Affiliations of authors: Z. von Marschall, A. Scholz, T. Cramer, G. Schäfer, B. Wiedenmann, M. Höcker, S. Rosewicz, Department of Hepatology, Gastroenterology, Endocrinology and Metabolism, Charité, Campus Virchow-Klinikum, Humboldt-University, Berlin, Germany; M. Schirner, Corporate Research, Schering AG, Berlin; K. Öberg, Department of Internal Medicine, University Hospital, Uppsala, Sweden.
Correspondence to: Stefan Rosewicz, M.D., Department of Hepatology, Gastroenterology, Endocrinology and Metabolism, Charité, Campus Virchow-Klinikum, Humboldt-University, Augustenburger Platz 1, 13353 Berlin, Germany (e-mail: stefan.rosewicz{at}charite.de).
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ABSTRACT |
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INTRODUCTION |
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Interferon alpha (IFN-) is a cytokine with pleiotropic cellular functions, including antiviral, antiproliferative, immunomodulatory, and antiangiogenic activities (6,7). Clinical studies demonstrate that IFN-
treatment can induce impressive responses in angioproliferative diseases, such as Kaposis sarcoma and hemangiomas (6). Preclinical data suggest that these antiangiogenic activities may be associated with the regulation of endothelial cell motility (8) and survival (9) and with inhibition of molecules involved in the angiogenic response, such as basic fibroblast growth factor (bFGF) (10), interleukin 8 (11), and matrix metalloproteinase 9 (MMP-9) (12).
In addition to its potential therapeutic effects on angioproliferative diseases, IFN- therapy is being clinically evaluated for the treatment of some malignancies, including malignant melanoma, renal cell carcinoma, myeloproliferative disorders [summarized in (13)], and neuroendocrine (NE) tumors (14). NE tumors of the gastroenteropancreatic tract provide a particularly valuable model to study the mechanism of IFN-
action because most NE tumors are extremely well vascularized (15), and the clinical benefit of IFN-
treatment in these patients has been well documented (14). Because the VEGF/VEGF receptor (VEGF-R) system is important for the induction of angiogenesis in numerous human solid tumors, we tested the hypothesis that the therapeutic effect of IFN-
might result from an inhibition of angiogenesis through a decrease in VEGF gene expression. Therefore, we analyzed the effects of IFN-
on VEGF gene expression in vitro by using human NE tumor cell lines. Biologic and clinical relevance of our findings were further validated in a xenograft mouse model and NE tumor patients treated with IFN-
.
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PATIENTS AND METHODS |
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Eight NE tumor patients with the following characteristics were included in this study: all patients had histologically proven midgut carcinoids with carcinoid syndrome and liver metastasis as documented by computed tomography (CT). The median age was 69.5 years (range = 5577 years). Six patients were male and two were female. All patients received 5 x 106 IU of IFN- subcutaneously three times per week. Distinct liver metastases were detected by ultrasound or CT-guided techniques, and fine-needle biopsy specimens were harvested before and after 6 months of IFN-
treatment. Tumor staging was performed by CT every 3 months. This study was approved by the ethics committee of the University Hospital of Uppsala, and all patients provided written informed consent before inclusion in the study.
Levels of VEGF and VEGF-R expression and angiogenesis were determined in surgical specimens from a different group of 10 patients with NE tumors. These patients underwent surgery with curative intent, and their diagnoses were proven histologically by a pathologist. Seven patients had NE tumors of the foregut, and three patients had NE tumors of the midgut. This study was approved by the ethics committee of the Charité (Humboldt-University, Berlin, Germany), and all patients gave written informed consent.
Cell Culture
The human pancreatic NE tumor cell line Bon, colon carcinoma cell line HT29, and hepatoma cell line HepG2 (all obtained from the American Type Culture Collection [ATCC], Manassas, VA) were grown in Dulbeccos modified Eagles medium with 10% fetal calf serum. The human NE tumor cell line QGP1 and the human pancreatic carcinoma cell line Capan-1 (obtained from ATCC) were cultured in RPMI-1640 with 10% or 15% fetal calf serum, respectively. The human NE tumor cell line LCC18 was grown as described (16). All cell cultures were grown as subconfluent monolayers in a humidified atmosphere containing 5% CO2 at 37 °C.
