Androgen Receptor is highly over-expressed by Castrate Resistant Prostate Cancer Cells
AR-expressing human prostate cancer xenografts derived from localized prostate cancers [i.e., PC82 (34
) and CWR22 (35
)] and cell lines from both localized prostate cancer [i.e., E006AA (36
)] and lymph node metastasis [i.e., PacMetUT1 (37
)] have been established derived from hormonally naïve patients. In addition, AR-expressing castrate resistant prostate cancer (CRPC) cell lines have been established from metastases harvested from patients progressing on androgen ablation therapy [i.e., LNCaP and LAPC4 from lymph node metastases and VCaP and MDA-PCA-2b from bone metastases (23
)]. The level of AR protein expression by these xenograft/cell lines was compared to localized prostate cancer cells (L-PCA) and normal prostate epithelial cells (i.e., N-PrEC) analyzed directly from hormonally naïve patients without culturing. These comparisons documented that AR protein levels in localized as well as metastatic prostate cancer cells from hormonally naïve patients range from 1–6 fold that of normal prostate epithelial cells, . In contrast, AR is >25 higher in all of the CRPC lines (i.e., ranging from 27–90 fold higher compared to N-PrEC and from 9–30 fold higher compared to L-PCA cells harvested from hormonally naïve patients not adapted to a castration resistant state, .
AR is characteristically increased in human castrate resistant prostate cancer (CRPC) cell lines and CRPC clinical specimens
To evaluate whether this increase in AR expression by CRPC cell lines is an in vitro artifact of cell culture, mRNA expression of AR was compared in 23 normal prostates obtained from organ donors, 6 normal prostate tissues obtained from prostatectomy specimens, 38 localized prostate cancers from prostatectomy specimens of hormonally naïve patients, and 18 prostate cancer metastases obtained at warm autopsy from castrate resistant patients. AR RNA is over-expressed by 4-fold in metastatic CRPC compared to localized prostate cancer from hormonally naïve patients and more than 4-fold compared to normal prostate, . These results document that increased AR expression in CRPC cell lines is not an artifact of culture.
Adaptive Androgen Receptor auto-regulation is an acquired ability by CRPC cells
AR gene is amplified in prostate cancers and this occurs more frequently in cancers progressing on hormonal therapy than in hormonally naïve patients (38
). AR amplification, however, only occurs in a minority of CRPCs (38
). This suggests that the characteristic > 25 fold increase in AR protein expression usually is not due to initiating genetic events in prostate carcinogenesis but to molecular changes acquired during progression required for CRPC cells to selectively grow in a low androgen environment in androgen ablated patients. To evaluate this possibility, two independently derived androgen dependent human xenografts (i.e., PC82 and CWR22) derived from localized prostate cancer tissue from hormone naïve patients (34
) were used.
When xenografted into ten intact adult male immune-deficient mice whose normal serum testosterone (T) is 3.5 +/− 1.5 nM, which is the low end of the physiological range for an intact adult human male (39
), both PC82 and CWR22 cancers grow in all animals, . While AR in these cancers is slightly elevated compared to normal prostates from hormonally naïve patients, (i.e., 1.6 +/− 0.4 in PC82 and 6.3+/− 1.5 fold in CWR22), this elevation is much lower than the >30 fold elevation characteristic of CRPCs, . Also unlike the CRPCs, none of ten PC82 cancers and only one of ten CWR22 cancers grew over a 1 year observation period when inoculated into castrated male mice whose serum T is < 300pM [i.e., high end of ablation range in castrated patients (39
)]. These results document that not all androgen dependent human prostate cancer cells have the ability to adapt and become castrate resistant. Interestingly, the one CWR22 cancer which did eventually grow in a castrate was not palpable until 6 months, but after this lag-time, it grew at a rate comparable to that of CWR22 cancers growing in intact males, . These results are consistent with cancer cells in this one tumor stochastically acquiring molecular changes allowing its progression to a castration resistant state.
