Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
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[Cancer Research 63, 2206-2215, May 1, 2003]
© 2003 American Association for Cancer Research

Molecular Biology and Genetics

Constitutive Activation of Nuclear Factor {kappa}B p50/p65 and Fra-1 and JunD Is Essential for Deregulated Interleukin 6 Expression in Prostate Cancer1

Luiz F. Zerbini, Yihong Wang, Je-Yoel Cho and Towia A Libermann2

BIDMC Genomics Center and New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To date, no effective treatment for patients with advanced androgen-independent prostate cancer is available, whereas androgen ablation therapy, surgery, and radiation therapy are effective in treating local, androgen-dependent tumors. The mechanisms underlying the differences between androgen-dependent and -independent prostate cancer remain elusive. Interleukin (IL)-6 is a pleiotropic cytokine whose expression under normal physiological conditions is tightly controlled. However, aberrant constitutive IL-6 gene expression has been implicated in prostate cancer progression and resistance to chemotherapy and has been directly linked to prostate cancer morbidity and mortality. Particularly striking is the large increase in the expression of IL-6 in hormone-refractory prostate cancer. IL-6, in addition to its role as an immunomodulatory cytokine, functions as a growth and differentiation factor for prostate cancer cells. To determine the molecular mechanisms that lead to deregulated IL-6 expression in advanced prostate cancer, we examined the regulatory elements involved in IL-6 gene expression in androgen-independent prostate cancer cells. We demonstrate that, in contrast to the androgen-sensitive LNCaP cells, androgen-insensitive PC-3 and DU145 cells express high levels of IL-6 protein and mRNA due to enhanced promoter activity. Deregulated activation of the IL-6 promoter is for the most part mediated by a combined constitutive activation of the nuclear factor (NF)-{kappa}B p50 and p65 and the activator protein 1 (AP-1) JunD and Fra-1 family members as demonstrated by electrophoretic mobility shift assays, site-directed mutagenesis, and transfection experiments. Mutation of the NF-{kappa}B and AP-1 sites drastically reduces IL-6 promoter activity in both androgen-independent prostate cancer cell lines. Additionally, inhibition of these transcription factors using adenovirus vectors encoding either the I{kappa}B{alpha} repressor gene or a dominant negative JunD mutant leads to a strong down-regulation of IL-6 gene expression at the mRNA and protein level as measured by real-time PCR and ELISA, respectively. Furthermore, the blockade of IL-6 gene expression results in drastic inhibition of the constitutively activated signal transducers and activators of transcription 3 signaling pathway in DU145 cells. Our data demonstrate for the first time that a combined aberrant activation of NF-{kappa}B p50 and p65 and AP-1 JunD and Fra-1 in androgen-independent prostate cancer cells results in deregulated IL-6 expression, suggesting a novel potential entry point for therapeutic intervention in prostate cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostate cancer has become the most common solid cancer in older men and is one of the most frequent causes of cancer deaths. Although local prostate cancer is slow-growing and many times asymptomatic, advanced prostate cancer, upon conversion from an initially androgen-dependent state into an androgen-independent state, becomes resilient to any known therapy and invariably results in death of the patient. The lack of effective therapies reflects in part the lack of knowledge about the molecular mechanisms involved in the development, progression, and metastasis of this disease. Especially little is known about how androgen-dependent prostate cancer gets converted into androgen-independent, highly metastatic prostate cancer.

IL-63 is a pleiotropic cytokine with a wide range of immune and hematopoietic activities. IL-6 expression is tightly regulated and can be induced in macrophages, synovial fibroblasts, endothelial cells, and other cell types. IL-6 plays a crucial role in autoimmune diseases because abnormally high quantities of IL-6 are found in several autoimmune diseases (1) . The majority of agents that induce IL-6 gene expression are inflammatory agents including cytokines such as IL-1, TNF-{alpha}, and IFN-{gamma}; bacterial endotoxins such as LPS; and viruses (1) . High levels of IL-6 have also been observed in a variety of human cancers such as prostate cancer, renal cancer, and multiple myeloma, raising the possibility that IL-6 may play a critical role in cancer development or progression (2, 3, 4, 5) . The importance of IL-6 for multiple myeloma growth has been established, and interference with IL-6 function is being exploited in clinical trials as a treatment for multiple myeloma.

Serum levels of IL-6 are significantly elevated in patients with hormone-refractory prostate cancer, and IL-6 has been suggested to be a mediator of morbidity and mortality in patients with metastatic disease (2 , 3 , 5 , 6) . Prostate cancer cells themselves are constitutively secreting a major portion of IL-6, although other sources such as stromal cells cannot be excluded. Because prostate cancer cells also express high levels of IL-6 receptor, IL-6 may elicit both paracrine and autocrine responses. Androgen-sensitive prostate cancer cell lines such as LNCaP do not express IL-6, but androgen-independent prostate cancer cell lines such as PC-3 and DU145 do express IL-6 (Ref. 2 and this report). However, LNCaP cells can be induced to express IL-6 by treatment with the cytokine IL-1ß (7) . This distinction between androgen-sensitive and androgen-insensitive prostate cancer cell lines is also reflected in their responses to IL-6. Whereas IL-6 induces G1 growth arrest and neuroendocrine differentiation of hormone-dependent LNCaP cells, IL-6 is an important growth factor for hormone-refractory PC-3 cells (2 , 8) .

Thus, prostate cancer cells may convert from a hormone-dependent IL-6-inhibited state into a hormone-refractory IL-6-dependent state. Elevated IL-6 levels in prostate cancer patients correlate with increased serum PSA levels (9) . Indeed, IL-6 up-regulates AR activity in DU145 cells transfected with an AR expression vector in a ligand-independent manner and induces AR-dependent PSA gene expression in LNCaP cells in the absence of androgen (10) . The exact mechanism of IL-6-mediated AR activation is not clear, but it appears to involve direct interaction of the IL-6 receptor with the ErbB2 and ErbB3 receptors, leading to tyrosine phosphorylation of ErbB2 and ErbB3 and stimulation of protein kinase A and mitogen-activated protein kinase signaling pathways (11) .

Another important clinical impact of constitutive IL-6 expression in hormone-refractory prostate cancer is the ability of IL-6 to block TGF-ß- and p53-mediated apoptosis. This effect of IL-6 also leads to protection of prostate cancer cells from the cytotoxic effect of chemotherapeutic agents (12) . Additionally, the constitutive IL-6 expression activates the JAK-STAT pathway, which has been implicated in multiple cytokine-mediated mechanisms of growth regulation. The JAK-STAT pathway is activated by binding of IL-6 to its receptor complex, consisting of the IL-6 {alpha}-receptor subunit and gp130, the ß-receptor subunit (13 , 14) . This event results in autophosphorylation and activation of JAK1, JAK2, and TYK2 (tyrosine kinase 2), leading to recruitment of STAT3, which gets phosphorylated and migrates to the nucleus to activate gene transcription (15, 16, 17) .

