Hypoxia and HIF-1 increase S100A8 and S100A9 expression in prostate cancer

Authors

  • Sina Grebhardt,

    1. Hormones and Signal Transduction Group, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany
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  • Christian Veltkamp,

    1. Hormones and Signal Transduction Group, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany
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  • Philipp Ströbel,

    1. Institute of Pathology, University Hospital Mannheim, Heidelberg University, Heidelberg, Germany
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  • Doris Mayer

    Corresponding author
    1. Hormones and Signal Transduction Group, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany
    • Hormones and Signal Transduction Group, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
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    • Tel.: +49-6221-423238, Fax: +49-6221-423237


Abstract

S100A8 and S100A9, two heterodimer-forming members of the cytosolic S100 Ca2+ signaling protein family, are overexpressed in various cancer types, including prostate cancer. They act as proinflammatory danger signals when secreted to the extracellular space and are thought to play an important role during tumorigenesis, affecting inflammatory processes, proliferation, invasion and metastasis of tumor cells. Despite this fact, little is known about tumor environmental factors influencing S100A8/A9 expression. The aim of this study was to test the effect of hypoxia and its master transcriptional regulator hypoxia-inducible factor 1 (HIF-1) on S100A8/A9 expression. Hypoxia treatment resulted in induction of S100A8/A9 protein and mRNA expression in prostate epithelial BPH-1 cells, the latter was also confirmed in the prostate cancer cell lines PC-3 and DU-145. Furthermore, overexpression of HIF-1α caused increase in S100A8/A9 protein and mRNA expression as well as secretion. Functional hypoxia response elements mediating promoter activation on HIF-1α overexpression were identified within the S100A8 and S100A9 promoters using promoter luciferase reporter constructs. Binding of HIF-1α to S100A8 and S100A9 promoters was confirmed by chromatin immunoprecipitation. Immunohistochemical analysis of a prostate cancer tissue array showed clear correlation of S100A8 and S100A9 with HIF-1α expression. Multivariate proportional hazard analysis revealed association of high S100A9 level with time to prostate cancer recurrence. In conclusion, we identified hypoxia and HIF-1 as novel regulators of S100A8/A9 expression in prostate cancer. S100A9 might be useful as prognostic marker for prostate cancer recurrence after radical prostatectomy.

S100A8 and S100A9 are two heterodimer-forming members of the S100 family of cytosolic Ca2+ binding proteins, which exhibit cell- and tissue type-specific expression.1, 2 While S100A8/A9 are constitutively expressed in myeloid cells, like neutrophils and monocytes, their expression is inducible in other cell types, for example, in different epithelial cells, like keratinocytes as well as in cancer cells.3, 4 As Ca2+ signaling proteins, they are involved in intracellular homeostatic processes, for example, regulating the tubulin cytoskeleton5 and NADPH oxidase activity6 in phagocytes. When secreted to the extracellular space by a still unknown nonclassical pathway, S100A8/A9 act as proinflammatory danger signals. Receptors mediating response to S100A8/A9 include toll-like receptor 4 (TLR-4)7 and receptor for advanced glycation end products (RAGE).8

S100A8/A9 are implicated and display marked changes in their expression in many types of cancer, including prostate cancer.3, 9 Different protumorigenic effects of S100A8/A9 have been identified.3, 4, 10 S100A8/A9 are thought to be involved in inflammation-associated carcinogenesis by creating an inflammatory microenvironment that promotes tumorigenesis.11 It has been shown that S100A8/A9 regulate accumulation and activity of myeloid-derived suppressor cells in the tumor microenvironment, leading to repression of host antitumor immune response.12, 13 In mouse, soluble tumor-derived factors induced S100A8/A9 expression in distant organs, thereby creating a premetastatic niche and facilitating tumor spread.14, 15 Furthermore, S100A8/A9 have been reported to activate key genes of protumorigenic pathways involved in tumor progression and metastasis.16 Direct tumor promoting effects of S100A8/A9, like stimulation of proliferation and migration have been shown for various tumor cell lines.3

In prostate cancer, S100A8/A9 are significantly overexpressed in tumor tissue compared to normal or benign hyperplastic tissue.9 S100A8/A9 overexpression increases with tumor grade, indicating a role of S100A8/A9 in prostate cancer progression.

Despite mounting evidence for their important role during tumorigenesis, little is known about S100A8/A9 expressional regulation. In skin, according to their role as danger signals, several stresses like detergent and UV exposure induce S100A8/A9 expression.17 In addition, various inflammatory mediators have been shown to regulate S100A8/A9 expression, mainly in immune and epithelial cells.11 Subsequently, S100A8/A9 were characterized as stress-induced proteins.17 Regarding the impact of tumor environmental factors on S100A8/A9 expression, information is scarce.

