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The Journal of Immunology, 2006, 177: 8266-8272.
Copyright © 2006 by The American Association of Immunologists, Inc.

IL-22-Mediated Tumor Growth Reduction Correlates with Inhibition of ERK1/2 and AKT Phosphorylation and Induction of Cell Cycle Arrest in the G2-M Phase

Georg F. Weber1,*, Florian C. Gaertner{dagger}, Wolfgang Erl{ddagger}, Klaus-Peter Janssen*, Birgit Blechert{dagger}, Bernhard Holzmann*, Heike Weighardt2,* and Markus Essler2,{dagger}

* Chirurgische Klinik und Poliklinik der Technischen Universität München, {dagger} Nuklearmedizinische Klinik und Poliklinik der Technischen Universität München, and {ddagger} Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Ludwig-Maximilians-Universität München, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-22 is a recently discovered cytokine of the IL-10 family that binds to a class II cytokine receptor composed of IL-22R1 and IL-10R2c and influences a variety of immune reactions. As IL-22 has also been shown to modulate cell cycle and proliferation mediators such as ERK1/2 and JNK, we studied the role of IL-22 in proliferation, apoptosis, and cell cycle regulation in EMT6 murine breast cancer cells in vitro and in vivo. In this study, we report that murine breast cancer cells express functional IL-22R as indicated by RT-PCR studies, immunoblotting, and STAT3 activation assays. Importantly, IL-22 exposure of EMT6 cells resulted in decreased levels of phosphorylated ERK1/2 and AKT protein kinases, indicating an inhibitory effect of IL-22 on signaling pathways promoting cell proliferation. Furthermore, IL-22 induced a cell cycle arrest of EMT6 cells in the G2-M phase. IL-22 reduced EMT6 cell numbers and the proliferation rate by ~50% as measured by [3H]thymidine incorporation. IL-22 treatment of EMT6 tumor-bearing mice lead to a decreased tumor size and a reduced tumor cell proliferation in vivo, as determined by 3'-deoxy-3'-fluorothymidine-positron emission tomography scans. Interestingly, IL-22 did not induce apoptosis, as determined in annexin V binding assay and caspase-3 activation assay and had no effect on angiogenesis in vivo. In conclusion, our results indicate that IL-22 reduced tumor growth by inhibiting signaling pathways such as ERK1/2 and AKT phosphorylation that promote tumor cell proliferation in EMT6 cells. Therefore, IL-22 may play a role in the control of tumor growth and tumor progression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immune reactions as well as direct proliferative and antiproliferative effects of cytokines or cytokine-related mediators participate in the natural course of cancer (1, 2, 3, 4, 5). The recently discovered cytokine IL-22, which is structurally related to IL-10 (6), was identified as a T cell-derived inducible factor produced by IL-9-activated murine T cells (7). IL-22 binds to a class II cytokine receptor that is composed of two chains, the tissue-specific receptor component IL-22R1 and the relatively ubiquitous receptor component IL-10R2c (8, 9). Binding of IL-22 to its receptor activates JAK1 and Tyk2 tyrosine kinases, mainly leading to phosphorylation of STAT3 as well as STAT1/STAT5. IL-22 was also found to activate the kinases ERK, JNK, and p38 MAPK in the rat hepatoma cell line H4IIE (10).

Although multiple immune effects, such as the acute-phase response, activation of the innate immune system, cell migration and differentiation as well as gene expression, have been shown to be regulated by IL-22 (11, 12, 13, 14), the potential role of IL-22 in cancer cell biology has not been studied so far. However, it is conceivable that IL-22 might play a role during tumor genesis because IL-22 stimulates signaling pathways that are involved in the regulation of cell growth, cell proliferation, and cell cycle control (15, 16, 17, 18, 19, 20).

Mammary adenocarcinoma EMT6 cells are a widely used model to study different aspects of growth control in cancer (21, 22, 23, 24, 25). A broad variety of chemical compounds (23), growth factor inhibitors, and other biomolecules, including cytokines either alone or in combination with endocrine modulation (22), have been studied using in vitro and in vivo settings in this model. So far, cytokines alone have not been found to be effective in EMT6 cell growth inhibition or reduction of tumor proliferation.

We report that EMT6 cells express functional IL-22R1 and IL-10R2c receptor chains, as evidenced by activation of STAT3 after IL-22 exposure. Importantly, we demonstrate that IL-22 treatment causes reduced phosphorylation of the growth stimulating mediators ERK1/2 and AKT in EMT6 cells and elicits a cell cycle arrest in the G2-M phase. Because inhibitors of ERK1/2 phosphorylation have been suggested previously to be effective in anticancer therapy (26, 27, 28, 29), our findings, which sustained IL-22 treatment of EMT6 cells-bearing mice reduces tumor growth, suggest that this recently discovered cytokine might function as a immunological inhibitor of tumor growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

Cells were grown in a 7% CO2 humidified atmosphere at 37°C in Waymouth medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. For all experiments, confluent cells were subjected to a limited trypsin-EDTA treatment. The cells were treated with trypsin-EDTA (Invitrogen Life Technologies) for 1 min, after which trypsin was inactivated by addition of medium containing 10% FCS. Cells were washed gently, and fresh medium was added to the cultures.

Immunoblotting

EMT6 (1 x 106) or eEND2 cells (30) were seeded onto 6-well plates. Cells were cultured for 6 h in serum-free medium to reduce endogenous levels of phosphorylated mediators. Then cells were treated with 50 ng/ml rIL-22 (R&D Systems) for different times. Total cell lysates were prepared in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, and 1 mM EDTA on ice. Samples were sonicated for 30 s and boiled at 95°C for 5 min. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Abs specific to STAT1, phospho-STAT1, STAT3, phospho-STAT3, cyclin D1, p27KIP1, p38 MAPK, phospho-p38 MAPK, p70/S6 kinase, phospho-p70/S6 kinase, retinoblastoma, phospho-retinoblastoma, ERK1/2, phospho-ERK1/2, AKT, phospho-AKT, caspase-3, cleaved caspase-3, and beta-actin (Invitrogen Life Technologies) were used in blocking solution. Specific binding was visualized with the ECL Western Blotting Detection System according to the manufacturer’s instructions (Amersham Biosciences).

