it shows what a bulldog can do with lots of tenacity and so I—I think this was a very important observation. And in fact, this reawakening of the steroid—steroidogenic pathway is creating drugs that are being tested in the clinic, and we’ve all heard about Abiraterone this morning. Co-activators are very important for transactivating the androgen receptor, and our problem with co-activators is we just got too darned many. There’s over 160 that have been characterized; I think it may be up 200 now—but this slide shows some of these co-activators in the regions on the androgen receptor with which they interact. And this slide shows one table from Nancy Weigel’s laboratory where they were looking at correlation of one of these co-activators, SRC-1, with different clinical pathological variables in tumors. And you can see that this was highly correlated with clinical stage and—and also extra-capsular extension and a number of co-activators have now been shown to be over—highly expressed in castration-resistant tumors.
What about these mutations? Some of these mutations in the androgen receptor itself exhibit a gain of function so they can bind to other steroids such as coming from the adrenal. Some of them can even now bind to anti-androgens. So the treatment that was being used for this particular tumor can now be driven by these anti-androgens. This is a—a relatively small number of—of percentage-wise but nonetheless I think it shows the power of these cells to morph.
Charles Sawyers has demonstrated a number of years ago the importance of amplification of the androgen receptor. These are data from xenografts and in every case where androgens were withdrawn or mice were castrated, there was an increase in expression of the androgen receptor. And this leads to a very interesting phenotype and that is that these cells now are not very responsive to anti-androgens, and you can see here the presence of bicalutamide which can inhibit PSA in these androgen-dependent cells—do not respond to bicalutamide in the castration-resistant state. Scott mentioned variants; this just shows these smaller variants with respect to the wild-type protein and conceptually we can think of these variants as truncated androgen receptor proteins that lack the controlling element of the androgen, but nonetheless something else is driving the expression and activity of these variants and I would submit that this N-terminal domain again is critical for that activity.
A number of laboratories have published on these variants. This shows Steve Plymate’s lab data where he shows nuclear localization in the absence of androgen and Yun Qiu’s laboratory showing a high expression of these variants in castration-resistant cells. There are other mechanisms that cross-talk with the androgen receptor. One is the TNF-alpha pathway. This shows TRADD-expression which is an integral part of that—that is down-regulated as tumors progress, also down-regulated as tumors progress to a castration-resistant state, and this program—this gene is regulated by androgens and androgen activity is regulated by TNF-alpha.
So there are a number of—of mechanisms by which these cells respond to the removal of androgen. I would submit to Dr. Sharp that this is an outstanding target to go after with this siRNA therapy. Thank you very much.