Hypoxia depicts a common stress factor in solid tumors, leading to onset of a variety of adaptive responses, including the transcriptional induction of a series of genes that participate in angiogenesis, glucose metabolism, cell proliferation, survival and migration.18 Thus, adaption to hypoxia promotes many key mechanisms of cancer progression and contributes to aggressive tumor behavior. Master regulator of the hypoxia response is transcription factor hypoxia-inducible factor 1 (HIF-1), comprising an oxygen sensitive subunit HIF-1α and a constitutively expressed subunit HIF-1β also known as aryl hydrocarbon receptor nuclear translocator (ARNT).19–21 Oxygen sensitivity is mediated via oxygen-dependent prolyl hydroxylases (PHDs). Under normal oxygen levels, HIF-1α is hydroxylated, which promotes binding of von Hippel-Lindau protein and subsequently leads to HIF-1α ubiquitinylation and targeted proteasomal degradation. Under hypoxic conditions, HIF-1α is stabilized and HIF-1 binds to hypoxia response elements (HREs) in the promoter regions of hypoxia-regulated genes. HREs are characterized by a conserved core sequence A/GCGTG and highly variable flanking sequences. Increased HIF-1 expression is found in the majority of human cancers, including prostate cancer, and correlates with increased patient mortality, tumor progression, invasiveness and metastasis.22

The purpose of this study was to test the impact of hypoxia and its master regulator HIF-1, as important stress factors of the tumor microenvironment, on S100A8/A9 expression in vitro and in vivo, using a prostate cancer tissue array with tumor samples of patients who underwent radical prostatectomy. Furthermore, we tested the capacity of S100A8/A9 as potential markers for prostate cancer progression.

Abbreviations

ARNT: aryl hydrocarbon receptor nuclear translocator; ChIP: chromatin immunoprecipitation; EV: empty vector; HIF-1: hypoxia-inducible factor 1; HRE: hypoxia response element; OV: overexpression vector; PBS: phosphate-buffered saline; PHD: prolyl hydroxylase; PSA: prostate-specific antigen; RAGE: receptor for advanced glycation end products; TLR-4: toll-like receptor 4

Material and Methods

Cell culture

Benign prostatic hyperplasia cell line BPH-1 (DSMZ, Braunschweig, Germany) and prostate cancer cell line PC-3 (ATCC-LGC Standards, Wesel, Germany) were maintained in RPMI-1640 (PAA, Cölbe, Germany). DU-145 prostate cancer cells (DSMZ) were cultured in Dulbecco's Minimal Essential Medium (DMEM) (Invitrogen, Karlsruhe, Germany). Media were supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom). Human origin and identity of the cell lines were verified by the Core Facility Genomics and Proteomics of the German Cancer Research Center using the multiplex human cell authentication assay.23 Cells were routinely cultured in 5% CO2 and 95% air in a humidified atmosphere at 37°C. For hypoxic exposure, cells were cultured in a hypoxia chamber (Billups Rothenberg, Del Mar, CA) in an atmosphere consisting of 1% O2, 5% CO2 and 94% N2.

Quantitative real-time PCR

Total RNA was isolated using RNeasy kit (Qiagen, Hilden, Germany) and reverse transcribed to cDNA with QuantiTect Reverse Transcription kit (Qiagen). Quantitative (q) real-time polymerase chain reaction (RT-PCR) was performed using a DyNAmo Flash SYBR Green qPCR kit (New England Biolabs, Frankfurt/Main, Germany) and a Chromo4 Real-Time Detector (Bio-Rad, München, Germany). Several housekeeping genes were tested for expression stability using geNorm software. Hydroxymethylbilane synthase (HMBS) was identified to be most stable under the tested experimental conditions and chosen for normalization. Relative mRNA expression was calculated by comparative Ct method ( equation image). All reactions were performed in triplicates. Sequence of the primers used was as follows: S100A8 forward primer: 5′-AAT TTC CAT GCC GTC TAC AG-3′; S100A8 reverse primer: 5′-CGC CCA TCT TTA TCA CCA G-3′; S100A9 forward primer: 5′-AAA AGG TCA TAG AAC ACA TCA TGG-3′; S100A9 reverse primer: 5′-GAA GCT CAG CTG CTT GTC TG-3′; HMBS forward primer: 5′-CGC ATC TGG AGT TCA GGA GTA-3′; HMBS reverse primer: 5′-CCA GGA TGA TGG CAC TGA-3′.