Densitometric analysis of immunoblots

The enhanced chemiluminescence signal was quantified using a scanner and a densitometry program (Scion Image). To quantify phospho-specific signals in activated samples, background was subtracted, signals were normalized to the amount of beta-actin or total target protein in the lysate, and values were plotted as fold induction over unstimulated samples.

Cell proliferation assay

EMT6 cells (5 x 104) per well were seeded into a 96-well plate. Cells were allowed to adhere and spread for 4–6 h and were then incubated for 72 h with rIL-22. Cells treated with PBS served as control. After 60 h, 1 µCi of [3H]thymidine was added into each well. After 72 h, cells were harvested and cell proliferation was determined using a beta counter.

Flow cytometric quantification of apoptosis

The rat anti-mouse Abs and control Igs used in this study were purchased from BD Biosciences. EMT6 cells (1 x 106) were seeded onto 6-well plates. After 6 h, cells were treated with 50 or 100 ng/ml rIL-22 for 6 or 24 h. After that EMT6 cells were trypsinized, washed with binding buffer, and incubated with FITC-labeled annexin V Ab in binding buffer (BD Pharmingen) for 20 min at 37°C in the dark. Cells were washed, resuspended in 200 µl of PBS with 1.5 mM Ca2+, and 1% FCS and 10 µl of propidium iodide (PI)3 solution (1 mg/ml) were added. Flow cytometry was performed in a FACScan (FACSCalibur flow cytometer and CellQuest software; BD Biosciences) using a single cell gate. The data were analyzed in FL-1/FL-2 dot plots to quantify the percentage of annexin V-positive/PI-negative or annexin V-negative/PI-negative cells, representing the apoptotic population.

Flow cytometric analysis of cell cycle status

Cell cycle status was determined by measuring cellular DNA content after staining with PI using flow cytometry (31). EMT6 cells (1 x 104) were plated on 10-cm dishes and incubated for 24 h with serum-free medium. Cells were treated with 50 ng/ml rIL-22 or PBS. After 1 h, 1% FCS was added to the cultures. Cells were removed 24 h later with trypsin, collected, washed twice in ice-cold PBS, fixed overnight in 70% ethanol at –20°C, and centrifuged at 300 x g for 5 min. Cells were then resuspended in 30 µl of phosphate/citrate buffer (0.2 M Na2HPO4/0.1 M citric acid (pH 7.5)) and incubated with PI (20 µg/ml) and RNase A (20 µg/ml) in PBS for 30 min. Additionally, we investigated the role of rIL-22 treatment regarding cell cycle markers. We therefore analyzed cyclin B1 and Cdc2. Abs specific to cyclin B1 and Cdc2 were used. The PI fluorescence and intensity of cyclin B1 and Cdc2 Abs were measured using a flow cytometer (FACSCalibur flow cytometer; BD Biosciences).

Immunofluorescence staining and microscopy of cultured cells

EMT6 cells (1 x 105) were seeded onto coverslips in a 6-well plate and incubated for 24 h in serum-free medium. The 50 ng/ml rIL-22 and 1 h later 1% FCS was added to the cultures. After 24 h, cells were fixed in 2% (w/v) paraformaldehyde/PBS (pH 7.4, 67% (v/v) PBS) for 1 h on ice. All subsequent steps were performed at 20°C. The cells were rinsed in PBS, incubated in 100 mM glycine/PBS for 30 min, and permeabilized by three washes in 0.1% Triton X-100/PBS. Ab incubations were performed sequentially for 1 h in 0.1% Triton X-100/PBS containing 2% (w/v) BSA. 4',6'-diamidino-2-phenylindole (DAPI, 1/1000; The Jackson Laboratory) and tetramethylrhodamine isothiocyanate (TRITC)-phalloidin Abs were used to visualize nuclei or F-actin. Finally, coverslips were mounted in 10% (w/v) Mowiol (Calbiochem), 15% (v/v) glycerol, 2.5% (w/v) NaN3, and 100 mM Tris-HCl (pH 8.5), and analyzed with a DM RB/E microscope (Leica). Digital images were obtained using a confocal laser scanning microscope (MRC 1000; Bio-Rad).

Matrigel plug assay

To determine the possible antiangiogenetic activity of IL-22, an in vivo angiogenesis assay was performed as previously described (32, 33). Briefly, rIL-22 (25 ng) and fibroblast growth factor (FGF, 30 ng; R&D Systems) was mixed with 500 µl of Matrigel (BD Biosciences) on ice and injected s.c. into athymic nude mice (Swiss nude; The Jackson Laboratory). Animals receiving Matrigel containing only FGF served as positive controls and animals receiving Matrigel containing no growth factors served as negative controls. Each group comprised of six animals, and the experiments were performed twice. Animals were sacrificed 7 days after injection. The Matrigel plugs were recovered, photographed, and analyzed for hemoglobin. The hemoglobin content of FGF plus Matrigel plugs has been reported to be directly proportional to the degree of neovascularization in each plug (34). Results from in vivo experiments are expressed as mean hemoglobin ± SE in grams per deciliter. The studies have been reviewed and approved by an appropriate institutional review committee.

Preparation of EMT6 xenograft tumors and in vivo IL-22 treatment

EMT6 cells (1 x 107) were injected in the neck region of each athymic nude mouse. After that an osmotic pump was implanted in the neck of each mouse filled with 3 µg of rIL-22. The amount of rIL-22 that diffused through the pump was in the range of 15–20 ng/hour. The blood level of active rIL-22 was ~5 ng/ml as measured by calculating the rIL-22 diffusion, animal weight, blood volume as well as IL-22 inactivation. Control mice were implanted with an osmotic pump filled with PBS. Mice were observed every day to investigate the growth of the tumor. At day 7, mice were sacrificed in deep anesthesia after measuring the in vivo tumor growth by [18F]3'-deoxy-3'-fluorothymidine (FLT) positron emission tomography (PET) scan and dissection of the tumors. The studies have been reviewed and approved by an appropriate institutional review committee.