Thus, elevated IL-6 expression in prostate cancer may result in enhanced proliferation of hormone-refractory prostate cancer metastases concomitant with increased drug resistance and inhibition of apoptosis. These results suggest that interference with IL-6 function or expression should be of high clinical relevance in the treatment of hormone-refractory, drug-resistant prostate cancer.

The IL-6 promoter is composed of a variety of overlapping regulatory elements (18 , 19) . We and others have demonstrated that binding sites for the inducible transcription factors NF-{kappa}B, NF-IL-6, cAMP-responsive element binding protein, and AP-1 are essential for induction of the IL-6 gene by LPS, TNF-{alpha}, IL-1, phorbol ester, phytohemagglutinin, and double-stranded RNA (18, 19, 20) . In addition p53, Rb, and glucocorticoid receptors are involved in inhibiting IL-6 gene expression. Although, a variety of regulatory elements are involved in the regulation of IL-6 gene expression, IL-6 promoter regulation in prostate cancer has not yet been explored.

Interference with IL-6 activity could occur either on the level of the protein or by blocking expression of IL-6 mRNA. Because IL-6 may be only one of a set of cytokines and other genes that are aberrantly expressed in prostate cancer with similar activities, blocking the IL-6 protein alone may have only limited success. We propose as an alternative to interfere with the transcription factors involved in constitutive IL-6 gene expression in prostate cancer, with the notion that the same transcription factors regulate a whole set of cytokines and other genes. Thus, blocking one of the critical transcription factors may interfere with transcription of a whole set of cytokines and other genes. Many cytokine promoters contain a similar set of transcription factor binding sites.

In this study, we determined the molecular mechanisms underlying the transcriptional activation of the IL-6 promoter and the deregulated IL-6 gene expression in androgen-independent prostate cancer cells that could provide new tools for therapeutic intervention. We show here that the IL-6 gene is constitutively expressed in two androgen-independent prostate cancer cell lines, PC-3 and DU145. Additionally, the data revealed that endogenous IL-6 gene expression correlates with IL-6 promoter activity and is at least partially mediated via constitutive activation of NF-{kappa}B and AP-1. Furthermore, we demonstrated that the blockade of these transcription factors down-regulates IL-6 expression at the mRNA and protein level and leads to inhibition of the phosphorylation of STAT3.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The prostate cancer cell lines DU145, PC-3, and LNCaP and the breast cancer cell lines MDA231, MDA435, MCF7, and SKBR3 were obtained from American Type Culture Collection (Manassas, VA). The CL1 prostate cancer cell line was kindly provided by Dr. Sun Paik and Dr. Arie Belldegrun (University of California Los Angeles School of Medicine, Los Angeles, CA). DU145 cells were grown in MEM (Life Technologies, Inc.), PC-3 cells were grown in Ham’s F-12 medium (BioWhittaker, Walkersville, MD), LNCaP and CL1 cells were grown in RPMI 1640 (Life Technologies, Inc.), and MDA231, MDA435, MCF7, and SKBR3 cells were grown in DMEM (Life Technologies, Inc.). The media were supplemented with 10% fetal bovine serum (Life Technologies, Inc.) or charcoal-treated serum (CL1), 50 units/ml penicillin, and 50 µg/ml streptomycin (all from Life Technologies, Inc.). PME cells and PRECs were purchased from Clonetics (Walkersville, MD). PME cells were grown in MEGM media (Clonetics), and PRECs were grown in PREGM media (Clonetics). Both media were supplemented with epithelial medium single quote kit, which contains growth factors (Clonetics). The cells were maintained in a 5% CO2-humidified incubator.

Plasmids.
The full-length IL-6 promoter was inserted into the BamHI-HindIII sites of the pXP2 luciferase vector (pXP2-IL6) as described previously (13) . The promoter was also cloned into the pUC-CAT plasmid, generating pIL6-CAT (13) , and plasmids carrying point mutations in the MRE, NF-IL-6, AP-1, and NF-{kappa}B regulatory elements of the IL-6 promoter (Fig. 1)Citation were generated using site-directed mutagenesis as described previously within the context of the CAT vector (13) . The plasmid pßGal-control (BD Biosciences) was used as control for transfection efficiency.



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  		Fig. 1. Regulatory elements of the hIL-6 promoter with their approximate locations relative to the major transcription start site (+1). GRE, glucocorticoid response element; SRE, serum response element; CRE, prostaglandin- and cAMP-responsive element; HLH, helix-loop-helix.

 
DNA Transfection Assays.
Transfections of 3–8 x 105 cells were carried out with 700 ng of reporter gene construct DNA using LipofectAMINE Plus (Life Technologies, Inc.) as described previously (21) . The cells were harvested 16 h after transfections and assayed for luciferase activity or CAT enzyme activity. Transfections were performed independently in duplicate and repeated three to four times with different plasmid preparations with similar results. Cotransfection with pßGal-control was used for controlling transfection efficiency. CAT enzyme activity in the transfected cells was determined using the CAT ELISA assay (Roche Molecular Biochemicals, Indianapolis, IN) with a specific antibody to CAT (anti-CAT) according to the protocol supplied by the manufacturer.

EMSA and Supershift Assay.
DNA binding reactions and EMSA assays were performed as described previously (22) . Whole cell extracts were made from DU145, PC-3, and LNCaP prostate cancer cells using as lysis buffer 1% Triton X-100, 25 mM glycylglycine (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 1% aprotinin (Sigma). Protein concentrations were measured by the Bio-Rad protein assay. The reactions were made using 3 µl of whole cell extract and 0.1–0.5 ng of 32P-labeled double-stranded specific oligonucleotides (5,000–25,000 cpm) and run on 4% polyacrylamide gels containing 0.5x Tris glycine EDTA as described previously (23) . For supershift analysis, the cell extracts were preincubated with specific antibodies against different members of the rel/NF-{kappa}B family or the AP-1 family (Santa Cruz Technologies) for 20 min before the addition of the labeled probe for an additional 20 min (23) . The oligonucleotides used as probes for direct binding studies are as follows: IL-6 promoter wild-type AP-1, 5'-TCGACGTGCTGAGTCACTAAC-3' and 3'-GCACGACTCAGTGATTGAGCT-5'; and IL-6 promoter wild type NF-{kappa}B, 5'-TCGACATGTGGGATTTTCCCATGAC-3' and 3'-GTACACCCTAAAAGGGTACTGAGCT-5'.

