Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoration of testosterone levels could suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets were implanted. Of 33 tumors, 24 adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen. We then studied the molecular mechanism underlying the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G1 cell cycle arrest through reduction of S-phase kinase-associated protein 2 (Skp2) and c-Myc, and induction of p27Kip1. 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with a slower growth rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27Kip1. Nuclear AR and prostate-specific antigen expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype; knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells through AR, Skp2, and c-Myc. (Cancer Sci 2011; 102: 2022–2028)
Prostate cancer is one of the most common carcinomas of men in United States. In 1941, Charles Huggins(1) reported that androgen ablation therapy caused regression of metastatic prostate cancer. Since then, androgen ablation therapy has become the primary treatment for metastatic prostate cancer.(2) However, 80–90% of the patients who receive androgen ablation therapy ultimately develop recurrent tumors in 12–33 months. The median overall survival of patients after tumor relapse is 1–2 years.(3,4) Androgen deprivation therapy is associated with several undesired side-effects, including sexual dysfunction, osteoporosis, hot flashes, fatigue, gynecomastia, anemia, depression, cognitive dysfunction, and increased risk of diabetes and cardiovascular diseases.(2,5–7) Therefore, shortening the period of androgen ablation therapy could protect patients.
A commonly used cell line, LNCaP was established from a human lymph node metastatic lesion of prostatic adenocarcinoma.(8) We previously established relapsed androgen-independent human LNCaP 104-R1 (passage >80, approximately 12 months) and 104-R2 cells (passage >150, approximately 23 months) from androgen-dependent LNCaP 104-S cells after culturing under long-term androgen-depleted conditions.(9,10) Compared with 104-S cells, 104-R1 and 104-R2 cells express higher androgen receptor (AR) protein and mRNA.(9–11) Upregulation of AR protein has been observed in many patients with recurrent hormone-refractory tumors.(12–14) Proliferation of 104-R1 and 104-R2 cells is not dependent on androgen (i.e. androgen-independent) but is suppressed by physiological concentrations of androgen both in vitro and in vivo (i.e. androgen-responsive),(9–11,15–17) in part by downregulation of c-Myc and induction of cyclin-dependent kinase inhibitor p27Kip1, thereby causing G1 cell cycle arrest.(9,10) In this study, we investigated whether AR-positive relapsed prostate tumors in patients receiving long-term (more than 3 years) androgen ablation therapy might be suppressed by physiological concentration of androgens after termination of the androgen ablation therapy. For this purpose, we used late passage (passage 285) of LNCaP 104-R2 cells, which were derived from androgen-dependent LNCaP 104-S cells after 43 months of androgen deprivation, both in xenograft model and in cell culture, to study the effect of androgens on these cancer cells.
Materials and Methods
Cell culture. LNCaP 104-S and 104-R2 cells were passaged and maintained as previously described.(9–11,18,19) R2Ad cells were maintained in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 1 nM dihydrotestosterone, 10% FBS (Atlas, Fort Collins, CO, USA) plus penicillin (100 U/mL), and streptomycin (100 μg/mL; Invitrogen). R1881 (17β-hydroxy-17α-methylestra-4,9,11-trien-3-one) was from Perkin Elmer (Boston, MA, USA). Bicalutamide (Casodex) was from AstraZeneca Pharmaceuticals (Wilmington, DE, USA).
Cell proliferation assay. Relative cell number was analyzed by measuring DNA content of cell lysates with the fluorescent dye Hoechst 33258 (Sigma, St. Louis, MO, USA) as described previously.(18,19)
Western blot analysis. Western blots were carried out as previously described.(16–19) Antibodies were against AR (AN-21 polyclonal rabbit antibody),(9–11) prostate-specific antigen (PSA; Dako, Elostrup, Denmark), p27Kip1 (BD Biosciences, Lexington, KY, USA), p21Cip, and S-phase kinase-associated protein 2 (Skp2; Santa Cruz Biotechnology, Santa Cruz, CA, USA), c-Myc (Abcam, Cambridge, MA, USA), and β-actin (Chemicon, Temecula, CA, USA). Measurement of β-actin expression was used as a loading control in all experiments.