PET imaging of tumor cell proliferation

PET was conducted on a MOSAIC animal PET scanner (Philips Medical Systems). The detector system consists of a full ring of 2 x 2 x 10-mm gadolinium-orthoxysilicate crystals without septa. Data were acquired in true three-dimensional volume imaging mode. [18F]FLT was obtained from the radiochemical facility at the Department of Nuclear Medicine (Klinikum Rechts der Isar, Munich, Germany). Mice were injected with 300 µCi of FLT i.v. via the tail vein and anesthetized 2 h later with ketamine and xylazine i.p. and measured in prone position. Acquisition time was 15 min using one bed position. Attenuation correction was not performed. Images were created using a three-dimensional iterative reconstruction algorithm resulting in voxels of 1 x 1 x 1 mm.

Histology and immunohistochemistry of tumor sections

Organs were collected in paraffin 7 days after injection of EMT6 cells. Sections were stained with Mayer’s H&E Y solution or underwent immunohistochemical staining with CD31 Ab, which was detected by EnVision Plus System Peroxidase DAB (DakoCytomation). Direct microscopy micrographs were captured using a Zeiss AxioCam MRc 5 video camera and elaborated using Zeiss AxioVision 4 software.

Gene expression analysis by real-time quantitative RT-PCR

The expression of IL-22R1 and IL-10R2c was analyzed using quantitative real-time PCR (ABI 7300 real-time PCR System; Applied Biosystems). Primers used in this study include: IL-22R1 forward 5'-CTA CGT GTG CCG AGT GAA GA-3', reverse 5'-AAG CGT AGG GGT TGA AAG GT-3'; IL-10R2c forward 5'-ACA TTC GGA GTG GGT CAA TGT C-3', reverse 5'-TCT GCA TCT CAG GAG GTC CAA T-3'; and beta-actin forward 5'-ACC CAC ACT GTG CCC ATC TAC-3', reverse 5'-AGC CAA GTC CAG ACG CAG G-3'. Total RNA was prepared using the Qiagen RNeasy kit according to recommendations of the manufacturer. Briefly, 2 µg of total RNA treated with RNase inhibitor (Fermentas) was reverse-transcribed using RevertAid H Minus M-MuLV Reverse Transcriptase (Fermentas). Specific primers corresponding to the target genes were used to generate the amplicons. To measure relative concentration of gene expression, 5 µl of cDNA of each sample (dilution 1/10) was analyzed in duplicate. Real-time PCR was performed in a 30 µl of volume with 5 µl of respective cDNA and 0.4 µM primers. Nucleotides, Taq polymerase, reaction buffer, and SYBR Green I dye were supplied in the iQ SYGR Green Supermix (Bio-Rad). The individual mRNA levels were obtained by quantitative RT-PCR. Changes in expression were calculated after normalization to levels of beta-actin in each sample.

Statistical analysis

Statistical analysis of the data was performed by the {chi}2 test, the Fisher’s exact test, or the Mann-Whitney U test where appropriate. The level of significance was p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-22 activates STAT3 and inhibits ERK1/2 and AKT phosphorylation in EMT6 breast cancer cells

IL-22R expression has been reported in cancer cells including C170 colon tumor cells, HepG2 hepatocyte carcinoma cells, A549 lung carcinoma cells, MDA-MB-231 breast adenocarcinoma as well as BxPC-3 pancreatic adenocarcinoma cells (14). Therefore, we studied the expression of the IL-22R chains IL-22R1 and IL-10R2c and functional consequences of IL-22 exposure in EMT6 breast cancer cells. The presence of IL-22R1 and IL-10R2c receptor chains on EMT6 cells was demonstrated by specific RT-PCR (Fig. 1a), immunoblot (Fig. 1c), and its biological function by time-dependent STAT3 activation after rIL-22 exposure (Fig. 1b). In contrast to STAT3 activation, IL-22 does not lead to increased phosphorylation of STAT1 in EMT6 cells (Fig. 1c). As a control we compared the effect of IL-22 in EMT6 cells with the effect on eEND2 cells. eEND2 cells do not express IL-22R1 mRNA as determined by RT-PCR and also do not express IL-22R protein as determined by immunoblotting (Fig. 1, c and d). Consequently, IL-22 does not induce STAT3 phosphorylation in eEND2 cells. We then studied signaling pathways involved in cell cycle control, cell proliferation and tumor growth such as ERK1/2 and AKT phosphorylation (1, 15, 16, 35, 36, 37, 38). IL-22 significantly inhibited these signaling pathways as shown by immunoblot analysis. ERK1/2 phosphorylation (Fig. 2, a and b) as well as AKT phosphorylation (Fig. 2, c and d) were markedly reduced by rIL-22 in a time-dependent manner. The level of phosphorylated ERK1/2 was reduced by ~45% as compared with control levels 40 min after rIL-22 stimulation (Fig. 2b). The level of phosphorylated AKT was reduced by ~85% as compared with control levels 40 min after rIL-22 stimulation (Fig. 2d). IL-22 had no effect on other signaling pathways such as phosphorylation of STAT1, p27KIP1, p38 MAPK, p70/S6 kinase, and retinoblastoma or on regulation of cyclin D1.


Figure 1
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  		FIGURE 1. IL-22 activates STAT3 in EMT6 cells. a, Analysis of EMT6 cDNA by RT-PCR showed high expression of IL-22R1 and IL-10R2c receptor chains, as demonstrated by RT-PCR product agarose separation. b, The immunoblot shows STAT3 activation 20, 40, and 60 min after stimulation of EMT6 cells with 50 ng/ml rIL-22. After 120 min the signal returned to prestimulation level. c, For IL-22R1 expression, STAT1 and STAT3 phosphorylation were determined by immunoblots in EMT6 and eEND2 cells. d, The expression of IL-22R1 was determined by RT-PCR in EMT6 and eEND2 cells.

