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* Departments of Oncology,
Pathology, and
Urology, Hadassah-University Hospital, Jerusalem, Israel; and
Division of Cardiology and Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
1Correspondence: Department of Oncology, Hadassah-University Hospital, POB 12000, Jerusalem 91120, Israel. E-mail: barshav{at}md.huji.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: PAR1 • protease-activated receptor-1 • prostate carcinoma • androgen hormone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
, these hormones accelerate the rise of prostate cancer progression. The mainstay standard therapy for advanced disease is therefore androgen ablation therapy. Androgen withdrawal causes cancer regression by decreasing cell proliferation and increasing apoptosis. A major limitation of the hormonal therapy, however, is that it offers only a temporary relief; the cancer eventually reappears in an androgen-independent form, characterized by aggressive growth and invasion to distal organs, predominantly to bones (4
5
6
7)
. One of the ongoing, major goals is to better understand molecular mechanisms of hormone-dependent and -independent prostate cancer progression. Thus, it is critical to identify genes that are expressed specifically in prostate malignancy and evaluate their hormonal regulation.
Genes that are expressed specifically in human prostate tissues are often regulated at the transcriptional level by testosterone, which with the more potent agonist dihydrotestosterone (DHT), exert their effects by binding to an androgen receptor (AR). Upon ligand activation, the AR is phosphorylated and forms a homodimer, which is transported to the nucleus, where it activates transcription by binding to androgen response elements (ARE) present in the promoters of target genes. Coactivators and corepressors bind to the AR, facilitating or suppressing its interactions with the general transcription apparatus. The ARE consensus sequence is composed of one nearly canonical half-site of TGTYCT and separated by three nucleotides from the other half-site (8)
.
Protease-activated receptor-1 (PAR1) is the first identified member of the PAR gene family, currently including four seven transmembrane G-protein-coupled receptors activated by proteolytic cleavage. Collectively, the release of N-terminal peptides from these receptors exposes otherwise masked ligands, which further initiate downstream signaling. We (9
, 10)
as well as others (11
12
13
14)
have addressed the involvement of human PAR1 (hPar1) in malignant and physiological (15)
invasion processes, showing a direct correlation and function between hPar1 and the invasion properties. The hPar1 gene was shown to play a role in tumor angiogenesis (16
17
18)
. A cDNA microarray analysis comparing two prostate cancer cell lines derived from the same patient, a bone-derived line VCaP and a soft tissue-derived line DuCaP, revealed high hPar1 expression in VCaP but not in the parental DuCaP cell line, where hPar1 levels are minimal (19)
. The hPar1 gene is organized in two exons and a single intron of 
15 kb in size. Exon I contains the 5'-regulatory region (located 2.8 kb upstream of the translation initiation start site ATG; refs. 20
, 21
). The hPar1 promoter appears to be regulated in part by the stimulating protein family (22)
and by activator protein-2 alpha (AP-2
) transcription factors (23)
. It has been reported that steroid hormones used in animal models increased the expression of PAR-1 by initiating glucocorticoid receptor signaling (24)
, pointing to the possibility that other critical regulatory sites may control its expression.
In the present study, we have identified a novel ARE domain within the hPar1 promoter. This was carried out by luciferase (Luc) promoter activities before and after androgen treatment, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) analyses, altogether demonstrating androgen hormone regulation of hPar1 gene expression during prostate tumor progression. In parallel, examinations of prostate biopsy specimens taken from prostate cancer patients reveal that androgen ablation therapy leads to the down-regulation of the otherwise highly expressed hPar1 gene in vivo. Conversely, in hormone-independent prostate cancer cell lines (e.g., CL1 and PC3), we find a surprisingly high hPar1 expression level, regardless of AR expression. The molecular mechanism leading to high hPar1 expression levels in aggressive prostate cell lines lacking AR remains to be elucidated.