Cell cycle analysis. Cell cycle distribution analysis of LNCaP 104-S, 104-R2, or R2Ad cells treated with different concentrations of R1881 (0, 0.1, 10 nM) for 96 h was carried out as described previously.(10,11,20)
Preparation of nuclear and cytoplasmic fractions of LNCaP cells. LNCaP cells were washed twice with cold PBS and incubated with 1 mL of 10 mM Tris HCl pH 7.5, 150 mM NaCl, and 1 mM EDTA for 2–3 min. Cells were removed by scraping and transferred suspension to a 1.5 mL tube on ice. Cells were pelleted at 3000g at 4°C for 2 min. The cell pellet was resuspended in 0.5 mL cold cell lysis buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 0.4% Nonidet P40, 1 mM PMSF, 1 μg/mL aprotinin, and 1 μg/mL leupeptin). After gentle mixing, nuclei were centrifuged at 3000g for 2 min. Supernatant was collected as the cytoplasmic fraction and resuspended in cell lysis buffer. Nuclei in the pellet were resuspended in 0.5 mL cell lysis buffer and centrifuged at 3000g for 2 min. Washed nuclei were resuspended in cell lysis buffer.
Tumor xenografts in athymic mice. Experiments involving mice were approved by the University of Chicago Institutional Animal Care and Use Committee. Male BALB/c nu/nu mice (National Cancer Institute, Frederick, MD, USA), 6–8 weeks of age, were injected s.c. in both flanks with 1 × 106 LNCaP 104-R2 cells suspended in 0.5 mL Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) 14 days after mice were castrated. Tumors were measured weekly using calipers and volume was calculated using the formula volume = length × width × height × 0.52.(16,17) Where indicated, mice were implanted s.c. with 20 mg pellets of testosterone or cholesterol.
Real-time quantitative PCR. RNA was isolated and specific mRNAs were quantified as described.(16–19,21,22) The sequences of primers and probes for AR and GAPDH were described previously.(11,17,21) Primer and probe sequences used for PSA quantification were described by Gelmini et al.(23) All transcript levels were normalized to GAPDH levels in each sample.
Protein overexpression and knockdown in LNCaP cells. Overexpression and shRNA knockdown of AR were carried out as previously described.(11) Ectopic expression of Skp2 and c-Myc was achieved by infecting LNCaP 104-R2 cells with pMV7 or pBabe retroviruses carrying the cDNA of the Skp2 and c-Myc, respectively. Antibiotic-resistant (G418 and puromycin) colonies were expanded and screened for increased target protein expression by Western blot. Cells infected with retrovirus carrying empty vectors were used as controls.
Regression of LNCaP 104-R2 xenografts. Six weeks after inoculation of LNCaP 104-R2 passage 285 (P285) cells, mice in the treatment group were implanted s.c. with testosterone pellets to mimic elevation of serum testosterone in patients after termination of androgen ablation therapy. Control mice were implanted s.c. with cholesterol pellets. Tumors in the treatment group started to regress within 1 week, whereas all tumors in the control group continued to grow (Fig. 1A). Six weeks after pellet implantation, 24 of 33 tumors relapsed and began to increase in size, but 9 of the regressed tumors never relapsed. Removal of the testosterone pellet did not affect the growth of these adapted R2Ad tumors (Fig. 1B), indicating that growth of R2Ad tumors was androgen-insensitive. Testosterone pellet implantation in mice bearing 104-R2 tumors initially increased the serum PSA level 5-fold (Fig. 1C,D), but as the growth of 104-R2 tumors was suppressed by testosterone and R2Ad tumors developed, the serum PSA level decreased 4-fold. Removal of the testosterone pellet from the mice bearing R2Ad tumors decreased the serum PSA level further to a level similar to that in intact mice bearing 104-S tumors (Fig. 1C,D).
On average, during the progression from 104-R2 to R2Ad tumors, AR mRNA reduced 7-fold (Fig. 1E). The AR protein level in 104-R2 tumors was dramatically increased compared to 104-S tumors, and AR protein in R2Ad tumors was reduced to a level comparable to that in 104-S tumors (Fig. 1E). The PSA mRNA and protein in 104-R2 tumors in castrated mice without testosterone pellets was 4- to 6-fold higher than that of 104-S tumors in intact mice (Fig. 1F). After testosterone pellet implantation, the PSA mRNA and protein increased another 2- to 3-fold. R2Ad tumor endogenous PSA mRNA and protein decreased slightly after removal of testosterone pellets (Fig. 1F).