 

Figure 2
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  		FIGURE 2. IL-22 reduces ERK1/2 and AKT phosphorylation in EMT6 cells. EMT6 cells were serum-deprived for 6 h to decrease endogenous levels of phosphorylated ERK1/2 and AKT. Cells were treated with 50 ng/ml rIL-22 for 120 min in medium with 0.5% FCS. Medium with 20% FCS served as positive control. Immunoblot analysis shows a time-dependent reduction of phosphorylated ERK1/2 and AKT. a and c, Immunoblots are representative of three independent experiments. For quantitative analysis of immunoblots, the background was subtracted, signals were normalized to the amount of total target protein in the lysate, and band intensity was quantified by Scion Image software. b and d, Values were plotted as a ratio over unstimulated samples.

 
IL-22 induces cell cycle arrest in the G2-M phase

ERK1/2 and AKT phosphorylation are central in cell cycle control (1, 17, 19, 36, 37, 38). Therefore, we studied the effect of IL-22 on cell cycle regulation in EMT6 cells. FACScan analysis of PI-stained EMT6 cells revealed that significantly more cells accumulated in the G2-M phase of the cell cycle after treatment with rIL-22 as compared with control cells (Fig. 3a). Microscopic analysis of fixed EMT6 cell cultures stained with DAPI/TRITC-phalloidin showed a significantly increased number of cells with two or more nuclei after rIL-22 treatment (Fig. 3b). To further characterize the molecular mechanism by which IL-22 leads to a G2-M phase arrest, we determined the expression of cyclin B1 and Cdc2 (Fig. 3c) in EMT6 cells stimulated with IL-22 using FACScan analysis. We found that cyclin B1 is not, but Cdc2 is, slightly down-regulated by IL-22, suggesting that this effect may contribute to the G2-M arrest but is unlikely to be the only factor involved. Taken together, these findings indicate that IL-22 treatment elicits a cell cycle arrest of EMT6 cells in the G2-M phase.


Figure 3
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  		FIGURE 3. IL-22 induces cell cycle arrest. a, A total of 1 x 105 cells were incubated for 24 h in a 6-well plate with serum-free medium. Cells were then stimulated with 1% FCS or 50 ng/ml rIL-22 plus 1% FCS for 24 h and analyzed by FACScan after staining with PI. Cell cycle analysis of EMT6 cells stimulated with 50 ng/ml rIL-22 showed a significantly increased amount of cells in the G2-M phase, as compared with control. The results indicate that cells stimulated with rIL-22 were arrested in the G2-M phase. b, For immunofluorescence microscopy, 1 x 105 cells were incubated for 24 h in a 6-well plate with serum-free medium. Cells were stimulated with 1% FCS or 50 ng/ml rIL-22 plus 1% FCS for 24 h. DAPI/TRITC-phalloidin stain was performed, and 400 cells were counted and analyzed for quantity of nuclei. The graphs (bottom left and right) show that cells stimulated with rIL-22 had significantly more nuclei compared with control. c, The expression of cyclin B1 and Cdc2 in EMT6 cells was analyzed 24 h after stimulation with 50 ng/ml rIL-22. *, p < 0.05; **, p < 0.01 (n = 3).

 
IL-22 reduces tumor cell proliferation in vitro and in vivo

ERK1/2 and AKT phosphorylation are known to be pivotal mediators for cell proliferation (18, 19, 20). As IL-22 reduces phosphorylation levels of ERK1/2 and AKT and induces cell cycle arrest in the G2-M phase, we investigated the role of IL-22 in the cell proliferation rate in vitro and in vivo. Proliferation of EMT6 cells in vitro, with or without rIL-22 treatment, was determined by measuring [3H]thymidine incorporation. Our results show a decreased proliferation of EMT6 cells during stimulation with rIL-22 (Fig. 4a). In addition, trypan blue staining of EMT6 cells confirmed the antiproliferative effects, showing decreased cell counts in rIL-22-treated samples as compared with control samples (Fig. 4b).


Figure 4
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  		FIGURE 4. Antiproliferative effect of IL-22 in vitro and in vivo. a, To investigate rIL-22 effects in vitro, 2 x 105 EMT6 cells per well were seeded into a 96-well plate. EMT6 cells were treated with 50 ng/ml rIL-22 for 72 h. For the last 12 h, 1 µCi of [3H]thymidine was added per well. Proliferation of EMT6 cells treated with rIL-22 was reduced to 48 ± 10%. b, Trypan blue staining of EMT6 cells 72 h after treatment with 50 ng/ml rIL-22 showed significantly reduced cell counts in 10-cm2 culture plates (n = 3). c, FLT-PET scan 7 days after injection of 1 x 107 EMT6 cells in the neck region of mice and implanting of an osmotic pump filled with rIL-22 or PBS shows decreased signal intensity of [18F]FLT in rIL-22-treated mice. d, The weight of tumor tissue from rIL-22-treated mice was significantly decreased as compared with weight of tumor tissue in PBS treated mice. e, Quantification of [18F]FLT per gram of tumor tissue demonstrates a significantly reduced cell proliferation in tumors of mice treated with rIL-22 (3.02 ± 0.42) as compared with PBS-treated control mice (5.46 ± 0.34) by analyzing the percentage of injected dose per gram of tumor tissue. f, EMT6 xenograft tumors were dissected and stained with anti-IL-22R1 or an isotype control Ab. *, p < 0.05; **, p < 0.01 (n = 6).

