Department of Surgery, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267
Submitted 5 August 2003 ; accepted in final form 20 November 2003
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ABSTRACT |
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epithelial; angiogenesis; angiostatic; oncogene; tumor
Cancer of the prostate is the leading cancer diagnosed in men and is the second leading cause of cancer-related death among men in the United States (24). Increased production of angiogenic chemokines by prostate tumor cell lines and in prostate cancer patients have been previously documented (6, 15, 30). Furthermore, tumor growth after injection of prostate cancer cells into SCID mice can be reduced by antibody neutralization of growth-related oncogene- (GRO-
, which is CXCL1) or IL-8 (CXCL8; Ref. 15). Prostate cancer cells that are engineered to overexpress IL-8 are significantly more tumorigenic and metastatic than control cells (11). These studies suggest that angiogenic CXC chemokines may be required for the growth and development of prostate tumors in humans.
The production of nearly all angiogenic ELR+ CXC chemokines, including GRO-, epithelial neutrophil-activating peptide (ENA-78; which is CXCL5), and IL-8, is regulated by the transcription factors nuclear factor-
B (NF-
B) and CCAAT/enhancer-binding protein (C/EBP; Refs. 2, 21, 25, 32). NF-
B appears to be the primary transcription factor for these genes, whereas C/EBP seems to play more of a role as a transcriptional enhancer. NF-
B is a ubiquitous transcription factor that has been best described as a major regulator of proinflammatory gene expression including genes for cytokines, cellular adhesion molecules, and chemokines (21). Aberrant activation of NF-
B has also been associated with increased production of angiogenic CXC chemokines by malignant cells. In the melanoma cell line Hs294T, it was found that activation of NF-
B was nearly 20-fold higher than in normal cells (23). There is also evidence that NF-
B is constitutively activated in prostate cancer cell lines (7, 19, 27), but it is currently unknown whether this activation is directly associated with augmented angiogenic chemokine production.
Less is known about the transcriptional regulation of the angiostatic ELR CXC chemokines. The production of these mediators is known to be induced by interferon (IFN)-, -
, and -
, and at least for MIG (CXCL9) and inducible protein-10 (IP-10, which is CXCL10), signal transducer and activator of transcription-1 (STAT1) has been implicated (9, 17). The transcriptional regulation of platelet factor 4 (PF4, which is CXCL4) and IFN-inducible T-cell
-chemoattractant (ITAC, which is CXCL11) have not been described but are likely to be similar to IP-10 due to their high homology (5, 13). STAT1 is activated by IFN-
, -
and -
, but its role as a regulator of angiostatic chemokine expression in cancer has not been assessed.
The primary goal of this study was to determine whether there are significant differences in angiogenic and angiostatic CXC chemokine production by normal prostate epithelial cells and prostate tumor cells derived from primary and metastatic tumor sites that may result in promotion of tumor growth. Second, we examined whether altered production of these mediators was caused by malignancy-related alterations in activation of the transcription factors NF-B and STAT1.
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MATERIALS AND METHODS |
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Cell culture. The cell line PZ-HPV-7 was used to represent normal human prostate epithelial cells. PZ-HPV-7 cells were derived from normal human prostate and transformed with human papillomavirus (HPV)-18. The cell line CA-HPV-10 was used to represent cancer cells from a primary prostate tumor. CA-HPV-10 cells were derived from a grade-4 prostatic adenocarcinoma and transformed with HPV-18. The cell line PC-3 was used to represent cancer cells from a metastatic prostate tumor. PC-3 cells were derived from a bone metastasis of a grade-4 prostatic adenocarcinoma. Cells were cultured in 5% CO2-95% humidity at 37°C in keratinocyte-serum-free medium (K-SFM, GIBCO-Invitrogen) with L-glutamine, human recombinant epidermal growth factor (5 ng/ml), bovine pituitary extract (50 µg/ml), penicillin (100 U/ml), and streptomycin (100 µg/ml).
For studies of chemokine production, 1 x 106 cells/well were seeded in 0.5 ml of medium in 24-well tissue-culture plates. Cells were allowed to grow to confluence (48 h), and at that time the medium was changed. Cells were then incubated, and the culture medium was collected after 0, 24, 48, 72, and 96 h for analysis of chemokine production. Culture medium was analyzed by ELISA for GRO-
, ENA-78, IL-8, MIG, IP-10, and ITAC. For studies using BAY 11-7085 (20) and MTA (16) for the inhibition of NF-
B and STAT1, respectively, these reagents were added to the fresh medium when the cells reached confluence and medium was harvested after 72 h for analysis of chemokine production.
