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Androgen independent prostate cancer growth and metastasis are a major cause of prostate cancer death. Aberrant androgen receptor activation due to androgen receptor mutation is an important mechanism of androgen independence. We determined the effectiveness and mechanism of 17α-estradiol (Sigma®) in blocking aberrant androgen receptor activation due to androgen receptor mutation.
We used LNCaP and MDA Pca-2b prostatic tumor cells (ATCC®) containing a mutated androgen receptor and WT estrogen receptor β to test 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression and cell growth. Cotransfection analysis was used to further elucidate the mechanism of 17α-estradiol action. Xenograft animals with an LNCaP prostate tumor were prepared to study the in vivo effect of 17α-estradiol on tumor growth inhibition.
In LNCaP cells 17α-estradiol produced a dose dependent inhibition of cyproterone acetate (Sigma) or dihydrotestosterone induced prostate specific antigen gene expression. In MDA Pca-2b cells 17α-estradiol inhibited cortisol (Sigma) induced prostate specific antigen expression and blocked dihydrotestosterone and cortisol induced cell proliferation in LNCaP and MDA Pca-2b cells, respectively. Cotransfection analysis showed that 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression was medicated via estrogen receptors. In xenograft mice with LNCaP prostate cancer 17α-estradiol but not 17β-estradiol (Sigma) significantly inhibited tumor growth, although each estrogen tended to decrease tumor growth.
Results suggest that 17α-estradiol with less classic estrogenic activity is a potential therapeutic agent for androgen independent prostate cancer due to androgen receptor mutation.
Prostate cancer is the second leading cause of cancer death in American men.1 After prostate cancer has metastasized there is currently no curable therapy available. Standard therapy for metastatic prostate cancer is androgen deprivation to withdraw androgen action.2–4 Although most patients clinically respond to androgen ablation with a high rate initially, unfortunately the tumor inevitably recurs in an androgen independent form and proceeds to further growth and metastasis. Thus, it is imperative to develop new therapeutic agents for androgen independent prostate cancer.
The mechanism of androgen independent prostate tumor growth is not fully understood but it may be categorized as AR positive and AR negative.4,5 More than 80% of androgen independent tumors are AR positive and their growth may be explained by aberrant AR activation due to AR amplification, mutation, abnormal coregulator expression and ligand independent activation.4–6 As many as half of metastatic prostate tumors that escape from androgen ablation therapy have some somatic mutations in the AR gene.6 Mutated ARs have been identified in various prostatic tumor cells, such as LNCaP and MDA Pca-2b.7 AR in LNCaP cells has a point mutation at codon 877 (T877A) of the hormone binding domain, which alters AR ligand binding specificity, and can be activated by progesterone, estrogens and antiandrogens as well as androgen.7 Mutated AR in MDA Pca-2b cells has a double mutation (L701H and T877A), resulting in high affinity to glucocorticoids. 8 Such AR mutations have been found in tumor tissue in patients with androgen independent prostate cancer.6 AR signaling in androgen independent prostate cancer with AR mutations remains intact and is even broadly activated by other stimuli.6–8 Agents with the capability to block aberrant AR activation may be effective for androgen independent prostate cancer therapy.
Historically estrogen has been used as advanced prostate cancer therapy.2 However, it is limited in clinical practice due to classic estrogenic related cardiovascular side effects.9,10 The stereoisomer of βE2, αE2, is a weaker estrogen analogue with less classic estrogenic activity.11,12 It binds weakly to ERα and its receptor complex only transiently binds to the estrogen responsive element.13,14 Recently we reported that αE2 inhibited DHT induced PSA gene expression and cell proliferation in LAPC-4 prostatic tumor cells containing a WT AR. Also, αE2 but not βE215,16 effectively inhibited tumor growth in a LAPC4 xenograft animal model.16
To explore the effectiveness of αE2 for androgen independent prostate cancer we examined the effect of αE2 on inhibiting the aberrant AR activation of PSA gene expression and tumor cell growth in cell cultures and in a xenograft animal model. Results indicate that αE2 significantly inhibited the aberrant AR activation of PSA gene expression and attenuated tumor cell growth.