 
IL-22 treatment reduces tumor proliferation in EMT6 cell-bearing mice

To investigate whether IL-22 also has antiproliferative effects in vivo, 1 x 107 EMT6 cells were injected s.c. in the neck region of T cell-deficient Swiss nude mice and were treated with 3 µg of rIL-22 over 7 days (15–20 ng of rIL-22 per hour) using an implanted osmotic pump at the same time. After 7 days, [18F]FLT was injected into the tail vein of the mice, and the proliferation rate of tumor cells was analyzed by PET scans in vivo. Mice were sacrificed after the scans, tumors were surgically removed, and the amount of FLT present was determined by a gamma counter. The PET signal in tumors treated with rIL-22 was significantly lower than in PBS-treated control tumors (Fig. 4c). The FLT concentration in tumors was also markedly lower in the rIL-22-treated mice as compared with PBS treated control mice (Fig. 4e). Importantly, the tumor weight of rIL-22-treated mice was significantly lower than in PBS-treated mice (Fig. 4d). To demonstrate that the IL-22R is present in EMT6 xenograft tumors we performed immunohistochemistry. We found that ~98% of cells in EMT6 xenografts stain positive for IL-22R1 (Fig. 4f). The percentage of IL-22R1-positive cells was not changed by rIL-22 treatment. Taken together, the results indicate that IL-22 inhibits EMT6 tumor cell proliferation in vitro and in vivo.

IL-22 does not induce apoptosis in EMT6 breast cancer cells

Next, we asked whether inhibition of tumor growth by IL-22 might also involve other mechanisms such as apoptosis induction. Therefore we measured annexin V binding by FACScan and determined caspase-3 activity by immunoblotting, using a specific Ab to cleaved caspase-3 in EMT6 cells treated with rIL-22 for 6 or 24 h. FACScan analysis showed that the fraction of annexin V-positive or PI-positive cells was not significantly increased upon incubation with rIL-22 (Fig. 5a). Immunoblot analysis showed that the amount of cleaved caspase-3 after stimulation of EMT6 cells with rIL-22 was not elevated (Fig. 5, b and c). Taken together, the results indicate that IL-22 does not induce apoptosis in breast cancer cells.


Figure 5
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  		FIGURE 5. IL-22 does not induce apoptosis in EMT6 cells. a, FACS analysis of EMT6 cells treated with 50 or 100 ng/ml rIL-22 for 24 h does not induce apoptosis as measured by annexin V- or PI-positive cells. b and c, Immunoblots using Abs specific to caspase-3 (b) and cleaved caspase-3 (c) show that caspase-3 is not activated in EMT6 cells incubated with 50 ng/ml rIL-22 for 6 or 24 h, indicating that apoptosis is not induced. Staurosporin (ST, 10 ng/ml) served as positive control and PBS (–) as negative control. Immunoblots are representative of three independent experiments yielding similar results.

 
IL-22 does not modulate angiogenesis and tumor blood vessel formation in vivo

As IL-22 treatment inhibits the growth of EMT6 tumors in vivo, we examined whether a reduced tumor angiogenesis may have contributed to this effect. We tested whether IL-22 modulates FGF-induced angiogenesis in Matrigel plug assays in vivo. IL-22 has no effect on angiogenesis in this model (Fig. 6a). We also analyzed the vessel density in EMT6 tumor sections stained with an Ab to CD31, a specific marker of vascular endothelial cells. Although tumors treated with rIL-22 were significantly smaller than control tumors, the vessel density was not different in both groups (Fig. 6b). Taken together, the results indicate that IL-22 reduces tumor growth, but does not modulate angiogenesis.


Figure 6
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  		FIGURE 6. IL-22 does not modulate angiogenesis in vivo. a, PBS, FGF (30 ng), or FGF (30 ng) in combination with rIL-22 (25 ng) were added to 500 µl of Matrigel matrix. Analysis of the hemoglobin content 7 days after injection of the Matrigel matrix in the neck of Swiss nude mice showed no influence of rIL-22 on FGF-induced angiogenesis. b, Immunohistological analysis of the xenograft tumor tissues regarding vessel density using an Ab specific to the endothelial cell marker CD31 showed no difference between rIL-22-treated mice as compared with PBS-treated control mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We report that IL-22, a member of the IL-10 cytokine family, reduces tumor growth in a murine xenograft model in vivo and proliferation of breast cancer cells in vitro. As we found IL-22R expression in breast cancer cells, the IL-22 effect is most likely induced by IL-22R activation and modulation of subsequent signaling mechanisms such as STAT3 phosphorylation, which is known to be central in IL-22-mediated cell activation. We also demonstrate that STAT3 phosphorylation is followed by reduced levels of ERK1/2 and AKT phosphorylation in EMT6 cells after IL-22 exposure. Phosphorylation of ERK1/2 and AKT promotes cell proliferation in different cell systems, including cancer cells (18, 19, 20, 36). Specific inhibition of ERK1/2 phosphorylation by a variety of natural and synthetic compounds has been shown to be effective in anticancer strategy in the treatment of breast cancer (26, 27, 28, 29). Other signaling molecules that are known to be activated by cytokines STAT1, p27KIP1, and cyclin D1 were not modulated by IL-22. Hence, IL-22 may mediate its tumor suppressive and antiproliferative effects, at least in part, by inhibition of ERK1/2 and AKT phosphorylation. The reduced levels in ERK1/2 and AKT phosphorylation were not found earlier than 40 min after stimulation with IL-22. Therefore, it cannot be excluded that additional, indirect mechanisms connect STAT3 activation and ERK1/2 and AKT inactivation. For example IL-22 may lead to expression of genes that indirectly modulate ERK1/2 or AKT. Interestingly it has been reported that IL-22 induces activation of ERK1/2 in hepatoma cells (10). As we report reduced levels of AKT and ERK1/2 phosphorylation in EMT6 cells after IL-22 treatment, it is conceivable that IL-22 exerts specific effects in different cell types.

We also show that inactivation of AKT by rIL-22 treatment is paralleled by a significant accumulation of cells in the G2-M phase of the cell cycle. As only ~30% of tumor cells accumulate in the G2-M phase, it is unlikely that IL-22 induces an irreversible cell cycle arrest, but rather leads to a slower progression through this phase of the cell cycle. The rIL-22 treatment does not lead to the formation or to development of a cell population that does not express the IL-22R, and thus escape the IL-22 treatment. This result potentially explains why only 30% of cells accumulate in the G2-M phase. The number of cells expressing the IL-22R, however, was not different in tumors treated or not treated with rIL-22.

