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Typically, the initial response of a prostate cancer patient to androgen-ablation therapy is regression of the disease. However, the tumor will progress to an “androgen-independent” (AI) stage that results in renewed growth and spread of the cancer. Both nuclear factor-kappa B (NF-κB) expression and neuroendocrine differentiation predicts poor prognosis but their precise contribution to prostate cancer progression is unknown. This report demonstrates that secretory proteins from neuroendocrine cells will activate the NF-κB pathway in LNCaP cells resulting in increased levels of active androgen receptor (AR). By blocking NF-κB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-κB signaling in vivo, by the absence of the IκBα inhibitor, prevents regression of the prostate after castration by sustaining high levels of nuclear AR, maintaining differentiated function and renewed proliferation of the epithelium. Furthermore, the NF-κB pathway was activated in the ARR2PB-myc-PAI (Hi-myc) mouse prostate by cross breeding into a IκBα +/− haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-κB is sufficient to maintain AI growth of prostate and prostate cancer by regulating AR action. Thus, the NF-κB pathway may be a potential target for therapy against AI prostate cancer.
With the development of the prostate-specific antigen (PSA) assay for screening, prostate cancer is detected at an earlier stage. However, with 218,890 new cases of prostate cancer, there were still 27,050 deaths in the United States in 2007 due to the disease. If prostate cancer remains localized, therapy such as prostatectomy or radiation therapy can cure the patient. For metastatic prostate cancer, standard treatment uses approaches to block androgen receptor activity. Androgen-ablation therapy (luteinizing hormone-releasing hormone analogs that block the production of testicular androgens and/or anti-androgens that directly block AR activation) in the majority of patients results in initial regression of disease and a dramatic decrease in serum PSA. Eventually, however, all patients will fail this therapy and the cancer is commonly referred to as “androgen refractory”, “Androgen Independent (AI)” or “castrate resistant prostate cancer”. At this stage, although there is modest proven benefit with docetaxel treatment, there is no curative treatment. A number of mechanisms have been proposed to explain the acquisition of AI; however, the emerging theme is that the tumor is still dependent upon AR signaling (1;2). The proposed mechanisms that explain continued AR signaling include AR gene amplification resulting in a response to low levels of circulating androgens (3-5), the local synthesis/concentration of androgens (6), AR mutations that allow activation by anti-androgens or weak androgens (7), AR activation by growth factors/kinase pathways (4;8), and/or changes in AR co-regulators (9). Since the AR is still a potential target in patients that fail androgen-ablation therapy, identifying the pivotal pathway(s) that regulate continued AR signaling can result in new therapeutic approaches to treat the advanced disease.
Neuroendocrine (NE) cells are present in the normal and neoplastic prostate (10). Increases in NE phenotype of the cancer and NE secretory products are closely correlated with tumor progression, androgen independence and failure of androgen-ablation therapy in the prostate cancer (11-13). The NE phenotype first appears as focal NE differentiation of adenocarcinoma in the clinically localized prostate cancers (14;15). This is not to be confused with NE prostate cancer, a rare form of androgen-independent prostate cancer that has the pathologic features of small cell carcinoma (16). Rather, in NE differentiation, the adenocarcinoma phenotype is broadly maintained but the cancer cells begin to also express markers of NE cells. For example, expression of chromogranin A, a NE protein, is one of five genes that are reported to serve as outcome predictors for tumor recurrence (17). Although there is no consensus agreement as to whether or not NE cells are important in advanced prostate cancer (18-20), we have previously published that secreted neuropeptides from NE tumor xenografts (NE-10) will support AR activation in adenocarcinoma (LNCaP cells) xenografted at a distant site (4). The virtue of having the NE cancer grafted in the same mouse as the otherwise androgen-dependent LNCaP adenocarcinoma line allowed us to follow the progression to AI in castrated mice (4). Therefore, a pathway allowing progression to AI can be activated by NE secretions. This conclusion is consistent with the recent report that shows PKA-differentiated NE cells enhance AI growth of prostate cancer (21).
