Reprinted from PCRI Insights February 2003 v 6.1
By Charles E. (Snuffy) Myers, M.D., Founder and Medical Director, The American Institute for Diseases of the Prostate, Charlottesville, VA, and Member of the PCRI Medical Advisory Board
|Part 1 of this article Part 3 of this article|
Treatment Options for Androgen Hypersensitization: Antiandrogens
In Part 1 of this paper, I reviewed how tumor cells can develop the capacity to grow well at low concentrations of androgen by increasing the androgen receptor level, increasing the sensitivity of the receptor through phosphorylation, or enhancing its action by altering the amount of co-activating proteins. In these studies, the anti-androgen Casodex® (bicalutamide) was commonly added. This drug consistently blocks the ability of androgen to stimulate prostate cancer cell growth, despite these enhancements in androgen receptor function.
The usual dose of Casodex® is 50 mg daily. This leads to sustained blood levels of 8-10 micrograms per milliliter of blood. Casodex® can be safely administered at doses as high as 450 mg a day, but doses over 200 mg are not absorbed well. Thus, the maximal sustained blood level is 30-35 micrograms per milliliter. Most of the laboratory studies I have cited used the equivalent of less than 10 micrograms of Casodex® per milliliter of blood. I found only one that used concentrations in excess of 35 micrograms per milliliter. Thus, Casodex® blood levels in patients are typically well within the range where this antiandrogen should block the development of most of the known mechanisms for enhancing androgen receptor function.
Why is it common that large randomized controlled trials fail to show a survival advantage to adding an anti-androgen to medical or surgical castration? In the case of Eulexin® (flutamide), the drug appears to induce mutations in the androgen receptor discussed in Part 1 of this paper. These mutant receptors then react to flutamide and its major metabolite as though these were testosterone. The result is that flutamide administration now fuels the growth of the cancer.
In one study of hormone-resistant bone marrow metastases in patients on complete androgen blockade with flutamide, five out of sixteen, or more than 30%, exhibited androgen receptor mutants stimulated by flutamide. In contrast, only one mutant receptor was found among seventeen patients who failed on surgical castration alone. The incidence of androgen receptor mutants in this study nicely match the common 20-30% response to flutamide-withdrawal in patients who progress on flutamide-containing programs of complete androgen blockade. The flutamide-withdrawal response is typically seen in patients who have been on flutamide for more than two years and is uncommon in those on therapy for less than one year. It seems very likely that the gradual appearance of androgen receptor mutations seriously limits the effectiveness of Eulexin® in clinical trials where hormonal therapy is administered continuously.
Casodex® appears to be much less likely to foster the emergence of androgen receptor mutants. Also, among the androgen receptor mutants that emerge after prolonged exposure to flutamide, most do not appear to have their growth stimulated by Casodex®. As a result, Casodex® is often effective in the treatment of men who have progressed on Eulexin®. Withdrawal of Casodex® appears to be less likely to induce a tumor response than withdrawal of Eulexin®, presumably because of the lower likelihood of androgen receptor mutants.
Recently, investigators at Johns Hopkins University (Laufer, et al) were only able to gather a series of five patients who exhibited rapid cancer progression associated with Casodex® administration. Four out of these five patients responded when Casodex® was withdrawn.
If Casodex® is unlikely to cause androgen receptor mutations, what happened in these men at Johns Hopkins that made their cancers grow when Casodex® was used? In laboratory experiments, culture of human prostate cancer cells in the absence of androgen for prolonged time periods led to the development of cells whose growth is stimulated by the addition of Casodex®. Where Eulexin® or its active metabolites were tested, they also stimulated the growth of these cells. These cell lines did not show androgen receptor mutants or an increased amount of androgen receptors. Cells that contain increased amounts of androgen receptor coactivators, especially ARA70, increase their growth when exposed to Casodex® and Eulexin®. Paradoxically, addition of testosterone or dihydrotestosterone suppresses the growth of some of these cell lines that grow when Casodex® is added.
This combination of laboratory and clinical observations suggest that prolonged complete androgen blockade leads to the emergence of tumor cells that will grow faster in the presence of anti-androgens and might be suppressed by normal concentrations of testosterone. These findings provide a rationale for intermittent hormonal therapy where androgen withdrawal and exposure to anti-androgens are limited to a year or less. Also, the fact that normal to high testosterone levels suppressed the growth of these prostate cancer cells provides a rationale for the use of testosterone in selected patients with hormone resistant disease and this concept is currently in clinical testing.