Cell cycle arrest may be caused by the inhibition of AKT, as it was reported recently that AKT activation promotes the transition through the G2-M phase of the cell cycle and inhibition of AKT leads to a G2-M phase arrest (39, 40, 41). It is widely accepted that Cdc2 and cyclin B1 regulate G2-M transition in a variety of cell types (42, 43). We found that IL-22 leads to a slight reduction of Cdc2. However, cyclin B1 expression seems not to be regulated by IL-22. Therefore, other regulators that remain to be identified may be involved in the inhibition of G2-M transition by IL-22.

IL-22 significantly reduces the proliferation rate of EMT6 cancer cells as measured by [3H]thymidine incorporation and cell counts. Moreover, the same effect was seen in EMT6 murine xenograft tumors in vivo, as measured by FLT-PET scans and by biodistribution experiments showing lower FLT concentrations in tumors from rIL-22-treated mice compared with tumors from PBS-treated mice. Most likely as a consequence of reduced proliferation, xenograft tumors from mice treated with rIL-22 were found to have 50% lower weights than tumors from control mice treated with PBS.

To exclude that mechanisms other than inhibition of tumor cell proliferation contribute to the tumor growth-reducing effect of sustained rIL-22 treatment (44), we studied the effect of IL-22 on EMT6 cell apoptosis and angiogenesis in vitro and on tumor vessel density in vivo. The rIL-22 treatment did not induce apoptosis in EMT6 cells in vitro as indicated by an unchanged number of annexin V-positive cells and an unchanged number of cleaved caspase-3 cells, making this mode of the antitumor effects of IL-22 unlikely. As angiogenesis is vital for tumor growth (45, 46) and because IL-24 was found to inhibit angiogenesis via the IL-20R2/IL-22R1 heterodimer receptor (47), we also asked whether IL-22 interferes with angiogenesis. The rIL-22 treatment did not alter angiogenesis in Matrigel plug assay and did not reduce the vessel density in xenograft tumors. These findings suggest that the effects of IL-22 on tumor growth are not mediated through a reduction of tumor angiogenesis or through increased levels of apoptosis.

Taken together, we demonstrate that IL-22 inhibited EMT6 tumor cell proliferation in vitro and in vivo, most likely by down-regulating the proproliferative signaling mediators ERK1/2 and AKT phosphorylation in EMT6 cells. This was associated with a cell cycle arrest in the G2-M phase. Apoptosis and angiogenesis were not found to be involved in the inhibitory IL-22 effects of tumor growth in EMT6 tumor-bearing mice. Further studies regarding IL-22R distribution and IL-22 responsiveness in different tumor cells are required to evaluate the potential of IL-22 in different tumor settings with regard to therapeutic options. It has been assumed that tumor cells may elicit a microenvironment allowing unopposed tumor growth (48). Therefore, reduced IL-22/IL-22R expression in tumor tissues as compared with normal tissue may contribute to a specific microenvironment enhancing tumor growth. As IL-22 does not inhibit angiogenesis, IL-22 treatment is unlikely to interfere with tumor oxygenation, which is a prerequisite of enhanced effectiveness in radiation therapy. The possibility to combine IL-22 and radiation therapy may be an interesting option because we have demonstrated that IL-22 effectively induces tumor cell cycle arrest in the G2-M phase, which is known to be most vulnerable to radiation (23). Because inhibition of MAPK phosphorylation has been described as a potent anticancer strategy (26, 27, 28, 29), IL-22 as an effective inhibitor of ERK1/2 phosphorylation may be used therapeutically as an extrinsic immune suppressor of tumors. IL-22 might also function as a natural immune inhibitor of tumor growth, e.g., during a pronounced immune response associated with high endogenous levels of IL-22.


   Acknowledgments
 
We thank B. Holzmann and M. Schwaiger for support, constructive discussions, and advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 Address correspondence and reprint requests to Dr. Georg F. Weber, Department of Surgery, Technische Universität München, Ismaningerstrasse 22, 81675 Munich, Germany. E-mail address: georgfweber{at}web.de Back

2 H.W. and M.E. contributed equally to this work. Back

3 Abbreviations used in this paper: PI, propidium iodide; DAPI, 4',6'-diamidino-2-phenylindole; FGF, fibroblast growth factor; FLT, 3'-deoxy-3'-fluorothymidine; PET, positron emission tomography; TRITC, tetramethylrhodamine isothiocyanate. Back