In Vivo adaptive AR auto-regulatory response of castrate resistant CWR22RH cancer to varying androgen environments
In this single castrate resistant cancer (i.e., termed CWR22RH), AR level is enhanced by 3.5 fold compared to the parental CWR22 tumors growing in intact mice, . Since CWR22 cancers in intact hosts already express a 6 fold higher level of AR than normal prostate epithelial cells, , this means that CWR22RH cancer cells growing in a castrate express AR at a > 20-fold higher level than normal prostate epithelial cells in an intact host. This raises the issue of whether such increased AR expression is constitutive in such CRPC cells or whether these cells acquire an adaptive ability to auto-regulate AR expression to optimize growth under varying androgen conditions. To address this question, the original castrate resistant CWR22RH cancer was serially passaged five times in castrated hosts. Such serial passage in castrated hosts results in CWR22RH cancers which eventually grow equally well in intact and castrated animals, . While their growth is similar in either low or high serum androgen, their level of AR is inversely related to the level of serum androgen (i.e., low androgen-high AR in castrate vs. high androgen-low AR in intact host), . These results document that the development of castration resistance can involve acquiring adaptive auto-regulation of AR to optimize growth under changing host levels of androgen.
Paradoxical death of castrate resistant prostate cancer cells constitutively expressing high AR in intact vs. castrated hosts
The previous results raise the issue of the consequence of disrupting this adaptive AR auto-regulation upon the growth of CRPC cells under different levels of systemic androgen. To test this, CWR22-Rv1 cells were transduced with an AR/GFP lenti-viral construct in which AR expression is driven by a strong constitutive promoter and GFP expressing clones isolated and screened for AR expression by Western blot analysis. A clone [i.e., termed CWR22-Rv1(4X/AR)] which constitutively expressed a 4-fold enhanced level of AR regardless of androgen level in the media was expanded and the cells inoculated into intact and castrated mice. Paradoxically, growth of CWR22-Rv1(4X/AR) cells is much better in castrates than in intact hosts, . This difference in growth is not due to a difference in the percentage of cancer cells in cycle (i.e.,> 40% of the cells express the cell cycle marker Ki-67 in both intact and castrated animals), but instead is due to a 3-fold (p<0.05) increase in the percentage of cancer cells dying in intact vs. castrated animals (i.e., 44+/− 7% dying in intact treated vs. 15+/− 2 % in castrated hosts) determined by flow cytometric analysis.
These results document that if AR expression is too high, exposure to a physiologic level of androgen paradoxically stimulates death of CWR22-Rv1 cells in vivo
. These results raise 2 issues. First, is this response unique to CWR22-Rv1 cells and second, if not, is such androgen-dependent stimulation of death in vivo
due to a direct effect on the cancer cells themselves or via indirect effects requiring other host cells? To answer these 2 questions, LNCaP cells was utilized since this additional CRPC line grows continuously in culture, , and secretes the AR-dependent marker, PSA (i.e.,> 200 ng PSA /106
cell/day) in the presence of the castrate level of androgen provided by 10% FBS-containing media (39
). In this low androgen environment, these CRPC cells express AR protein at >10-fold higher level than hormonally responsive primary cancers from untreated patients and >30-fold higher than normal prostate epithelial cells, . The level of AR in these cells is not a constitutive characteristic, however, but can be dynamically regulated. For example, when these cultured cells are rapidly exposed to the anti-androgen, bicalutamide, AR is down regulated by >3 fold, . Associated with AR downregulation is a profound (p<0.05) inhibition of growth, , a 10-fold inhibition in PSA secretion (i.e., reduced to < 20 ng PSA/106
cell/day), and decreased expression of the cell cycle marker Ki-67 from 79+/− 11 % to 11+/− 4% following exposure to bicalutamide. In contrast to AR downregulation by bicalutamide, when LNCaP cells are acutely exposed to a supraphysiological level of the non-metabolizable synthetic androgen, methyltrienolone [aka R1881] (i.e., 10nM R1881 which is biologically equivalent to >25nM testosterone), AR is stabilized and its nuclear level is rapidly increased by another 2-fold, , resulting in AR being 60-fold higher than in normal prostate epithelial cells. This results in enhanced AR signaling as documented by a significant (p<0.05) 4-fold increase in PSA secretion (i.e., to a level >800ng/ PSA /106
cell/day) detectable within 1 day of androgen exposure. Paradoxically, however, such androgen-induced 2-fold increase in nuclear AR results in profound (p<0.05) growth inhibition, . Importantly, unlike the growth inhibition produced by bicalutamide, growth inhibition induced by acute exposure to supraphysiologic androgen is not associated with a decrease in the percentage of cells in cycle (i.e., 72+/− 14% Ki-67 positive). Instead, flow cytometric analysis documented that this growth inhibition induced by acute exposure to high androgen is due to a 3-fold increase (p<0.05) in cell death ( i.e., going from 11+/−3 % dying without the high androgen to 34+/−6% of cells dying with 10nM R1881).