Western blotting

Cells were washed with ice-cold phosphate-buffered saline (PBS), scraped and lysed using lysis buffer containing 50 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.6, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1.5% Triton X-100 and complete protease inhibitor cocktail (Roche, Mannheim, Germany) for 1 hr on ice. Lysates were cleared by centrifugation (16,000g, 15 min, 4°C). Protein concentration was determined using the DC protein assay kit (Bio-Rad). Ten micrograms of protein was subjected to 10% (HIF-1α) or 15% (S100A8/A9) sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, followed by electrotransfer to polyvinylidene fluoride (PVDF) membranes (Immobilon P, Millipore, Eschwege, Germany). Immunoblot analysis was performed with mouse monoclonal anti-HIF-1α (Novus Biologicals, Littleton, CO), rabbit polyclonal anti-S100A8 and anti-S100A924 (kindly provided by Dr. C. Kerkhoff, Institute for Experimental Dermatology, University of Münster, Münster, Germany), mouse monoclonal anti-β-actin (Abcam, Cambridge, UK) and horseradish peroxidase-labeled secondary antibodies (Dianova, Hamburg, Germany). Immunoreactive bands were detected by ECL plus reagent (VWR, Darmstadt, Germany). For quantitative analysis signal intensities were determined using ImageJ software and normalized on β-actin.

Plasmids and transfection

HIF-1α overexpression plasmid (HIF-1α OV) was generated by subcloning HIF-1α cDNA from Gateway entrance vector pENTR221 (Invitrogen; Clone ID: 190122276) into Gateway destination vector pFLAG-CMV-D11 using Gateway LR clonase II Enzyme Mix (Invitrogen) and following supplier's protocol.

For construction of luciferase reporter plasmids, whole genomic DNA was isolated from BPH-1 cells with QIAamp DNA kit (Qiagen). Promoter regions were amplified using Phusion Hot Start polymerase (New England Biolabs) and inserted into pGL3-Basic vector (Promega, Mannheim, Germany). Point mutations of putative HREs were performed using QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Waldbronn, Germany) and the following primers: A8Luc/U forward primer: 5′-GCC ATC AGC CAG GAC AAT AGT TCT CAA CCC TTT TGC GGT C-3′, A8Luc/U reverse primer: 5′-GAC CGC AAA AGG GTT GAG AAC TAT TGT CCT GGC TGA TGG C-3′, A9Luc/D forward primer: 5′-GTT GCA GCC TGT GCT TTC TTT TTA TTT TGG CGT TCC CCC AC-3′, A9Luc/D reverse primer: 5′-GTG GGG GAA CGC CAA AAT AAA AAG AAA GCA CAG GCT GCA AC-3′. Exchanged nucleotides are underlined.

For transient transfection, 3 × 105 cells per well were seeded into six-well plates. After 24 hr, plasmids were transfected using Effectene transfection reagent (Qiagen) following manufacturer's protocol.

Luciferase assay

BPH-1 cells were cotransfected with luciferase reporter plasmids and HIF-1α overexpression vector (OV) or empty vector (EV) as described before. After 48 hr, cells were washed with PBS and lysed using Cell Culture Lysis Reagent (Promega). Lysates were cleared by centrifugation (16,000g, 15 min, 4°C), and luciferase activity was analyzed using Bright-Glo firefly luciferase assay system (Promega). Protein concentration was determined and used for calculation of specific luciferase activity.

Chromatin immunoprecipitation

BPH-1 cells were plated in 100 mm dishes and grown for 24 hr, followed by hypoxia (1% O2) or normoxia treatment for 24 hr. Cells of two 100 mm dishes per treatment were cross-linked using 0.75% formaldehyde for 10 min at room temperature (RT) and cross-linking was arrested by addition of glycine (125 mM final concentration). Cells were washed with PBS, scraped off in PBS and harvested by centrifugation at 1,000g for 5 min. Pellets were resuspended in FA lysis buffer containing 50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS and complete protease inhibitor cocktail (Roche). DNA was sheared to fragments of 200–1,000 bp by sonication. Lysates were cleared by centrifugation (16,000g, 15 min, 4°C). Chromatin preparation was diluted in RIPA buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, complete protease inhibitor cocktail (Roche) and incubated with or without 7.5 μg ChIP-grade rabbit polyclonal anti-HIF-1α antibody (Abcam) on a rotary shaker overnight at 4°C. Chromatin was cleared (12,000g, 4°C, 10 min) and incubated with 20 μl preblocked Protein A agarose beads (0.25 μg/μl beads single-stranded salmon sperm DNA, 0.1 μg/μl beads bovine serum albumin (BSA)) for 1 hr at 4°C. Beads were washed with high salt, low salt and LiCl buffer. Immunoprecipitated DNA was recovered by incubation in 1% SDS and 100 mM NaHCO3 at RT for 30 min. Cross-linking was reversed by addition of NaCl (0.5 M final concentration) and incubation at 65°C for 6 hr, followed by purification using PCR Purification Kit (Qiagen). PCR was performed using primers spanning potential HREs in the S100A8 (forward primer: 5′-TGG ATA TAG CCC TTG GCA AC-3′; reverse primer: 5′-CAT TCC TCC ATG GCT TCT GT-3′) and S100A9 promoter (forward primer: 5′-GAC AGG CCA TCT CCC AGT TA-3′; reverse primer: 5′-TGC GAC ATT TTG CAA GTC AT-3′).