Received for publication February 14, 2006. Accepted for publication September 21, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Shen, W. H., S. T. Jackson, S. R. Broussard, R. H. McCusker, K. Strle, G. G. Freund, R. W. Johnson, R. Dantzer, K. W. Kelley. 2004. IL-1beta suppresses prolonged Akt activation and expression of E2F-1 and cyclin A in breast cancer cells. J. Immunol. 172: 7272-7281. [Abstract/Free Full Text]
  2. Dudley, M. E., J. R. Wunderlich, P. F. Robbins, J. C. Yang, P. Hwu, D. J. Schwartzentruber, S. L. Topalian, R. Sherry, N. P. Restifo, A. M. Hubicki, et al 2002. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 298: 850-854. [Abstract/Free Full Text]
  3. Négrier, S., B. Escudier, F. Gomez, J.-Y. Douillard, A. Ravaud, C. Chevreau, M. Buclon, D. Pérol, C. Lasset. 2002. Prognostic factors of survival and rapid progression in 782 patients with metastatic renal carcinomas treated by cytokines: a report from the Groupe Francais d’Immunotherapie. Ann. Oncol. 13: 1460-1468. [Abstract/Free Full Text]
  4. Rosenberg, S. A.. 2001. Progress in human tumour immunology and immunotherapy. Nature 411: 380-384. [Medline]
  5. Fulda, S., W. Wick, M. Weller, K. M. Debatin. 2002. Smac agonists sensitize for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo. Nat. Med. 8: 808-815. [Medline]
  6. Dumoutier, L., E. Van Roost, G. Ameye, L. Michaux, J. C. Renauld. 2000. IL-TIF/IL-22: genomic organization and mapping of the human and mouse genes. Genes Immun. 1: 488-494. [Medline]
  7. Dumoutier, L., J. Louahed, J. C. Renauld. 2000. Cloning and characterization of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9. J. Immunol. 164: 1814-1819. [Abstract/Free Full Text]
  8. Xie, M. H., S. Aggarwal, W. H. Ho, J. Foster, Z. Zhang, J. Stinson, W. I. Wood, A. D. Goddard, A. L. Gurney. 2000. Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2–4 and IL-22R. J. Biol. Chem. 275: 31335-31339. [Abstract/Free Full Text]
  9. Kotenko, S. V., L. S. Izotova, O. V. Mirochnitchenko, E. Esterova, H. Dickensheets, R. P. Donnelly, S. Pestka. 2001. Identification of the functional interleukin-22 (IL-22) receptor complex: the IL-10R2 chain (IL-10Rbeta) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes. J. Biol. Chem. 276: 2725-2732. [Abstract/Free Full Text]
  10. Lejeune, D., L. Dumoutier, S. Constantinescu, W. Kruijer, J. J. Schuringa, J.-C. Renauld. 2002. Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line: pathways that are shared with and distinct from IL-10. J. Biol. Chem. 277: 33676-33682. [Abstract/Free Full Text]
  11. Dumoutier, L., E. Van Roost, D. Colau, J. C. Renauld. 2000. Human interleukin-10-related T cell-derived inducible factor: molecular cloning and functional characterization as an hepatocyte-stimulating factor. Proc. Natl. Acad. Sci. USA 97: 10144-10149. [Abstract/Free Full Text]
  12. Boniface, K., F. X. Bernard, M. Garcia, A. L. Gurney, J. C. Lecron, F. Morel. 2005. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J. Immunol. 174: 3695-3702. [Abstract/Free Full Text]
  13. Ikeuchi, H., T. Kuroiwa, N. Hiramatsu, Y. Kaneko, K. Hiromura, K. Ueki, Y. Nojima. 2005. Expression of interleukin-22 in rheumatoid arthritis: potential role as a proinflammatory cytokine. Arthritis Rheum. 52: 1037-1046. [Medline]
  14. Wolk, K., S. Kunz, E. Witte, M. Friedrich, K. Asadullah, R. Sabat. 2004. IL-22 increases the innate immunity of tissues. Immunity 21: 241-254. [Medline]
  15. Romerio, F., D. Zella. 2002. MEK and ERK inhibitors enhance the anti-proliferative effect of interferon-{alpha}2b. FASEB J. 16: 1680-1682. [Abstract/Free Full Text]
  16. Wu, J. J., X. D. Zhang, S. Gillespie, P. Hersey. 2005. Selection for TRAIL resistance results in melanoma cells with high proliferative potential. FEBS Lett. 579: 1940-1944. [Medline]
  17. Bernardi, R. J., D. L. Trump, W. D. Yu, T. F. McGuire, P. A. Hershberger, C. S. Johnson. 2001. Combination of 1{alpha},25-dihydroxyvitamin D3 with dexamethasone enhances cell cycle arrest and apoptosis: role of nuclear receptor cross-talk and Erk/Akt signaling. Clin. Cancer Res. 7: 4164-4173. [Abstract/Free Full Text]
  18. Fang, J. Y., B. C. Richardson. 2005. The MAPK signalling pathways and colorectal cancer. Lancet Oncol. 6: 322-327. [Medline]
  19. Johnson, G. L., R. Lapadat. 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298: 1911-1912. [Abstract/Free Full Text]
  20. Zhang, W., H. T. Liu. 2002. MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res. 12: 9-18. [Medline]
  21. Zhang, G., W. Li, L. Holle, N. Chen, W. Y. Chen. 2002. A novel design of targeted endocrine and cytokine therapy for breast cancer. Clin. Cancer Res. 8: 1196-1205. [Abstract/Free Full Text]
  22. Muraoka, R. S., N. Dumont, C. A. Ritter, T. C. Dugger, D. M. Brantley, J. Chen, E. Easterly, L. R. Roebuck, S. Ryan, P. J. Gotwals, et al 2002. Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases. J. Clin. Invest. 109: 1551-1559. [Medline]
  23. Gorodetsky, R., L. Levdansky, I. Ringel, A. Vexler. 1998. Paclitaxel-induced modification of the effects of radiation and alterations in the cell cycle in normal and tumor mammalian cells. Radiat. Res. 150: 283-291. [Medline]
  24. Gauthier, N., S. Lohm, C. Touzery, A. Chantôme, B. Perette, S. Reveneau, F. Brunotte, L. Juillerat-Jeanneret, J.-F. Jeannin. 2004. Tumour-derived and host-derived nitric oxide differentially regulate breast carcinoma metastasis to the lungs. Carcinogenesis 25: 1559-1565. [Abstract/Free Full Text]