Nuclear factor kappa B (NF-κB) proteins are an important class of transcriptional regulators. The mammalian NF-κB family contains five members: NF-κB1 (p105 and p50), NF-κB2 (p100 and p52), c-Rel, RelB, and RelA (p65) (22). These proteins share a Rel homology domain (RHD), which mediates DNA binding, dimerization, and interactions with specific inhibitory factors, the IκBs, which retain NF-κB dimers in the cytoplasm (22;23). Many stimuli activate NF-κB, mostly through IκB kinase–dependent (IKK-dependent) phosphorylation and subsequent degradation of IκB proteins. The inhibitor of κB (IκB) kinase (IKK) complex activates via its two catalytic subunits, IKKβ and IKKα, the classical and alternative NF-κB-signaling pathways, respectively [reviewed in (24)]. In prostate cancer, activity of NF-κB is higher in AI cell lines and AI xenografts compared to androgen dependent grafts (25) as well as in metastatic prostate cancer compared to localized disease (26). Further, elevation of NF-κB activity in primary prostate cancer correlates with a poor prognosis (27;28) and predicts biochemical (PSA) relapse (29;30). By the analysis of multiple microarray studies, the NF-κB pathway was identified as significantly dysregulated in metastatic prostate cancer (31). However, the precise contribution of NF-κB to prostate cancer progression is unknown.
Thus, both NE differentiation and increased expression of nuclear NF-κB in prostate cancer correlates with poor prognosis. This study demonstrates that neuropeptides can activate the NF-κB pathway in LNCaP prostate cancer cells. In addition, activation of NF-κB in the prostate prevents regression after castration of the normal prostate by maintaining both high levels of nuclear AR and continued cell proliferation. Further, activation of NF-κB in the ARR2PB-myc-PAI transgenic model results in continued growth of the prostatic adenocarcinoma after castration. Therefore, the activation of the NF-κB pathway results in progression of prostate cancer to androgen independence.
The human prostate carcinoma cell line LNCaP was obtained from ATCC (Manassas, VA). The cells were cultured in RPMI 1640 (Gibco-BRL) medium containing 5% fetal calf serum (FBS) (Hyclone), 0.1% ITS and 0.1% Glutamine (Gibco-BRL).
NE-10 tumor tissue was cut into 1- 2 mm3 pieces. The small tissue fragments were placed into 100 mm Primaria tissue culture dishes (Becton Dickinson Labware), cultured in RPMI 1640 containing 5% FBS, 10% heat-inactivated horse serum (Hyclone), 1% antibiotic-antimycotic (Gibco-BRL), 50μg/ml gentamicin (Gibco-BRL), 1% L-glutamine, 1% sodium pyruvate, 1M Hepes at 37 °C, in a 5% CO2 incubator. When the explants displayed an initial outgrowth of NE cells (usually one week after plating), the culture medium was changed every two days. Fibroblast cells that contaminated the cultured NE cells were removed by differential trypsinization.
The NGL vector [a NF-κB responsive reporter vector which has Luciferase and Green Fluorescent Protein (GFP) reporter genes] (32) and ARR2PB-Luc vector (an AR responsive reporter vector, which does not respond directly to NF-κB) (33) was used to measure androgen receptor activity were used in the transfection and infection experiments. LNCaP cells were plated at an initial density of 2.5 × 104/well in 24-well tissue culture plates. After 24 hours, the cells were transfected with Lipofectamin (Invitrogen) for four hours according to the manufacturer's protocol. After transfection, the cells were treated with conditioned media (containing NE extracts) and NE peptides [Bombesin (BBS) and Gastrin-Rleasing Peptide (GRP), 10−8 M each)] (Sigma). To generate the conditioned media containing NE secretions, RPMI 1640 medium (containing 5% dextran-charcoal-stripped serum, 0.1% ITS and 0.1% Glutamine) was added to the NE cell culture dish. After 24 hours, the medium (containing NE secretions) was harvested and transfered to targeted cells (LNCaP cells). RPMI 1640 medium (containing 5% dextran-charcoal-stripped serum, 0.1% ITS and 0.1% Glutamine) was added to the targeted cells as the control. All experimental groups were tested with a dose response curve for DHT (10−9 - 10−8 M) with or without Bicalutamide (10−5 M) (Zeneca). The transfection efficiency was determined by co-transfecting pRL-CMV containing the Renilla luciferase reporter gene (Promega). Luciferase activity was determined using the Promega Corp luciferase assay system 24 hours after transfection. The values plotted represent the mean of at least three individual samples ± SD. IκBα-DN adenovirus (a mutant avian IκBα with serine to alanine substitutions that prevent phosphorylation and degradation) (34) was used to block NF-κB signaling in the infection experiments and the empty adenovirus was used as control.