Establishment of LNCaP R2Ad cells. To study the molecular mechanism of androgenic growth inhibition, we cultured LNCaP 104-R2 P285 in medium containing physiological concentrations of androgen to generate R2Ad cells. 104-R2 P285 cells treated with 10 nM synthetic androgen R1881 for 6–8 days remained attached but enlarged. By day 14, slow-growing colonies appeared. After passaging, the enlarged 104-R2 cells could not re-attach. Colony counts indicated that the attached cells, called R2Ad cells, arose from 104-R2 populations at a frequency of approximately 1 in 1000. The morphologies of 104-R1, 104-R2, and R2Ad cells were similar, but were very different from 104-S cells (Fig. 2A). The proliferation rate of the R2Ad cells was very slow during the first three passages, but increased afterwards. Treatment with R1881 at 0.1 nM and 1 nM increased growth of 104-S cells by 50% and 10%, respectively (Fig. 2B). In the absence of androgen, 104-R2 cells proliferated 50% faster than 104-S cells; however, 0.1, 1, and 10 nM R1881 caused 45%, 57%, and 57% inhibition of 104-R2 cell proliferation, respectively (Fig. 2B). In the presence of 5.5 μM bicalutamide, 0.1 nM and 1 nM R1881 increased 104-S cell population by 6% and 41%, respectively, indicating that the most optimal growing condition for 104-S cells shifted from 0.1 nM R1881 to 1 nM R1881 when co-treated with bicalutamide (Fig. 2C). R1881 at 10 nM suppressed proliferation of 104-S cells when 5.5 μM bicalutamide was present. Bicalutamide at 5.5 μM blocked the suppression of 0.1 nM R1881 treatment in 104-R2 cells but could not block the suppressive effect of androgens at higher concentrations. These results may reflect the low affinity of bicalutamide for AR. In the presence of 5.5 μM bicalutamide, treatment of 1 nM R1881 and 10 nM R1881 caused 64% and 93% reduction of the proliferation of 104-R2 cells, respectively (Fig. 2D). It is interesting that cotreatment of anti-androgen bicalutamide and high concentrations of androgen almost completely inhibited the proliferation of LNCaP 104-R2 cells. Cotreatment with the anti-androgen bicalutamide (Casodex) blocked the androgenic effect of R1881, confirming that the effect of androgen on cellular proliferation was mediated through AR. The proliferation rate of R2Ad cells was only 53% of parental 104-R2 cells (Fig. 2B). Neither R1881 nor bicalutamide affected the proliferation of R2Ad cells, suggesting that R2Ad cells are androgen-insensitive (Fig. 2B,C).
Cell cycle regulation by androgens in LNCaP 104-S, 104-R2, and R2Ad cells. R1881 increased the percentage of cells in S phase and decreased the percentage of cells in G1 phase in 104-S cells, but R1881 caused G1 cell cycle arrest in 104-R2 cells (Fig. 2D). The S-phase population of 104-R2 cells in the absence of R1881 was 4.8-fold higher than that of 104-S cells. In the presence of R1881, the S-phase population in 104-R2 cells dropped 21% and was slightly higher than that of 104-S cells. However, R1881 had no effect on cell cycle distribution of R2Ad cells (Fig. 2D). R1881 increased c-Myc and Skp2 but decreased p27Kip1 protein in 104-S cells. In contrast, R1881 decreased c-Myc and Skp2 but increased p27Kip1 protein in 104-R2 cells (Fig. 2E). R1881 did not alter the protein abundance of c-Myc, p27Kip1, or Skp2 in R2Ad cells. R1881 had very little effect on p21Cip in any of these LNCaP sublines (Fig. 2E).