Immunohistochemistry and Microvessel Density
Fine-needle biopsy and surgical tissue specimens were fixed in acetone as frozen tissues, and cryosections (5- to 8-µm thick) were stained for immunohistochemistry by the alkaline phosphatase/anti-alkaline phosphastase (APAAP) method with new fuchsin as a developer, as described (17). The following primary antibodies were used: mouse anti-human kinase insert domain-containing region (KDR) (1 : 200; Sigma, Deisenhofen, Germany), mouse anti-human fms-like tyrosine kinase (Flt-1) (1 : 100; Sigma), rabbit anti-human synaptophysin (1 : 200; DAKO Diagnostika GmbH, Hamburg, Germany), and rat anti-mouse pan endothelial cell antigen (MECA-32) (1 : 50; PharMingen, Heidelberg, Germany). If more than 90% of a given cell population stained, then the antigen expression was graded as "strong."
To quantify angiogenesis, microvessel density was determined by staining tissue sections immunohistochemically for the pan endothelial cell antigen as described (18). The entire sections were scanned under low magnification, and vascularization was subjectively graded. Three highly vascularized areas per tumor were then evaluated at high magnification (x400). Any red-staining endothelial cell cluster distinct from adjacent microvessels, tumor cells, or other stromal cells was considered a single countable microvessel. The total number of microvessels was determined for each area, and the average number was recorded for each tumor. Two independent approaches were used to confirm the specificity of the observed immunohistochemical signals: 1) serial dilutions of the primary antibody until the signal disappeared, and 2) substitution of the primary antibody with preimmune rabbit immunoglobulin G. Slides were analyzed by two independent investigators.
In situ Hybridization
Sense and antisense 35S-labeled cRNA probes were prepared from human cDNAs coding for VEGF121, Flt-1, and KDR as described (17). Prehybridization and hybridization conditions, washing procedures, RNase digestion of mismatched sequences, and autoradiography were done on cryosections as described (17).
Quantification of VEGF in Cell Culture Samples and Human Plasma
Bon cells (5 x 104 per well) were cultured in 24-well plates under serum-free conditions in UltraCulture medium (BioWhittaker, Walkersville, MD). The cells were treated with IFN--2a (Hoffmann-La Roche, Grenzach-Wyhlen, Germany) at 1000 IU/mL or vehicle (phosphate-buffered saline [PBS]), and the conditioned medium was collected after 24, 48, 72, or 96 hours. Extracts from IFN-
- or vehicle-treated cells were prepared as previously described (17). VEGF was analyzed using the Quantikine human VEGF enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany), following the instructions supplied by the manufacturer. VEGF concentration was then normalized to protein content.
Blood samples were collected into tubes containing EDTA from NE tumor patients before and 6 months after initiation of the IFN- treatment. Plasma was separated by centrifugation (15 minutes at 1000g at 4 °C) and stored at 80 °C. All samples were measured on the same ELISA plate.
Quantification of VEGF mRNA by Quantitative Competitive Reverse TranscriptionPolymerase Chain Reaction
VEGF mRNA concentrations in NE tumor cell lines were analyzed after treatment for 96 hours (unless otherwise indicated) with IFN- (1000 IU/mL) or vehicle (PBS). Total RNA from the tissue samples was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) in 30 µL of RNase-free water, and 1 µg of RNA was subjected to reverse transcription. The quantitative competitive reverse transcriptionpolymerase chain reaction (RTPCR) for the VEGF isoform VEGF165 (corresponding to the 165-amino-acid splice variant) and
-actin was carried out as described (17). Expression of VEGF mRNA in NE tumor cell lines and in NE tumor tissue was normalized to the expression of
-actin mRNA or to the amount of total cDNA, respectively. cDNA was quantified with Hoechst 33258 DNA binding dye (Sigma), following the manufacturers instructions.