hPar1 is overexpressed in the two phases of prostate cancer cells, the hormone-dependent and -independent phases. Although our data point to a role for androgen-dependent, increased transcription in the hormone-dependent phase, the mechanism of hPar1 over increased expression in the hormone-independent phase is not yet known. Nevertheless, we propose that hPar1 could serve as a target for therapy in the two phases, as it is overexpressed in both.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was prepared using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA), as described by the manufacturer. RNA (1 µg) was used for cDNA synthesis, using Molony murine leukemia virus reverse transcriptase and oligo dT (Promega, Madison, WI, USA). hPar1 transcripts were amplified, using Taq polymerase (Bioline, London, UK), in a 20-µl total PCR. Initial melting was carried out at 95°C for 3 min, followed by 32 cycles of 95°C for 1 min, 56°C for 30 s, and 72°C for 45 s; 5 min at 72°C was used for final extension after cycling. PCR primers were as follows: upstream hPar1: 5'-GCCAGAATCAAAAGCAACAA-3', downstream hPar1: 5'-GAGATGAATGCAGGAAGTTGTTT-3'; upstream human glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5'-CCACCCATGGCAAATTCCATGGCA-3', downstream human GADPH: 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (26 cycles); upstream AR: 5'-CAA GCT CCT GGA CTC CGT GCA-3', downstream AR: 5'-TAGATGGGCTTGACTTTCCC-3'. PCR products were separated on a 1.5% agarose gel stained with ethidium bromide and visualized under ultraviolet light.
Northern blot analysis
Total RNA (20 µg) was electrophoresed on 1% formaldehyde-agarose gels and transferred to Hybond-N+ membranes (Amersham Pharmacia Biotech UN, Little Chalfont, UK). The membranes were hybridized (42°C, 18 h) with an -32P-deoxycytidine 5'-triphosphate (dCTP) -labeled (Rediprimer II, Amersham Biosciences UK, Little Chalfont, UK) probe for hPar1. After hybridization, membranes were washed and exposed to X-ray films. We used the 28S RNA as a control for RNA loading.
Transfection and Luc expression assay
JEG-3 cells at 60–80% confluency, grown for 48 h in steroid-depleted cell-culture medium, were transfected with 2 µg of the different hPar1 promoter fragment plasmids DNA: Poluc-HTR/–4.1(F1), Poluc-HTR/–1.6 (F5), and Poluc-HTR/0.7 (F6) in Fugene 6 transfection reagent (Boehringer Mannheim, Germany) according to the manufacturer’s instructions. After 48 h, the cells were lysed in 0.1 mL lysis buffer (Promega, Madison, WI, USA). Cell lysate was transferred to a 1.5 mL microcentrifuge tube and cleared by centrifugation at 12,000 rpm for 2 min at 4°C. Luc activity was measured by mixing 20 µL supernatant in a 96-well microtiter plate with 100 µL Luc assay substrates (Promega, Madison, WI, USA) and read using a luminometer (Mithras LB 940, Berthold Technologies, Germany). Cytomegalovirus (CMV)/ß-galactosidase (ß-gal) plasmid was cotransfected as an internal control for transfection efficiency.
Prostate tissue specimens and pathological evaluation
Human materials used in this study were from archival paraffin-embedded tissue blocks. Cases were selected for this study based on medical history and AR expression levels. Patients were treated for hormone ablation before radical prostatectomy by luteinizing a hormone-releasing hormone agonist [Zoladex (Goserelin), Zeneca Pharmaceuticals, Wilmslow, UK] in combination with antiandrogen [Eulexin (Flutamide), Schering-Plough, Madison, New Jersey, USA]. With this treatment, serum androgen levels are effectively eliminated after 4 wk, and treatment was continued for an additional 4–8 wk before surgery.