ChIP.
PC-3 human prostate cancer cells (2 x 107) were plated on two 100-mm dishes. Cross-linking was performed by adding formaldehyde directly to tissue culture medium to a final concentration of 1% and incubating for 10 min at room temperature. Cells were rinsed twice with ice-cold PBS and collected into 1 ml of PBS 100 and centrifuged for 5 min at 2,000 x g. Cells were washed sequentially with 1 ml of ice-cold PBS, buffer I [0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES (pH 6.5)] and buffer II [200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES (pH 6.5)]. Cells were then resuspended in 0.3 ml of lysis buffer [1% SDS, 5 mM EDTA, 50 mM Tris-HCl (pH 8.1), 1x protease inhibitor mixture (Roche Molecular Biochemicals)], incubated on ice for 10 min, and sonicated three times for 30 s each with 1-min interval at the maximum setting to generate 400-bp to 1-kb DNA fragments, followed by centrifugation for 10 min at 14,000 x g at 4°C. Supernatants were collected, and 100 µl of chromatin preparation were aliquoted as the input fraction. The remainder of the supernatants was diluted in buffer [1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, 1x protease inhibitor mixture (pH 7.9)], followed by immunoclearing with 2 µg of sheared salmon sperm DNA, 20 µl of normal rabbit serum, and protein A-Sepharose [45 µl of 50% slurry in 10 mM Tris-HCl (pH 8.1), 1 mM EDTA (Amersham Pharmacia Biotech)] for 2 h at 4°C. Immunoprecipitation was performed overnight at 4°C with 0.5 µg of specific antibody, anti-NF-{kappa}B p65 (Santa Cruz Technologies), or normal rabbit serum as a negative control. After immunoprecipitation, 45 µl of protein A-Sepharose and 2 µg of sheared salmon sperm DNA were added, and the incubation was continued for another 1 h. Precipitates were washed sequentially for 10 min each in TSE I [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl], and TSE III [0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)]. Precipitates were then washed three times with T10E1 buffer (pH 8.0) and extracted three times with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated at 65°C overnight to reverse the formaldehyde cross-linking. DNA fragments were purified with the QIAquick PCR purification kit (Qiagen). IL-6 promoter-specific PCR was performed using 2 µl of a 50-µl DNA extraction in Tris EDTA buffer with Hi-Fi Taq polymerase (Invitrogen). PCR mixtures were amplified for 1 cycle at 94°C for 2 min; followed by 32 cycles at 94°C for 30 s, annealing temperature of 55°C for 30 s, and 68°C for 1 min; and then subjected to a final elongation at 68°C for 5 min. The primers used, hIL6P-F3 (5'-GCTAGCCTCAATGACGACCT-3') and hIL6P-R3 (5'-GCCTCAGACATCTCCAGTCC-3'), amplify 222 bp of the hIL-6 promoter surrounding the NF-{kappa}B site.

Adenovirus Construction and Infection.
The adenovirus encoding the I{kappa}B gene was kindly provided by Fionula Brennan (24) . The adenovirus encoding dominant negative JunD and the ß-gal gene were generated using the ADENO-X system from Clontech Laboratories, Inc. The dominant negative JunD mutant was obtained from the pCMV5-Jund{Delta} plasmid, kindly provided by Dr. Lester F. Lau, University of Illinois (25) . The fragment encoding the ß-gal gene was from pCMVß plasmid (Clontech Laboratories, Inc.). Briefly, the dominant negative JunD mutant cDNA was inserted into appropriate restriction sites inside the polylinker of the pShuttle vector (3.9 kb), creating an independent expression cassette. The cassette was transferred to pAdeno-X (32.6 kb) containing adenoviral DNA by means of an in vitro ligation at the sole restriction sites I-CeuI and PI-SceI. The ligation was digested by SwaI to linearize nonrecombinant (self-ligated) pAdeno-X DNA. The cosmids were then used to transform DH5{alpha} electrocompetent bacterial cells, using standard molecular biology techniques. Because pShuttle and pAdeno-X carry different antibiotic selection markers, purification of the expression cassette fragment before ligation was not required. The recovered ampicillin-resistant clones were screened by PCR analysis and restriction endonuclease digestion. Clones containing the desired construct were amplified, and large scale DNA purification was performed. The recombinant adenovirus vectors were then linearized by digestion with PacI to correctly expose the two inverted terminal repeats, thus allowing adenovirus replication and bacterial plasmid sequence excision. The DNAs were transfected in HEK 293 cells using LipofectAMINE Plus reagent (Life Technologies, Inc.). Cytopathic effects were evident 10–12 days after transfection. When most of the cells were detached, the suspension was collected, and the viruses were isolated using freeze/thaw cycles (-20°C/37°C). The expression of the transgenes is under the control of the strong human cytomegalovirus immediate early promoter/enhancer (PCMV IE) and the polyadenylation signal from the bovine growth hormone gene. For virus purification, HEK 293 cells were plated on a large scale (approximately 30 plates, 150-cm2 dishes, 80% confluent) and infected with the recombinant adenovirus obtained after transfection. Following the cytopathic effect, cells were collected in a centrifuge tube (15 ml) and centrifuged at 6000 x g for 20 min, and the supernatant was discarded. The cell pellet was then resuspended in PBS, submitted to five freeze/thaw cycles, and centrifuged at 6000 x g for 10 min. The supernatant was recovered, and the virus was purified by two steps of CsCl gradient (26) . The virus band was recovered and dialyzed against 10 mM Tris (pH 7.4), 1 mM MgCl2, 10% glycerol solution. Determination of virus particle titer was accomplished spectrophotometrically by the method described by Maizel et al. (27) with a conversion factor of 1.1 x 1012 viral particles per unit at 260 nm. To determine the titer of infectious viral particles on HEK 293 cells, a plaque assay was used as described by Mittereder et al. (26) . The infection experiments were carried out in serum-free medium during 1 h at 37°C with a MOI of 1000.

IL-6 ELISA.
DU145 and PC-3 cells at 106 cells/ml were infected with Ad5I{kappa}B, Ad5-DNJunD, or both together using Ad5-ß-gal as a negative control for 24, 48, and 72 h. IL-6 in the supernatant was assayed by ELISA (BioSource International Inc., Camarillo, CA) using a monoclonal antibody specific for hIL-6 according to the protocol supplied by the manufacturer.

RT-PCR Analysis.
Total RNA was harvested using QIAshreder (Qiagen) and RNeasy Mini Kit (Qiagen). cDNA was generated from 2 µg of total RNA using Ready-to-Go You-Prime First-Strand Beads (Amersham Pharmacia Biotech). RT-PCR amplifications of 0.1 µg of cDNA were carried out using a MJ Research thermal cycler PTC-100 as follows: 5 min at 94°C; 35 cycles of 30 s at 94°C, 45 s at 50°C, and 1 min at 72°C; followed by 5 min at 72°C. The sequences of the IL-6 primers were 5'-GGGAACGAAAGAGAAGCTCT-3' (sense) and 5'-ACCAGAAGAAGGAATGCCCA-3' (antisense), with an expected size of 730 bp. The sequences of the primers for hGAPDH were 5'-CAAAGTTGTCATGGATGACC-3' (sense) and 5'-CCATGGAGAAGGCTGGGG-3' (antisense), with an expected size of 195 bp.