For studies of the activation of the transcription factors NF-B and STAT1, 1 x 107 cells were seeded in 15 ml of medium in 100-mm cell culture dishes. Cells were allowed to grow to confluence (
5 days) and at that time the medium was replaced with fresh medium. The cells were harvested for nuclear protein extraction and analysis of NF-
B and STAT1 activation 24 h later. For studies using BAY 11-7085 and MTA for the inhibition of NF-
B and STAT1, respectively, the fresh medium was supplemented with or without 0.1 mM BAY 11-7085 or 1 mM MTA.
Transgenic mouse model of prostate cancer. Transgenic mice that spontaneously develop prostate cancer have been previously described (8). These mice express the simian virus 40 small and large T antigens under the control of the rat probasin promoter, which confers prostate specificity. These mice are referred to as the "transgenic adenocarcinoma of the mouse prostate" (TRAMP) model of prostate cancer. This project was approved by the University of Cincinnati Animal Care and Use Committee and conforms to the National Institutes of Health guidelines. Male TRAMP mice and wild-type mice on a C57BL/6 background were used for all experiments. At 30 wk of age, mice were killed, and prostate tissues and/or tumors were removed for analysis. Frozen prostate tissues or tumors were used for mRNA studies. Extraction of proteins from prostate tissue or tumor samples was performed as described elsewhere (31). Tissue or tumor extracts were analyzed by ELISA for MIP-2 and IP-10 proteins using reagents from R&D Systems.
Nuclear protein extraction and transcription factor activation assays. Nuclear extracts were prepared using the methods described by Dignam et al. (4). Protein concentrations were determined by bicinchoninic acid assay with TCA precipitation using BSA as a reference standard. For electrophoretic mobility shift assays, double-stranded NF-B consensus oligonucleotide was end labeled with [
-32P]ATP (3,000 Ci/mmol at 10 mCi/ml). Binding reactions containing equal amounts of nuclear protein extract (20 µg) and 35 fmol (
50,000 cpm, Cherenkov counting) of oligonucleotide were incubated at room temperature for 30 min in binding buffer that contained 4% glycerol, 1 mM MgC12, 0.5 mM EDTA, pH 8.0, 0.5 mM DTT, 50 mM NaC1, 10 mM Tris·HCl (pH 7.6), and 50 µg/ml poly(dI·dC). Binding-reaction products were separated in a 4% polyacrylamide gel and analyzed by autoradiography. For ELISA-based DNA-binding assays for NF-
B and STAT1, nuclear extracts were assayed using the methods outlined in the manufacturer's instructions.
CXC chemokine mRNA expression. Quantitative analysis of mRNA expression was performed using Quantikine mRNA kits (R&D Systems). These kits utilize a quantitative hybridization method that includes colorimetric signal amplification. Probes for analysis of human IL-8, IP-10, and ITAC as well as murine MIP-2 and IP-10 were obtained from R&D Systems. Total RNA was extracted from cell and tissue samples using TRIzol (Invitrogen, Carlsbad, CA) and analyzed according to the manufacturer's instructions. Data are expressed as attomoles of gene-specific mRNA per microgram of total RNA.
Statistical analyses. All data are expressed as means ± SE. Data were analyzed with a one-way ANOVA with subsequent Student-Newman-Keuls test. Differences were considered significant when P < 0.05.
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RESULTS |
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Dysregulated activation of NF-B and STAT1 in prostate cancer cells. Because the transcription factors NF-
B and STAT1 have been linked to the production of angiogenic and angiostatic CXC chemokines, respectively (9, 17, 21), we assessed their activation states using DNA-binding assays. Using an ELISA-based DNA-binding assay, we found that NF-
B activation was significantly increased in CA-HPV-10 cells compared with PZ-HPV-7 cells (Fig. 3A). In PC-3 cells, NF-
B activation was markedly greater than in both PZ-HPV-7 and CA-HPV-10 cells (Fig. 3A). To verify the ELISA-based assay and to provide more detailed information regarding the NF-
B complex, we conducted electrophoretic mobility shift assays of nuclear extracts. Similar results were found using this assay, which confirms the progressive increase in NF-
B activation in CA-HPV-10 and PC-3 cells over PZ-HPV-7 cells (Fig. 3B). Supershift assays were performed to determine the composition of the NF-
B complex in PC-3 cells. Supershift bands were detected only with antibodies to p50 and p65 (Fig. 3C), which indicates that p50/p65 heterodimers are the predominant form of NF-
B. We next evaluated the activation of STAT1 in PZ-HPV-7, CA-HPV-10, and PC-3 cells using an ELISA-based DNA-binding assay. Similar to results of angiostatic chemokine production, we found that PZ-HPV-7 cells had the highest STAT1 activation (Fig. 4). STAT1 activation in CA-HPV-10 cells was not statistically different from PZ-HPV-7 cells, but PC-3 cells had significantly less STAT1 activation than both PZ-HPV-7 and CA-HPV-10 cells.
