Androgen Resistance, Part 1

Reprinted from PCRI Insights November 2002 v 5.2
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

Introduction
The development of hormone resistance is the event most patients with prostate cancer (PC) fear most. They fear the side effects of chemotherapy and worry that their survival may be short. I have seen this fear cause men to simply give up and ask only to be kept comfortable. This is unfortunate because the best of modern chemotherapy can be highly effective and the side effects are usually quite manageable. What I find particularly tragic is that a majority of men who are diagnosed with hormone-refractory PC are still hormonally responsive. All too often I have seen men who have progressed on Lupron® or Zoladex® alone be diagnosed as hormone-refractory and then be placed on one of the older, toxic chemotherapy protocols, have a short response of less than a year’s duration and then be placed on hospice care. This grim state of affairs is quite unnecessary. The purpose of this article is to describe what I think is a much more effective approach.

Is Androgen Withdrawal Complete?
The basis of hormonal therapy in treating PC is the reduction of testosterone (T) levels to a range typically found after surgical castration. The mechanism of action of androgens is shown in Figure 1. While there is some controversy about the specific T level that must be attained, most specialists would accept that the T level must be below 50 ng/dl. I think a more appropriate goal would be a T level below 20. When a physician is faced with a patient who is progressing on hormonal therapy, his or her first step should be to make sure that the patient’s T level is in the castrate range. Unfortunately, many physicians assume that administration of Lupron® or Zoladex® automatically means that castrate androgen levels have been attained. This is often not the case. If T levels are still elevated, the next step is to determine whether this is the result of inadequate suppression of LH by the LHRH agonist/antagonist or because of increased production of androgen precursors by the adrenal gland. In the October 2001 issue of Insights, Dr. Stephen B. Strum’s article, “Listening to the Biology of Prostate Cancer,” provided detailed information on how to determine why medical castration has failed and how to deal with it.

Androgen Receptor Mutations

Figure 2. Human prostate stained with Androgen Receptor Ab-1.

An androgen receptor (shown in Figure 2) is very specific; while it binds avidly to testosterone or dihydrotestosterone (DHT), it does not react with a wide range of other steroid hormones. For example, the female sex hormones estradiol and progesterone are chemically similar to T, but the androgen receptor does not react with either of them. The specificity of this receptor is critical for normal biology and allows the prostate and other tissues to respond selectively to T. The specificity of these receptors for their various hormones is based on the structure of these proteins. The androgen receptor has a portion that fits like a lock and key with the T molecule.

A minor change in the structure of the androgen receptor can have profound effects on its function. For example, there is a strain of chickens called the Sebright bantam. The cocks of this strain look like hens. While they have normal T levels, their androgen receptors do not function, causing their feminine appearance. In humans, a similar genetic change causes what is called testicular feminization syndrome in which males assume the external appearance of females, including the development of breasts and female external genitalia.

The first indication that androgen receptor mutations might be important in PC came from an experiment conducted by Dr. George Wilding at the University of Wisconsin. Wilding was testing the antiandrogen Eulexin® (flutamide) against a human PC cell, LNCaP. In the test tube, LNCaP responds to T or DHT by increasing its growth rate. Wilding anticipated that Eulexin® would block the growth stimulation caused by T or DHT. Instead, Eulexin® and its active metabolite, hydroxyflutamide, actually stimulated the growth of LNCaP as if it were testosterone.

Wilding then went on to show that progesterone and estradiol also stimulated LNCaP growth. The explanation for this unusual behavior was that the androgen receptor in LNCaP was mutated in such a way that it reacted promiscuously with androgen, estradiol, progesterone, and drugs like Eulexin®. LNCaP reacted to all of these compounds as if they were androgens, because of this altered receptor. These observations led to the prediction that Eulexin® would stimulate the growth of the cancer in a patient whose tumor possessed an altered androgen receptor like LNCaP. If the Eulexin® was stopped, the tumor might shrink. We now know that approximately 25% of men who have been on Eulexin® for several years and then develop apparent hormone-resistant PC will have a significant response when the Eulexin® is stopped.