Real-Time PCR.
Total RNA and cDNA were generated as described in RT-PCR analysis. SYBR Green I-based real-time PCR was carried out on a MJ Research DNA Engine Opticon Continuous Fluorescence Detection System (MJ Research Inc., Walthan, MA). All PCR mixtures contained PCR buffer [final concentration, 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2 mM MgCl2, and 0.1% Triton X-100], 250 µM deoxynucleoside triphosphate (Roche Molecular Biochemicals), 0.5 µM of each PCR primer, 0.5x SYBR Green I (Molecular Probes), 5% DMSO, and 1 unit of Taq DNA polymerase (Promega, Madison, WI) with 2 µl of cDNA in a 25-µl final volume reaction mix. The samples were loaded into wells of Low Profile 96-well microplates. After an initial denaturation step for 1 min at 94°C, conditions for cycling were 35 cycles of 30 s at 94°C, 30 s at 50°C, and 1 min at 72°C. The fluorescence signal was measured right after incubation for 5 s at 75°C following the extension step, which eliminates possible primer dimer detection. At the end of the PCR cycles, a melting curve was generated to identify specificity of the PCR product. For each run, serial dilutions of hGAPDH plasmids were used as standards for quantitative measurement of the amount of amplified DNA. For normalization of each sample, hGAPDH primers were used to measure the amount of hGAPDH cDNA. All samples were run in triplicates, and the data were presented as ratio of IL-6:GAPDH and then as a percentage of ß-gal control sample or LNCaP cells control sample. The primers used for real-time PCR are described in RT-PCR analysis.

Western Blot Analysis.
Whole cell lysates were prepared in lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na PPi, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride]. Thirty µg of protein were electrophoresed in a 10% acrylamide-SDS gel. Proteins were electroblotted onto a polyvinylidene difluoride membrane in a 50 mM Tris-base, 20% methanol, 40 mM glycine electrophoresis buffer. Membranes were incubated in 5% nonfat dry milk in TBST (60 mM Tris-base, 120 mM NaCl, and 0.2% Tween 20) for 1 h. Blots were probed with anti-STAT3 antibody (Cell Signaling) or anti-phospho-STAT3 antibody (Cell Signaling) overnight at 4°C in 2% BSA in TBST and then incubated with a horseradish peroxidase-conjugated secondary antibody (Cell Signaling) in 5% dry milk in TBST for 1 h at room temperature. Bound antibodies were detected by chemiluminescence with ECL detection reagents (Amersham Pharmacia Biotech) and visualized by autoradiography.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Androgen-independent Prostate Cancer Cell Lines Constitutively Express IL-6.
Highly elevated constitutive expression of IL-6 has been observed in advanced prostate cancer as well as a variety of other cancers. To establish a prostate cancer model system in which to study deregulated IL-6 gene expression, we tested a variety of prostate cancer cell lines as well as PRECs for endogenous IL-6 gene expression by RT-PCR (Fig. 2)Citation . At the same time, we also evaluated breast cancer cell lines and primary mammary gland epithelial cells for IL-6 expression (Fig. 2A)Citation . As reported previously, androgen-independent PC-3 and DU145 prostate cancer cells expressed high levels of IL-6 mRNA. PC-3 cells expressed significantly higher levels of IL-6 than DU145 cells. In contrast to the androgen-insensitive prostate cancer cells, androgen-sensitive LNCaP prostate cancer cells were devoid of IL-6 mRNA (Fig. 2A)Citation . Multiple PCR products were observed in PC-3 cells, most likely indicating the expression of alternatively spliced transcripts of the IL-6 gene because the IL-6 PCR primers used in this experiment anneal in the 5'-untranslated region and 3'-untranslated region, respectively. It has been demonstrated previously that the hIL-6 gene can transcribe at least six different alternatively spliced transcripts (28) . Low levels of IL-6 mRNA were also detected in PRECs and mammary gland epithelial cells that represent relatively undifferentiated, proliferating epithelial cells of the prostate and mammary gland, respectively (Fig. 2A)Citation . Of the four breast cancer cell lines examined (MDA231, MDA453, MCF7, and SKBR3), only MDA231 cells expressed IL-6 (Fig. 2A)Citation .



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  		Fig. 2. Expression of IL-6 mRNA in prostate and breast cancer cell lines. mRNA was analyzed by RT-PCR and real-time PCR using IL-6- and GAPDH-specific primers as described in "Materials and Methods." A, total RNA isolated from DU145, PC-3, and LNCaP prostate cancer cells; MDA231, MDA435, MCF7, and SKBR3 breast cancer cells; PME cells; and PRECs was analyzed by RT-PCR using IL-6- and GAPDH-specific primers as described in "Materials and Methods." B, total RNA isolated from CL1 and LNCaP prostate cancer cells was analyzed by RT-PCR using IL-6- and GAPDH-specific primers as described in "Materials and Methods." C, total RNA isolated from PC-3, DU145, CL1, and LNCaP prostate cancer cells was analyzed by real-time PCR. The reactions were carried out in triplicates using the SYBR Green I methodology and primers specific for hGAPDH and hIL-6. Normalization of each sample was carried out by measuring the amount of hGAPDH cDNA. Data were presented as fold increase over the LNCaP cell control sample.

 
Although DU145 and PC-3 cells have been used in many studies as models for advanced, androgen-independent prostate cancer, both cell lines do not exactly reflect advanced, hormone-refractory prostate cancer in vivo. In contrast to DU145 and PC-3 cells, hormone-refractory prostate cancer cells do express PSA and AR and preferentially metastasize to the bone. To confirm that deregulated IL-6 expression in DU145 and PC-3 cells can be used as a model system for studying IL-6 expression and indeed reflects some aspects of the conversion of androgen-dependent to androgen-independent prostate cancer, we evaluated IL-6 expression in a clonal cell line, CL1, generated upon conversion of the androgen-sensitive LNCaP cells to an androgen-insensitive state by the removal of androgen in the media. As described previously, the LNCaP cells were devoid of IL-6 mRNA (Fig. 2B)Citation . However, CL1 cells showed high levels of IL-6 expression, similar to the DU145 and PC-3 cells (Fig. 2B)Citation . These data provide direct evidence that androgen-sensitive prostate cancer cells start to express IL-6 upon loss of androgen dependence. To quantify the amount of IL-6 mRNA present in the cells, we tested LNCaP, CL1, DU145, and PC-3 prostate cancer cell lines for endogenous IL-6 gene expression by real-time PCR (Fig. 2)Citation . Androgen-independent PC-3, DU145, and CL1 prostate cancer cells expressed 22, 12.8, and 6.4 times higher levels of IL-6 mRNA than androgen-sensitive LNCaP cells, further supporting the data obtained by RT-PCR (Fig. 2C)Citation . Due to the low transfection efficiency in CL1 cells, we decided to use the DU145 and PC-3 cells to further define the molecular mechanisms of IL-6 gene expression in prostate cancer cells.

IL-6 Expression in Prostate Cancer Cell Lines Correlates with IL-6 Promoter Activity.
To determine whether constitutive IL-6 expression in prostate cancer cells is due to enhanced transcription of the IL-6 promoter, we assessed whether endogenous IL-6 expression correlates with IL-6 promoter activity. The full-length human 1.2-kb IL-6 promoter was inserted into the pxp2 reporter gene construct containing the luciferase gene and transiently transfected into the PC-3, DU145, and LNCaP prostate cancer cell lines. Transfection of pxp2-IL6 resulted in a 2000-, 270-, and 6-fold transcriptional stimulation of the IL-6 promoter construct as compared with the parental pxp2 vector in PC-3, DU145, and LNCaP cells, respectively (Fig. 3)Citation . These data demonstrate that constitutive expression of IL-6 in prostate cancer cells directly correlates with its promoter activity and that PC-3 cells express the highest IL-6 promoter activity.