Growth and AR response of parental LNCaP cells vs. low androgen adapted LNCaP/A- cells to acute exposure to either anti-androgen or androgen supplementation
These results document that acute exposure to a high level of androgen directly induces death of CRPC cells initially expressing a high level of AR. To further evaluate whether this association between high AR expression and androgen induced death is causally linked, LNCaP cells were chronically maintained in phenol red free RPMI-1640 media with 10% DCC stripped FBS which has an even lower level of androgen than in androgen ablated patients (39
). These cells, termed LNCaP/A- cells adapt to growth in the lower androgen media by additionally increasing their expression of AR protein by 2.5-fold compared to the parental LNCaP cells, . This results in LNCaP/A- cells expressing an AR level that is 75 times higher than normal prostate epithelial cells and 25 times higher than hormone naïve prostate cancers. Associated with this 75-fold increase in AR is complete resistance to growth inhibition by bicalutamide, , even though anti-androgen treatment does lower PSA secretion 2.6-fold (p<0.05) from 54+/−7ng PSA/106
cell/day to 21+/−5ng PSA/106
cell/day. While resistant to bicalutamide, the growth of these LNCaP/A- cells is still profoundly (p<0.05) inhibited by acute exposure to a supraphysiologic level of androgen (i.e., 10nM R1881), . This paradoxical androgen induced growth inhibition is not associated with a decrease in the percent of cells in cycle based upon expression of the cell cycle marker Ki-67 on day 8 (i.e., 85+/− 9% in R1881 treated vs. 91+/− 5% in untreated cells), but is associated with a greater than 3-fold (p<0.05) increase in cell death index (i.e., 8+/− 2 % in untreated cells vs. 27+/− 6% dying in R1881 media). Associated with this paradoxical androgen-induced increase in death is a dramatic increase (p<0.05) in the percent of flow sorted mitotic cells which retain detectable AR expression (i.e., 85+/− 11% vs. > 0.5% in untreated cells). These growth inhibition changes, however, are not associated with decrease secretion of the AR-dependent PSA gene into the media (i.e., 54+/−7ng PSA/106
cells/day in untreated cells vs. 84+/− 35 ng PSA/106
cells/day in R1881 treated cells) documenting that AR-dependent transcription is not inhibited by added androgen. These combined results document that CRPC cells have the ability to adaptively up-regulate their AR level for optimal growth under decreasing androgen environment, but if the androgen level is acutely increased, paradoxically, such adaptive auto-regulation is a liability though over-stabilization of AR in mitosis.
Paradoxical death of castrate resistant prostate cancer cells induced by acute exposure to supraphysiologic androgen involves AR binding to replication complexes
Previous studies documented that in CRPC cells whose proliferation is stimulated by AR signaling, AR must be degraded sufficiently in mitosis or these cells do not completely re-license their DNA for replication completely and thus die in the next cell cycle (12
). These results suggest that AR is a licensing factor needed for DNA replication in CRPC cells. This would explain why these malignant cells adaptively auto-regulate to maintain an optimal level of nuclear AR for DNA replication under varying environmental androgen levels. This would also provide a mechanism for the paradoxical death induced by acute switching of CRPCs from castrate to a supraphysiologic level of androgen via over stabilizing AR/RC complexes preventing full re-licensing in the next cell cycle.