Total RNA from LNCaP cells at 48 hours after infection with RelA expression vector or empty adenovirus (as control) was extracted using Trizol (Gibco-BRL), and residual genomic DNA was removed by DNaseI (Invitrogen) treatment. The RNA was reverse transcribed using random primers and Superscript II (Gibco-BRL) according to the manufacturer's protocol. The primers used to amplify AR were 5'-ATCAGGGGCGAAGTAGAGCATC-3' (forward), 5'-AGCCCCACTGAGGGGACAAC C-3' (reverse). Real-time PCR reactions were carried out in a 20μl volume using a 96-well plate format and fluorescence was detected utilizing the Bio-Rad I-Cycler IQ Real-time detection system.
We extracted cytoplasmic and nuclear proteins from LNCaP cells at 48 hours after infection with RelA expression adenovirus vector (35) or empty vector (as control) using a nuclei extraction kit (Pierce) according to the manufacturer's instruction. A 20μg aliquot of each protein sample was separated on a 4 to 12% Trisglycine gradient gel (NOVEX™), and then transferred to nitrocellulose membranes (Schleicher & Schuell). The membranes were blocked with 5% skim milk in TBS-T (Tris-buffer saline, 1% Tween-20) buffer. The AR antibody (clone N20, Santa Cruz) was added at the optimal concentration (1:1000) and the blots were incubated 1 hour in room temperature. After washing three times for 10 minutes each in TBS-T, incubation was performed for 1 hour with the secondary horseradish-peroxidase-conjugated goat anti-rabbit antibody. The signals were detected using the ECL system (Amersham Biosciences).
All animal studies were conducted in accordance with the principles and procedures outlined by the NIH guide and the Vanderbilt Institutional Animal Care & Use Committee. A small fragment (about 50mg) of NE tumor from the NE-10 allograft model (36) was implanted subcutaneously into the right flank of six week-old male athymic nude mice (BALB/c strain). Mice were separated into two different groups. Eight mice with or without NE tumor were subsequently castrated via scrotal approach two weeks after NE tumor implantation. The mice were sacrificed four weeks after NE tumor implantation (two weeks after castration). Prostates were excised and fixed in 10% buffered formalin and paraffin-embedded for immunohistochemical analysis.
The IκBα −/− mice die at 6-9 days after birth due to constitutive NFκB activation (37;38). Therefore, to examine a mature −/− prostate requires rescuing the prostate from a newborn IκBα −/− mouse. Prostatic rescue is achieved from −/− mice that are either embryonic lethal or die shortly after birth by grafting the prostate under the kidney capsule of male athymic nude mice. Prostates from newborn mice (IκBα−/−, IκBα+/− and wild type) were grafted under the kidney capsule of male athymic nude mice and allowed to mature for 6 weeks in the male host. Then, host mice were castrated for two additional weeks. Each experimental group consisted of at least 4 mice. Prostates were excised and fixed in 10% buffered formalin and paraffin-embedded for histological and immunohistochemical analysis.
We developed a constitutively activated NF-κB prostate cancer mouse model (Myc/IκBα+/− mouse) by crossing the IκBα+/− mouse with the ARR2PB-myc-PAI (Hi-Myc) line which develop invasive adenocarcinoma in the prostate by 6 months of age (39). Both Myc and Myc/IκBα+/− transgenic mice were castrated at 6 months and the prostates were harvested at two weeks after castration for analysis. Each experimental group consisted of at least 4 mice. Prostates were excised and fixed in 10% buffered formalin and paraffin-embedded for histological and immunohistochemical analysis.