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  		Fig. 3. Transcriptional activity of the IL-6 promoter in DU145, PC-3, and LNCaP prostate cancer cell lines. The cells were transfected with either the full-length IL-6 promoter luciferase construct (pXP2-IL6) or the parental vector (pXP2). Luciferase activity in the lysates was determined 16 h later, as described. Data shown are means ± SDs of duplicates of one representative transfection and normalized by measuring the amount of ß-gal gene expression. The experiment was repeated three times with different plasmid preparations with comparable results. The luciferase activity of the IL-6 promoter is shown as fold induction of the parental vector as indicated on the left. A, androgen-independent PC-3 cells; B, androgen-independent DU145 cells; C, androgen-sensitive LNCaP cells.

 
Deregulated IL-6 Gene Expression in Prostate Cancer Cells Is Predominantly a Result of Constitutive NF-{kappa}B p50/p65 and AP-1 Fra-1/JunD Activation.
To determine which regulatory elements in the IL-6 promoter mediate deregulated IL-6 gene expression in prostate cancer cells, the wild-type IL-6 promoter CAT construct and IL-6 promoter constructs with point mutations in either the NF-{kappa}B, AP-1, MRE, or NF-IL-6 binding sites were transiently transfected into PC-3 and DU145 cells. Mutations of either the NF-{kappa}B or AP-1 site reduced IL-6 promoter activity in both cell lines drastically but not completely, indicating that NF-{kappa}B and AP-1 are constitutively active in androgen-independent prostate cancer cells and are critical transcription factors for IL-6 gene expression in prostate cancer cells (Fig. 4)Citation . A less pronounced reduction in IL-6 promoter activity was observed in PC-3 cells, but not in DU145 cells, when the MRE was mutated, whereas the NF-IL-6 element appears not to play any role in prostate cancer cells (Fig. 4)Citation . Similar experiments were carried out using plasmids containing combined mutations in both the NF-{kappa}B and AP-1 binding sites. Combined NF-{kappa}B/AP-1 mutation within the context of the IL-6 promoter did not significantly enhance the decrease in IL-6 promoter activity when compared with single mutations in either the NF-{kappa}B or AP-1 site, suggesting cooperativity between NF-{kappa}B and AP-1 (data not shown). Thus, mutation of either site appears to affect activity of the other site. In an effort to confirm that NF-{kappa}B binds to the IL-6 promoter in vivo, we performed a ChIP assay in PC-3 human prostate cancer cells. Immunoprecipitation of the cross-linked protein-DNA complexes with an antibody against NF-{kappa}B p65 was followed by analysis of the immunoprecipitated DNA by PCR using IL-6 promoter-specific primers spanning -220 to +2 surrounding the NF-{kappa}B site of the hIL-6 promoter. As shown in Fig. 4CCitation , immunoprecipitation of chromatin with anti-NF-{kappa}B p65 antibody, but not control normal rabbit serum, specifically enriched for the endogenous IL-6 promoter DNA, demonstrating that NF-{kappa}B p65 binds in vivo to the IL-6 promoter in PC-3 cells.



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  		Fig. 4. AP-1 and NF-{kappa}B elements are the predominant elements for IL-6 promoter activity in androgen-insensitive prostate cancer cells. A and B, site-directed mutations within the AP-1 and NF-{kappa}B elements diminish IL-6 promoter activity in androgen-independent prostate cancer cells. PC-3 (A) and DU145 (B) cells were transiently transfected with either the full-length IL-6 promoter CAT construct (pIL6-CAT) or the IL-6 promoter-CAT construct in which the specified cis-regulatory element had been eliminated by site-directed mutagenesis. The CAT activity in the lysates was determined 16 h later, as described above. Data shown are means ± SDs of duplicates of one representative transfection. The experiment was repeated three times with different plasmid preparations with comparable results. The CAT activity of the IL-6 promoter is shown as the percentage of induction of the wild-type IL-6 promoter as indicated on the left. C, anti-NF-{kappa}B p65 antibody specifically enriches IL-6 promoter DNA sequences in a ChIP assay. Chromatin proteins were cross-linked to DNA in PC-3 cells by formaldehyde, and purified nucleoprotein complexes were immunoprecipitated using either Anti-NF-{kappa}B p65 antibody (Lane 3) or nonspecific rabbit IgG (Lane 2). The precipitated DNA fractions were analyzed by PCR for the presence of the IL-6 promoter region. In each case, the input DNA (1:100 dilution) was used as a positive control (Lane 1). Amplification products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining.

 
NF-{kappa}B is a ubiquitous transcription factor that is activated by a variety of proinflammatory cytokines, bacterial endotoxins, viral infection, DNA damage, and free radicals. In unstimulated cells under normal physiological conditions, NF-{kappa}B is maintained in the cytoplasm as an inactive complex with members of the I{kappa}B inhibitor family (29, 30, 31, 32, 33) . This interaction inhibits nuclear translocation and the DNA binding activity of NF-{kappa}B. Cellular stimulation leads to phosphorylation, ubiquitination, and subsequent proteolysis of I{kappa}B in proteasomes, enabling NF-{kappa}B to translocate into the nucleus and to activate a variety of genes including cytokines, cell cycle-regulatory genes, and antiapoptotic genes.

To evaluate whether prostate cancer cells that express IL-6 differ in their NF-{kappa}B activity from prostate cancer cells that do not express IL-6, we performed EMSAs using whole cell extracts from IL-6-expressing PC-3 and DU145 cells and non-IL-6-expressing LNCaP cells. Our EMSA data clearly demonstrated that NF-{kappa}B is constitutively activated in PC-3 and DU145 cells, but not in LNCaP cells (Fig. 5A)Citation , directly correlating with IL-6 expression and promoter activity. A strong protein-DNA complex comigrated with the NF-{kappa}B complex formed in IL-1-stimulated U-138 MG human glioma cells. To further delineate which members of the NF-{kappa}B/rel family are activated in prostate cancer cells, we performed a supershift assay with antibodies against different members of the NF-{kappa}B/rel family. These experiments established that the NF-{kappa}B family members p50 and p65 make up the constitutively active NF-{kappa}B complex in both PC-3 and DU145 cells (Fig. 5B)Citation , whereas c-rel, p52, and rel B are not involved (Fig. 5C)Citation . Previous experiments with NF-{kappa}B elements in other promoters and cell extracts from other types of cells had demonstrated that all antibodies are able to shift the specific family member in nuclear extracts (data not shown). Additional EMSA analysis using cell extracts from DU145 and PC-3 cells overexpressing the I{kappa}B inhibitor gene demonstrated that I{kappa}B overexpression abolished NF-{kappa}B binding in PC-3 and DU145 cells (data not shown).