X. Miyamoto, et al. “Promotion of agonist activity of antiandrogen by the androgen receptor coactivator, ARA70, in human prostate cancer DU145 cells” Proceedings National Academy of Science USA95: 7379, 1998.
M.E. Taplin, et al. “Selection for androgen receptor mutations in prostate cancers treated with androgen antagonist” Cancer Research 59: 2511, 1999.
M. Laufer, et al. “Rapid disease progression after the administration of bicalutamide in patients with metastatic prostate cancer” Urology 54: 745, 1999.
C. Wang, et al. “Isolation and characterization of the androgen receptor mutants with divergent transcriptional activity in response to hydroxyflutamide” Endocrine 12: 69, 2000.
A. Hobisch, et al. “Antagonist/agonist balance of the nonsteroidal antiandrogen bicalutamide (Casodex®) in a new prostate cancer model” Urology International 65: 73, 2000.
Z. Culig, et al. “Androgen receptor gene mutations in prostate cancer. Implications for disease progression and therapy” Drugs Aging 10: 50, 1997. (continued) Androgen Resistance continued from page 1
L. Denis, et al. “Pharmacodynamics and pharmacokinetics of bicalutamide: defining an active dosing regimen” Urology 47: 26, 1996.
E.J. Dole, et al. “Nilutamide: an antiandrogen for the treatment of prostate cancer” Annals Pharmacotherapy 31: 65, 1997.
G.R. Blackledge “High-dose bicalutamide (Casodex®) monotherapy for the treatment of prostate cancer” Urology 47: 44, 1996.
H.I. Scher, et al. “Bicalutamide for advanced prostate cancer: the natural versus treated history of disease” Journal Clinical Oncology 15: 2928, 1997.
When the androgens are removed, both normal and cancerous prostate cells die. For normal prostate cells, death is fairly rapid and the gland can shrink to 90% of its original size within one month of surgical castration. Hormone-sensitive prostate cancer cells, however, die more slowly; their deaths are prolonged for over nine months.
In contrast, there are patients whose cancers stop growing when the androgen level is reduced by hormonal therapy, but whose tumor cells do not die. Consequently, tumor masses in the prostate gland, lymph nodes, and bone don’t disappear or even shrink. These simple observations illustrate the apparent major changes that occur in the speed and completeness of cell death in normal prostate cells when compared to metastatic prostate cancer cells. Recently, major advances have broadened our understanding of how the suicide program is altered when prostate cancer cells develop the ability to survive hormonal therapy.
There is growing evidence that the cytokine, IGF-1, plays a major role in promoting the survival of prostate cancer cells. IGF-1 can stimulate growth in these prostate cells, but, more importantly, it sends a powerful signal to the prostate cells, informing them not to activate their suicide program. Current evidence supports the theory that IGF-1 triggers one of the most important survival signals for prostate cells — second only to androgen. How does IGF-1 stimulate the survival of prostate cancer cells? When IGF-1 binds to its receptor on the surface of the prostate cells, it triggers changes in protein phosphorylation that lead to the activation of a protein called Akt. (Editor’s note: the naming convention for signaling proteins allow nonsense three letter names.
Thus, Akt and bcl-2 are not abbreviations, but full names for these proteins.) In turn, Akt inhibits prostate cancer cell suicide by blocking many of the key components of the suicide “machinery.”
Perhaps the best evidence supporting the importance of IGF-1 comes from work on prostate cancer cells that survive well in the absence of IGF-1. These cells often show changes that ensure activation of Akt when IGF-1 is absent. The most important mutation involves a protein called PTEN. Under normal conditions, if IGF-1 levels are not optimal PTEN deactivates Akt and renders prostate cancer cells more susceptible to suicide. Genetic changes in prostate cancer cells can lead to a loss of PTEN. Without PTEN, it takes much less IGF-1 to trigger maximal activation of Akt and ensure cancer cell survival.
The human prostate cancer cell line, LNCaP, has proved very useful in deciphering the role of Akt. Since they lack any active PTEN, LNCaP cells have fully activated Akt. These cells will survive in the absence of androgen and will grow slowly under these conditions. In the laboratory, when drugs inhibit Akt, these cells will live if androgen is present but die if it is removed. While LNCaP cells survive if either Akt is activated or androgen is present, growth is faster in the presence of both circumstances. Additionally, activation of Akt makes prostate cancer cells less sensitive to chemotherapy. Clearly, these experiments illustrate the critical role that Akt activation plays in prostate cancer biology.
