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The Journal of Immunology, 2008, 180, 5699 -5706
Copyright © 2008 by The American Association of Immunologists, Inc.
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Depletion of β-Arrestin-2 Promotes Tumor Growth and Angiogenesis in a Murine Model of Lung Cancer1

Sandeep K. Raghuwanshi*, Mohd W. Nasser*, Xiaoxin Chen*, Robert M. Strieter{dagger} and Ricardo M. Richardson2,*

* Julius L. Chambers Biomedical/Biotechnology Research Institute and Department of Biology, North Carolina Central University, Durham, NC 27707; and {dagger} Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Virginia Health System, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that β-arrestin-2 (βarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of βarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in βarr2-deficient mice (βarr2–/–) and control littermates (βarr2+/+) were used. βarr2–/– mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to βarr2+/+ mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR+ chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-{kappa}B activity was also enhanced in βarr2–/– mice, whereas hypoxia-inducible factor-1{alpha} expression was decreased. Inhibition of CXCR2 or NF-{kappa}B reduced tumor growth in both βarr2–/– and βarr2+/+ mice. NF-{kappa}B inhibition also decreased ELR+ chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that βarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-{kappa}B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chemokines represent a family of small-molecular-weight cytokines that induce leukocyte accumulation and activation at sites of inflammation (1). These functions are mediated via G protein-coupled receptors (GPCRs).3 Structurally, chemokines are classified into four major families (CXC, CC, CX3C, and C) based on arrangements of four conserved cysteine amino acid residues in the amino terminus (2). The CXC family of chemokine is subdivided into two groups based on the presence or absence of the amino acid sequence glutamic acid-leucine-arginine (ELR) at their N terminus (3). The ELR+ chemokines (CXCL1, 2, 3, 5, 6, and 8) promote angiogenesis in vivo, whereas the ELR– ones (CXCL4, 9, and 10) inhibit angiogenesis mediated by the ELR+ chemokine (4, 5).

The ELR+ chemokines activate the CXCR2 receptor to mediate cellular responses (6, 7). CXCR2 couples to the pertussis toxin-sensitive G{alpha}i proteins to activate phospholipase C, resulting in the generation of the intracellular messengers diacylglycerol and inositol triphosphate (8, 9). As with GPCR, CXCR2 functions are regulated by desensitization and internalization. Several adaptor/scaffold proteins, including adaptor protein-2 and β-arrestins, are shown to play a critical role in CXCR2 activation and regulation (10, 11, 12). β-arrestins first complex with the agonist occupied and phosphorylated form of CXCR2 to promote its desensitization and down-regulation (13). We have recently shown that leukocytes from β-arrestin-2 (βarr2)-deficient (βarr2–/–) mice displayed delayed CXCR2 internalization but enhanced cellular responses, including G protein activation and respiratory burst (14). In a wound healing model, neutrophil infiltration, granulation tissue angiogenesis, and wound reepithelization were also enhanced in βarr2–/– mice relative to control littermates (14). This study was designed to investigate the role of βarr2 in tumorigenesis and metastasis. To that end, we have used two different murine models: heterotopic Lewis lung cancer (LLC) and tail vein lung metastasis in βarr2–/– and βarr2+/+ mice. The data herein indicate that βarr2 depletion enhances the expression of proangiogenic factors (i.e., CXCL1/keratinocyte-derived chemokine (KC), CXCL2/MIP-2, and vascular endothelial growth factor (VEGF)), thereby promoting angiogenesis, tumor development, and metastasis. Inhibition of CXCR2 or NF-{kappa}B activation blocked the effect of βarr2. Altogether, the data indicate that the axis CXCR2/β-arrestin modulates tumor progression and metastasis via an NF-{kappa}B dependent pathway.