Even while on hormonal therapy, men continue to produce the female sex hormones, estradiol and progesterone. Indeed, this is the reason men on hormonal therapy often experience breast tenderness and enlargement. The mutated androgen receptor in LNCaP that is activated by Eulexin® also responds to estradiol and progesterone. Withdrawal of Eulexin® does not remove estradiol or progesterone, and it seemed possible that its presence may have limited the number of patients who responded to Eulexin® withdrawal. At low doses, the drug Cytadren® works by blocking aromatase, the enzyme that converts T to estradiol. At high doses, it blocks the production of most steroid hormones, including progesterone and all androgens. This led us to substitute Cytadren® in men who were discontinuing Eulexin®. Close to 45% of the patients responded to this treatment.

With Cytadren®, it was not possible to determine whether these responses were the result of the ability of this drug to block estradiol synthesis because it inhibited aromatase or the result of its ability at high dose to block nearly all steroid hormone synthesis. Recently, very specific aromatase inhibitors have been developed, and we tested one of these, Arimidex®, in hormone-refractory PC. We saw no responses, and I have concluded that the effectiveness of Cytadren® is the result of its capacity to block nearly all steroid hormone synthesis at high doses.

Casodex® is the other widely used antiandrogen. While it is more expensive than Eulexin®, it offers the convenience of once-a-day administration with a lower risk of liver damage and diarrhea. It also appears that the incidence of androgen withdrawal response is much lower than with Eulexin®. In fact, Casodex® can successfully block the growth of PC cells bearing the mutant androgen receptor found in LNCaP.      Continued

References

G. Wilding, et al. “Aberrant response in vitro of hormone responsive prostate cancer cells to antiandrogens” Prostate 14: 103, 1989

W. K. Kelly and H.I. Scher. “Prostate specific antigen decline after antiandrogen withdrawal: the flutamide withdrawal syndrome” Journal Urology 149:607, 1993.

M.A. Fenton, et al. “Functional characterization of mutant androgen receptors from androgen-independent prostate cancer” Clinical Cancer Research 3: 1383, 1997.

S. McDonald, et al. “Ligand responsiveness in human prostate cancer: structural analysis of mutant androgen receptors from LNCaP and CWR22 tumors” Cancer Research 60: 2317, 2000.

O. Sartor, et al. “Surprising activity of flutamide withdrawal, when combined with aminoglutethimide, in treatment of “hormone-refractory” prostate cancer” Journal of the National Cancer Institute 86: 222, 1994.

R.K. Tyagi, et al. “Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells” Molecular Endocrinology 14: 1162, 2000.

Androgen Hypersensitization
In the laboratory, the most widely studied androgen-responsive human PC cell line is LNCaP. The growth of this cell line slows or stops when T is removed. Over a period of about six months, cells emerge that do grow slowly without any T. Despite adaptation to the absence of T for up to six months, these cells will invariably grow better when T is added and they still have androgen receptors. However, it now takes between hundreds to thousands of times less T to stimulate these cells to grow to their maximal potential. Instead of becoming hormone-resistant, these cells have become extraordinarily responsive to testosterone!

These results have profound implications for PC treatment. Surgical castration or treatment with drugs like Lupron® or Zoladex® typically causes a 90–95% drop in blood T levels. Interestingly, surgical castration causes only a 75% drop in the T content of human prostate tissue. Even complete androgen blockade, including medical or surgical castration plus an antiandrogen such as Eulexin® or Casodex®, does not cause a decrease in androgen in the blood or prostate much beyond 99%. The implication is that given sufficient time, hormone-responsive PC cells can adapt to grow in the small amounts of androgen remaining – even after what has been called complete androgen blockade has been achieved. In fact, a large number of drug combinations have been tested for their ability to reduce T and DHT levels in prostate tissue. None come close to reaching levels that would be effective against cancer cells able to grow in up to 10,000 times less androgen. Yet, we now know that human PC cells are able to adapt to such low androgen concentrations.