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  		Fig. 5. EMSA of prostate cancer cell extracts for the IL-6 promoter NF-{kappa}B and AP-1 elements. Five µg of DU145, PC-3, and LNCaP whole cell lysates were incubated with 32P-labeled oligonucleotides encoding the IL-6 promoter NF-{kappa}B and AP-1 sites, and DNA-protein complexes were visualized on nondenaturing polyacrylamide gels. A, interaction of the IL-6 promoter NF-{kappa}B binding site with prostate cancer cell lysates. Extracts of U-138 MG cells treated with IL-1ß were used as a positive control for activated NF-{kappa}B. The arrow indicates the specific DNA-protein complex. B and C, supershift assay for NF-{kappa}B/rel family members. The DU145, PC-3, and LNCaP whole cell extracts were incubated with the labeled IL-6/NF-{kappa}B oligonucleotide probe in the absence or presence of antibodies against p50, p65, p52, c-rel, and relB. The arrows indicate the NF-{kappa}B DNA-protein complex and the supershifted antibody-protein-DNA complex. D, interaction of the IL-6 promoter AP-1 binding site with prostate cancer cell extracts and supershift assay for AP-1 family members. The DU145, PC-3, and LNCaP whole cell extracts were incubated with the labeled IL-6/AP-1 oligonucleotide probe with either no antibody or antibodies against c-jun, JunB, JunD, and JAB1. E, supershifts with antibodies against c-fos, FosB, Fra-1, and Fra-2. The arrows indicate the AP-1 DNA-protein complex protein and the supershifted antibody-protein-DNA complex.

 
AP-1 has been implicated in a variety of biological processes including cell differentiation, proliferation, apoptosis, and oncogenic transformation. AP-1 activity is modulated both by enhanced expression and by interactions with other transcriptional regulators and is further controlled by upstream kinases that link AP-1 to various signal transduction pathways (34, 35, 36) . AP-1 proteins contain basic region leucine zipper domains and act as a variety of homo- or heterodimers composed of members of the Fos, Jun, and ATF families (2 , 37, 38, 39) . Whereas the Fos proteins can only heterodimerize with members of the Jun family, the Jun proteins can both homo-dimerize and heterodimerize with Fos members to form transcription active complexes (2 , 18 , 40) .

Similarly to NF-{kappa}B, EMSA analysis demonstrated constitutive activation of AP-1 in IL-6-positive PC-3 and DU145 cells and the lack of AP-1 in IL-6-negative LNCaP cells (Fig. 5)Citation , further enhancing the notion that constitutive activation of IL-6 gene expression in prostate cancer cells is due to constitutive activation of NF-{kappa}B and AP-1. Surprisingly, supershift analysis using antibodies recognizing various members of the Jun and Fos families identified JunD and Fra-1 as the predominant proteins present in the complex (Fig. 5, D and E)Citation . These results clearly demonstrate that deregulated IL-6 gene expression in prostate cancer cells is for the most part mediated by constitutive activation of NF-{kappa}B p50 and p65 and AP-1 JunD and Fra-1.

Inhibition of NF-{kappa}B and AP-1 Strongly Down-Regulates IL-6 Gene Expression and IL-6 Protein Secretion in Androgen-independent Prostate Cancer Cells.
Having demonstrated that NF-{kappa}B and AP-1 play major roles in deregulated IL-6 promoter activity in prostate cancer, we went on to determine whether interference with NF-{kappa}B and/or AP-1 activity would down-regulate IL-6 expression in androgen-independent prostate cancer cells. Previous experiments have shown that overexpression of the I{kappa}B inhibitor blocks NF-{kappa}B activity. Inhibition of NF-{kappa}B by the superrepressor form of I{kappa}B has also been shown to block the antiapoptotic activity of NF-{kappa}B, thereby facilitating apoptosis through a variety of therapeutic agents and TNF-{alpha} (29 , 32 , 33 , 41 , 42) . To determine the importance of the NF-{kappa}B and AP-1 binding sites for endogenous IL-6 gene expression in prostate cancer cells, we infected androgen-independent PC-3 and DU145 prostate cancer cells with an adenovirus encoding the I{kappa}B gene (Ad5CMVI{kappa}B), an adenovirus encoding the dominant negative mutant JunD gene (AD5CMV-DNJunD), or both adenoviruses together (Ad5CMVI{kappa}B/Ad5CMVDNJunD), or an adenovirus carrying the ß-gal gene (Ad5CMVß-gal) as a control at a MOI of 1000. Total RNA and tissue culture supernatant were obtained from cells infected with Ad5CMVI{kappa}B, Ad5CMVDNJunD, Ad5CMVI{kappa}B/Ad5CMVDNJunD, or Ad5CMVß-gal at 24, 48, and 72 h after infection. Real-time PCR revealed that overexpression of I{kappa}B and dominant negative JunD led to a strong down-regulation of IL-6 gene expression in both cell lines that was sustained for at least 72 h (Fig. 6)Citation , further supporting the notion that the NF-{kappa}B and AP-1 binding sites are crucial for activation of the IL-6 gene in prostate cancer cells. Ad5CMVI{kappa}B infection reduced IL-6 mRNA expression down to 43.2%, 53.2%, and 65.4% of the control for the different time points in DU145 cells and down to 46.6%, 59.6%, and 58.7% of the control for the different time points in PC-3 cells (Fig. 6)Citation . Overexpressing the dominant negative JunD mutants reduced the levels of IL-6 gene expression down to 48.8%, 34.2%, and 52.1% of the control in DU145 cells and down to 43.3%, 39.9%, and 66.3% of the control in PC-3 cells. Combined infection with both adenoviruses resulted in an additive effect on IL-6 down-regulation. In this case, IL-6 gene expression was down to 25.7%, 24.1%, and 37.7% of the control in DU145 cells and 40.2%, 32.9%, and 39.2% of the control in PC-3 cells. However, the total expression of IL-6 mRNA molecules in DU145 cells was approximately three time less than that in PC-3 cells after infection with the viruses (data not shown). Because IL-6 expression was not completely blocked, it is possible that additional transcription factors play roles in IL-6 expression in prostate cancer cells.



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  		Fig. 6. Blockage of NF-{kappa}B and AP-1 activity down-regulates IL-6 gene expression in androgen-independent prostate cancer cells. DU145 and PC-3 cells were infected with an adenovirus expressing I{kappa}B, an adenovirus encoding dominant negative JunD, or both adenoviruses together, using an adenovirus expressing the ß-gal gene as a control at a MOI of 1000. Total RNA was isolated at 24, 48, and 72 h after infection. Real-time PCR reactions were carried out in triplicates using the SYBR Green I methodology and primers specific for hGAPDH and hIL-6. Normalization of each sample was carried out by measuring the amount of hGAPDH cDNA. Data were presented as fold increase over the ß-galactosidase control sample.