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Effects of NF-B and STAT1 inhibition on prostate cancer cell chemokine expression. To assess whether inhibition of NF-
B or STAT1 alters the production of angiogenic or angiostatic chemokines, we treated cells with various concentrations of NF-
B inhibitor BAY 11-7085 or the STAT1 inhibitor MTA. The addition of 10 µM BAY 11-7085 had no effect on the production of IL-8, GRO-
, or ENA-78 in PZ-HPV-7, CA-HPV-10, or PC-3 cells (Fig. 5). The addition of 30 or 100 µM BAY 11-7085 significantly reduced the production of IL-8, GRO-
, and ENA-78 in CA-HPV-10 and PC-3 cells but had no effect on the low baseline production of these chemokines by PZ-HPV-7 cells (Fig. 5). Interestingly, 100 µM BAY 11-7085 also significantly reduced the expression of IP-10 and ITAC (Table 1), which suggests that NF-
B activation may be involved in the expression of IP-10 and ITAC. Next we examined whether treatment with BAY 11-7085 may have reduced angiogenic CXC chemokine protein expression by inhibiting the expression of mRNA. Because PC-3 cells had the highest expression of angiogenic CXC chemokine proteins and because IL-8 was produced in the greatest abundance by these cells, we determined the effects of BAY 11-7085 on IL-8 mRNA expression in PC-3 cells. We found that treatment with 100 µM BAY 11-7085 significantly reduced IL-8 mRNA expression in PC-3 cells after 24 and 48 h (Fig. 6). To confirm that BAY 11-7085 was indeed inhibiting the activation of NF-
B, we assessed NF-
B activation in PC-3 cells 24 h after the addition of fresh medium or fresh medium that contained 100 µM BAY 11-7085 (Fig. 7). Treatment with BAY 11-7085 significantly reduced the activation of NF-
B (Fig. 7).
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We next determined the effects of the STAT1 inhibitor MTA on the production of the angiostatic chemokines IP-10 and ITAC. The addition of 0.1, 0.3, and 1 mM MTA resulted in a dose-dependent reduction in IP-10 production in PZ-HPV-7 and CA-HPV-10 cells but did not alter the production of IP-10 by PC-3 cells (Fig. 8). Similarly, MTA dose-dependently inhibited the production of ITAC in PZ-HPV-7 cells but did not alter the low level of production in either CA-HPV-10 or PC-3 cells (Fig. 8). MTA had no effect on the production of IL-8, GRO-, or ENA-78 (data not shown). To examine whether reductions in IP-10 and ITAC protein expression induced by MTA were associated with reduced mRNA expression for these chemokines, we determined the effects of MTA on IP-10 and ITAC mRNA expression. For these experiments, we used PZ-HPV-7 cells because they expressed the highest amounts of IP-10 and ITAC. Treatment of PZ-HPV-7 cells with 1 mM MTA resulted in significant reductions in mRNA expression for both IP-10 and ITAC after 24 and 48 h (Fig. 9). To determine whether the inhibitory effects of MTA on the production of angiostatic chemokines were due to suppression of STAT1 activation, nuclear extracts from PZ-HPV-7 cells were analyzed 24 h after the addition of fresh medium or fresh medium that contained 1 mM MTA. Treatment with MTA was associated with a significant reduction in STAT1 activation (Fig. 10).
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Proangiogenic chemokine profile in murine prostate tumors. Because our in vitro studies showed that prostate cancer cells produced more angiogenic and less angiostatic chemokines than normal prostate epithelial cells, we next examined whether these findings were relevant in an in vivo model of prostate cancer. The TRAMP mouse model of prostate cancer has been shown to be similar biochemically and histologically to human prostate cancer (8). We examined the mRNA expression of the murine angiogenic CXC chemokine MIP-2 (a mouse homolog to human GRO-) and the angiostatic chemokine IP-10 from prostate tissues from normal 30-wk-old C57BL/6 and TRAMP mice. We found similar mRNA expression in prostate tissues from C57BL/6 mice and noncancerous prostate tissues from TRAMP mice for both MIP-2 and IP-10 (Fig. 11). In contrast, prostate tumor tissues from TRAMP mice had significantly higher mRNA expression for MIP-2 and significantly lower mRNA expression for IP-10 (Fig. 11). To assess whether these alterations in mRNA expression corresponded with increased protein production, we next analyzed these tissues for MIP-2 and IP-10 proteins. In parallel with mRNA expression, protein levels of MIP-2 and IP-10 were similar in normal prostate tissues from C57BL/6 and TRAMP mice (Fig. 12). However, in prostate tumor tissue from TRAMP mice, there was a three- to fourfold increase in the amount of MIP-2 protein expressed and a nearly 40-fold reduction in the expression of IP-10 protein (Fig. 12).