Reporter Gene Plasmids
The following plasmids were provided by G. Finkenzeller (GeneScan AG, Freiburg, Germany) and have been previously described (19): human VEGF gene promoter 5'-deletion luciferase constructs -2018-VEGF-Luc, -1286-VEGF-Luc, -789-VEGF-Luc, -414-VEGF-Luc, -267-VEGF-Luc, -85-VEGF-Luc, and -52-VEGF-Luc (previously named pLuc 2068 to pLuc 102); plasmids pTATA and pTATA-85/-50 (sequence -85/-50 in front of a heterologous thymidine kinase promoter luciferase construct); and GCmut and AP-2/Egr-1mut (previously named pLuc 135/Sp1 mut and pLuc 135/Egr-1 mut, respectively).
A reporter gene construct containing the interferon-stimulated response element (ISRE) fused to a luciferase reporter gene (ISRE-Luc) (20) was provided by S. Bhattacharya (Department of Cardiovascular Medicine, University of Oxford, Oxford, U.K.) and was used as a positive control for IFN-induced transactivation in transfection experiments. Vector pT81-based constructs containing various GC box deletions and pGL3-based constructs containing 5' deletions within the 115 to 50 region of the VEGF promoter will be described in detail elsewhere (G. Schäfer and M. Höcker, personal communication). Constructs used for Gal4 reporter assays were Gal4-Luc (five Gal4 binding sites upstream of a minimal promoter linked to luciferase), Sp1-Gal4, and Sp3-Gal4 (activator constructs containing the Gal4 DNA binding domain linked to the Sp1 or Sp3 transactivation domain) and were provided by G. Suske (Institut für Molekularbiologie und Tumorforschung, Marburg, Germany). Two plasmids that do not contain ISREs were used for normalization in cotransfection experiments: the
-galactosidase expression construct
-gal pSV2 (Promega, Mannheim, Germany) and the renilla luciferase expression construct pRL-TK (Promega).
Transient Transfection Experiments
Bon, HT29, and HepG2 cells were transiently transfected with plasmids using the calcium phosphate mammalian cell transfection kit (5 Prime 3 Prime, Boulder, CO), following the manufacturers instructions, and Capan-1 cells were transiently transfected using the Effectene Transfection Reagent Kit (Qiagen), according to the instructions supplied by the manufacturer. In brief, cells were plated at a density of 2 x 105 cells/35-mm well and transfected the next day with 2 µg per well of plasmid. To determine the transfection efficiencies of the test plasmids, normalization was performed by cotransfecting 1 µg per well of a -galactosidase expression construct or 0.01 µg per well of a renilla luciferase expression construct. After the transfection protocols, cells were washed, incubated in serum-free UltraCulture medium, and treated with IFN-
(1000 IU/mL) or vehicle for 24 hours. Cell extracts were made and subsequently analyzed for luciferase activity (Lumat LB 9501 by EG & G Berthold, Promega, Bad Wildbad, Germany), according to the manufacturers recommended protocol. Luciferase activity was expressed as relative light units (RLU).
Electrophoretic Mobility Shift Assays
Approximately 2 x 106 Bon cells per 100-mm dish were incubated in UltraCulture medium with IFN- (1000 IU/mL) or vehicle for various times, and nuclear extracts were prepared using a non-ionic detergent method as described (19). Equal amounts (10 µg) of nuclear extracts were incubated with radiolabeled double-stranded (ds)-oligonucleotides 88/50 (5'-CC CGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAG3'), 63/47 (5'-CCGGCGGGGCGGAGCCA-3'), or 88/47 (5'-CCGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCG GAGCCA-3') of the human VEGF promoter in the presence of 20 µL of binding buffer (20 mM HEPES [pH 8.4], 1 µg of poly(dI-dC), 10 µg of bovine serum albumin, 60 mM KCl, 5 mM dithiothreitol, 1 mM ZnCl2, and 10% glycerol) for 30 minutes at 30 °C. For competition experiments, extracts were preincubated for 30 minutes at 25 °C with a 100x molar excess of wild-type or mutant competitor oligonucleotides (Santa Cruz Biotechnology, Heidelberg, Germany) before the addition of labeled probes. For supershift experiments, extracts were preincubated for 30 minutes at 25 °C with 1 µL of antibodies against human Sp1, Sp3, AP-2, or Egr-1 (Santa Cruz Biotechnology) before the addition of labeled probes. Nuclear extracts derived from HeLa cells (Santa Cruz Biotechnology) served as a positive control for the binding activity of the AP-2 antibody. DNAnuclear protein complexes were separated and visualized as described (19).