Patients and prostate cancer tissue array

With approval from the local ethics committee, 167 radical prostatectomy specimens were collected from the University Hospital Würzburg, Germany. Patient characteristics are listed in Table 1. For two patients, no clinical data were available. Among the remaining 165 patients, median age at diagnosis was 64 years. Median follow-up was 55 months. Eighteen patients developed tumor recurrence, defined as rise in serum prostate-specific antigen (PSA) level, within a median time of 34 months. Tumor stage ranged from T2a to T3b, Gleason scores were between 4 and 10.

Table 1. Patient characteristics
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A tissue array was constructed taking five cores (1-mm diameter) of cancerous regions and three cores of normal prostate regions from each formalin-fixed and paraffin-embedded specimen.

Immunohistochemistry

For immunohistochemical staining of proteins, 4-μm thick tissue array sections were deparaffinized with xylene and rehydrated using a descending concentration series of ethanol in H2O. Endogenous peroxidases were blocked with 3% H2O2 for 20 min and epitopes were retrieved by microwaving in 0.01 M citrate buffer pH 6.0 for 2 × 7 min. After blocking with 5% BSA in tris(hydroxymethyl)-aminomethane-buffered saline (TBS), sections were incubated with rabbit polyclonal anti-HIF-1α antibody (Abcam) or mouse monoclonal antibodies targeting S100A8 or S100A9 (BMA, Augst, Switzerland) at 4°C overnight. Immunohistochemical staining was performed using Dako LSAB+ HRP-System (Dako, Hamburg, Germany). Sections were counterstained with hematoxylin and mounted in glycerol/gelatine. Protein expression was determined semiquantitatively, taking into account staining intensity (scored 0–2) and percentage of stained epithelial cells, yielding four categories: no (0), weak (Score 1, 1–60% positive cells), moderate (Score 1, 60–100% positive cells and Score 2, 1–30% positive cells) or strong (Score 2, 30–100% positive cells). Exclusive nuclear staining of HIF-1α was scored as intensity 1, in accordance with reports that cytoplasmic staining is a more important predictor for outcome.25

Statistical analysis

Data are presented as mean ± SD of three independent experiments and were analyzed using GraphPad Prism Version 5.0 and InStat Version 3.10 (GraphPad Software). Differences between groups were statistically evaluated using one sample one-tailed Student's t-test. A p value <0.05 was considered significant.

For tissue array evaluation, correlation of protein expression was analyzed using Pearson's correlation coefficient test. Correlation of S100A8, S100A9 and HIF-1α with time to tumor recurrence was tested by univariate and multivariate (adjusted for Gleason score, tumor stage and lymph node stage) Cox hazard regression modeling.

Results

Hypoxia induces S100A8/A9 protein and mRNA expression

BPH-1 cells were subjected to hypoxic environment (1% O2) for several time periods to investigate the effect of hypoxia on S100A8/A9 expression. Western blot analysis revealed strong induction of HIF-1α in hypoxia-treated cells compared to control cells after 24 hr, which was accompanied by upregulation of S100A8 as well as S100A9 protein levels (Fig. 1a). After 48 hr, S100A8 and S100A9 protein levels were increased twofold and 1.6-fold, respectively, in hypoxia-treated compared to normoxia-treated cells (Fig. 1b).

Figure 1.