We used αE2, βE2, DHT, cyproterone acetate and cortisol dissolved in absolute ethanol at a stock concentration of 10−2 M. The PSA5.8-pGL3 reporter construct, and WT ERα and ERβ expression vectors were described previously.15,17
LNCaP cells were cultured in RPMI-1640 medium (Sigma) and MDA Pca-2b cells were cultured in BRFF-HPC1 ™ medium with supplements, as previously described. 18,19 For cell proliferation assay cells were plated in 96-well plates with the density indicated in each experiment in phenol red-free RPMI-1640 medium supplemented with 5% stripped fetal bovine serum. At 24 hours cells were treated with various hormones or vehicle controls. Medium and hormonal agents were replenished every 3 days. We determined the number of viable cells using a CellTiter One Solution Cell Proliferation Assay kit (Promega, Madison, Wisconsin).
PSA in cell culture medium was measured by ELISA (Diagnostic Systems Laboratory, Webster, Texas). Total cellular RNA was extracted using TriPure® reagent. The RNA concentration was determined by ultraviolet absorbance.
RT-PCR was done using the Titan™ One Tube RT-PCR System with 1 µg total cellular RNA, as previously described. 15 We used a pair of specific primers, 5′-GATGAGGGGAAATGCGTAGA-3′ and 5′-CTTGTTACTCGCATGCCTGA-3′, for the human ERβ gene. RT-PCR conditions were 50C for 30 minutes, 94C for 2 minutes, and 40 cycles at 94C for 30 seconds, 60C for 30 seconds and 68C for 45 seconds with a final cycle at 68C for 7 minutes. PCR products were fractionated on 2% agarose gel and visualized by ethidium bromide staining. Human glyceraldehyde-3-phosphate dehydrogenase served as an internal control and yeast tRNA served as a negative control for RT-PCR.
Western blot was done as previously described.20 Briefly, 20 µg total cellular proteins were fractionated on sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, Piscataway, New Jersey). Blots were blocked and incubated with specific primary antibody against AR (Upstate Biotech, Lake Placid, New York) or ERβ (Affinity BioReagents, Rockford, Illinois). After secondary antibody incubation the signal was visualized using the SuperSignal® West Pico Chemiluminescent kit and exposed to film (Kodak, Rochester, New York). We used α-tubulin as an internal control by incubating a specific antibody against α-tubulin (Sigma).
CV-1 cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium (Sigma) with supplements. Cells were cotransfected using the calcium phosphate precipitation method, as described previously.15,17 Briefly, CV-1 cells seeded in 6-well plates were cotransfected with 1.5 µg PSA5.8-pGL3 luciferase reporter, 0.25 µg mutated AR expression vector with or without 0.25 µg ERα or β, or 1 µg of the empty vector pRL-null containing Renilla luciferase as an internal control with pBluescript-SK plasmid (Stratagene, LaJolla, California) to a total of 4 µg DNA per well, as indicated in experiments. At 16 hours after transfection medium was replaced and cells were treated with various hormones or vehicle controls for 48 hours. Luciferase activity was determined using a dual luciferase assay system (Promega) and expressed in rlu to indicate PSA transcription activity.
An LNCaP prostatic tumor xenograft tumor model was prepared and monitored, as previously described.16 Briefly, LNCaP cells with more than 90% viability were mixed with a 50% volume of Matrigel™ to a final concentration of 5 × 106 cells per 0.2 ml. A 0.2 ml aliquot was injected subcutaneously into the left flank of adult male nude mice (Harlan™). Animals were housed aseptically at the animal facility at our institution. We assessed tumor growth, body weight and mobility change twice weekly. Tumor development was followed until the end of the experiment by caliper measurement of its length and width. Tumor volume/size was calculated using the formula, volume = (width)2 × length/2. The animal study was approved by the institutional animal care and use committee, and done in accordance with accepted standards of humane animal care.