Determination of Nuclear Sp1 and/or Sp3 Levels
Nuclear extracts from Bon cells previously incubated with IFN- (1000 IU/mL) in UltraCulture medium for 0, 6, 12, or 24 hours were analyzed by immunoblotting (17) using mouse anti-human Sp1 antibody (1 : 1000; PharMingen) and rabbit anti-human antibodies against Sp3 (1 : 1000; Santa Cruz Biotechnology) and STAT1 (1 : 5000; PharMingen).
Nude Mouse Human Tumor Xenograft Model
Capan-1 or Bon cells (106 cells per animal) were injected subcutaneously into the left flank of female 6- to 8-week-old BALB/c mice (Bomholtgard, Rye, Denmark). Tumor growth was monitored by measuring tumor area, calculated as the product of the largest diameter and its perpendicular diameter. After tumor areas of 25 mm2 were attained (after approximately 10 days), the mice were randomly assigned to one of three groups of five animals, and either a dose of IFN- (5 x 104 or 5 x 105 IU) or 0.9% NaCl was injected subcutaneously once a day until the mean tumor area in the control group reached 100 mm2. The mice then were killed, and the tumors were processed for immunohistochemical analysis. Animal care was in accordance with institutional guidelines. The experiments were approved by local animal research authorities.
Statistical Analysis
Nonparametric tests for unpaired and paired observations (MannWhitney U test and Wilcoxon matched paired test, respectively) were done using the Statistical Package for Social Sciences 11.0 software package (Statistical Package for Social Sciences, Inc., Chicago, IL). All tests were two-sided, and data were considered to be statistically significant at P<.05. Data represent the mean and 95% confidence intervals (CIs) from at least three independent experiments.
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RESULTS |
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To test the hypothesis that IFN- treatment regulates VEGF gene expression, we first analyzed the expression of VEGF and VEGF-Rs in 10 surgically resected NE tumors from humans: seven derived from the foregut and three derived from the midgut. By using immunohistochemistry, we observed strong VEGF protein expression in the cytoplasm of all tumor samples that were positive for the neuroendocrine marker synaptophysin (compare Fig. 1, A, a and b
). In all tumor specimens, immunohistochemistry for the endothelial cell marker CD31 demonstrated an abundance of endothelial cells surrounding the tumor cells (Fig. 1, A, c
), which is consistent with the clinical observation of a highly vascularized tumor (15). Nine of the 10 tumor samples also expressed the VEGF-Rs KDR/flk-1 (Fig. 1, A, d and e
) and Flt-1 (Fig. 1, A, f
). To confirm the cellular origin of VEGF and the VEGF-Rs, we used in situ hybridization and determined that VEGF mRNA was expressed exclusively in NE tumor cells, whereas KDR and Flt-1 mRNAs were expressed exclusively in tumor endothelia (data not shown).
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Effect of IFN- on VEGF Gene Expression
To explore whether IFN- directly affects VEGF expression in NE tumor cells, we determined VEGF165 mRNA concentrations in Bon, LCC18, and QGP1 cells incubated for 96 hours with IFN-
at 1000 IU/mL or vehicle by quantitative competitive RTPCR. This method is highly sensitive and particularly valuable for measuring the relative abundance of a particular mRNA (17). VEGF165 mRNA levels in all three cell lines treated with IFN-
were lower than those in untreated cells (Fig. 2, A
). Using Bon cells as a representative cell model, we found that the decrease in mRNA levels was cumulative, occurring in a time-dependent manner and continuing to decrease at 96 hours (Fig. 2, B
). The IFN-
-mediated decrease in VEGF mRNA levels was reflected by a concomitant decrease in VEGF protein secretion. Compared with VEGF levels from untreated control cells, those from IFN-
-treated cells were statistically significantly lower (difference = 38% [95% CI on the difference = 22% to 61%; n = 9; P = .008] after 96 hours) (Fig. 2, C
).