Role of AR, Skp2, and c-Myc in androgenic suppression. The AR protein expression level was higher in 104-R2 cells than in 104-S cells in the absence of androgen. The AR expression level was barely detectable in R2Ad cells (Fig. 3A). Androgen receptor translocates to the nucleus upon binding androgen to activate gene transcription, thus we compared the subcellular localization of AR in response to androgen in these cells. In the absence of androgen, no AR was detected in nuclei of 104-S cells but a significant amount of AR was present in the nuclei of 104-R2 cells (Fig. 3B). R1881 treatment increased the amount of nuclear AR in 104-S, 104-R2, and R2Ad cells. However, AR was undetectable in the nuclei of R2Ad cells treated with 0 or 0.1 nM R1881, suggesting that nuclear AR was no longer required for proliferation and survival of R2Ad cells. R1881 treatment increased the amount of cytoplasmic AR in 104-S and R2Ad cells but not 104-R2 cells (Fig. 3B). R1881 slightly increased expression of AR mRNA in 104-S and 104-R2 cells but not R2Ad (Fig. 3C). The AR mRNA level was very low in R2Ad cells and may be responsible for the low level of expression of AR in these cells. The effect of androgen on the various cells lines may reflect transcriptional activity of AR. Transcriptional activity of AR in 104-S, 104-R2, and R2Ad cells was assessed by examining androgenic induction of protein and mRNA expression of the AR target gene, PSA. The PSA protein levels increased with R1881 treatment but were much higher in 104-R2 than in 104-S cells (Fig. 3A). The expression level of PSA mRNA correlated with protein expression levels in 104-S, 104-R2, and R2Ad cells treated with R1881 (Fig. 3D). When R1881 was absent, the PSA mRNA level was higher in 104-R2 cells compared to 104-S or R2Ad cells (Fig. 3D).
To determine if the AR expression level was responsible for the response of R2Ad cells to androgen, R2Ad cells were transduced with a pLNCX2-AR retroviral expression vector. This restored androgenic stimulation of PSA expression (Fig. 4A) and androgen repression of cell proliferation (Fig. 4B). The knockdown of AR by shRNA in clones 1, 6, and 10 was approximately 90%, 90%, and 100%, respectively, whereas knockdown of AR in clone 16 was approximately 50% (Fig. 4C). All shRNA AR-knockdown clones expressed reduced the level of PSA (Fig. 4C). Therefore, knockdown of AR by shRNA in 104-R2 cells reduced androgen-stimulated PSA expression. Clone 10, which expressed no detectable AR protein level, expressed the least PSA; in contrast, clone 16, which expressed 50% of AR protein compared to control, expressed approximately half the amount of PSA compared to control. Proliferation of clones 1, 6, and 10 was androgen-insensitive (Fig. 4D). Proliferation of clone 16 was suppressed by androgen, however, the androgenic suppression was much less compared to control 104-R2 cells. These observations indicated that reduction of 50% AR protein expression in 104-R2 was not enough to generate androgen-insensitive phenotype but partially blocked the growth inhibition caused by androgens. In contrast, reduction of 90% of AR protein expression in 104-R2 cells generated a totally androgen-insensitive phenotype. These results also suggested that downregulation of AR allowed the 104-R2 cells to escape from androgenic suppression. (Fig. 4C) and generated an androgen-insensitive phenotype (Fig. 4D). We next determined whether Skp2 or c-Myc is essential for growth inhibition caused by androgen. Overexpression of Skp2 and c-Myc both partially blocked the androgenic growth inhibition (Fig. 4E,F). A better rescue effect was observed in c-Myc overexpressed 104-R2 cells.