Evidence from patient samples suggests that IGF-1 and Akt activation play important roles at various points in the development of prostate cancer. Most of the well-designed studies show that the higher the IGF-1 blood level, the greater the risk of developing prostate cancer. Studies performed on prostate biopsies and radical prostatectomy specimens reveal that PTEN is absent in 10-20% of prostate cancers. Since this results in cancers where Akt is chronically active, these patients would not be expected to do well. In fact, PTEN absence generally occurs in prostate cancers with Gleason Grade of 7 or greater, confirming the association between loss of PTEN and high-risk cancer. PTEN was also more likely to be absent in locally advanced cancer (involving both sides of the gland or invading into surrounding tissues) than in cancers that were smaller and limited to one area of the prostate gland.
The absence of PTEN can speed the growth of prostate cancer. It can also allow the cancer to survive hormonal therapy as well as chemotherapy, equipping the cells to survive further treatment and eventually kill patients. One recent study analyzed fifty metastatic prostate cancer lesions in nineteen fatal PC cases. In 80% of these patients, PTEN was absent from at least one metastatic lesion.
A number of approaches have been taken to solve the problem presented by the IGF-1 survival pathway. Most of the IGF-1 in the blood is produced in response to growth hormone. The drug Sandostatin® blocks the release of growth hormones and causes a drop in IGF-1 levels. In laboratory models, Sandostatin® (as well as other drugs that block growth hormone action) shows impressive activity against human PC cell lines. Human clinical trials of these drugs yield a mixed picture: some investigators report promising results and others see no activity at all. I think that these clinical differences may result from patient characteristics. For example, heavily pretreated patients may well have fully active Akt independent of IGF-1 levels, and they would not be expected to have a significant response to drugs designed to suppress growth hormone and IGF-1 production. The few trials that have used the growth-hormone antagonists as part of initial hormonal therapy report antitumor activity that warrants further investigation in a patient population whose tumors are most likely to still be responsive to circulating IGF-1.
A more promising approach is to identify drugs that work directly on Akt or on PI3 kinase, the protein that activates Akt. The drugs wortmannin and LY294002 are widely used in the laboratory to block activation of Akt by inhibiting PI3 kinase. These drugs are very effective in triggering the suicide program in prostate cancer cells. I am aware of several major pharmaceutical firms who are developing Akt inhibitors with the hope of finding a useful anticancer agent. One drug already on the market, Celebrex®, has been reported to block Akt function and cause the death of human prostate cancer cell lines. Celebrex® is widely used (and is FDA-approved) for treating arthritis; it is also much less toxic than most anticancer agents.
Rapamycin doesn’t alter Akt activation but does block one of the survival pathways under Akt control. Charles Sawyers, from University of California, Los Angeles, has shown that rapamycin is able to kill cells lacking PTEN at concentrations that appear to be well tolerated. Rapamycin is currently available for clinical use and is used as an immunosuppressive drug in organ transplant patients. Continued
T. Nickerson, et al. “In Vivo Progression of LAPC-9 and LNCaP Prostate Cancer Models to Androgen Independence is Associated with Increased Expression of IGF-1 and IGF-1 Receptor” Cancer Research 61: 6276, 2001.
A. W. Hsing, et al. “Prostate Cancer Risk and Serum Levels of Insulin and Leptin: a population- based study” Journal National Cancer Institute 93: 783, 2001.
A, Di Cristofano, et al. “PTEN and p27KIP1 Cooperate in Prostate Cancer Tumor Suppression in the Mouse” Nature Genetics 27: 222, 2001.
A.P. Chokkalingam, et al. “Insulin-like growth factors and prostate cancer: a -population-based case-control study in China” Cancer Epidemiology Biomarkers Prevention 10: 412, 2001.
L. A. Mucci, et al. “Are dietary influences on the risk of prostate cancer mediated through the insulin-like growth factor system?” BJU International 87: 814, 2001.
H.K. Lin, et al. “Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor” Proceedings National Academy Sciences USA98: 7200, 2001.
O. Kucuk, et al. “Phase II randomized clinical trial of lycopene supplementation before radical prostatectomy” Cancer Epidemiolgy Biomarkers Prevention 10: 861, 2001.
P. Li, et al. “Antagonism between PTEN/MMAC1-1 and androgen receptor in growth and apoptosis of prostatic cancer cells” Journal Biologic Chemistry 276: 20444, 2001.