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

LLC cell line (CRL-1642) was purchased from the American Type Culture Collection. Anti-CD3, CD4, CD8a, CD45, NK1.1, and Ly6 were purchased from BD Pharmingen. Anti-CXCR2, CXCL1/KC, CXCL2/MIP-2, VEGF Abs, recombinant murine CXCL1/KC, CXCL2/MIP-2, and VEGF proteins were purchased from R&D Systems. Mouse monoclonal anti-βarr1/2 Ab was a generous gift from Dr. M. Oppermann (Georg-August-University, Göttingen, Germany). Monoclonal anti-phospho-NF{kappa}B-p65 (Ser536) and polyclonal anti-p65-NF{kappa}B Abs were purchased from Cell Signaling Technology. Monoclonal anti-β-actin, anti-hypoxia-inducible factor (HIF)-1{alpha}, and anti-factor VIII-related Ag Abs, 3,3',5,5'-tetramethylbenzidine, ExtrAvidin peroxidase, DNase, and collagenase were purchased from Sigma-Aldrich. NF-{kappa}B inhibitor BAY 11–7085, microtiter plates, and Bouin’s fixative were purchased from VWR International. All the other reagents were obtained from commercial sources.

Animals

All experiments were approved by and conformed with the guidelines of the Animal Care Committee of North Carolina Central University (Durham, NC). βarr2–/– mice (C57BL/6 background) were kindly provided by Dr. R. J. Lefkowitz (Duke University Medical Center, Durham, NC). Male and female mice were evaluated, and controls were age- and sex-matched littermates. All mice were genotyped at the age of 21 days; DNA samples were prepared from the tail tips with a tissue PCR kit from Sigma-Aldrich and subjected to triplex PCR as described (15, 16, 17).

Heterotopic LLC model

LLC is a spontaneously occurring lung cancer in C57BL/6 mice. The LLC cell line (CRL-1642) was routinely maintained in DMEM supplemented with 10% heat-inactivated FBS, 1.5 g/L sodium bicarbonate, 4 mM/L glutamine, 100 µg/ml streptomycin, and 100 IU/ml penicillin. βarr2+/+ and βarr2–/– mice (6–8 wk, n = 14) were injected s.c. with LLC cells (5 x 105 cells in 100 µl). After each week, mice were sacrificed by CO2, and tumors were dissected from mice and measured with a Thorpe caliper. Tumor volume was calculated using the formula: volume = (d1 x d2 x d3)0.5236, where dn represents the three orthogonal diameter measurements (18). Tumor and tissue specimens were either fixed in buffered formalin or processed for ELISA or FACS analysis.

Tail vein metastasis model

The LLC cells (3 x 105 viable cells/100 µl) were injected via the tail vein of βarr2+/+ and βarr2–/– mice (6–8 wk, n = 8). The mice were observed daily for any sign of respiratory distress and were euthanized by CO2 after 4 wk. The lungs were removed and inflated with Bouin’s fixative. The number of metastatic nodules on the lungs was counted with the aid of a dissecting microscope (19). For survival experiments, βarr2+/+ and βarr2–/– mice (6–8 wk, n = 14) were injected as described above and observed daily for mortality. The experiment was terminated when all the mice died from either group.

FACS analysis of single-cell isolates from heterotopic LLC tumors

Tumors were isolated from mice (n = 6), minced with scissors to fine slurry, and incubated in digestion buffer (RPMI 1640, 5% FBS, 1 mg/ml collagenase, and 30 µg/ml DNase) at 37°C for 45 min. Cells were washed and then cell counts and viability were determined using trypan blue exclusion on a hemocytometer. Cells (2 x 106) were resuspended in FACS analysis buffer and stained with PE-conjugated anti-mouse CD3, CD4, CD45, CD8a, NK1.1, or Ly6 Abs. Cells were also stained with rat anti-mouse CXCR2 and goat anti-rat PE-conjugated secondary Abs. Cells were analyzed on a FACScan flow cytometer using CellQuest software (BD Biosciences).

