Androgen causes growth suppression and reversion of androgen-independent prostate cancer xenografts to an androgen-stimulated phenotype in athymic mice

Most prostate cancer patients develop androgen-independent recurrent prostate tumors a few years after androgen ablation therapy. No therapy, however, has been shown to substantially extend survival in these patients. Previously, we reported that androgen suppresses the growth of androgen-independent LNCaP prostate tumor cells both in vitro and in vivo. In cell culture, androgen receptor (AR)-rich androgen-independent LNCaP 104-R1 cells adapt to growth suppression by androgen and then their growth is androgen stimulated. Because maintaining androgen dependency of prostate tumor cells should prolong the usefulness of androgen ablation therapy, we determined if androgen-independent prostate tumors would revert to an androgen-stimulated phenotype in vivo upon androgen treatment. Growth of the LNCaP 104-R1 tumors was suppressed by androgen, but tumors then adapted to suppression by androgen and growth became androgen stimulated. Tumor AR and prostate-specific antigen mRNA and protein were initially high in 104-R1 tumors but decreased during adaptation. Subsequent removal of androgen decreased the serum prostate-specific antigen level further and stopped the growth of the adapted tumors. Because androgen caused growth suppression and then reversion of androgen-independent tumors to an androgen-stimulated phenotype and because the growth of androgen-stimulated tumors could be restrained by androgen ablation, these results suggest a novel therapy for AR-positive androgen-independent prostate cancer.     

Identification of steroid derivatives that function as potent antiandrogens

We have hypothesized that some steroid derivatives bind to the androgen receptor (AR) with very low androgenic activity and therefore potentially function as better AR antagonists than clinically used antiandrogens, such as flutamide. Indeed, we previously found such a compound, 3beta-acetoxyandrosta-1,5-diene-17-one ethylene ketal (ADEK), with some estrogenic activity. Here we report the identification of 2 additional steroid derivatives, 3beta-hydroxyandrosta-5,16-diene (HAD) and androsta-1,4-diene-3,17-dione-17-ethylene ketal (OAK), as new potent antiandrogens. Like ADEK, HAD and OAK could interrupt androgen binding to the AR and suppress both dihydrotestosterone- and androstenediol-induced transactivations of wild-type and mutant ARs in prostate cancer cells. These 2 compounds also inhibited prostate-specific antigen expression in LNCaP as well as growth of different AR-positive prostate cancer cell lines stimulated by androgen. Significantly, HAD and OAK had only marginal agonist effects, as compared to hydroxyflutamide. More importantly, in contrast to ADEK, OAK was shown to possess marginal estrogenic activity. These results strengthen our hypothesis and suggest that selective steroid derivatives could be potent antiandrogenic drugs with less unfavorable effects for the treatment of prostate cancer.     

Functional analysis of 44 mutant androgen receptors from human prostate cancer

Mutations of the androgen receptor gene are believed to contribute to the androgen-independent growth of prostate cancer cells. To date, 56 missense mutations of the androgen receptor have been identified in human prostate cancer. The functional status of most of these mutants has not yet been investigated. To address their functional properties, we generated 44 androgen receptor mutants that have been identified in human prostate cancer and used a colorimetric yeast reporter assay to analyze their transactivational activities in response to seven different ligands. We found that these mutant androgen receptors exhibited diverse transactivational activity: seven (16%) showed loss of function, three (7%) had wild-type function, 14 (32%) had partial function, and 20 (45%) had gains of function. Five of 20 gain-of-function mutants had promiscuous activity, being transactivated by non-androgens. We also found that the combination of estradiol and progesterone at physiological concentrations weakly or moderately activated an additional seven mutant androgen receptors. Our findings provide essential information for understanding the role of mutant androgen receptors in prostate cancer.     