A. Berruti, et al. “Effects of the somatostatin analog lanreotide on the circulating levels of chromogranin A, prostate-specific antigen, and insulin-like growth factor-1 in advanced prostate cancer patients” Prostate 47: 205, 2001.
J. Khosravi, et al. “Insulin-like growth factor 1 (IGF-1) and IGF-binding protrein-3 in benign prostatic hyperplasia and prostate cancer” Journal Clinical Endocrinology Metabolism 86: 694, 2001.
P. Stattin, et al. “Plasma insulin-like growth factor-1, insulin-like growth factor-binding proteins and prostate cancer risk: a prospective study” Journal National Cancer Institute 92: 1910, 2000.
L. N. Thomas, et al. “Prostatic involution in men taking finasteride (Proscar) is associated with elevated levels of insulin-like growth factor binding proteins –2, -4, and –5” Prostate 42: 203, 2000.
J. R. Graff, et al. “Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27kip1 expression” Journal Biologic Chemistry 275: 24500, 2000.
A.L. Hsu, et al. “The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2” Journal Biologic Chemistry 275: 11397, 2000.
N.E. Allen, et al. “Hormones and diet: low insulin-like growth factor 1but normal bioavailable androgens in vegan men” British Journal Cancer 83: 95, 2000.
Y.Wen, et al. “HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway” Cancer Research 60: 6841, 2000.
Mitochondria are the cell’s major energy generators; they are the powerhouses of the cell. They also play a major role in the function of cell suicide programs. When removal of androgen, lack of IGF-1, and other forces that push the cell toward suicide reach a critical point, the mitochondria release the compound cytochrome C, which initiates cell death. In this sense, the mitochondria act as a switch that determines the fate of the cancer cell.
A small protein, called Bcl-2 (see Figure 1), acts to prevent mitochondria from releasing cytochrome C. Laboratory techniques that can increase the amount of bcl-2 make prostate cancer cells resistant to a wide range of treatments, including radiation, removal of androgen, and various chemotherapy drugs. Prostate cancer cell lines with increased amounts of bcl-2 grow faster.
|Figure 1: Cancer Cells Making bcl-2. This is a picture of prostate cancer under the microscope. The cancer cells making bcl2 are stained brownish red. Arrow 1 points to a cluster of these bcl-2-containing cancer cells. As with most prostate cancers, the cancer cell clusters are surrounded by broad bands of collagen (arrow 2) that are white in this picture. As you can see, the intensity of the bcl2 staining ranges from mild to strong. This reflects the fact that within any population of cancer cells, the amount of bcl-2 will vary. In most newly diagnosed prostate cancers, the cancer cells would not stain with bcl-2.|
In animal models, castration causes an increase in bcl-2 in prostate cancer cells and may limit the speed and magnitude with which cancer cells die. In the same animal models, prostate cancer cell lines genetically engineered to have a higher bcl-2 content show increased resistance to hormonal therapy. Bcl-2 is undetectable in about 70% of patients with hormone responsive cancers. In contrast, hormone resistant tumors showed high levels of the protein. Like the animal model, the amount of bcl-2 found in the remaining cancer increased during the course of hormonal therapy. A number of agents have been identified that decrease the amount or activity of bcl-2. Three of these drugs look particularly interesting:
1. Indol 3-carbinol
Indol 3-carbinol, which normally abounds in cabbage, broccoli, cauliflower, kale, collard greens, and related plants, may play a major role in cancer prevention. For instance, diets high in these vegetables, and hence Indol 3-carbinol, are associated with a low risk of cancers of the breast, prostate, and other organs. It is also available in pill form from Life Extension Foundation (www.lef.org). Although the compound appears to be relatively nontoxic, there are still no clinical trials testing Indol 3-carbinol in prostate cancer treatment.
Phenylbutyrate is approved by the FDA for the treatment of children with certain genetic abnormalities that cause mental retardation and early death. Phenylbutyrate is also relatively nontoxic, and, in the laboratory, it shows activity against prostate cancer. But, overall, the clinical trials testing the activity of this drug against advanced prostate cancer were not impressive.
Finally, the herbal combination PC-SPES suppressed the amount of bcl-2 present in prostate tumor cells in laboratory settings. However, the anticancer activity of PC-SPES is complex, and suppression of bcl-2 may play a role in the activity of this herbal preparation.