In situ hybridization
Hybridization was carried out as described previously (9)
. Briefly, RNA probes were transcribed and labeled by T7 RNA polymerase (for antisense, orientation) or T3 RNA polymerase (for sense, control orientation) using digoxigenin (DIG)–uridine 5'-triphosphate-labeling mixture (Boehringer Mannheim). Probes were labeled by using a plasmid containing a 462-bp fragment of the hPar1 (pBhPar1-462S) inserted into the EcoRI-HindIII site. Final concentration for hybridization was 1 µg/mL, according to the manufacturer’s instructions for a nonradioactive in situ hybridization application. Hybridization was performed overnight at 45°C. Slides were washed three times in 0.2x sodium chloride, sodium phosphate and EDTA (SSPE) at 50°C, 1 h for each washing, and blocked with blocking reagent (Boehringer Mannheim). Detection was done by incubation with alkaline phosphatase-conjugated anti-DIG antibodies (Fab fragment, diluted 1:300, Boehringer Mannheim) overnight at room temperature. Alkaline phosphatase was detected using 4-nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate reagents (Boehringer Mannheim) according to the manufacturer’s instructions.
EMSA
Nuclear extracts were prepared as described previously (25)
. In brief, cells were scraped in phosphate-buffered saline (PBS), and after centrifugation, the cell pellet was reconstituted in a hypotonic lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCI, and 0.1 mM EDTA), supplemented with 1 mM dithiothreitol (DTT) and a broad-spectrum cocktail of protease inhibitors (Sigma-Aldrich, Israel). The cells were allowed to swell on ice for 15 min, then Nonidet P-40 (NP-40) was added, and cells were lysed by vortexing. After centrifugation, nuclear extracts were obtained by incubating nuclei in a hypertonic nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.42 M KCI, 1 mM EDTA, supplemented with 1 mM DTT) for 15 min at 4°C. The supernatant was collected after centrifugation. Complementary oligonucleotide probes were synthesized (Hy Labs, Germany) for the hPar1-putative ARE –1791 to –1777, 5'-CAACTTCTATGTACA-3', and –1380 to –1366, 5'-CCAAGC GAGTGTCCC-3', and for a mutated version of the putative hPar1 ARE, 5'-CAACTT CTA TtTACA-3'. Oligonucleotides were annealed by heating them to 95°C in Tris-EDTA (TE) buffer and cooling slowly to room temperature. The double-stranded probes were labeled with -32P-dCTP using Rediprimer II (Amersham Biosciences UK). The labeled probes (0.3 ng) were incubated in a total volume of 20 µL with 5 µg LNCaP cells nuclear extract, 2 µg poly[d(I-C)], and 2 µL 10x binding buffer (binding buffer: 100 mM Tris-HCl, pH 8.0, 200 mM KCl, 10 mM MgCl2, 10 mM EDTA, 10 mM DTT, 40% glycerol) at room temperature for 20 min. For competition experiments, 100-fold of unlabeled, double-stranded oligonucleotide was added 15 min before incubation. The core II sequence of the first intron of the C3 gene (5'-AGTACGTGATGTTCT-3') and a mutated C3 oligo (5'-AGTACGTGATtTTCT-3') were used for specific competition experiments, and an oligonucleotide containing a vitamin E-binding site was used in nonspecific competition experiments. The samples were loaded on a prerun (100 V for 1 h at room temperature) 6% polyacrylamide gel (acryl:bisacryl=29:1) and electrophoresed in 0.25x 0.1 M Tris, 0.083 M boric acid, 1 mM EDTA at 100 V for 3 h at room temperature. The gel was then dried and exposed to X-ray films.