Increased Androgen Receptor Expression
There are several paths that PC cells can follow to become more sensitive to low T levels. These include increasing the number of androgen receptors and using one of several means to enhance androgen receptor efficiency. It is easy to understand the importance of increasing the number of androgen receptors. PC cell growth is controlled by the number of T- or DHT-androgen receptor complexes that are present. The higher the concentration of androgen receptors, the more likely it is that enough receptor complexes will form to fuel cancer cell growth.

Examination of the androgen receptors content of human PC specimens shows that increased expression of androgen receptors is quite common in patients who have failed medical or surgical castration. In one study, the androgen receptor content of 33 untreated PC cases was compared with 13 cases in which hormonal therapy had failed. All of the hormone-resistant tumor specimens contained androgen receptors, and the average amount was six times higher than in the untreated patients.

In another study, androgen receptor expression was measured in each patient who failed castration; the patients were then placed on complete androgen blockade. Of the ten patients with increased androgen receptor levels in their cancers, nine responded or experienced disease stabilization because of this increase in the androgen withdrawal intensity. In contrast, those patients who had no increase in androgen receptor content of their tumors were quite unlikely to respond.

While these results are quite provocative, the study involved only a small number of men with cancers that exhibited an increased number of androgen receptors. Much larger numbers of patients need to be studied to determine the effectiveness of complete androgen blockade in that group of men whose tumors have developed increased androgen receptor levels. Nevertheless, it is clear that increased androgen receptor content is common in patients on hormonal therapy, and that complete androgen blockade will provide improved tumor control for many of these patients.      Continued

References:
J. Geller, “Basis for hormonal management of advanced prostate
cancer,” Cancer 71: 1039, 1993.

J.D. McConnell, et al. “Finasteride, an inhibitor of 5-alpha-reductase,
suppresses prostatic dihydrotestosterone in men with benign
prostatic hyperplasia,” Journal Clinical Endocrinology Metabolism
74: 505, 1992.

G. Forti, et al. “Three-month treatment with a long-acting
gonadotropin-releasing hormone agonist of patients with benign
prostatic hyperplasia: effects on tissue androgen concentration, 5-
alpha-reductase activity and androgen receptor content,” Journal
Clinical Endocrinology and Metabolism 68: 461, 1989.

P.S. Rennie, et al. “Relative effectiveness of alternative androgen
withdrawal therapies in initiating regression of rat prostate,” Journal
of Urology 139: 1337, 1988.

M. Linja, et al. “Amplification and over expression of androgen
receptor gene in hormone-refractory prostate cancer” Cancer
Research 61: 3550, 2001.

C. Palmberg, et al “Androgen receptor gene amplification in a
recurrent prostate cancer after monotherapy with the nonsteroidal
potent antiandrogen Casodex (bicalutamide) with a subsequent
favorable response to maximal androgen blockade” European
Urology 31: 216, 1997.

Increased Androgen Receptor Efficiency
Androgen receptor complexes form and break down rapidly. (See Figure 3.) The number of androgen-receptor complexes present at any point in time represents a balance between the rate of complex formation and the rate at which these complexes break down. A few papers have described human PC cells able to grow at low levels of T where the mechanism seems to involve the formation of much more stable androgen receptors. For example, Gregory, et al examined human PC cell lines adapted to grow at low T levels. They found that the combination of increased androgen receptor content, increased receptor stability and enhanced nuclear translocation (See Figure 1) resulted in a 10,000-fold decrease in the dihydrotestosterone concentration required for cancer growth! The information is too incomplete for me to tell whether this occurs with any frequency in patients, but it illustrates the capacity of PC cells to adapt to extremely low androgen levels rather well.

Figure 3. Dual-color FISH analysis of prostate cancer xenografts. The clustering of red signals indicates amplification of the AR gene.