When the tumor was approximately 350 mm3, 6 animals each were randomly assigned to 1 of the 3 treatment groups and implanted subcutaneously with a placebo (20 mg cholesterol), αE2 (2 mg αE2 plus 18 mg cholesterol) or βE2 (2 mg βE2 plus 18 mg cholesterol) pellet in the opposite flank for 4 weeks. Tumor size was recorded twice weekly after treatment.
Data are shown as the mean ± SEM. We used 1-way ANOVA and the post hoc Student-Newman-Keuls test to determine differences among multiple groups with p <0.05 considered statistically significant.
DHT and the AR antagonist cyproterone acetate produced dose dependent induction of PSA expression in LNCaP cells (fig. 1). DHT (50 nM) or cyproterone acetate (1 µM) for 72 hours produced an approximately 6-fold increase in PSA compared to the corresponding control in LNCaP cell medium while treatment with the pure AR antagonist CX (5 and 10 µM) did not alter PSA levels in LNCaP cell medium, in agreement with previous reports.7,19 DHT induced PSA expression was partially inhibited by concomitant administration of cyproterone acetate (1 µM) or CX (10 µM) in LNCaP cells (fig. 1, B). Cyproterone acetate and DHT induced PSA expression was attenuated by αE2 or βE2 (fig. 1, C and D). Notably βE2 alone produced significant, dose dependent induction of PSA expression while αE2 only moderately increased PSA at the highest dose of 1 βM (fig. 1, C and D).
In MDA Pca-2b cells containing a double point mutation in the AR ligand binding domain with high affinity to glucocorticoid8 treatment with cortisol (100 nM) for 72 hours significantly increased PSA in cell medium. This was completely blocked by αE2 or βE2 in a dose dependent manner (fig. 2).
To understand the molecular mechanisms underlying the inhibitory modulation of aberrant AR activation of PSA gene expression by αE2 and βE2 we investigated the involvement of ERs in αE2 and βE2 actions using in vitro cotransfection assays in CV-1 cells. Figure 3 shows that PSA transcription activity was significantly induced by DHT (50 nM) and cyproterone acetate (1 µM) when a mutated AR (ARL) was cotransfected with a PSA promoter directed reporter construct, which was partially attenuated by the presence of ERα or ERβ compared to that in mock transfected cells (fig. 3, A). Most importantly αE2 and βE2 completely inhibited cyproterone acetate and DHT induced PSA transcription activity in a dose and ER dependent manner since αE2 and βE2 slightly potentiated the PSA transcription induced by cyproterone acetate via ARL transfected cells without ER expression (fig. 3, C to F).
Estrogen modulation of aberrant AR activation of PSA transcription was further evaluated using the mutated AR (ARccr) detected in MDA Pca-2b prostatic tumor cells.8 Cortisol produced dose and ARccr dependent induction of PSA transcription activity (fig. 4, A). This cortisol induced ARccr transactivation was significantly inhibited by αE2 and βE2 in a dose and ER dependent manner since αE2 and βE2 failed to modulate cortisol action in the absence of ERs in mock transfected cells (fig. 4, D to F). We noted similar αE2 and βE2 inhibition of ARccr transactivation when ARccr was activated by DHT (data not shown).
Since the point mutation of L701H in AR confers AR responsiveness to cortisol,8 we assessed the effects of estrogen on the modulation of cortisol induced ARL701H transactivation. Cortisol induced PSA transcription activity was completely inhibited by αE2 and βE2 via ERα, and attenuated by approximately 50% via ERβ (fig. 5, B and C). This estrogen effect depended on ERs since in the absence of ERs βE2 significantly potentiated the cortisol effect, while αE2 failed to modulate cortisol action (fig. 5, A).