ChIP and PCR analysis
LNCaP cells were grown in medium with 10% CS FCS for 48 h prior to steroid treatment. After 48 h, the media was replaced with CS FCS media supplemented with or without DHT (10 nM). After treatment, LNCaP cells were treated with formaldehyde and added directly to culture medium (to a final concentration of 1%) at room temperature for 10 min to cross-link histone proteins to DNA, then glycine (to a final concentration of 0.125 M) was added to plates to quench formaldehyde. Soluble chromatin was made as follows: Cells were washed and detached from the dish by scraping after addition of ice-cold PBS, then pelleted by centrifugation for 4 min at 700 g. The resultant cell pellet was then lysed, pelleted, and lysed in two consecutive lysis buffers: LB1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitor cocktail) and LB2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). After the second lysis, the pellet was suspended in LB3 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate) and sonicated. Samples were then centrifuged at 13,000 rpm for 10 min, and supernatant was collected. For immunoprecipitation, 10 µg antibodies, prebound protein A, were added to 500 µL of the purified chromatin sample and incubated overnight at 4°C. Immunocomplexes with the beads were washed with radioimmunoprecipitation assay buffer followed by a wash with TE, then the immunocomplexes were recovered by adding elution buffer [1% sodium dodecyl sulfate (SDS), 1%, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0] for 10 min at 65°C, then by centrifugation at 14 K rpm for 10 min. Antibody-immunocomplexed DNA was then recovered by phenol/chloroform extraction and ethanol precipitation and resuspended in TE. PCR primer sets were designed to overlap and span the androgen response region of the Par1 promoter: primer set forward, 5'-TCTTGGGTATGTTTCCAGAGG-3'; reverse, 5' AGAGCCCGGACACTTACATC-3', and of fibroblast growth factor 8 (FGF8; positive control); primer set forward, 5'-AGTTGGAAAGATGGGGCACA-3; reverse, 5'-GTCTTCACTTACAACCTCCC-3'. These primers were first evaluated using the Par1-Luc construct as DNA template. Quantitative PCR was then performed with eluted AR immunocomplexed DNA, Titanium Taq PCR kit (Clontech Laboratories, Palo Alto, CA, USA). PCR was performed on unprecipitated chromatin as a positive control and to correct for input volume. Amplification was carried out for 35 cycles (28 cycles for unprecipitated chromatin input lanes) with denaturation at 94°C for 1 min, annealing at 58°C (64°C for FGF8 primers) for 30 s, and extension at 72°C for 1 min. PCR products were run in 2% agarose gel.
Fluorescein-activated cell sorter (FACS) analysis
After a PBS wash, cultured cells were detached from plates by treatment with 0.5 mM EDTA. After being washed twice in PBS, the cells were resuspended in 200 µL PBS, and the anti-hPar1 antibody (WEDE15-PE) was added (20 µL/sample). Extensive washing in PBS followed this reaction, performed at 4°C for 60 min. After washes, cells were resuspended in 100 mL PBS and analyzed by FACS. Mouse immunoglobulin G (IgG) -phycoerythrin (Dako Cytomation, Glostrup, Denmark) was used as a negative control.
Western blot analysis
Cells were solubilized in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and broad spectrum protease inhibitor cocktails (Sigma-Aldrich) at 4°C for 30 min. The cell lysates were subjected to centrifugation at 10,000 g at 4°C for 20 min. The supernatants were saved, and their protein contents were measured; 50 µg of each lysate was loaded onto 10% SDS-polyacrylamide gels. After the proteins were separated, they were transferred to an immobilon-P membrane (Millipore, Beford, MA, USA). Membranes were blocked and probed with anti-AR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 0.5 µg/mL. The antibody was suspended in 3% bovine serum albumin in 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20. After washes with 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20, the blots were incubated with secondary antibodies conjugated to horseradish peroxidase. Immunoreactive bands were detected using the enhanced chemiluminescence reagent SuperSignal (Pierce, Rockford, IL, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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a). Examination of the regulatory region of the hPar1 promoter revealed several candidate ARE sites. The sequences of these putative ARE are shown in Fig. 1f
, including 5'-CAACTTctaTGTACA-3' at site –1791, 5'-CCAAGCgagTGTCCC-3' at position –1380, and 5'-CTTTGCcggTGTCCC at site –646 as compared with known AREs (26–31). Promoter activity of full-length hPar1 promoter or deletion constructs of the promoter was carried out in JEG3 cells expressing AR, similar to LNCaP cells (and in contrast to prostate PC3 cells lacking AR), as shown by RT-PCR analysis (Fig. 1c
). Although in LNCaP cells, DHT treatment results with an approximate threefold increase in hPar1 levels, in PC3 cells, highly aggressive cells, the levels of hPar1 are initially high. No effect is seen after treatment with DHT (Fig. 1b
). We transfected JEG3 cells with the full-length hPar1 promoter (Luc-F1) or deletion constructs of the Luc-promoter including bp from –1601 to the ATG start site (Luc-F5; hPar1) and from –702 to the ATG site (Luc-F6; hPar1). After 4 h DHT treatment (10–8M), there was a threefold increase in Luc activity in cells containing the full-length promoter but not in cells containing deleted promoter constructs (Fig. 1e
). After 8 h of DHT treatment, Luc activity was still high in Luc-F1-transfected cells but to a slightly smaller degree, and there is no change in Luc activity observed in cells transfected with Luc-F5 or Luc-F6 (Fig. 1d, e
). This suggests that a functional ARE site is present in the vicinity of –1791 bp in the hPar1 promoter located upstream of –1601 bp, and the two proximal ARE sites are not functional.