There are other proteins in PC cells that bind to the androgen receptor. Some of these act to enhance and others to suppress the effectiveness of the androgen receptor. This has become one of the “hot” areas of PC research, and it appears likely that shifts in the spectrum 4 of androgen receptor helpers and suppressors play a role in allowing these cancer cells to grow at low T levels. For example, Gregory, et al reported that a majority of hormone resistant prostate cancers not only show elevated levels of androgen receptors, but also show increased levels of proteins, called coactivators, that make androgen receptors more efficient. Three activators have been identified that appear to play a role in hormone-resistance: (1) transcriptional intermediary factor 2, (2) steroid receptor coactivator 1, and (3) ARA70.     Continued 

References:

C.W. Gregory, et al. “Androgen receptor stabilization in recurrent
prostate cancer is associated with hypersensitivity to low androgen”
Cancer Research 61: 2892, 2001.

C.W. Gregory, et al. “A mechanism for androgen receptor-mediated
prostate cancer recurrence after androgen deprivation therapy”
Cancer Research 61: 4315, 2001.

A. Bubulya, et al. “c-Jun targets amino terminus of androgen
receptor in regulating androgen-responsive transcription”
Endocrine 13: 55, 2000.

Activation of the Receptor at Low Androgen Levels
After the androgen receptor is made, phosphate must be added to the protein at certain sites before it can form effective complexes with T or DHT.

This process of adding a phosphate to a protein is called phosphorylation. After the androgen receptor binds to T, additional phosphate groups are added to the protein to facilitate prostate cell growth. Changes in the cancer cell that significantly foster the addition of these phosphate groups can markedly enhance the ability of the androgen receptor to respond to low levels of T. This process of androgen receptor phosphorylation appears to play a major role in allowing PC cells to elude hormonal therapy.

The epidermal growth factor (EGF) is part of a family of cytokines that share a capacity to control the growth of cells. This family of cytokines plays a major role in the biology of prostate cells. When the T- or DHT-androgen receptor complex triggers the growth of human prostate cells, it does so, in part, because it causes the prostate cells to release the EGF-family cytokines. These EGF-family cytokines then bind to the surface of the prostate cells and trigger growth. In at least some hormone resistant cancer cells, EGF-family cytokine release can occur in the absence of T or DHT, thus supporting androgen-independent growth.

There are a series of receptors specific for EGF-family cytokines. One of these, HER-2, is expressed in both androgen-sensitive and androgen resistant PC cells. An antibody that blocks HER-2 is able to prevent the growth of hormone-sensitive and hormone-resistant PC cells in mice. One antibody against HER-2, Herceptin®, is already on the market as a treatment for breast cancer.

EGF-family members also appear to play important roles in the growth of breast, lung, head, neck, and other cancers. Consequently, these cytokines and their receptors have become popular targets for pharmaceutical companies. Several agents that block this cytokine family are in late-stage clinical testing for head and neck cancer and lung cancer. One of these, Iressa®, has received the support of the FDA advisory committee for cancer drugs and is likely to become available for the treatment of lung cancer within the next year. However, once it is on the market, physicians will be able to use it for a wide range of other cancers, including PC. I should mention that this drug is already available in Japan. This drug has two desirable features. First, it can be given orally, once a day. Second, its side effects are generally mild and usually limited to an acne-like skin rash and diarrhea.

IL-6 is another cytokine that appears to play a role in androgen receptor function. (See Figure 4.) IL-6 is a cytokine that is released during infections and other inflammatory diseases. This cytokine is also released by PC cells, especially in the more aggressive, life-threatening forms of this disease. In PC patients, elevated IL-6 blood levels occur in association with widespread metastatic cancer and the development of hormone-resistant disease. PC cells have IL-6 receptors, and the adding of IL-6 increases the phosphorylation of the androgen receptor. Simply adding IL-6 also stimulates the growth of hormone-resistant PC cells in response to T or DHT.

Figure 4. Androgen receptor sensitization by EGF and IL6.

to block the production of IL-6 by PC cells or to block the action of this cytokine on PC cells. However, the body generally produces IL-6 in response to inflammation at any site. For example, arteriosclerosis or hardening of the arteries caused by cholesterol commonly causes widespread inflammation in the arteries, causing IL-6 elevation. If you are worried about this, the cardiac C-reactive protein represents the most sensitive and specific test for widespread inflammation in the body. If it is positive, you should ask your physician to determine why your C-reactive protein is elevated and treat this. For example, if it is caused by hardening of your arteries, the combination of one baby aspirin and a statin drug, like Lipitor, can be highly effective.