Paraffin-embedded tissue sections of the prostate were stained immunohistochemically with antibodies against AR (clone N20, Santa Cruz), Probasin (M-18, Santa Cruz) and Ki67 (clone TEC-3, DACO). The primary antibody was incubated at the appropriate concentration (AR: 1:1000; Probasin: 1:1000; Ki67: 1:1000) for one hour at room temperature. The secondary antibody was incubated for 60 minutes,being either horseradish-peroxidase-conjugated goat anti-rabbit or goat anti-mouse(1:1000), respectively. Slides were rinsed extensively in tap water, counterstained with Mayer's hematoxylin and mounted. For quantitation of the prostate proliferation, the cells were counted as positive for Ki67 when nuclear immunoreactivity was observed. The positive cells for ki67 were counted by monitoring at least 200 luminal epithelial cells from 3-5 different fields of each sample. Each group had at least 4 mice. The results are reported as mean value (%).
Where appropriate, experimental groups were compared using Student's two-tailed t-test, with significance defined as P <0.05. For quantitation of immunoblot data, images were analyzed using Scion Image software, version 1.62 (Scion Corp., Frederick, MD).
Many studies have indicated that BBS and GRP are NE cell secretory peptides, and the serum BBS levels are significantly elevated in AI prostate cancer patients (11;40;41). In addition, our studies have demonstrated that NE-10 tumors secrete BBS and GRP. In order to understand how NE cells influence prostate/prostate cancer growth and progression, especially after androgen-ablation therapy, we investigated the mechanisms by which NE secreted neuropeptides affect AR action using BBS and GRP, the known NE secreted peptides, and NE conditioned medium (obtained from primary cultured NE cells). To investigate whether the NE secreted peptides affect activation of NF-κB signaling, LNCaP cells were treated with or without BBS and GRP after transient transfection with the NGL vector, an NF-κB responsive luciferase reporter. The neuropeptides (BBS and GRP, 10−8 M each) increased the activation of the NGL-luciferase reporter by 3-fold in LNCaP cells in the absence and 2-fold in the presence of androgen (DHT; Fig. 1A). These results suggested that neuropeptides increase functional activation of NF-κB signaling in LNCaP cells both in the presence and absence of androgen.
To further understand how the NE secreted neuropeptides affect AR action. ARR2PB-Luc, an AR responsive reporter vector, which does not respond directly to NF-κB, was used to measure AR activity. LNCaP cells were transfected with the ARR2PBLuc construct and infected with adenoviral vectors expressing a dominant inhibitor of the NF-κB pathway (IκBα-DN) or empty adenovirus (control) (Fig. 1B). In the absence of androgen, ARR2PB promoter activity was not detected in the cells either in the presence or absence of NE peptides (BBS and GRP) and NE secretions (conditioned medium containing NE extracts). In the presence of androgen (10−9–10−8 M), the activity of the ARR2PB promoter, when treated with NE peptides (BBS and GRP) or conditioned media from NE cultured cells, was 1.5 to 3-fold higher respectively than that when NE peptides or NE secretions were absent (white bars) (Fig. 1B). This effect was blocked by Bicalutamide, an inhibitor of the LNCaP mutated AR. Statistically significant induction of the reporter occurred even at lower concentrations of androgen (10−9 M of DHT) plus neuropeptides than with androgens alone. However, the greatest inductions occurred at higher concentrations of androgens (10−8 M of DHT) plus neuropeptides. The slightly higher activation by NE secretions relative to BBS plus GRP suggests that other neuropeptides may be present in this conditioned media. In all cases, the increased AR activity can be blocked by IκBα-DN (gray bars) ie. by inhibiting of NF-κB activity (Fig. 1B). These results indicate that NE secreted factors increase functional activity of AR through the NF-κB pathway in LNCaP cells.
To investigate how NF-κB signaling affects activation of the AR, LNCaP cells were infected with adenoviral vectors expressing RelA, the transactivating subunit of NF-κB (an empty adenovirus was used for the control). The data from real time RT-PCR shows that AR mRNA levels are increased in LNCaP cells about 2 to 3-fold after infection with RelA adenovirus independent of the presence of androgen (DHT) (P <0.05) (Fig. 2A). When androgens were absent, western blot analysis showed that AR protein levels are only increased in the cytoplasm with no change in nuclear protein levels (Fig. 2B and C-a). However, when androgens and RelA were both present, the AR protein levels showed a small increase in cytoplasm and a large increase in the nuclear compartment (Fig. 2B and C-b). This affects were further confirmed by immunocytochemistry staining of AR (Supplementary Fig. S1). These results suggest that NF-κB signaling increases the transcription and/or stability of AR in LNCaP cells, resulting in increased AR levels. This data is consistent with our published in vivo observation that neuropeptides increase AR levels 2-fold in LNCaP grafts in castrated mice (4).