The possibility that AR becomes a licensing factor in CRPC cells is supported by co-IP/IB analysis which have documented that AR binds RCs in proliferating human LNCaP cells (13
). To evaluate whether this is a general observation or unique to LNCaP cells, similar co-IP/IB analysis of AR binding to RC was performed on additional well-characterized human CRPC in vitro
lines were evaluated, all of which are routinely maintained in vitro
in media supplemented with 10 % FBs containing a castrate level of androgen (39
). To evaluate whether AR binds with the RC in CRPCs, a proprietary protocol was used to extract RC proteins from the nuclei of 3 additional CRPC lines (i.e., LAPC4, VCaP, and CWR22Rv1), beside LNCaP, whose growth is also stimulated by AR even when grown in a low androgen environment (i.e., 10% FBS containing media). This protocol fragments nuclear DNA into sizes of less than 100 base pairs (bp), , allowing efficient extraction of DNA-bound multi-protein complexes. The fact that the DNA is less than 100bp is significant since this limits non-specific co-IP of unrelated multi-protein complexes bound to different, but closely located DNA binding locations. Nuclear extracts were initially analyzed by IB for AR protein. These results (i.e., first lane in ) confirm that AR is present in the nuclei of these growing CRPCs even in media containing only castrate level of testosterone. Additional aliquots of these chromatin extracts were then incubated with the validated anti-Orc2, Mcm2, and Cdc6 antibodies to co-IP RC associated proteins. In all 4 human CRPC lines tested, AR is associated with RC licensing proteins Orc2, Mcm2, and Cdc-6, . The association of AR with RC proteins is similarly detected using AR specific antibody for co-IP followed by IB for the RC licensing proteins, .
AR binds RC proteins in AR-dependent but not AR-independent human castrate resistant prostate cancer cells
To determine the biological specificity of association of AR with RC proteins in CRPC lines, similar co-IP studies were preformed with E006AA cells. This line was established from a localized prostate cancer from a hormonally naïve patient (36
). Like the other lines derived from hormonally naïve patients, E006AA cells expresses AR at a level comparable to normal PrECs, , even though these cells have amplified their AR gene and are AR-independent due to a mutation in the AR dimerization domain rendering AR non-functional for growth stimulation or PSA expression (24
). Despite this mutation, however, AR is present within the nuclei of E006AA cells even when grown in castrate level testosterone, . There is no binding between AR and RC licensing proteins in these AR-independent E006AA cells regardless of whether antibodies for the RC proteins, , or AR, , are used for co-IP.
To further document that binding of AR with RC proteins is not an artifact of cellular fractionation, LNCaP cells were exposed to the cell-permeable cross-linker dithiobis[succinimdylpropionate] (DSP) before isolating nuclei. DSP was used because previous studies documented that exposure of cells to this agent results in crosslinking RC proteins without crosslinking protein to DNA (32
). DSP treated cells were lysed in RIPA buffer which contains 0.1 % SDS and a series of detergents to disrupt non-specific protein-protein interactions. These RIPA buffered lysates were then evaluated by IP/IB for the co-incident presence of RC proteins and AR. DSP treatment cross links Orc2 to RC proteins, like Mcm2, in AR dependent LNCaP confirming previous reports with other cell types (32
). Orc2 is also cross linked to AR in the AR dependent LNCaP cells, , documenting that AR is bound to RC even before the cells are fractionated.
AR binding to Orc2 is chemically cross-linkable and present in castration resistant metastatic prostate cancer tissue directly from patients
To investigate whether AR binds with RC licensing factors is an artifact of cell culture, metastatic prostate cancers harvested at rapid autopsy from CRPC patients with no in vitro
culturing was initially evaluated for expression of AR and Orc2 proteins, . Seven out of 12 castration-resistant metastatic tissues had a sufficient growth fraction to allow detection of nuclear Orc2 expressed by the small subset of malignant cells in cycle. This remarkably low growth fraction (i.e., median % Ki-67 positive cancer cells is 4.6) and variable (i.e., range % Ki-67 positive cancer cells is 0.25–26) is characteristic of metastatic CRPC (5
, 40). Interestingly, the 7 tissues which had detectable nuclear Orc2 were the tissues with the highest AR expression, . In the 4 metastatic tissues with the highest nuclear AR, co-IPs of nuclear extracts with anti-Orc2 antibody demonstrated specific binding between AR and Orc2, , confirming that the previous cell line based results are not an artifact of cell culturing.