Neuroendocrine Cells and Hormone-Resistance
There are a group of specialized cells, called neuroendocrine cells, which release a wide range of cytokines and hormones, including epinephrine, serotonin, calcitonin, gastrin-releasing peptide, and parathormone-related peptide. These cells are normally found in such normal tissues as the lining of the lung airways, the gut, breast ducts, and prostate gland ducts. The products of these neuroendocrine cells promote fluid secretion and muscle contraction by the ducts in these organs.

Neuroendocrine cells are commonly found scattered throughout PC masses. In newly diagnosed patients, the greater the number of these neuroendocrine cells, the more likely a patient is to develop life-threatening PC. In men on hormonal therapy, the appearance of large numbers of neuroendocrine cells in the PC deposits commonly precede the development of hormone-resistant PC.

For several years, we have known that the cytokines and hormones produced by neuroendocrine cells stimulate the growth of PC cells in the test tube. More recently, several of these neuroendocrine cell products have been shown to activate the androgen receptor through phosphorylation, leading to a receptor able to act at low levels of T or DHT. While there is no generally accepted treatment designed to suppress these neuroendocrine cells, Sandostatin® has been used with some clinical benefit in a small series of patients.

In some men, the neuroendocrine cells become the dominant cell in the cancer and become very aggressive. These cancers are very similar to small cell cancer of the lung and produce little or no PSA. This clinical presentation has recently been characterized by Chris Logothetis and his colleagues at M.D. Anderson. These patients typically present with rapidly progressing cancer in the presence of a relatively low or normal serum PSA. While there is no treatment associated with a high response rate, individual patients have responded well to combinations of paclitaxel (Taxol®) or docetaxel (Taxotere®) with carboplatin.

It is possible to monitor the appearance of neuroendocrine cells in men with PC because these cells release markers into the blood stream. The most generally useful test is the serum chromogranin A (CgA), but neuron specific enolase (NSE), calcitonin and bombesin can be of value in individual patients.

References:

Part 2 of this article                               Part 3 of this article

A. Hobisch, et al. “Interleukin-6 regulates prostate-specific protein expression in prostate carcinoma cells by activation of the androgen receptor” Cancer Research 4640, 1998.

M.D. Sadar “Androgen-independent induction of prostate-specific antigen gene expression via cross-talk between the androgen receptor and protein kinase A signal transduction pathways” Journal Biologic Chemistry 274: 7777, 1999.

L.G. Wang, et al. “Phosphorylation/dephosphorylation of androgen receptor as a determinant of androgen agonistic or antagonistic activity” Biochemistry Biophysics Research Communications 259: 21, 1999.

Z. Culig, et al. “Synergistic activation of androgen receptor by androgen and luteinizing hormone-releasing hormone in prostatic carcinoma cells” Prostate 32: 106, 1997.

T. Ikonen,et al. “Stimulation of androgen-regulated transactivator by modulators of protein phosphorylation” Endocrinology 135: 1359, 1994.

C. Culig, et al. “Androgen receptor activation in prostatic tumor cell lines by insulin- like growth factor 1, keratinocyte growth factor and epidermal growth factor” Cancer Research 54: 5474, 1994.

D.B. Agus, et al. “Response of prostate cancer to anti-Her-2/neu antibody in androgen-dependent and –independent human prostate xenograft models” Cancer Research 19: 4761, 2000.

S. Signoreti, et al. “Her-2 neu expression and progression toward androgen independence in human prostate cancer” Journal National Cancer Institute 92: 1918, 2000.

N. Craft, et al. “A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase” Nature Medicine 5: 280, 1999.

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.

S. Yeh, et al. “From Her2/Neu signal cascade to androgen receptor and its coactivators: a novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells” Proceedings National Academy Sciences USA 96: 5458, 1999.

J. Jongsma, et al. “Androgen-independent growth is induced by neuropeptides in human prostate cancer cell lines” Prostate 42:34, 2000.

 

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