To investigate the effect of the NE cells on NF-κB signaling in the normal prostate, a small fragment (about 50 mg) of a NE tumor from the NE-10 allograft model (36) was implanted subcutaneously into the flank of six week-old male athymic nude mice. Mice with or without the NE tumor were castrated two weeks after NE tumor implantation. Prostate tissues were harvested two weeks after castration for analysis (Fig. 3). The results showed that after castration, AR staining was more intense in nuclei of prostate tissue from mice hosting NE allografts, relative to AR in the prostate of mice not carrying the allograft. Castrated allografted mice had proliferative luminal epithelial cells as determined by Ki67 staining, but significantly fewer proliferative luminal epithelial cells were detected in castrated mice without a NE tumor graft (Fig. 3A and B). Further, heterogeneous staining for androgen regulated probasin occurred in castrated mice bearing the NE grafts (Fig. 3A), as well as general secretory dorsolateral prostate proteins as detected by the DLP antibody (42) (data not shown). However, some areas in the prostate were negative for these markers of differentiation (data not shown) suggesting that certain populations of cells specifically respond to neuropeptides. These findings indicate that factors secreted from NE cells can act systemically to stimulate the continued activation of AR signaling in the absence of testicular androgens, thus maintaining prostatic differentiation and proliferation.
In order to determine that activation of the NF-κB pathway is sufficient for AI growth of the prostate, we utilized a knockout mouse model of IκBα (37), the major inhibitor of NF-κB function (43). IκBα−/− mice die at 6-9 days after birth due to constitutive NF-κB activation (37;38). Therefore, to examine a mature IκBα−/− prostate we rescued the prostate from newborn mice. Prostatic rescue was achieved by grafting the urogenital sinus from 20 day embryonic or newborn mice under the kidney capsule of male athymic nude mice, as previously described (44). Urogenital sinus from wild type, haploid insufficient (IκBα+/−) and IκBα−/− were grafted and allowed to mature for 6 weeks in the male athymic nude mouse host. The host mice were then castrated and after two additional weeks, killed, and prostatic grafts removed from the kidney capsule. As expected, wild type control prostatic grafts regressed after castration showing atrophic glands, with limited to undetectable nuclear AR and no Ki67 staining (Fig. 4A). However, after castration, IκBα+/− and IκBα−/− prostatic grafts had strong nuclear AR staining (Fig. 4A) and significantly greater numbers of luminal Ki67 positive cells than wild type control prostates (Fig. 4A and B).
In order to further understand the response of NF-κB activation in the prostate to long term castration, IκBα+/− and wild type mice were castrated at 7-8 weeks of age and the prostates were harvested at six weeks after castration. The prostates from wild type mice showed characteristic features of castration, including involution and fibrosis of the gland. The wild type prostate had only a few atrophic glands with limited to undetectable nuclear AR staining. The prostates from IκBα+/− mice, however, still maintained more typical glandular structure and strong nuclear AR staining (Fig.4C). These results suggest that constitutive NF-κB signaling prevents the mouse prostate from regressing and maintains prostatic epithelial cell proliferation even six weeks after castration.
ARR2PB-myc-PAI (Hi-Myc mouse), a transgenic mouse model, was generated using a probasin promoter (ARR2PB) to target high levels of the human c-myc gene to the mouse prostate. These mice develop androgen dependent invasive adenocarcinoma in the prostate by 6 months of age. With castration, these tumors regressed and had no apparent regrowth (39). To further determine that activation of the NF-κB pathway is sufficient for AI growth of the prostate cancer, we developed a constitutively NF-κB activated prostate cancer mouse model (Myc/IκBα+/− mouse). The Myc/IκBα+/− mouse model was developed by crossing the ARR2PB-myc-PAI with the IκBα+/− mouse. Since androgen-ablation therapy is the primary clinical treatment for prostate cancer patients with advanced stage disease, we examined the effect of castration on disease progression in our Myc/IκBα+/− prostate cancer mouse model. Myc and Myc/IκBα+/− mice were castrated at 6 months and the prostates were harvested two weeks after castration for analysis. Our results showed that Myc and Myc/IκBα+/− transgenic mice develop invasive adenocarcinoma at 6 months of age (all dorsal and lateral lobes, and some anterior and ventral lobes) (Fig. 5A). The prostates from Myc mice regressed after castration showing atrophic glands, with limited to undetectable nuclear AR staining in all lobes (Fig. 5A). The number of luminal Ki67 positive cells significantly decreased (p<0.01) as a result of castration (Fig. 5B). In contrast, the prostates from Myc/IκBα+/−mice following castration still had strong nuclear AR staining and luminal Ki67 staining in all lobes (Fig. 5A) with no significant decrease (p=0.269) in the number of luminal Ki67 positive cells between intact and castrated mice (Fig. 5B). These results indicate that NF-κB signaling controls progression of prostate cancer to AI growth in the mouse.