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DISCUSSION |
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The current study and previous reports by others (15) demonstrating increased angiogenic CXC chemokine production by prostate cancer cells are consistent with clinical studies of prostate cancer. Increased expression of IL-8 has been found in prostate tumors and in the serum of patients with prostate cancer but not in normal patients or patients with benign prostatic hyperplasia (6, 30). We show here that prostate tumors in the TRAMP mouse model have significantly greater expression of MIP-2, which is an analog to human GRO-. Furthermore, blockade of angiogenic CXC chemokines in animal models inhibits tumor growth, whereas injection into mice of tumor cells engineered to overexpress IL-8 results in augmented tumor growth (11). Angiogenic CXC chemokines may also play a significant role in the predisposition of African-American men to developing prostate cancer. The Duffy antigen receptor for chemokines (DARC) is a promiscuous chemokine receptor expressed on erythrocytes that also serves as a receptor for infection by Plasmodium vivax malaria (10). The majority of the population of African descent lack expression of the DARC on erythrocytes as a natural selection against malarial infection (14). The DARC has been shown to bind only to angiogenic and not to angiostatic chemokines, and it functions to remove these mediators from a site of production (3, 28). As such, individuals who lack erythrocyte expression of the DARC, as occurs in >95% of African men in endemic regions and in
70% of African-American men, may have a higher concentration of angiogenic CXC chemokines in the microenvironment of a developing prostate tumor (12). Thus angiogenic CXC chemokines may contribute to the greater risk of developing prostate cancer in African-American men.
We found that increased production of angiogenic CXC chemokines in prostate cancer cells was caused by progressive dysregulation of NF-B. Constitutive activation of NF-
B in prostate cancer cells has been previously demonstrated (7, 19, 27). Some of these studies have linked constitutive activation of NF-
B with increased activity of the inhibitor-
B (I
B) kinase complex, which leads to increased phosphorylation and degradation of I
B
(7, 19). We show here that normal prostate epithelial cells (PZ-HPV-7) had little activation of NF-
B, whereas cells derived from primary prostate tumors (CA-HPV-10) had slight but significant NF-
B activation and cells from a bone metastasis (PC-3) had marked activation of NF-
B. Furthermore, we demonstrated that this progressive activation of NF-
B is responsible for the production of angiogenic CXC chemokines. When we blocked NF-
B activation with the pharmacological agent BAY 11-7085, production of angiogenic CXC chemokines in prostate cancer cells was inhibited. Interestingly, treatment of cells with BAY 11-7085 also decreased the production of angiostatic chemokines IP-10 and ITAC, which suggests that NF-
B may contribute to the expression of these chemokines. Our findings are consistent with a previous report of potential synergism between STAT1 and NF-
B for transcription of IP-10 (18).
Our studies are the first to assess the production of angiostatic CXC chemokines by prostate cancer cells. Interestingly, we found that normal prostate epithelial cells (PZ-HPV-7) produce substantial amounts of these mediators, which was facilitated by activation of STAT1. However, prostate cancer cells from primary tumors (CA-HPV-10) produced markedly less of these mediators and those from metatstatic tumors (PC-3) produced almost none. In the TRAMP mouse model, prostate tumor tissue had virtually no detectable expression of the angiostatic CXC chemokine IP-10, which is in contrast to normal prostate tissue that expressed IP-10. In our in vitro studies, we found that decreased production of angiostatic CXC chemokines occurred concomitantly with a decrease in STAT1 activation. STAT1 has not been previously studied in the context of prostate cancer. Our data suggest that STAT1 plays an important role in the regulation of angiostatic CXC chemokine production, and this transcription factor may represent a potential therapeutic target. Treatment of the prostate cancer cell line JCA-1 with IFN- was shown to activate STAT1 and reduce cellular proliferation (29). However, IFN-
may have other significant effects on cell function in vivo and immune cell activation.
In conclusion, our data demonstrate that prostate cancer cells have a progressive dysregulation of the transcription factors NF-B and STAT1, which leads to increased production of angiogenic CXC chemokines and decreased production of angiostatic CXC chemokines. These findings identify a potential mechanism by which prostate cancer cells promote their own growth and suggest potential points of therapeutic intervention.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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The United States Army Medical and Materiel Command under DAMD 17-01-1-0243 supported this work.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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