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Binding properties of a hPar1-ARE motif
The Luc-promoter transfection results suggested that the putative ARE located at –1791 is functional. To verify this, we prepared oligonucleotides (oligos) containing wild-type ARE sequences of –1791 (–1791 ARE) and –1380 (–1380 ARE) in the hPar1 promoter or mutated sequences lacking binding activities. Labeled oligos were incubated with nuclear extract (NE) from LNCaP cells and used in EMSA to determine their ability to bind nuclear proteins. Wild type –1791 ARE but not –1380 ARE showed significant binding (Fig. 2
a). This binding of –1791 ARE oligos were found to be dose dependent, increasing with the increased amounts of NE present in the incubation mixture (Fig. 2b
), and was inhibited efficiently by excess nonlabeled oligos (Fig. 2c
). These data demonstrate that the –1791 ARE is capable to bind proteins specifically present in LNCaP NE.
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We next determined the specificity of ARE binding and whether binding is dependent on the presence of AR. To this end, nuclear extracts were prepared from two prostate-derived cell lines, LNCaP (expressing AR) and PC3 cells (devoid of AR). EMSA (Fig. 3
) demonstrated that factors present in LNCaP retarded the mobility of –1791 ARE domain in a specific manner, similar to the pattern obtained for the C3 consensus site. No binding however, was observed with NEs obtained from PC3 cells, suggesting that AR is required for binding to this element (Fig. 3)
. Further evidence for the specificity of binding to the –1791 ARE and for its identity as an ARE was provided by competition studies. Although unlabeled wild-type –1791 ARE and unlabeled wild-type C3 consensus oligos were able to compete effectively with labeled –1791 ARE binding to LNCaP NE, mutated –1791 ARE and mutated C3 consensus oligos did not compete with their corresponding ARE binding (Fig. 3)
. Moreover, unlabeled –1791 ARE oligos were able to compete effectively with C3 consensus site binding in this assay, as unlabeled C3 consensus ARE competed effectively with –1791 ARE binding (Fig. 3a
).
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Specific anti-AR antibodies inhibited the interaction of AR with ARE (Fig. 3b
), suggesting that these antibodies recognize AR epitopes that interact or interfere with ARE association to AR. A similar inhibitory effect of the anti-AR antibodies was observed when a labeled C3 consensus ARE site was used. Control rabbit IgG did not affect AR-ARE interactions.
Taken together, these results strongly suggest that the ARE sequence located at –1791–1777 in the hPar1 promoter is functional, capable of interacting in a sequence-specific manner with its cognate receptor, similar to a well-defined ARE sequence of the C3 consensus site (27)
. This indicates that AR may regulate hPar1 expression by binding directly to the ARE motif in the promoter and thereby increase transcription.
In vivo ChIP analysis demonstrates AR binding to a specific androgen response region in the hPar1 promoter
We next asked if the identified androgen response region in the hPar1 promoter interacted in vivo with AR. To confirm an association between the AR protein and the identified androgen responsive region in the hPar1 promoter, we performed PCR on chromatin DNA specifically immunocomplexed to AR.