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To further elucidate the molecular mechanisms underlying the IFN--mediated decrease in VEGF gene expression, we tested whether IFN-
inhibits VEGF transcription by using a luciferase reporter gene construct containing the promoter sequences 2018/+50 of the human VEGF gene (2018-VEGF-Luc) in Bon cells. Compared with the control treatment, IFN-
treatment resulted in a statistically significant, dose-dependent decrease of VEGF promoter activity, with half-maximal effects of 23% inhibition observed at a therapeutically relevant concentration of 10 IU/mL (P = .012) and maximal effects of 47% (95% CI = 30% to 61%; n = 12; P = .002) observed at 1000 IU/mL (Fig. 3, A
). This transcriptional inhibition was not the result of a nonspecific inhibition of gene transcription, because IFN-
increased luciferase activity 3.90-fold (95% CI = 2.78- to 5.02-fold) in Bon cells transfected with the ISRE-Luc reporter construct (Fig. 3, B
) but had no effect on luciferase activity in Bon cells transfected with the viral thymidine kinase promoter construct that was used to normalize the cotransfection experiments (data not shown).
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Next, we investigated whether IFN--mediated inhibition of VEGF gene transcription is cell type-specific. Using 2018-VEGF-Luc, which contains the promoter sequences 2018/+50 of the human VEGF gene, we observed a statistically significant IFN-
-mediated decrease in VEGF promoter activity in cancer cell lines from different origins, including those from the pancreas (Capan-1, n = 5, 43% of untreated control; P = .031), liver (HepG2, n = 9, 45% of untreated control; P = .002), and colon (HT29, n = 8, 78% of untreated control; P = .008) (Fig. 3, D
). These results suggest that the inhibition of VEGF gene transcription by IFN-
is not restricted to NE tumor cells but can also be observed in tumors from different origins.
Identification of Minimal Promoter Requirements and Involved Transcription Factors
Analysis of the 85/50 region of the human VEGF gene promoter reveals putative binding sites for the transcription factors AP-2 (21) and Egr-1 (19) and for three GC boxes (19), which are known to bind Sp1 and Sp3 with similar affinities in different promoters (22) (Fig. 4, A). To identify transcription factors involved in the IFN-
-dependent decrease of VEGF promoter activity, we performed reporter gene transfection experiments in which the constructs contained inactivated transcription factor binding sites. The basal promoter activity and the ability to respond to IFN-
were comparable among Bon cells transfected with constructs containing functionally inactivated Egr-1 and Egr-1/AP-2 binding sites and Bon cells transfected with the intact construct (85-VEGF-Luc) (Fig. 4, B
). By contrast, the basal promoter activity and the ability to respond to IFN-
were reduced in Bon cells transfected with constructs containing functionally inactivated GC boxes (Fig. 4, B
). Sequential 5' deletion analysis within region 115/50 (Fig. 4, C
) and deletions of GC box III, GC boxes II and III, or GC box I (Fig. 4, D
), showed that the sequence 63/50 corresponding to the proximal GC box I was sufficient to confer IFN-
responsiveness to Bon cells.
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The VEGF promoterreporter gene experiments had demonstrated that the proximal GC box I (63/50) is sufficient to confer IFN- responsiveness (Fig. 4, D
). By using a 32P-labeled 63/47 promoter region (Fig. 5, A
) for gel shift assays, we observed two DNAprotein complexes. On the basis of results obtained from competition (lanes 22, 23, 28, and 29) and supershift (lanes 2426 and 2931) analyses, Sp1 and Sp3 but not Egr-1 or AP-2 (data not shown) bound to this minimal promoter region required for IFN-
-mediated promoter inhibition (Fig. 5, B
). In summary, Sp1 and Sp3 bind to the VEGF promoter sequence located at positions 88/50 and to the sequence 63/50, which is sufficient but not absolutely required to confer IFN-
responsiveness.