Prostate tumors are heterogeneous where multiple foci may be genotypically distinct from each other. These multifocal cancers will respond differently to androgen-ablation therapy resulting in selection of androgen-independent cells and/or adaptative changes that result in altered gene expression giving a subpopulation of cells a selective advantage to survive therapy. For example, focal NE differentiation has been observed in the majority of clinically localized prostate cancers (14;15). During progression to androgen-independent prostate cancer, both nuclear NF-κB localization (45-47) and neuroendocrine differentiation (11-13;17) occurs. Both events predict poor prognosis for the patient but their precise contribution to prostate cancer progression to AI is unknown.
NE secretions enhance AI growth of prostate cancer by increased AR activity (4). In this study, we found that NE peptides (BBS and GRP) increased functional activation of NF-κB and AR. In addition, RelA, a transactivating subunit of NF-κB, increased the transcription and/or stability of AR in the prostate cancer cells. These data demonstrate that the NF-κB-signaling pathway, which can be activated by NE secretory factors, is responsible for AI growth of prostate cancer. In cell culture, NE peptides increased functional activation of NF-κB as detected by the NGL reporter both in the presence and absence of androgen. However, in vitro, NE peptides increased functional activation of AR only in the presence of androgen. In addition, although NF-κB (RelA) increased AR expression independent of the presence of androgen, NF-κB increased functional activation of nuclear AR only in the presence of androgen. These results suggest that NE peptides increase functional activation of AR in the prostate yet still require the presence of androgen, although the androgen levels may be low or there may be weak adrenal androgens such as dehydroepiandrosterone that can now activate the AR. Our in vivo experiments showed that prostatic grafts in the kidney capsule from IκBα−/−and IκBα+/−mice continue to function normally in castrated mice. In addition, continuous activation of NF-κB signaling converted androgen dependent prostate cancer to AI growth in the ARR2PB-myc-PAI transgenic mouse after castration. This suggests that non-testicular androgens are sufficient to activate the AR when NF-κB is constitutively expressed in the castrated mouse. These results are consistent with a previous study that showed very low levels of testosterone from the adrenal gland and higher levels of weak androgens such as DHEA would be present in castrated mice (4). Further, NE cancers promote LNCaP tumor growth in castrated mice mediated through increased AR expression (4). AR over-expression is associated with increased sensitivity to the growth-stimulating effects at low androgen concentrations in recurrent prostate cancer-derived cell lines (5) and xenografts (48).
In summary, our findings suggest that NE secretory proteins will activate the NF-κB pathway in prostate cancer cells and the activation of NF-κB signaling is sufficient to maintain AI growth of prostate cancer via regulation of AR action. Our report provides the basis to develop a new therapeutic strategy to treat prostate cancer patients after they fail androgen-ablation therapy. Thus, the NF-κB pathway may be a potential target for therapy against AI prostate cancer.
We thank Drs. Simon W. Hayward and Peter E. Clark for discussion and comments on the manuscript. This research was supported by National Institutes of Health grants to RJM (R01-CA76142 and R01-AG023490) and the Prostate Cancer Foundation (PCF), to TSB (R01-HL61419) and Frances Williams Preston Laboratories of the T.J. Martell Foundation.
This research was supported by National Institutes of Health grants to RJM (R01-CA76142 and R01-AG023490) and the Prostate Cancer Foundation (PCF), to TSB (R01-HL61419) and Frances Williams Preston Laboratories of the T.J. Martell Foundation.