 
Protein expression and mRNA expression do not necessarily correlate. To determine whether the blockage of NF-{kappa}B and AP-1 and the subsequent reduction in IL-6 mRNA levels have an effect on IL-6 protein secretion, we analyzed IL-6 protein levels in tissue culture supernatants obtained from DU145 and PC-3 cells infected with the same virus combination as described above at 24, 48, and 72 h. IL-6-specific ELISA analysis revealed that overexpression of I{kappa}B and dominant negative JunD leads to drastic inhibition of IL-6 protein secretion in both cell lines (Fig. 7)Citation . I{kappa}B overexpression showed a 3.8-, 4.0-, and 2.7-fold inhibition of IL-6 secretion in DU145 cells and a 1.8-, 2.9-, and 3.9-fold inhibition of IL-6 in PC-3. Dominant negative JunD overexpression resulted in a 2.0-, 1.31-, and 1.73-fold inhibition of IL-6 secretion in DU145 and a 4.3-, 4.2-, and 2.2-fold inhibition of IL-6 secretion in PC-3 cells. The highest fold inhibition was obtained when the cell lines were infected with both viruses together (Fig. 7)Citation . In DU145 cells, the simultaneous blockage of NF-{kappa}B and AP-1 showed a 12.5-, 11-, and 8.23-fold inhibition of IL-6 protein levels in comparison with the control at 24, 48, and 72 h, respectively. The same experiment in PC-3 revealed a 5.4-, 8.5-, and 7.4-fold inhibition of IL-6 secretion. These data corroborate the IL-6 mRNA expression experiments and most vividly show the importance of constitutively activated NF-{kappa}B and AP-1 for deregulated IL-6 gene expression in prostate cancer cells.



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  		Fig. 7. Overexpression of the I{kappa}B repressor and dominant negative JunD inhibits IL-6 secretion in DU145 and PC-3 androgen-independent prostate cancer cells. DU145 and PC-3 cells were infected with an adenovirus expressing I{kappa}B, an adenovirus expressing dominant negative JunD, or both adenoviruses together, using an adenovirus encoding ß-galactosidase gene as a control. The immunoreactivity in the cell supernatant was determined by an IL-6-specific ELISA at 24, 48, and 72 h after infection. A, IL-6 ELISA measurement in PC-3 cells; B, IL-6 ELISA measurement in DU145 cells.

 
Inhibition of IL-6 Secretion and Gene Expression by I{kappa}B Overexpression Blocks the Autocrine, STAT3-activating Trigger by IL-6.
IL-6 utilizes JAK-STAT as major mediators of signal transduction (16 , 17 , 43) . To further delineate the effect of inhibition of IL-6 gene expression and IL-6 secretion due to overexpression of I{kappa}B gene on the autocrine activity of IL-6, we tested the activity of STAT3, one of the signal transducers of IL-6, in PC-3 and DU145 cells by immunoblotting with an anti-phospho-Tyr705 antibody specific for the tyrosine-phosphorylated active form of STAT3 in a time course experiment. We observed that down-regulation of IL-6 gene expression blocks STAT3 phosphorylation efficiently at 48 and 72 h in DU145 cells, correlating with the reduction in IL-6 levels at those time points. In contrast to DU145 cells, PC-3 cells expressed only very low levels of STAT3 protein and had no STAT3 activity (Fig. 8)Citation .



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  		Fig. 8. Overexpression of the I{kappa}B repressor blocks constitutive activation of STAT3 in DU145 prostate cancer cells. PC-3 and DU145 cells were infected with either an adenovirus expressing I{kappa}B or an adenovirus encoding the ß-galactosidase gene as a control. The total cell lysate was collected at 24, 48, and 72 h after infection. The extracts were then subjected to immunoblot analysis using antibodies against either tyrosine-phosphorylated active STAT3 (Tyr705) or total STAT3.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although androgen ablation therapy, surgery, and radiation therapy are effective for the treatment of local prostate cancer, there is no effective treatment available for patients with the metastatic androgen-independent disease. The poor prognosis for androgen-independent advanced prostate cancer reflects in part the lack of knowledge about the tumor’s basic biology. A variety of differences between androgen-sensitive and androgen-insensitive prostate cancer have been revealed over the last few years. However, despite that accumulation of additional and new data about these differences, the crucial steps that lead to progression and conversion of androgen-dependent to androgen-independent, metastatic prostate cancer remain elusive. One feature of advanced prostate cancer that particularly triggered our interest is the sudden deregulated activation of the IL-6 gene in advanced, androgen-independent prostate cancer, but not in primary, androgen-dependent prostate cancer. Furthermore, the level of IL-6 found in prostate cancer patients directly correlates with morbidity and mortality of the patients, further enhancing the notion that the IL-6 gene may be a key to understanding and treating advanced prostate cancer.

Deregulated IL-6 expression has been implicated in a variety of other cancers, particularly in multiple myeloma, where it is clear that IL-6 is a major growth factor for cancer cells and a major target for potential therapies. In addition to its potential growth and differentiation effects in prostate cancer, IL-6 is also antiapoptotic and interferes with chemotherapies. IL-6 could also affect neighboring prostate tumor cells by a paracrine mechanism that could affect prostate tumor cell invasion as well as growth (44) . Nevertheless, the importance of IL-6 per se for prostate cancer progression is not entirely clear, and it is possible that IL-6 is only one of several genes that act in a similar fashion in synergy but are regulated by the same upstream signals. Therefore, IL-6 may represent a surrogate marker for prostate cancer progression, but interference with IL-6 as a therapeutic modality may not work because IL-6 is only one of several factors with a similar function. We therefore argued that understanding the regulation of IL-6 gene expression in prostate cancer may elucidate the upstream factors that deregulate expression of a whole set of genes involved in advanced prostate cancer. These upstream factors should be far more desirable points of interference for novel therapies than IL-6 itself.

Although IL-6 expression in advanced prostate cancer has been previously observed, very little attention has been focused until now on the mechanism of this deregulated IL-6 expression. We confirm here that indeed only androgen-insensitive prostate cancer cell lines such as LNCaP-derived CL1 cells and PC-3 and DU145 cells, but not androgen-sensitive prostate cancer cell lines such as LNCaP, constitutively express IL-6. This result is consistent with previous reports and enables us to use these prostate cancer cell lines as a cell culture model for the study of aberrant IL-6 gene expression (2 , 3 , 5 , 45) . As a first step to delineate the molecular mechanisms leading to deregulated IL-6 gene expression in advanced prostate cancer we defined the transcription factors that constitutively activate the IL-6 gene in androgen-independent prostate cancer. We now demonstrate that deregulated IL-6 expression in prostate cancer cells directly correlates with IL-6 promoter activity, although changes in RNA stability or protein secretion may also play some role. This result immediately suggests that activation of IL-6 expression upon progression from an androgen-sensitive, IL-6-negative prostate cancer to a hormone-refractory, IL-6-positive prostate cancer is for the most part due to constitutive activation of a transcription factor or transcription factors that turn on the IL-6 gene and/or constitutive repression of a transcriptional suppressor of the IL-6 gene. Systematic analysis of IL-6 promoter activity in hormone-refractory prostate cancer cell lines enabled us to identify two regulatory elements, NF-{kappa}B and AP-1, whose activity correlates with IL-6 expression in prostate cancer cells and is essential for constitutive IL-6 gene expression in prostate cancer cells. Point mutations in the NF-{kappa}B and AP-1 binding sites drastically reduced IL-6 promoter activation in androgen-independent prostate cancer cell lines, although there was some residual promoter activity left, leaving the possibility open that additional regulatory elements may be involved. We identified the members of the NF-{kappa}B and AP-1 family that are constitutively active in hormone-refractory prostate cancer cells as the NF-{kappa}B heterodimer of p50 and p65 and the AP-1 heterodimer of JunD and Fra-1, and we demonstrated that p65 binds in vivo to the IL-6 promoter.