IFN- treatment did not appear to change Sp1 or Sp3 DNA binding properties (Fig. 5, B
). To assess whether Sp1- and Sp3-dependent inhibition of VEGF promoter activity resulted from an IFN-
-induced change of nuclear Sp1 or Sp3 content, we analyzed nuclear levels of both transcription factors by immunoblotting. Compared with Sp1 and Sp3 nuclear protein levels from untreated cells, levels in IFN-
-treated Bon cells did not change over time (Fig. 6, A
). By contrast, levels of STAT1, which translocates to the nucleus in response to IFN-
, did increase over time (Fig. 6, A
).
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In vivo Effects of INF- on VEGF Expression and Angiogenesis
To evaluate the biologic relevance of our in vitro observations, we investigated whether IFN- could regulate the angiogenic response elicited by Bon and Capan-1 tumor cells in a nude mouse xenograft model. In response to IFN-
, constitutive expression of VEGF was transcriptionally inhibited in both cell lines (Figs. 2 and 3
). Compared with tumors in vehicle-treated mice, tumors in INF-
-treated mice were dose dependently growth inhibited (Fig. 7, A
, and data not shown). Growth inhibition of Capan-1 xenografts was statistically significant at days 17 and 19 (n = 5; tumor area was reduced by approximately 36% on day 19; P = .008 and P = .016, respectively) for mice who received IFN-
at 5 x 105 IU/day (Fig. 7, A
). Compared with microvessel density in tumors from vehicle-treated mice, that in Capan-1 tumors from IFN-
-treated mice was statistically significantly decreased. The mean microvessel density for the control group was 56 (95% CI = 49 to 69), whereas the mean microvessel density for the 5 x 104 IU/day group and the 5 x 105 IU/day group was 38 (95% CI = 32 to 44; P = .026) and 37 (95% CI = 32 to 41; P = .015), respectively (Fig. 7, B and C
). The Bon cell xenograft experiments showed similar results regarding IFN-
-mediated growth inhibition and decreased microvessel density (data not shown). However, Bon cells do not form tumors consistently, which is necessary for appropriate statistical analyses.
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DISCUSSION |
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We chose human NE tumors as a model to analyze the effects of IFN- on VEGF expression and angiogenesis because therapeutic efficacy of IFN-
in these highly vascularized (15) tumors is well documented (14). VEGF and the VEGF-Rs KDR and flt-1 were strongly overexpressed in all the human tumor samples relative to expression in adjacent nontransformed tissue samples. The tumor samples were also extensively vascularized, suggesting that the VEGFVEGF-R system plays a central role in the vascularization of NE tumors.
Using several human NE tumor cell lines, we demonstrated that IFN- can inhibit VEGF gene transcription. Although many factors have been identified that stimulate VEGF gene expression, including hypoxia, cytokines, growth factors, oncogenes, and tumor suppressor genes (28), IFN-
is, to the best of our knowledge, the first therapeutically relevant agent to inhibit VEGF gene transcription. Of note, the inhibitory effect of IFN-
was not restricted to NE tumor cells and could also be demonstrated in other tumor cell types, suggesting that our observations are potentially applicable to a variety of other cancers.