To determine the effects of αE2 and βE2 on aberrant AR activation of cell growth we treated LNCaP and MDA Pca-2b cells with various doses of DHT, cortisol, αE2 or βE2 alone or in combination for various times. In LNCaP cells DHT produced a time and dose dependent dual effect on the number of viable cells (fig. 6, A). This dose dependent, dual action of DHT on LNCaP cell growth, which was reported previously,21 is distinct from DHT induction of PSA expression for unidentified mechanisms.19
Since 0.1 nM DHT consistently stimulates cell growth, this DHT dose was used in all subsequent cell growth experiments. DHT induced cell growth was significantly inhibited by αE2 at doses of 0.01 to 5 µM. However, the effect of βE2 varied and it only inhibited DHT action at high doses (greater than 3 µM) in LNCaP cells (fig. 6, C). In MDA Pca-2b cells cortisol produced dose dependent induction of cell growth, which was significantly inhibited by αE2 but partially attenuated by βE2 in a dose dependent manner (fig. 7). Also, βE2 alone induced cell growth in dose dependent fashion in LNCaP and MDA Pca-2b cells while αE2 slightly induced cell growth in LNCaP but had no effect in MDA Pca-2b cells (figs. 6, B, and 7, A and C).
Consistent with previous reports,15,22,23 AR and ERβ were expressed in LNCaP and MDA Pca-2b cells, as shown by Western blot and RT-PCR (fig. 8, A and B). However, ERα expression was undetectable by Western blot and barely detectable by RT-PCR in LNCaP and MDA Pca-2b cells (data not shown).15
In xenograft animals with LNCaP prostate cancer αE2 inhibited tumor growth. To evaluate the in vivo effectiveness of αE2 for inhibiting prostate tumor growth we treated xenograft animals with LNCaP prostate tumors with a placebo, αE2 or βE2 pellet for 4 weeks and recorded tumor size twice weekly. Initial tumor size was similar in the 3 groups at approximately 350 mm3 (fig. 8, C). Beginning 1 week after pellet implantation αE2 produced notable inhibition of tumor growth and significantly reduced tumor size at the end of the 4-week treatment (αE2 vs placebo mean 1,492 ± 383 vs 2,704 ± 382 mm3, p <0.05). However, tumor size in the βE2 treated group was not significantly different from that in the placebo group at all time points, although we noted moderate reduction. As in our previous study,16 animal body weight and mobility were not significantly altered by αE2 and βE2 vs those in the placebo group (data not shown).
Aberrant activation of AR due to AR amplification, AR mutation, coregulator dysregulation and hormone independent AR activation are often linked to prostate cancer progression and androgen independence. 4,5 However, the mechanisms of androgen independent tumor growth and PSA expression are not entirely understood. Somatic AR mutations with various characteristics have been identified in prostate cancer tissues and cell lines. Some AR mutations broaden its ligand binding specificity, such as ARL (T887A) and ARccr (T877A and L701H). ARL with a mutation of T877A in the AR ligand binding domain is stimulated strongly by βE2 and progesterone, and the AR antagonists 4-hydroxyflutamide and cyproterone acetate.24 The doubly mutated ARccr originally detected in MDA Pca-2b cells has the properties of the ARL and ARL701H mutants as well as high affinity to cortisol and cortisone.8 In agreement with previous studies8,24 we report that ARL cells were activated by DHT, cyproterone acetate and estrogen, and ARccr was strongly stimulated by cortisol, as shown by PSA expression and cell proliferation assays (figs. 1, ,2,2, ,66 and and7).7). These results support the concept that aberrant AR activation of cell proliferation and PSA gene expression by hormones other than androgen in LNCaP and MDA Pca-2b cells is a potential cause of androgen independence in prostate cancer cases.