Although the activation of NF-{kappa}B in prostate cancer cell lines has been observed previously, no direct link between constitutive activation of NF-{kappa}B and IL-6 had been demonstrated up to now. The blockage of NF-{kappa}B activity in human prostate cancer cells has been associated with invasion and metastasis (46) and enhanced TNF-{alpha}-induced apoptosis (47 , 48) , and our results indicate that one of the mechanisms may involve the inhibition of IL-6 expression. Similarly, AP-1 activation in prostate cancer cell lines has been reported, and it has been suggested that NF-{kappa}B and AP-1 may play a role in the survival of prostate cancer cells (42 , 47 , 49, 50, 51) . However, the particular members of the NF-{kappa}B and AP-1 family that are active in prostate cancer cells had not been identified until now. Indeed, this is the first report indicating activation of JunD and Fra-1 in advanced prostate cancer cells, although sporadic activation of these factors in some other cancer types has been reported. Furthermore, our results are the first evidence that activated JunD and Fra-1 play a critical role in IL-6 gene expression in prostate cancer cells. Similarly to p50 and p65, JunD and Fra-1 form heterodimers that act as activating transcription factors on a variety of target genes. Strikingly, blocking either NF-{kappa}B or JunD drastically inhibits IL-6 expression and secretion as well as the downstream signaling pathways such as activation of STAT3 in prostate cancer cells, and a combination of both inhibitors further enhances this blockade. This inhibition is most likely direct inhibition of IL-6 promoter activation but could also involve indirect mechanisms of action via other genes regulated by NF-{kappa}B or AP-1 that affect IL-6 expression. However, in contrast to DU145 cells, PC-3 cells express only very low levels of STAT3, suggesting that IL-6 in PC-3 cells acts via a different mechanism that may include other members of the STAT family such as STAT1. Activation of STAT3 has been shown to enhance growth of prostate cancer cells (52 , 53) and neuroendocrine differentiation (54) , and constitutive STAT3 activation has been observed in prostate cancer, indicating that blocking STAT3 activation due to inhibition of IL-6 expression via AP-1 and NF-{kappa}B may be beneficial. Thus, we envision that a rational drug design with the purpose to intervene efficiently with advanced prostate cancer should target both NF-{kappa}B and JunD/Fra-1 concomitantly. To prove this hypothesis, we are now determining whether targeting AP-1 and NF-{kappa}B will interfere with prostate cancer growth and survival. Indeed, both AP-1 and NF-{kappa}B have been implicated in growth and survival in many different cell types (55, 56, 57, 58) , although there is significantly less known about the JunD and Fra-1 AP-1 family members than about p50 and p65. To further our understanding of the role of AP-1 and NF-{kappa}B in prostate cancer, we are in the process of evaluating the functional consequences of AP-1 and NF-{kappa}B activation in prostate cancer.

AP-1 and NF-{kappa}B are targets for a variety of different external stimuli that elicit their biological responses via a wide array of signal transduction pathways. Activators of AP-1 and NF-{kappa}B include, among others, various cytokines, growth factors, tumor promoter 12-O-tetradecanoylphorbol-13-acetate, reactive oxygen species, bacterial endotoxin LPS, viral infection, and UV light (59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71) . The specific trigger leading to constitutive activation of AP-1 and NF-{kappa}B in prostate cancer is not yet known, but it may include the constitutive activation of a cytokine or growth factor that activates AP-1 and NF-{kappa}B via an autocrine mechanism. To elucidate this mechanism is an important goal of our current efforts, and this upstream regulator of AP-1 and NF-{kappa}B may turn out to be a master switch for prostate cancer progression.

It does not come as a surprise that both AP-1 and NF-{kappa}B are activated in prostate cancer because AP-1 and NF-{kappa}B have been implicated in the pathogenesis of a variety of human cancers, and several cancer types have been reported to concomitantly activate AP-1 and NF-{kappa}B (43 , 72, 73, 74, 75, 76) . For example, transformed keratinocytes contain constitutively active AP-1 and NF-{kappa}B, and both are required for maintaining the transformed phenotype. Similarly, AP-1 and NF-{kappa}B are activated in pancreatic cancer and head and neck squamous cell carcinoma cell lines (14 , 59 , 75 , 77 , 78) . Furthermore, a variety of genes involved in cancer are regulated by the combined action of AP-1 and NF-{kappa}B such as IL-6, IL-8, MMP-9, COX-2, and MCP-1, suggesting that AP-1 and NF-{kappa}B cooperate synergistically in regulating a wide array of genes. However, in contrast to prostate cancer cells, in most cases the activated AP-1 complex contains c-jun and c-fos rather than Fra-1 and JunD.

In conclusion, our results have elucidated some of the major transcriptional regulatory mechanisms leading to deregulated IL-6 expression in advanced prostate cancer and have proven that blockage of these regulatory mechanisms can inhibit IL-6 expression and signal transduction in prostate cancer. Because IL-6 and possibly other genes with similar functions may be regulated by the same set of transcription factors, namely, AP-1 and NF-{kappa}B, combined interference with these transcription factors may give rise to novel therapeutic modalities in the fight against prostate cancer.


   ACKNOWLEDGMENTS
 
We acknowledge fruitful discussions with Drs. Ellen Gravallese, Xuesong Gu, Franck Grall, Gina Napoleone, Jon Jones, Mehmet Inan, Rosana Kapeller-Libermann, and Glen Bubley.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grant 1RO1 CA85467 and United States Army Medical Research Center Grant DAMD17-01-1-0023 (to T. A. L.). Back

2 To whom requests for reprints should be addressed, at BIDMC Genomics Center and New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115. Phone: (617) 667-3393; Fax: (617) 975-5299; E-mail: tliberma{at}bidmc.harvard.edu Back

3 The abbreviations used are: IL, interleukin; NF, nuclear factor; AP-1, activator protein 1; STAT, signal transducers and activators of transcription; TNF, tumor necrosis factor; LPS, lipopolysaccharide; PSA, prostate-specific antigen; AR, androgen receptor; JAK, Janus kinase; PME, primary mammary epithelial; PREC, primary prostate epithelial cell; CAT, chloramphenicol acetyltransferase; ß-gal, ß-galactosidase; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; hIL, human interleukin; MOI, multiplicity of infection; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; MRE, multiple response element. Back

Received 10/ 3/02. Accepted 2/25/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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