Molecular analysis revealed an unexpected and novel finding regarding the mechanism of IFN--mediated inhibition of VEGF gene transcription. The VEGF promoter does not contain classical IFN-
-sensitive enhancer elements, such as the gamma-activated sequence (GAS) or ISRE targeted by IFN-stimulated DNA binding factors (7,29). Instead, we found that the proximal GC box I (VEGF promoter region 66/50) was sufficient to confer IFN-
responsiveness. Gel shift analysis confirmed that this region specifically bound the transcription factors Sp1 and Sp3. Several activators of the VEGF promoter have been described that act via an Sp1- and/or Sp3-dependent mechanism, such as interleukin 1
, tumor necrosis factor-
, and platelet-derived growth factor (19); however, Sp1-mediated inhibition of the VEGF promoter has not been previously observed. Furthermore, there is no previous documented evidence describing Sp1 and Sp3 as downstream mediators of IFN-
-induced inhibition of gene expression. Therefore, the role of the transcription factors Sp1 and Sp3 in IFN-
-mediated inhibition of VEGF gene expression is novel. Although Sp1 and Sp3 can activate some GC-rich target genes, such as p21waf1/cip1 or the human alpha1 (I) collagen gene (30), Sp3 has been shown to either activate or repress promoter activity of other genes, depending on the cellular and molecular context (22,31,32). We and others [G. Schäfer and M. Höcker, (32)] have recently introduced the VEGF promoter into Sp1- and/or Sp3-deficient Sf9 Schneider insect cells and shown that Sp1 and Sp3 are both able to transactivate the VEGF promoter. Therefore, inhibition of VEGF promoter activity by IFN-
is most likely mediated by inhibition of Sp1 and/or Sp3 transactivation activity. We tested this hypothesis using Gal4 reporter assays and found that, indeed, IFN-
inhibits Sp1 and/or Sp3 transactivation activity. The molecular mechanisms underlying Sp1- and/or Sp3-dependent transactivation have not been completely elucidated, although several mechanisms have been proposed: 1) modulation of nuclear Sp1 and/or Sp3 concentrations (22), 2) phosphorylation changes in Sp1 and/or Sp3 proteins (33), 3) glycosylation changes in Sp1 and/or Sp3 proteins (34), and 4) interaction of Sp1 and/or Sp3 with specific co-activators or co-repressors (35). We are currently investigating which of these mechanisms is operative for IFN-
and, at this point, can exclude alterations in the nuclear concentration of Sp1 and Sp3.
Two of the goals of translational research are the validation of in vitro observations in a preclinical model and then further validation in the clinical setting in patients. To relate our in vitro observations to clinical observations, we analyzed the effects of IFN- on VEGF expression and angiogenesis in a xenotransplanted mouse model and on VEGF plasma levels and angiogenesis in liver metastases of patients with NE tumors treated with IFN-
. In mice, IFN-
treatment resulted in an inhibition of tumor growth and a marked reduction of angiogenesis demonstrated by reduced microvessel density within the tumor. In humans, patients with NE tumors treated with IFN-
demonstrated decreased VEGF plasma levels, decreased VEGF mRNA concentrations, and a pronounced reduction of microvessel density in biopsy specimens obtained from liver metastases. Together, these observations strongly suggest that IFN-
treatment results in inhibition of VEGF gene expression in an experimental in vivo model and in patients, at least in patients with NE tumors. The observations should, however, be considered cautiously because IFN-
might also regulate other factors involved in the angiogenic response, as suggested by several preclinical models (1012). However, by contrast with our data, there is currently no evidence for functional relevance of these mechanisms in human therapy. Although we did not examine all angiogenic factors, we did not detect changes in bFGF plasma levels in response to IFN-
treatment.
Our results have implications for the optimization of some aspects of antiangiogenic therapy. For example, circulating VEGF concentrations paralleled microvessel density during IFN- therapy. Therefore, VEGF plasma levels might prove a valuable surrogate marker to optimize response prediction as well as duration and dosage of IFN-
treatment (36). This monitoring appears essential when balancing the clinical benefit associated with IFN-
therapy with its costs and side effects. Furthermore, having defined the antiangiogenic mode of action now allows us to develop rationales for combining IFN-
with other antiangiogenic drugs to achieve synergistic therapeutic benefit.
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NOTES |
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S. Rosewicz was supported by grants from the Deutsche Krebshilfe, Wilhelm Sander-Stiftung, Deutsche Forschungsgemeinschaft (DFG), Berliner Krebsgesellschaft, Else Kröner Fresenius Stiftung, and Sonnenfeld Stiftung. M. Höcker was supported by DFG and the Bundesministerium für Bildung und Forschung (BMBF).
We thank G. Finkenzeller (GeneScan AG, Freiburg, Germany), G. Suske (Institut für Molekularbiologie und Tumorforschung, Marburg, Germany), and S. Bhattacharya (Department of Cardiovascular Medicine, University of Oxford, Oxford, U.K.) for the generous gifts of plasmids.
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Manuscript received July 30, 2002; revised December 30, 2002; accepted January 9, 2003.
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