To determine when during the cell cycle androgen occupied AR binds with RCs, LNCaP cells were synchronized via culturing in isoleucine-free media containing 6% dialyzed FBS plus 1nM testosterone for 40 hrs (33
). This protocol arrests cells in G0
as documented by the decline (p<0.05) in the percent of cells expressing Ki-67 [i.e., a marker present in all parts of the cell cycle except G0
(41)] from 86 +/− 11% to 5+/− 1% within 40 hrs of synchronization. This induced G0
-arrest is also confirmed by the determination that < 5% of the cells incorporate BrdUrd as opposed to 38+/− 7% of unsynchronized cells incorporating BrdUrd. Upon addition of regular growth media containing isoleucine, 10% FBS and 1nM testosterone, the G0
arrested cells are released and progress through the cell cycle undergoing peak DNA replication as determined by BrdUrd incorporation between 24–36 hrs. Thus cells were harvested at various times during the first 48-hour post G0
release and lysates analyzed by IB, . These results document that Orc2, Cdt1, and Mcm2 are expressed in G0
and their expression remains constant throughout G1-progression, as reported previously (42). In contrast, AR, Orc1, Cyclin D1
, Cdk4, Cdc6, and Cdk2 expression are low in early-G1
and increases during progression through G1
, as reported previously (11
, 42–44). AR and Orc1 reach maximum expression within two hours of G1-progression while Cyclin D1
and Cdk4 peak at eight hr, . Induction of maximal expression of Cdc6 and Cdk2, which are markers of mid-G1
and entrance into S-phase respectively (19
), occurs by twenty-four hours of release, .
AR binds with RCs in early-G1 in AR-dependent castration-resistant prostate cancer cells
Based upon these temporal expression studies, LNCaP cells were arrested in G0/early-G1 and harvested at 0, 8 and 24 hours post release into cycle. Using anti-Orc2 and anti-Cdc6 antibodies, co-IPs were performed on nuclear lysates and analyzed for the presence of AR at these specified time points. These studies documented that nuclear AR is bound with Orc2 at all of the time points; however, nuclear AR is only associated with Cdc6 as cells enter mid-G1 phase (i.e., at 24-hours post release), . These results demonstrate that nuclear AR initially binds with RCs in early-G1 as the pre-RC licensing complexes are forming.
To determine whether such AR binding to RCs is required for AR-dependent CRPC’s proliferation, LNCaP cells were exposure for 4 days to 10uM of the anti-androgen antagonist, bicalutamide which inhibited AR stimulated growth, . Using anti-Orc2 antibody, co-IPs were performed on nuclear lysates from these bicalutamide treated vs. untreated cells and analyzed for the presence of AR. This treatment results in the inhibition of AR binding to Orc2, , even though Orc2 and AR are still present within the cell nuclei, . Coincident with this bicalutamide-induced inhibition of AR binding to Orc2, 90% of the cells arrest in G0/early-G1as documented by only 10+/−3% of the bicalutamide treated cells express Ki-67. Such bicalutamide-induced G0/early-G1arrest is also confirmed by the determination that < 5% of the cells incorporate BrdUrd. These results support that AR binding to RC is required for proliferation of LNCaP cells.