Chromatin fragments were immunoprecipitated from cultured LNCaP cells before and after DHT treatment using the following antibodies: anti-AR, anti-PAR3, or a control rabbit IgG. DNA from the immunoprecipitated complex was then isolated. From this DNA, a 197-bp fragment of the hPar1 promoter was amplified by PCR using a set of primers directed to cover the region around –1791 bp ARE in the promoter. The signal level obtained in the noncomplexed chromatin PCR assay was used to confirm that equal amounts of DNA had been loaded. This PCR product exhibited a 2.9-fold induction in the presence of androgen compared with untreated cells. The signal intensity ratio for AR binding before and after treatment was 3324 and 9778, respectively, for the hPar1 gene. When an irrelevant antibody (anti-PAR3) or a control rabbit IgG was used to immunoprecipitate the chromatin from cell lysates (with and without androgen), only minimal levels of expression with no difference between treatments were obtained (1828 and 1683 before and after treatment, respectively, after PAR3 and 1629 and 1511 before and after treatment, respectively, for IgG), probably as a result of nonspecific, residual immunoprecipitation products (Fig. 4
). In contrast, a 2.2-fold induction (5263 and 11579 before and after treatment, respectively) was seen after androgen treatment in FGF8b (33)
, a known AR-regulated gene, which serves as a positive control. The specific induction was observed using an appropriate set of primers directed to the ARE region in the FGF8b promoter, after immunoprecipitation with anti-AR antibodies (Fig. 4)
. These data demonstrate that upon addition of androgens (DHT treatment), AR protein binds to the hPar1 promoter in a ligand-dependent manner. This protein-DNA complex was immunoprecipitated by an anti-AR antibody but not by irrelevant or control IgG antibodies, pointing to the specificity of the immunocomplex formed. The functional significance of this binding was further demonstrated by PCR analysis showing androgen-regulated hPar1-induced expression.
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Androgen-ablation therapy in prostate cancer leads to loss of hPar1 gene expression
hPar1 expression was analyzed in biopsy specimens from cancerous tissues and non-neoplastic prostate tissues. In situ hybridization reveals high levels of hPar1 expression in neoplastic glandular prostate epithelia and low-to-minimal levels in non-neoplastic prostate epithelia (Fig. 5
a). Semiquantitative RT-PCR analysis yielded a similar result, showing high expression levels in the cancerous tissue compared with minimal expression in non-neoplastic prostate tissue (Fig. 5b
).
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In prostate cancer patients, total androgen ablation therapy is a common practice with the goal of reducing the tumor mass to an operable size. The availability of paraffin-embedded clinical tissue specimens representing a wide spectrum of neoplastic prostate tissue before and after androgen ablation provided a unique opportunity to examine the consequences of this treatment on the level of hPar1 expression. A similar approach used in other studies showed that androgen ablation therapy leads to loss of vascular endothelial growth factor (VEGF) and obliteration of immature blood vessels in xenografted tumors (34)
and clinical biopsy specimens (35)
.
In situ hybridization analysis of these archival samples using a hPar1 DIG-labeled riboprobe showed that in nine out of nine grade- and age-matched pairs (all biopsies exhibiting AR expression, data not shown), there was a marked reduction in hPAR1 expression in the androgen-ablated specimens compared with prostate tumor specimens before the ablation treatment. This was obtained regardless of whether poorly (Fig. 6
A, B for hPar1 levels using antisense and sense probes, respectively; Fig. 6C, D
for hPar1 expression after hormone ablation) or well-differentiated tumors (Fig. 6E-H
before and after hormone ablation, respectively) were analyzed. We conclude that hPar1 expression is reduced markedly after hormone deprivation, and this is compatible with the fact that a functional ARE sequence is present in the hPar1 promoter.