Since more than 80% of androgen independent prostate cancers are AR positive and mostly AR mediated,5,25 inhibiting aberrant AR activation in tumor cells is considered a major strategy to control androgen independent prostate cancer progression. To our knowledge we report for the first time that αE2 significantly inhibits ARL activation of PSA expression induced by cyproterone acetate and DHT, LNCaP cell growth induced by DHT, and cortisol induced ARccr and ARL701H activation of PSA expression and cell growth in MDA Pca-2b cells (figs. 1, ,2,2, ,66 and and7).7). Furthermore, αE2 but not βE2 significantly inhibited tumor growth in a xenograft animal model of LNCaP prostatic tumors (fig. 8, C). The doses of αE2 and βE2 used in the current animal study were based on a previous study in which the circulating βE2 level attained the physiological concentration of βE2 in females.26 These doses were also used in our recent study in xenograft animals with LAPC-4 prostate tumors, in which αE2 effectively attenuated LAPC-4 xenograft tumor growth and βE2 significantly decreased PSA expression.16
Historically estrogens such as diethylstilbestrol and βE2 have been used to treat advanced prostate cancer, presumably by inhibiting testosterone biosynthesis through negative feedback of the hypothalamus-pituitary-gonadal axis.2 However, using estrogens for androgen ablation therapy is limited in clinical practice due to cardiovascular side effects. 9,10 Discovery of the ER isoform ERβ and its identification in prostate epithelial cells and prostatic tumor cells promote the concept that ligand ERs may directly modulate AR actions on prostate gene expression and cell growth. This is supported by our recent studies15,16 and a previous report.27 In agreement with reports that ERβ is the dominant ER in prostate epithelial and prostate tumor cells,22,28 we found that ERβ is expressed in LNCaP and MDA Pca-2b cells while ERα is undetectable (fig. 8, A and B).15 Our current findings that αE2 and βE2 inhibited aberrant AR activation by interacting with ER further support the direct modulation of AR action by estrogen in prostatic tumor cells.
The stereoisomer of βE2, αE2, is a weak estrogen compared to potent endogenous βE2.29 In mammalian model systems αE2 has no carcinogenic effects11,12 and is less effective in the vascular smooth muscle system.30 However, αE2 is as potent as βE2 for protecting neuronal cells29 and inhibiting DHT induced PSA expression and cell growth in LAPC-4 cells containing a WT AR.15,16 In an LAPC4 xenograft animal model αE2 but not βE2 effectively inhibited tumor growth.15,16 Our study shows that αE2 was more effective than βE2 to inhibit the aberrant AR activation of PSA expression, and cell growth in LNCaP and MDA Pca-2b cells containing mutated ARs, and αE2 but not βE2 significantly inhibited tumor growth in a xenograft animal model with LNCaP prostatic tumors. Together these data suggest that αE2 is a potential therapeutic agent for advanced prostate cancer.
To our knowledge we report for the first time that αE2 mediated via ERβ effectively inhibits the aberrant AR activation of PSA expression and cell growth in LNCaP and MDA Pca-2b cells, and significantly attenuates tumor growth in a xenograft animal model with LNCaP prostatic tumors. Given the efficacy of αE2 for inhibiting aberrant AR activation and the safety of αE2 with its less classic estrogenic actions, we suggest that αE2 may be a potential therapeutic agent for androgen independent prostate cancer due to AR mutation.
Dr. Feldman, Stanford University School of Medicine, Stanford, California, provided AR mutants. Drs. Mosselman and Jansen, N.V. Organon, Oss, The Netherlands, provided the ERβ expression vector. Dr. Chambon, Strasbourg, France, provided ERα expression vectors. CX was obtained from Toronto Research Chemicals, North York, Canada. Hormone pellets were obtained from Hormone Pellet Press, Leawood, Kansas.
Supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Grant DK061004 (YSZ), and a KL2 Award supported by National Institutes of Health Grant KL2RR024997 of the Weill Cornell Medical College Clinical and Translation Science Center (YMQ).
Study received institutional animal care and use committee approval.