Hypoxia induces S100A8 and S100A9 protein and mRNA expression. (a) Western blot analysis of HIF-1α, S100A8 and S100A9 protein levels in BPH-1 cells after 24, 48 and 72 hr of normoxic or hypoxic treatment. β-Actin was detected as loading control. (b) Quantitative analysis of S100A8 and S100A9 protein level after 48 hr hypoxia. Protein levels were normalized on β-actin and data are shown as fold of control (FOC). Results represent mean ± SD of three independent experiments; *p < 0.05 (Student's t-test, compared to respective normoxia treated control). (c) S100A8 and S100A9 mRNA expression in BPH-1 cells after hypoxia treatment for different time periods. Expression levels were normalized on mRNA expression of housekeeping gene HMBS and related to normoxia treated control. Data represent mean ± SD of three independent experiments; *p < 0.05; **p < 0.01 (Student's t-test). (d) S100A8 and S100A9 mRNA expression in BPH-1, PC-3 and DU-145 cells after 48 hr hypoxia incubation. Expression levels were normalized on mRNA expression of housekeeping gene HMBS and related to normoxia incubated control. Data represent mean ± SD of three independent experiments; *p < 0.05; **p < 0.01 (Student's t-test, compared to respective normoxia treated control). (e) Western blot analysis of HIF-1α protein levels in BPH-1, PC-3 and DU-145 cells after 24 hr of normoxic or hypoxic treatment. β-Actin was detected as loading control.

To investigate the mechanism underlying S100A8/A9 upregulation during hypoxia, RNA was isolated from BPH-1 cells after hypoxic treatment for different time periods and subjected to qRT-PCR. S100A8 and S100A9 mRNA levels showed time-dependent increase in hypoxia treated compared to normoxia-treated control cells, indicating a regulation at the transcriptional level (Fig. 1c). These results were confirmed in two prostate cancer cell lines, namely PC-3 and DU-145. In these cell lines, hypoxia induced S100A8 mRNA expression 4.9-fold and 11.3-fold, and S100A9 mRNA expression 2.7-fold and 4-fold, respectively (Fig. 1d). In BPH-1 cells, increase of S100A8 and S100A9 mRNA expression in hypoxia-treated compared to normoxia-treated control cells was 1.9-fold and 1.5-fold, respectively. Induction of HIF-1α protein level in response to hypoxia treatment was stronger in PC-3 and DU-145 cells than in BPH-1 cells (Fig. 1e).

HIF-1 is involved in regulation of S100A8/A9 expression

Master regulator of the cellular response to hypoxic conditions is the transcription factor HIF-1. Therefore, we were interested whether HIF-1 is involved in transcriptional regulation of S100A8/A9. We overexpressed the oxygen-sensitive subunit HIF-1α in BPH-1 cells and tested the effect on S100A8/A9 expression by Western blotting and qRT-PCR. We found clear induction of S100A8/A9 protein (2.5-fold and 2.1-fold for S100A8 and S100A9, respectively) (Figs. 2a and 2b) as well as mRNA expression (1.6-fold and 1.4-fold for S100A8 and S100A9, respectively) (Fig. 2c) in HIF-1α overexpressing (HIF-1α OV) compared to EV transfected cells. These results indicate that HIF-1 is involved in transcriptional regulation of S100A8/A9 under hypoxic conditions.

Figure 2.

HIF-1α overexpression increases S100A8 and S100A9 protein and mRNA expression as well as secretion. (a) Western blot analysis of HIF-1α, S100A8 and S100A9 protein levels in BPH-1 cells after HIF-1α OV or EV transfection. (b) Quantitative analysis of S100A8 and S100A9 protein level after overexpression of HIF-1α. Protein levels were normalized on β-actin and data are shown as FOC. Results represent mean ± SD of three independent experiments; *p < 0.05 (Student's t-test, compared to respective EV transfected control). (c) S100A8 and S100A9 mRNA expression in BPH-1 cells after overexpression of HIF-1α. Expression levels were normalized on mRNA expression of housekeeping gene HMBS and related to EV transfected control. Data represent mean ± SD of three independent experiments; **p < 0.01 (Student's t-test, compared to respective EV transfected control). (d) S100A8 and S100A9 proteins detected in conditioned medium of BPH-1 cells in response to HIF-1α OV or EV transfection.

Furthermore, we tested the effect of HIF-1α overexpression on secretion of S100A8/A9. Western blot analysis of conditioned medium of BPH-1 cells revealed a strong increase in secretion of S100A8 as well as S100A9 protein in response to HIF-1α OV compared to EV transfection (Fig. 2d).

HIF-1 binds to HREs and mediates activation of S100A8/A9 promoters

HIF-1 exerts its effects via binding to HREs in the promoter regions of its target genes. We analyzed S100A8 (GenBank accession no.: NM_002964.4) and S100A9 (GenBank accession no.: NM_002965.3) promoters for potential HREs using Transfaq database. We identified seven potential HREs in the S100A8 promoter, three located on the sense strand (consensus motif 5′-CGTG-3′, located at position −468, +289 and +338) and four located on the antisense strand (consensus motif 5′-GTGC-3′, located at position +58, +201, +221 and +298). Analysis of the S100A9 promoter revealed one HRE motif on the sense strand (located at position −1,010) and one on the antisense strand (located at position +311).