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hPar1 expression in aggressive androgen-independent cell lines
To develop new therapeutic strategies and drug screening for prostate cancer treatment, it is imperative to understand the molecular events that take place during the progression of a tumor from a hormone-dependent to a hormone- independent stage. An aggressive subtype of a slow, androgen-dependent, parental LNCaP cell line was generated through in vitro androgen-deprivation and selection (36)
, termed CL1. Among the molecular differences observed between CL1 and the parent LNCaP cells is the down-regulated expression of tumor suppressor-like genes in contrast to elevated expression of an array of growth and angiogenic factors. These changes reflect a state of advanced hormone-resistant prostate cancer, closely linked to the aggressive behavior of the tumor (30)
. When hPar1 expression was examined in CL1 cells, high hPar1 was observed using FACS (Fig. 7
a) and RT-PCR analyses (Fig. 7b
, lane B) for protein and mRNA levels, respectively. AR expression analyzed by Western blot analysis revealed only minimal levels in CL1 cells (Fig. 7c
, lane B). CL1 behaves similarly to another known, aggressive prostate human adenocarcinoma cell line PC3, showing high hPar1 expression (Fig. 7b
, lane C) and lacking AR (37)
. In contrast, the slow-growing, androgen-dependent, parental LNCaP cells express low levels of hPar1 (Fig. 7b
, lane A) and high AR levels (Fig. 7c
, lane A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5
6
7)
. The primary goal of this study was to determine whether hPar1, previously shown to overexpress in malignant epithelia (9
10
11
12
13
, 19)
, is modulated by androgens. We find that the high levels of hPar1 mRNA, which are localized abundantly in neoplastic prostate epithelium, are reduced markedly after hormone ablation therapy. Very little to nearly no hPar1 is expressed in normal prostate tissue. This regulation is demonstrated by comparing radical prostatectomy specimens from the same patient before and after androgen-ablation therapy for several weeks before surgery. Human Par1 thus joins a list of genes that were previously shown to be down-regulated after androgen deprivation, such as VEGF (35)
and FGF8b (33)
. VEGF levels are markedly reduced, as shown in retrospective analysis of surgically removed human tumors (35)
and in established animal models after castration and hormone ablation (34)
.
In LNCaP, an AR-positive prostate cell line, treatment with DHT induced an increase in hPAR1 expression, similar to the regulation of several other proteins such as prostate-specific antigen, T cell receptor 
-chain alternate reading frame protein, FGF8b, and human glandular kallikrein 2 overexpressed in prostate carcinoma and modulated by androgens in vivo and in vitro (27
, 32
, 33)
. After ligand (androgen) binding, the AR interacts directly with an ARE present at –1791–1777 upstream of the ATG start site in the hPar1 promoter. Transfection of the Luc–hPar1 promoter to AR-expressing cells showed 2.5- to threefold stimulation by DHT treatment. A similar increase in hPar1 mRNA levels in LNCaP cells after DHT suggests that the endogenous promoter is also responsive to DHT. Although sequence analysis revealed several potential androgen-response regions located in the hPar1 promoter, functional analysis of these regions using full-length Luc-promoter hPar1 and deleted Luc-promoter hPar1 constructs of varying size in vitro revealed that only the site located at –1791–1777 is functional. These studies, however, do not rule out the possibility that the proximal ARE sites located downstream of –1791–1777 are functionally active only in the presence of the –1791–1777 domain. The fact, however, that oligonucleotides of the proximal, putative ARE sites show no binding in EMSA, as indicated for the –1380 ARE (Fig. 2a
) and –646 ARE (data not shown), supports the conclusion that these sites are indeed not functional. Conversely, the –1791–1777 ARE does bind strictly to AR in LNCaP NE. This binding is inhibited specifically in the presence of AR antibodies, excess, unlabeled hPar1-ARE oligos and by excess, unlabeled oligos of a known C3 consensus ARE site, supporting the functionality of this domain further.
In addition to the specifically identified ARE site, the DNA sequence flanking ARE is known to be essential for AR-mediated transcriptional activity (39)
. It is reasonable to postulate that in the presence of its ligand, the AR translocates to the nucleus, where it recognizes and binds directly or as part of a transcriptional complex to the –1791 ARE site in the hPar1 promoter. Interactions between the AR and ARE may be facilitated by the recruitment of AR coactivators, which can act as bridging molecules between steroid hormone receptors and general transcription factors, resulting in increased polymerase II activity (40
41
42)
.