Now that PC-SPES is gone, many patients are looking for herbal preparations with similar effectiveness. While most of the effort seems to have focused on preparations that contain extracts from the same plants, the alternative is to identify how PC-SPES functioned and to duplicate those functions with the best agents possible. I think this is a more reasonable approach, and work on bcl-2 inhibitors may be a good place to start.
The bcl-2 protein can also undergo phosphorylation, but, unlike the androgen receptor, phosphorylation of bcl-2 renders it inactive.
Two drugs that have been proposed to deactivate bcl-2 by phosphorylation are paclitaxel (taxol®) and docetaxel (Taxotere®); their ability to alter bcl-2 phosphorylation may explain why they can enhance the anti-tumor activity of radiation therapy and interact synergistically with a range of other agents.
There is a third approach to the problem posed by bcl-2’s propensity to increase when responding to hormonal therapy, thereby decreasing the effectiveness of radiation and chemotherapy. Bcl-2 is one of the many cell survival proteins under the control of Akt. Increased active Akt means increased amounts of bcl-2, thus promoting cancer cell survival. PTEN blocks Akt activation, decreasing the amount of bcl-2 and promoting tumor cell death. Drugs able to block Akt (e.g., wortmannin and LY294002) will also be likely to decrease bcl-2, simultaneously disposing of two mechanisms that reduce androgen withdrawal response. In Part 3 of this article, I will be dealing with the importance of genetic damage in prostate cancer progression. I will discuss the role that the protein p53 plays in detecting and repairing gene damage and the significance of an abnormal p53. I will also discuss the role that the protein Rb plays in the evolution of hormone-resistant prostate cancer. I will conclude with a summary of the American Institute for Disease of the Prostate’s efforts to find the best way to combine androgen withdrawal with agents that block the known pathways to hormone resistance.
|Part 1 of this article Part 3 of this article|
H. Huang, et al. “PTEN induces chemosensitivity in PTEN-mutated prostate cancer cells by suppression of Bcl-2 expression” Journal of Biologic Chemistry 8: 8, 2001.
A. Y. Ng, et al. “Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53 and Fas in human prostate cancer cell lines” Ana. Quant. Cytol. Histol 22: 45, 2000. R. Buttyan, et al. “Regulation of Apoptosis in the Prostate Gland by Androgenic Steroids” Trends Endocrinol. Metab 10: 47, 1999.
T.J. McDonnell, et al., “Expression of the protoncogene bcl-2 in the prostate and its association with emergence of androgen-independent prostate cancer” Cancer Res 52: 6940, 1992.
P. Westin, et al., “Castration therapy rapidly induces apoptosis in a minority and decreases cell proliferation in a majority of human prostatic tumors” Am J Pathol 146: 1368, 1995.
C.J. Li, et al., “Induction of apoptosis by beta-lapachone in human prostate cancer cells” Cancer Res 55: 3712, 1995.
M.V. Blagosklonny, et al., “Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c- Raf-1 and represents a novel c- Raf-1 signal transduction pathway” Cancer Res 56: 1851, 1996.
I. Apakama, et al., “bcl-2 overexpression combined with p53 protein accumulation correlates with hormone-refractory prostate cancer” Br J Cancer 74: 1258, 1996.
M. Tsuji, et al., “Immunohistochemical analysis of Ki-67 antigen and Bcl-2 protein expression in prostate cancer: effect of neoadjuvant hormonal therapy” Br J Urol 81: 116, 1998.
B. An, et al., “Novel dipeptidyl proteasome inhibitors overcome Bcl- 2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts” Cell Death Differ 5: 1062, 1998.
M. Gleave, et al., “Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model” Clin Cancer Res 5: 2891, 1999.
R.F. Paterson, et al., “Immunohistochemical Analysis of Radical Prostatectomy Specimens After 8 Months of Neoadjuvant Hormonal Therapy” Mol Urol 3: 277, 1999.
D.S. Scherr, et al., “BCL-2 and p53 expression in clinically localized prostate cancer predicts response to external beam radiotherapy” J Urol 162: 12-6; discussion 16, 1999.
M.M. Rafi, et al., “Modulation of bcl-2 and cytotoxicity by licochalcone- A, a novel estrogenic flavonoid” Anticancer Res 20: 2653, 2000.
S. Chenn, “In vitro mechanism of PC SPES” Urology 58: 28, 2001.
S.R. Chinni, et al., “Indol-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells” Oncogene 20: 2927, 2001.