If this conclusion is correct, then in LNCaP cells acquiring resistance to bicalutamide inhibition of their growth, AR should still bind to RCs even in the presence of bicalutamide. To test this prediction, the bicalutamide-resistant LNCaP/A- variant described earlier was used. These cells adapted to growth in extremely low androgen by up-regulating their AR expression by 2.5 fold, , allowing these cells to grow at an identical rate in media with or without the addition of bicalutamide, . These bicalutamide-resistant LNCaP/A- cells were exposed for 4 days to bicalutamide during which time neither their growth nor AR binding to Orc2 is inhibited, , documenting that such AR binding to RC is not a non-specific effect but is tightly coupled with proliferation of these CRPCs. In addition, these results document that adaptive AR elevation allows AR in LNCaP/A- cells to continue to bind to RCs even in an extremely low androgen environment.
Bipolar Androgen Therapy (BAT) Increase Death of CRPCs in Xenografts
The previous results document that the characteristic AR auto-regulation by CRPCs paradoxically becomes a liability, when a supraphysiological level of androgen is acutely replaced since it prevents timely and complete degradation of AR bound at the ORS, stalling DNA re-licensing in the next cycle. These results provide a rationale for testing whether sequential cycling between periods of acute supraphysiologic followed by acute ablated androgen can be used to take advantage of the unique vulnerability produced by adaptive auto-regulation of AR and it’s binding to RC in CRPC cells. To determine whether such bipolar androgen therapy (BAT) is therapeutic against CRPC in vivo
, the previously described LNCaP/A- cells were xenografted into castrated NOG mice and half of the animals were implanted with two testosterone (T) filled-silastic capsules. These T-implants rapidly elevate (p<0.05) serum testosterone by more than 60-fold going from< 300 pM in the untreated castrates (n=10) vs.18.3+/−1.7nM in the T-implanted mice (n=10) within 1 day. This elevated androgen levels is supraphysiologic for NOG mice since serum T in intact adult NOG mice (n=50) is only 3–4nM, but this elevated level would be in the high normal range (i.e., 10–35nM) for humans (39
). After two weeks of high androgen replacement, these implants were removed causing the high serum T to acutely decrease to <0.3nM within 1day. After two weeks of low androgen, the group initially given T-implants were re-implanted to cyclically re-elevate the serum T to >18nM and a week later, serum and tumor tissue was harvested. This cyclic BAT results in greater than 70% inhibition (p>0.05) of LNCaP/A- tumor growth, . However, in the setting of this androgen induced growth inhibition, serum PSA level normalized per gram of tumor tissue increases nearly 4-fold when the supraphysiological level of androgen is present (i.e., from 24.5+/− 3.3 ng/ml per gram of tumor in un-supplemented castrates to 95.5 +/− 25.2 ng/ml per gram of tumor in androgenized animals).
Morphologic, Ki-67, and AR Response of casodex resistant LNCaP/A-to Bipolar Androgen Therapy
The growth inhibited cancers in the animals receiving BAT had a slightly lower mitotic index which was 0.32+/− 0.04 % vs. 0.51+/− 0.06% in the non-cycled castrate mice, , but a similar percentage of cancer cells in cell cycle (i.e., both had >75% of cells expressing Ki-67), . These results are consistent with growth inhibition in the animals treated with BAT being due not to a decrease in proliferation but instead to an increase in tumor cell death as the cells progress through cell cycle. This was confirmed based upon an increase (p<0.5) in the cell death index which was 15+/− 4.3% in un-supplemented vs. 39+/−8.2% in cycled mice. In addition, this enhanced death response is associated with an increase (p<0.5) in the percentage of cells in S-phase which was 15+/− 7 % in castrated animals vs. 30+/−6% in androgen-cycled animals; coupled with a decrease (p<0.05) in cells in G0/G1 which was 49+/−9% vs. 25+/− 8 % and G2/M which was 17+/−2% vs. 5 +/−3% in castrated vs. androgen cycled host, respectively. Immunocytochemistry documented that AR is present within the nuclei of LNCaP/A- cells even when they are growing in un-supplemented castrated hosts, . While bipolar androgen cycling did not increase the percentage of cells with nuclear AR (i.e., >90% for both situations), it did induced the aberrant present of AR in mitotic cells, . This is unlike the situation in un-supplemented castrates where AR is never present in any mitotic cells, .