In the present study, we have identified a functional ARE domain within the hPar1 promoter and demonstrated that hPar1 expression is modulated by androgens. This modulation is evidenced by induction of hPar1 mRNA by DHT, Luc-promoter activities, EMSA, and ChIP assays characterizing binding properties and in vivo association between AR and hPar1-ARE after DHT. In parallel, reduced levels of hPar1 portray this outcome by the following hormone ablation treatment in clinically obtained biopsy specimens in vivo.
In CL1, an aggressively metastasizing subclone of LNCaP exhibiting androgen-independent properties, hPar1 is expressed in high levels. Although androgen resistance does not necessarily indicate lack of AR (43
44
45
46)
, in the case of CL1 cells, Western blot analysis reveals minimal expression levels of AR. These data are compatible with studies by Patel et al. (36)
, showing by RT-PCR analysis, minimal AR levels. CL1 cells express lower levels of tumor suppressor-like genes (bcl-2, p53, pTEN, and E-cadherin) than the parental LNCaP and a higher levels of an array of growth and angiogenic factors including epidermal growth factor receptor, VEGF, transforming growth factor-ß, interleukin (IL) -8, and IL-6. These changes reflect a state of advanced hormone-resistant prostate cancer. These distinct molecular characteristics of CL1 cells appear closely linked to a tumor-aggressive behavior. A similar outcome is seen in another malignant prostate cell line PC3, exhibiting high hPar1 levels and lacking AR (37)
. The high level of hPar1 in hormone-independent cell lines with minimal AR expression shows that AR expression is not essential for this expression. The mechanism of hPar1 mRNA elevation in a hormone-independent phase is unknown and remains yet to be fully elucidated. These properties of hPar1 are similar to the described VEGF levels in animal models. VEGF levels are of dual phase: Initially, it is found reduced during hormone-regression phase, when Shionogi tumors are injected to severe combined immunodeficiency mice (after castration), and is then increased in expression after relapse, where the hormone-independent phase is taking over (34)
.
One possibility is that overexpression of hPar1 in malignant prostate adenocarcinoma is the result of loss of expression of a suppressor transcription factor gene such as AP-2. AP-2 expression is lacking in a range of neoplasia, such as metastatic melanoma and breast and colorectal cancers (23
, 47
48
49)
in an inverse correlation with hPar1 expression levels (9
, 23)
. Indeed, the loss of AP-2 causes up-regulation in the expression of PAR1, as demonstrated in melanoma, in addition to the expression of several genes including c-kit, MUC18, matrix metalloproteinase-2 (23)
, and VEGF (48)
.
Altogether, our data indicate that hPar1 expression correlates with prostate cancer progression in androgen-dependent and -independent phases. In the androgen-dependent phase, hPAR1 expression appears to be controlled directly via AR-induced transcriptional activation. In contrast, we have no current knowledge as to what induces hPar1 expression in the hormone-independent phase (beyond the scope of the present study). Based on the above data, and previous findings, suggesting a causative role for hPAR1 in tumor progression, we propose now that the modulation of hPar1 expression by controlling androgen levels in the hormone-dependent phase or by silencing hPar1 gene expression in the hormone-independent phase may provide an efficient tool for treating prostate cancer.
It is needless to point out that silencing hPar1 gene expression may provide only one approach for prostate therapy, as other genes such as FGF8b (33)
and VEGF (34)
are up-regulated as well during a hormone-independent, more aggressive phase of prostate cancer. Therefore, silencing one significant gene in the process might not be sufficient. However, the direct neutralization of the hPar1 gene may prove beneficial on the currently used hormone ablation therapy treatment, which is useful only for a limited period of time (during the hormone-dependent phase).
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ACKNOWLEDGMENTS |
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Received for publication June 10, 2004. Accepted for publication September 23, 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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