To test whether the identified HRE motifs contribute to activation of S100A8 and S100A9 promoters in response to HIF-1, we designed two promoter luciferase reporter constructs for each S100A8 and S100A9 promoter, respectively, containing the promoter sequence upstream (A8Luc/U, A9Luc/U) or downstream (A8Luc/D, A9Luc/D) of the transcription start site, and analyzed the effect of HIF-1α overexpression on luciferase activity (Fig. 3).

Figure 3.

HIF-1α binds to S100A8 and S100A9 promoters and induces promoter activity. Two promoter luciferase reporter constructs covering sequences upstream (A8Luc/U and A9Luc/U) or downstream (A8Luc/D and A9Luc/D) of transcription start site were generated for S100A8 and S100A9 promoter, respectively. Putative HRE1 in the S100A8 promoter and HRE2 in S100A9 promoter were mutated by site-directed mutagenesis. BPH-1 cells were transfected with S100A8 (a) or S100A9 (b) promoter luciferase reporter construct and HIF-1α OV or EV, respectively. After 48 hr, luciferase activity was determined. Data are shown as FOC and represent mean ± SD of three independent experiments; **p < 0.01 (Student's t-test, compared to respective EV transfected control). (c) ChIP was performed using anti-HIF-1α antibody or Protein A agarose b/o as control for unspecific binding of chromatin isolated from BPH-1 cells incubated for 24 hr under normoxia or hypoxia. Precipitated DNA was purified and subjected to PCR, using primers spanning potential HRE1 in upstream promoter region of S100A8 and HRE2 in downstream promoter region of S100A9, respectively.

Activity of A8Luc/U, containing one potential HRE (HRE1: 5′-CACGTG-3′), was induced 2.1-fold by HIF-1α OV compared to EV-transfected control. This effect was abolished by mutation of the HRE motif. A8Luc/D activity was not increased by HIF-1α OV, suggesting that the six potential HREs located in this promoter region of S100A8 are not functional (Fig. 3a).

Activity of A9Luc/D (2.4-fold) but not of A9Luc/U was increased by HIF-1α OV. Mutation of the potential HRE in A9Luc/D (HRE2: 5′-TTGCAC-3′) prevented promoter activation by HIF-1α overexpression (Fig. 3b). These data indicate that both the S100A8 and S100A9 promoters contain at least one HRE that induces promoter activation in response to HIF-1.

Next, we tested for interaction of HIF-1 with S100A8 and S100A9 promoters by chromatin immunoprecipitation (ChIP). S100A8 and S100A9 promoter fragments spanning HRE1 or HRE2, respectively, were efficiently precipitated by HIF-1α antibody under hypoxic conditions but not under normoxic conditions or by Agarose A beads only (b/o) (Fig. 3c). These data demonstrate that HIF-1 binds to the S100A8 and S100A9 promoters.

S100A8 and S100A9 expression correlates with HIF-1α expression in prostate cancer tissue

To test whether HIF-1 is also associated with S100A8/A9 expression in vivo, we performed immunohistochemical staining of the proteins using a prostate cancer tissue array (Fig. 4). Pearson correlation analysis adjusted for patient and tissue type effects revealed clear correlation of S100A8 as well as S100A9 with HIF-1α expression (r = 0.31 and r = 0.54, p < 0.0001). Overall, we identified 11 prostatic intraepithelial neoplasia in the tissue array showing weak (one), moderate (two) or strong (eight) HIF-1α expression. This number was too small for further analysis.

Figure 4.

S100A8, S100A9 and HIF-1α show similar expression patterns in prostate cancer tissue. Proteins were stained by immunohistochemistry in a prostate cancer tissue array, comprising five tumor tissue cores (be) and three normal prostate tissue cores (a) per case (167 cases in total). Staining was classified into four categories: no (a, b), weak (c), moderate (d) and strong (e) staining.

S100A9 expression is associated with time to prostate cancer recurrence after radical prostatectomy

To investigate the capacity of S100A8, S100A9 and HIF-1α as potential prognostic factors for prostate cancer, their expression was correlated with time to tumor recurrence using both univariate and multivariate Cox proportional hazard modeling.