We used testosterone pellet and R1881 treatment to mimic the elevation of serum testosterone levels due to termination of long-term androgen ablation therapy. Using LNCaP 104-R2 P285 xenograft and cell culture as a model, we studied the effect of androgen on the proliferation and cell cycle of relapsed AR-positive prostate cancer cells. We found that androgen caused G1 cell cycle arrest in 104-R2 P285 cells through AR, Skp2, and c-Myc. Overexpression of both Skp2 and c-Myc (Fig. 4F) in 104-R2 partially blocked the growth inhibition caused by androgen treatment, which suggested that other signaling molecules might also be involved in the androgenic suppression. Elevation of serum testosterone in mice bearing 104-R2 tumors initially increased the serum PSA level, but as the growth of 104-R2 tumors was suppressed by testosterone and R2Ad tumors developed, the serum PSA level decreased by several fold. Our observations may provide molecular mechanisms to explain why two clinical studies observed that patients with relapsed prostate tumor expressing elevated PSA responded to termination of androgen ablation therapy.(24,25) When the serum testosterone level increased, the serum PSA level first increased, then dropped and remained normal for a long period of time.(24,25)
Androgen binding induces a conformational change in the AR, which facilitates the formation of AR homodimer complexes and allows AR to enter the nucleus and bind to androgen-response elements in the promoter regions of target genes.(26) Nuclear localization of AR in the absence of androgen in 104-R2 cells may thus promote androgen-independent proliferation and survival (Fig. 3B). Prostate-specific antigen is the most common marker used for detecting prostate cancer growth in patients.(27) Nuclear AR may also be involved in PSA mRNA and protein expression in 104-R2 cells without androgen treatment (Fig. 3A). Ligands other than androgens or phosphorylation of AR may contribute to this androgen-independent AR transcription activity. The observations that androgen-independent 104-R2 cells expressed PSA in the absence of androgen (Fig. 3A) and that serum PSA level in castrated mice bearing 104-R2 tumors without a testosterone pellet implant was 8-fold higher than in intact mice with 104-S tumors (Fig. 1C) may be similar to the clinical situation where hormone refractory prostate cancer develops in patients after androgen deprivation therapy. Often, the serum levels of PSA in these patients increase several fold but the serum testosterone level is very low due to the androgen ablation therapy.(28,29)
The level of p21Cip in 104-R2 and R2Ad cells was higher than that in 104-S cells (Fig. 2B). p21Cip positivity is reported to be correlated with lower disease-free survival in prostate cancer patients(30) and elevation of p21Cip protein expression is associated with progression to hormone refractive growth in both prostate cancer patients and a mouse xenograft model,(31,32) indicating that 104-R2 and R2Ad cells represent a more advanced disease phenotype compared to 104-S cells. Skp2 is an F-box protein that targets p27Kip1 for degradation(33) and the observed changes in Skp2 and p27kip1 are consistent with the function of Skp2 (Fig. 2E). c-Myc protein, a well-known proto-oncoprotein, is an important transcriptional regulator of the androgenic response and cell proliferation in prostate cancer.(9,34) c-Myc mRNA and protein expression increase during progression of prostate cancer.(28,35) c-Myc was found to be downstream of AR and overexpression of c-Myc promoted androgen-independent growth of LNCaP cells.(36) c-Myc levels in R2Ad cells were several fold higher than that in 104-S and 104-R2 cells (Fig. 2E). Elevation of c-Myc may compensate for the requirement of AR signaling for cell proliferation in R2Ad cells.
LNCaP cells express a mutant AR (T877A) that displays relaxed ligand binding specificity,(37,38) however, androgenic suppression is not limited to LNCaP cells. PC-3 is a commonly used human prostate cancer cell line established from a bone-derived metastasis but does not express AR.(39) Our unpublished data about experiments using PC-3 cells overexpressing wild-type AR indicated that androgen also suppressed these cells through reduction of Skp2 and c-Myc, suggesting that the molecular mechanism we observed is possibly a universal phenomenon in AR-positive castration-resistant prostate cancer cells. Other groups also observed that physiological concentrations of androgens caused growth inhibition and G1 cell cycle arrest caused in PC-3 cells overexpressing full-length wild-type AR(40–42) or ARCaP cells. ARCaP is another AR-positive prostate cancer cell line derived from the ascites fluid of a patient with advanced metastatic disease.(43–45) Their observations suggested that the androgenic suppression phenomenon was not limited to LNCaP cells. We believe that our study may have potential applications in the clinical treatment of AR-positive castration-resistant prostate cancers.
This work was supported by CS-100-PP-12 (National Health Research Institutes), DOH100-TD-C-111-014 (Department of Health), and NSC 99-2320-B-400-015-MY3 (National Science Council) in Taiwan for C.-P.C. and US National Institutes of Health grant CA58073 for S.L.