Regulation of growth and epidermal growth factor receptor levels of LNCaP prostate tumor cells by different steroids

The growth of LNCaP cells, derived from a lymph-node carcinoma of the human prostate, was stimulated by different hormones. Optimal growth (3- to 4-fold increase in DNA content per culture versus controls) was observed at 0.1 nM R1881 (a synthetic androgen), 1 nM progesterone or 10 nM estradiol. Triamcinolone acetonide had no effect. Dihydrotestosterone maximally stimulated cell growth at 10 nM. When the culture medium was changed 4 times in 6 days instead of twice, optimal growth was observed at 1 nM dihydrotestosterone. This indicates that a rapid metabolism of dihydrotestosterone influenced growth response. LNCaP cells contained considerable amounts of androgen receptors (920 fmol/mg cytosol protein) while progestagen, estrogen and glucocorticoid receptors were absent. The affinity of steroids for the androgen receptor decreased in the order of: R1881 (relative binding affinity: 100.0) greater than dihydrotestosterone (67.7) greater than progesterone (29.4) greater than testosterone (23.8) greater than estradiol (4.3) greater than triamcinolone acetonide (less than 0.1). Effects on cell growth of these steroids paralleled their affinity for the androgen receptor. The number of EGF receptors per cell increased in a dose-dependent manner upon treatment with various hormones. Again the amount of steroid needed for maximal effects reflected the affinity of the steroid for the androgen receptor. An approximately 2-fold increase in EGF receptor number was observed within 24 hr and before an increase in growth could be detected. Actinomycin-D and cycloheximide inhibited the hormonally induced increase in EGF receptor numbers.     

Prostate cancer stem cells: the role of androgen and estrogen receptors

Prostate cancer is one of the most commonly diagnosed cancers in men, and androgen deprivation therapy still represents the primary treatment for prostate cancer patients. This approach, however, frequently fails and patients develop castration-resistant prostate cancer, which is almost untreatable.Cancer cells are characterized by a hierarchical organization, and stem/progenitor cells are endowed with tumor-initiating activity. Accumulating evidence indicates that prostate cancer stem cells lack the androgen receptor and are, indeed, resistant to androgen deprivation therapy. In contrast, these cells express classical (α and/or β) and novel (GPR30) estrogen receptors, which may represent new putative targets in prostate cancer treatment.In the present review, we discuss the still-debated mechanisms, both genomic and non-genomic, by which androgen and estradiol receptors (classical and novel) mediate the hormonal control of prostate cell stemness, transformation, and the continued growth of prostate cancer. Recent preclinical and clinical findings obtained using new androgen receptor antagonists, anti-estrogens, or compounds such as enhancers of androgen receptor degradation and peptides inhibiting non-genomic androgen functions are also presented. These new drugs will likely lead to significant advances in prostate cancer therapy.     

Epidermal growth factor receptor activation in androgen-independent but not androgen-stimulated growth of human prostatic carcinoma cells.

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.     

Id-1 expression in androgen-dependent prostate cancer is negatively regulated by androgen through androgen receptor

This study discovered that Id-1 expression in androgen-dependent prostate cancer decreased immediately after androgen deprivation but increased after longer androgen deprivation both in vivo and in vitro. Id-1 expression in androgen-independent LNCaP cells was about 6 fold as that in their parental cells. As was the case with LNCaP cells, when androgen receptor (AR) was introduced into AR-negative PC-3 cells, dihydrotestosterone inhibited while flutamide increased Id-1 expression. Thus, Id-1 expression in androgen-dependent prostate cancer was negatively regulated by androgen in a receptor-dependent way. The re-increased Id-1 might partially contribute to the emergence of androgen-independent prostate cancer after longer androgen deprivation therapy.     

Raloxifene,a selective estrogen receptor modulator,induces apoptosis in androgen-responsive human prostate cancer cell line LNCaP through an androgen-independent pathway