High S100A9 as well as HIF-1α expression was significantly associated with shorter time to tumor recurrence (rise in serum PSA level) in univariate analysis (Table 2). Multivariate analysis, adjusted for known prognostic factors (Gleason score, tumor and lymph node stage), also showed relevant correlation of S100A9 and HIF-1α expression with time to tumor recurrence, albeit not significant. S100A8 was not significantly associated with time to recurrence in both models.

Table 2. Univariate and multivariate Cox hazard regression analysis for correlation with time to tumor recurrence
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Discussion

In this study, we identified hypoxia as a novel regulator of S100A8/A9 mRNA as well as protein expression in benign epithelial cells and cancer cells of the prostate. Upregulation of S100A8/A9 mRNA was mediated via direct binding of HIF-1 to HREs within the S100A8 and S100A9 promoters. Furthermore, we found that S100A8 and S100A9 expression correlates with HIF-1α expression in prostate cancer tissue samples, indicating a role of HIF-1 in the regulation of S100A8/A9 expression in vivo. Despite their important role during different diseases including cancer, little is known about factors influencing S100A8/A9 expression. Thus, our findings represent important new insights into S100A8/A9 expressional regulation.

Although intracellularly involved in homeostatic processes, S100A8/A9 exhibit cytokine-like function when secreted to the extracellular space by a nonclassical pathway, amplifying and modulating inflammatory processes.4 They are classified as damage-associated molecular patterns, which activate cells of the innate immune system by binding to pattern-recognition receptors, like TLR-4 and RAGE. According to their role as danger signals, S100A8/A9 expression is induced by several stress factors. Proinflammatory mediators like lipopolysaccharide (LPS),26 tumor necrosis factor (TNFα)27 and interferon (IFNγ) and members of the interleukin (IL) family like IL-1α,28 IL-1029 and IL-2230 have been shown to increase S100A8/A9 expression in myeloid or epithelial cells. In human skin epidermis, several stresses like tape stripping, UV or detergent exposure lead to induction of S100A8/A9 expression.31 S100A8/A9 is furthermore upregulated in wounds of human and mouse skin,32 and UV exposure has been shown to induce S100A8 expression in mouse keratinocytes.33 Hypoxia represents a common stress factor of the tumor environment, ultimately leading to cell death when severe or prolonged.18 Thus, our results seem to be in line with previous findings, classifying S100A8/A9 as danger signals, induced in and released by cells on stress stimuli.

In this study, we show for the first time a direct regulation of S100A8/A9 expression by hypoxia. Previously, it has been shown that knockout of ARNT in mouse epidermis leads to oxygen-independent stabilization of HIF-1α, probably mediated via downregulation of Egln3/PHD, followed by substitutional dimerization with ARNT2.34 This caused upregulation of conventional HIF-1 target genes as well as S100A8/A9. However, the mechanisms underlying the induction of S100A8/A9 were not studied.

Despite the impact for cancer progression, induction of S100A8/A9 by hypoxia could also play an important role during ischemic events. Ziegler et al. reported upregulation of S100A8/A9 mRNA in mouse brains after induction of focal cerebral ischemia and suggested a contribution of S100A8/A9 to neuroinflammation and progression of ischemic damage.35 Furthermore, the effect of hypoxia on S100A8/A9 expression was tested in the context of acute myocardial infarction.36 In this study, S100A8/A9 protein could be neither detected in isolated rat hearts subjected to ischemia nor in hypoxia-treated rat cardiac myocytes. Therefore, the authors concluded that ischemia does not induce S100A8/A9 in myocardial tissue. Nevertheless, in vivo induction of ischemia in rat hearts by left coronary artery ligation caused strong S100A8/A9 upregulation, which was attributed by the authors to neutrophils infiltrating the infarcted myocardium. Exposure to hypoxic conditions was short, ranging from 10 to 240 min. In our study, we found significant increase of S100A8/A9 expression after 48 hr of hypoxic treatment. Thus, differences in study design or tissue and cell type studied may explain the differing study outcome.

We found induction of S100A8/A9 protein as well as mRNA expression in response to hypoxia treatment both in benign prostatic epithelial cells and in two prostate cancer cell lines, PC-3 and DU-145. Induction of S100A8/A9 mRNA expression was even more pronounced in PC-3 and DU-145 cells than in BPH-1 cells. Interestingly, this correlated with stronger induction of HIF-1α protein level in response to hypoxia in the respective cell lines. Therefore, higher sensitivity of the tested prostate cancer cell lines toward hypoxia could explain the stronger induction of S100A8/A9 mRNA expression. Overexpression of HIF-1α also caused upregulation of S100A8/A9 mRNA as well as protein expression in BPH-1 cells, indicating a direct transcriptional regulation via the HIF-1 pathway. Subsequent promoter analysis by database screening and promoter luciferase reporter constructs led to identification of functional HREs within the S100A8 and S100A9 promoters, located at position −474 and +311, respectively. Binding of HIF-1 to S100A8 and S100A9 promoters in response to hypoxia was confirmed by ChIP. Interestingly, the HRE in the S100A8 promoter is located at the sense strand, whereas the S100A9 promoter HRE is positioned on the antisense strand. Although the majority of identified HREs are located at the sense strand, binding of HIF-1 to antisense strand HREs and subsequent promoter activation has also been reported, for example, for RANTES37 and endothelin-1.38