  25. Amber, I. J., J. B. Hibbs, Jr, R. R. Taintor, Z. Vavrin. 1988. The L-arginine dependent effector mechanism is induced in murine adenocarcinoma cells by culture supernatant from cytotoxic activated macrophages. J. Leukocyte Biol. 43: 187-192. [Abstract]
  26. Kronblad, A., I. Hedenfalk, E. Nilsson, S. Pahlman, G. Landberg. 2005. ERK1/2 inhibition increases antiestrogen treatment efficacy by interfering with hypoxia-induced downregulation of ER{alpha}: a combination therapy potentially targeting hypoxic and dormant tumor cells. Oncogene 24: 6835-6841. [Medline]
  27. Rinehart, J., A. A. Adjei, P. M. Lorusso, D. Waterhouse, J. R. Hecht, R. B. Natale, O. Hamid, M. Varterasian, P. Asbury, E. P. Kaldjian, et al 2004. Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients with advanced non-small-cell lung, breast, colon, and pancreatic cancer. J. Clin. Oncol. 22: 4456-4462. [Abstract/Free Full Text]
  28. Sebolt-Leopold, J. S., R. Herrera. 2004. Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat. Rev. Cancer 4: 937-947. [Medline]
  29. Sebolt-Leopold, J. S., D. T. Dudley, R. Herrera, K. Van Becelaere, A. Wiland, R. C. Gowan, H. Tecle, S. D. Barrett, A. Bridges, S. Przybranowski, et al 1999. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat. Med. 5: 810-816. [Medline]
  30. Williams, R. L., S. A. Courtneidge, E. F. Wagner. 1988. Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene. Cell 52: 121-131. [Medline]
  31. Ye, K., Y. Ke, N. Keshava, J. Shanks, J. A. Kapp, R. R. Tekmal, J. Petros, H. C. Joshi. 1998. Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells. Proc. Natl. Acad. Sci. USA 95: 1601-1606. [Abstract/Free Full Text]
  32. Passaniti, A., R. M. Taylor, R. Pili, Y. Guo, P. V. Long, J. A. Haney, R. R. Pauly, D. S. Grant, G. R. Martin. 1992. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab. Invest. 67: 519-528. [Medline]
  33. Taraboletti, G., G. Micheletti, M. Rieppi, M. Poli, M. Turatto, C. Rossi, P. Borsotti, P. Roccabianca, E. Scanziani, M. I. Nicoletti, et al 2002. Antiangiogenic and antitumor activity of IDN 5390, a new taxane derivative. Clin. Cancer Res. 8: 1182-1188. [Abstract/Free Full Text]
  34. Nicosia, R. F., Y. J. Lin, D. Hazelton, X. Qian. 1997. Endogenous regulation of angiogenesis in the rat aorta model: role of vascular endothelial growth factor. Am. J. Pathol. 151: 1379-1386. [Abstract]
  35. Song, G., G. Ouyang, S. Bao. 2005. The activation of Akt/PKB signaling pathway and cell survival. J. Cell. Mol. Med. 9: 59-71. [Medline]
  36. Krop, I., M. T. Parker, N. Bloushtain-Qimron, D. Porter, R. Gelman, H. Sasaki, M. Maurer, M. B. Terry, R. Parsons, K. Polyak. 2005. HIN-1, an inhibitor of cell growth, invasion, and AKT activation. Cancer Res. 65: 9659-9669. [Abstract/Free Full Text]
  37. Yin, X., H. Zhang, F. Burrows, L. Zhang, C. G. Shores. 2005. Potent activity of a novel dimeric heat shock protein 90 inhibitor against head and neck squamous cell carcinoma in vitro and in vivo. Clin. Cancer Res. 11: 3889-3896. [Abstract/Free Full Text]
  38. Osaki, M., M. Oshimura, H. Ito. 2004. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 9: 667-676. [Medline]
  39. Lee, S. R., J. H. Park, E. K. Park, C. H. Chung, S. S. Kang, O. S. Bang. 2005. Akt-induced promotion of cell-cycle progression at G2-M phase involves upregulation of NF-Y binding activity in PC12 cells. J. Cell. Physiol. 205: 270-277. [Medline]
  40. Katayama, K., N. Fujita, T. Tsuruo. 2005. Akt/protein kinase B-dependent phosphorylation and inactivation of WEE1Hu promote cell cycle progression at G2-M transition. Mol. Cell. Biol. 25: 5725-5737. [Abstract/Free Full Text]
  41. Chandrasekar, N., S. Mohanam, M. Gujrati, W. C. Olivero, D. H. Dinh, J. S. Rao. 2003. Downregulation of uPA inhibits migration and PI3k/Akt signaling in glioblastoma cells. Oncogene 22: 392-400. [Medline]
  42. Dvory-Sobol, H., E. Cohen-Noyman, D. Kazanov, A. Figer, S. Birkenfeld, L. Madar-Shapiro, R. Benamouzig, N. Arber. 2006. Celecoxib leads to G2-M arrest by induction of p21 and downregulation of cyclin B1 expression in a p53-independent manner. Eur. J. Cancer 42: 422-426. [Medline]
  43. Innocente, S. A., J. L. Abrahamson, J. P. Cogswell, J. M. Lee. 1999. p53 regulates a G2 checkpoint through cyclin B1. Proc. Natl. Acad. Sci. USA 96: 2147-2152. [Abstract/Free Full Text]
  44. Nagakawa, H., O. Shimozato, L. Yu, Y. Takiguchi, K. Tatsumi, T. Kuriyama, M. Tagawa. 2004. Expression of interleukin-22 in murine carcinoma cells did not influence tumour growth in vivo but did improve survival of the inoculated hosts. Scand. J. Immunol. 60: 449-454. [Medline]
  45. Presta, M., P. Dell’Era, S. Mitola, E. Moroni, R. Ronca, M. Rusnati. 2005. Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev. 16: 159-178. [Medline]
  46. Carmeliet, P., R. K. Jain. 2000. Angiogenesis in cancer and other diseases. Nature 407: 249-257. [Medline]
  47. Ramesh, R., A. M. Mhashilkar, F. Tanaka, Y. Saito, C. D. Branch, K. Sieger, J. B. Mumm, A. L. Stewart, A. Boquoi, L. Dumoutier, et al 2003. Melanoma differentiation-associated gene 7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor. Cancer Res. 63: 5105-5113. [Abstract/Free Full Text]
  48. Kortylewski, M., M. Kujawski, T. Wang, S. Wei, S. Zhang, S. Pilon-Thomas, G. Niu, H. Kay, J. Mule, W. G. Kerr, R. Jove, et al 2005. Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity. Nat. Med. 11: 1314-1321. [Medline]



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