Raloxifene, a selective estrogen receptor (ER) modulator, is a mixed estrogen agonist/antagonist that has been shown to prevent osteoporosis and breast cancer in women. Because the prostate contains a high level of ER-beta, the present study investigated the effect of raloxifene in the androgen-sensitive human prostate cancer cell line LNCaP. Previously, it has been demonstrated that LNCaP cells express ER-beta but not ER-alpha and that tamoxifene induces apoptosis in these cells. After treatment with raloxifene, a dramatic increase in cell death occurred in a dose-dependent manner (10(-9) to 10(-6) M range). Using the terminal deoxynucleotidyl transferase-mediated nick end labeling apoptotic assay, we demonstrated that the nuclear fragmentation was due to apoptosis. The dramatic change in cellular morphology after treatment with raloxifene was no longer observed when cells were pretreated with a pan-caspase inhibitor, Z-VAD-FMK, and a specific caspase-9 inhibitor, Z-LEHD-FMK. Furthermore, immunoblot demonstrated an activation of caspase-9 in LNCaP cells. Because LNCaP cells contain a mutated androgen receptor that allows cellular proliferation in the presence of antiandrogens, prostate-specific antigen assay and transfection with a reporter construct containing luciferase gene under the control of androgen response element (pARE) were carried out. The results demonstrated that raloxifene does not significantly alter androgen receptor activity in LNCaP cells. Taken together, these results demonstrate that raloxifene, a selective ER modulator, induces apoptosis in the androgen-sensitive human prostate cancer cell line LNCaP through an androgen-independent pathway.     

Modulation of androgen receptor transactivation by gelsolin: a newly identified androgen receptor coregulator

The partial agonist effect of antiandrogens has been well documented, and such effect is amplified by derived mutant androgen receptors (ARs) in prostate cancer cells. Here we report the identification of gelsolin (GSN) as an AR-associated protein. Hydroxyflutamide (HF), as well as androgens, can promote the interaction between AR and GSN in a dose-dependent manner. GSN interacts with AR DNA-binding domain and ligand-binding domain via its COOH-terminal domain. Immunolocalization studies show that GSN interacts with AR during nuclear translocation. Functional analyses additionally demonstrate that GSN enhances AR activity in the presence of either androgen or HF. Two peptides representing partial regions of the AR DNA-binding domain and the ligand-binding domain can block the GSN-enhanced AR activity. The expression of GSN is enhanced in LNCaP cells, LNCaP xenografts, and human prostate tumors after androgen depletion. Increasing expression of GSN enhances the AR activity in the presence of HF. Together, these data suggest that the weak androgenic effect of HF may be amplified by increasing the amount of GSN after androgen ablation treatment. Therefore, blockage of the interaction between AR and GSN could become a potential therapeutic target for the treatment of prostate cancer.     

Steroid receptor regulation of epidermal growth factor signaling through Src in breast and prostate cancer cells: steroid antagonist action

Under conditions of short-term hormone deprivation, epidermal growth factor (EGF) induces DNA synthesis, cytoskeletal changes, and Src activation in MCF-7 and LNCaP cells. These effects are drastically inhibited by pure estradiol or androgen antagonists, implicating a role of the steroid receptors in these findings. Interestingly, EGF triggers rapid association of Src with androgen receptor (AR) and estradiol receptor alpha (ERalpha) in MCF-7 cells or ERbeta in LNCaP cells. Here, we show that, through EGF receptor (EGFR) and erb-B2, EGF induces tyrosine phosphorylation of ER preassociated with AR, thereby triggering the assembly of ER/AR with Src and EGFR. Remarkably, experiments in Cos cells show that this complex stimulates EGF-triggered EGFR tyrosine phosphorylation. In turn, estradiol and androgen antagonists, through the Src-associated receptors, prevent Src activation by EGF and heavily reduce EGFR tyrosine phosphorylation and the subsequent multiple effects, including DNA synthesis and cytoskeletal changes in MCF-7 cells. In addition, knockdown of ERalpha or AR gene by small interfering RNA (siRNA) almost abolishes EGFR tyrosine phosphorylation and DNA synthesis in EGF-treated MCF-7 cells. The present findings reveal that steroid receptors have a key role in EGF signaling. EGFR tyrosine phosphorylation, depending on Src, is a part of this mechanism. Understanding of EGF-triggered growth and invasiveness of mammary and prostate cancer cells expressing steroid receptors is enhanced by this report, which reveals novel aspects of steroid receptor action.     

NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.