Furthermore, we found a clear correlation of S100A8 and S100A9 with HIF-1α expression in prostate cancer tissue samples. This finding suggests HIF-1 to be one factor regulating S100A8/A9 expression in prostate cancer tissue. Some additional factors have been reported to be implicated in S100A8/A9 expressional regulation during tumorigenesis. On the one hand, the genes of S100A8 and S100A9 are located at chromosomal region 1q21, which is often affected by chromosomal rearrangements and DNA amplification in several cancer types, including prostate cancer.39 Furthermore, several inflammatory factors have been identified to regulate S100A8/A9 expression in immune and epithelial cells.11 Recently, the cytokine oncostatin M was shown to induce S100A9 expression in breast cancer cells via the STAT3 signaling cascade.40 These findings suggest a high complexity of regulation of S100A8/A9 expression. Nevertheless, studies about factors relevant for S100A8/A9 expression during tumorigenesis are limited. Therefore, our results provide important new information regarding mechanisms driving S100A8/A9 upregulation during tumorigenesis.

Prostate cancer is one of the major health problems in the male population, representing the second most frequently diagnosed cancer type in men worldwide.41 Usually an indolent disease, early stages can be treated effectively by radiotherapy or radical prostatectomy or can even be managed conservatively by “watchful waiting.”42 Nevertheless, a substantial fraction will progress rapidly and develop into aggressive, metastatic tumors. About one-third of patients who undergo radical prostatectomy will experience tumor recurrence.43 Current prognostic markers including PSA serum levels, Gleason score and tumor stage are limited in their predictive value.44 Therefore, additional prognostic markers helping to predict disease outcome more accurately are urgently needed. S100A8/A9 mRNA and proteins were found overexpressed in prostate tumor tissue compared to benign prostatic tissue.9 Furthermore, S100A9 serum levels were significantly increased in prostate cancer patients compared to patients with benign prostatic hyperplasia or healthy individuals, suggesting S100A9 as potential diagnostic serum marker. S100A8/A9 have been suggested as prognostic factors for different cancer types, like nonsmall lung cancer,45 cervical46 and breast cancer.47 HIF-1 and different target genes were proposed as prognostic markers for prostate cancer. In a recent study, HIF-1α, VEGF and osteopontin expression were found to be significantly correlated with time to prostate cancer recurrence.25 In this study, we were interested whether S100A8/A9 could serve as prognostic markers for prostate cancer. We found that S100A9 as well as HIF-1α expression were significantly correlated with time to prostate cancer recurrence (determined by rise in PSA serum levels), whereas the correlation was not significant for S100A8 expression. The results of the multivariate analysis also suggested an association of S100A9 and HIF-1α expression with time to tumor recurrence. Unfortunately, the multivariate analysis did not achieve statistically significant results, not even for known prognostic factors like Gleason score and tumor stage, due to the low number of recurrences in our study (18 out of 167 patients). Therefore, our results indicate an association of S100A9 expression with time to prostate cancer recurrence but further studies will be necessary to prove the prognostic value of S100A9.

In conclusion, we identified hypoxia and HIF-1 as novel regulators of S100A8/A9 expression. Correlation of HIF-1α and S100A8 as well as S100A9 expression in tissue samples indicates HIF-1 to be one factor regulating S100A8/A9 expression in prostate cancer. Furthermore, S100A9 could be useful as prognostic marker for tumor recurrence after radical prostatectomy.

Acknowledgements

The authors thank PD Dr. Claus Kerkhoff, Münster, for providing anti-S100A8 and anti-S100A9 antibodies for Western blot analysis. The authors are grateful to Dr. Tim Holland-Letz, Biostatistics, DKFZ Heidelberg, for his help with the statistical analysis. The authors also thank Prof. Sebastian Müller, Heidelberg, for advice on hypoxic treatment, Dr. Nibedita Gupta for helpful suggestions and critical reading of the manuscript and Dorothee Albrecht for excellent technical assistance.

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