CXCL1, CXCL2, and VEGF levels in tumors

Heterotopic LLC tumors (n = 8) were harvested 4 wk postinoculation. One gram of dissected tumors was homogenized in 10 ml PBS. The quantity of murine CXCL1, CXCL2, and VEGF present in tissue homogenates was determined by specific ELISA, using a modification of the double-ligand method, as previously described (18). Briefly, flat-bottom 96-well microtiter plates were coated with 100 µl/well of specific polyclonal anti-mouse CXCL1, CXCL2, or VEGF (1 µg/ml in coating buffer (pH 9.5)) for 24 h at 4°C and then washed three times with PBS (pH 7.5) plus 0.05% Tween 20 (wash buffer). Plates were blocked with 1% BSA in PBS for 90 min at 37°C and then washed three times with wash buffer. A total of 100 µl of supernatant from each homogenate was added in plates and then incubated at 37°C for 90 min. Plates were washed three times and 100 µl of biotinylated polyclonal anti-murine CXCL1, CXCL2, or VEGF (diluted in PBS (pH 7.5) plus 0.05% Tween 20) was added and then incubated at 37°C for 45 min. Plates were washed three times, 100 µl of ExtrAvidin peroxidase conjugate was added, and plates were incubated for another 45 min at 37°C. Plates were washed again and 100 µl of 3,3',5,5'-tetramethylbenzidine chromogenic substrate was added. Plates were incubated at room temperature for 20–30 min, and the reactions were terminated by the addition of 100 µl/well of 1 M H2SO4. Plates were read at 450 nm in an automated microplate reader (PerkinElmer). The amount of mouse CXCL1, CXCL2, or VEGF present was determined by interpolation of a standard curve generated by known amounts of recombinant mouse CXCL1, CXCL2, or VEGF, respectively.

Hypoxia treatment to LLC cells

LLC cells (1 x 106) were cultured in complete growth medium in 35-mm culture dishes for 48 h. Next, complete growth medium was replaced by starvation media (growth medium without FBS) in culture dishes and exposed to either normoxia (5% CO2 and ambient oxygen tension) or hypoxia (5% CO2, 1% oxygen, and 94% nitrogen) for 16 h. Subsequently, conditioned media were collected and assayed for ELISA as described above.

Treatments in vivo

βarr2+/+ and βarr2–/– mice (6–8 wk) were injected s.c. with LLC cells as outlined above. Mice (n = 6) were injected i.p. with 500 µl of either neutralizing goat anti-mouse CXCR2, control (preimmune) serum, or no treatment every day for 4 wk. In subsequent experiments, LLC tumor-bearing βarr2+/+ and βarr2–/– mice (n = 12) were treated i.p. with an NF-{kappa}B inhibitor, BAY 11–7085 (5 mg/kg), or vehicle three times a week for 4 wk. Tumor volume was measured as described above.

Quantitation of microvessel density

Paraffin-embedded tumor tissues from βarr2+/+ and βarr2–/– mice were processed for immunohistochemical localization of factor VIII-related Ag, as previously described (20). Briefly, tissue sections were dewaxed with xylene and rehydrated through graded series of alcohol. Tissue sections were treated with 0.10 M citric acid buffer in a heated pressure cooker for 10 min. Slides were blocked with normal goat serum and overlaid with 1/500 dilution of either control (rabbit) or anti-factor VIII-related Ag Ab overnight at 4°C. Slides were then rinsed and overlaid with secondary biotinylated goat anti-rabbit IgG (1/200) and incubated for 60 min. After washing with PBS, slides were overlaid with a 1/200 dilution of ExtrAvidin peroxidase conjugate and incubated for 60 min. For chromogenic localization of factor VIII-related Ag, 3,3'-diaminobenzedine tetrahydrochloride was used. After optimal color development, sections were washed with sterile water, counterstained with Mayer’s hematoxylin, and dehydrated into the graded series of alcohol and mounted. Quantitation of microvessel density was performed using a modification of the previously described method with slight modifications (20). Tumor specimens (n = 10) were scanned at low magnification (x20) to identify vascular hot spots. Areas of greatest vessel density (six from each specimen) were then examined under higher magnification (x200) and counted with the help of a grid eyepiece. Any distinct areas of positive staining for factor VIII-related Ag were counted as single vessels. Results were expressed as the number of microvessels/mm2.

Western blot analysis of HIF-1{alpha}, p65, and βarr2

For HIF-1{alpha} expression analysis, LLC tumor- and zymosan-elicited PMN (1 x 106) isolated from βarr2+/+ and βarr2–/– mice were lysed in radioimmune precipitation assay buffer (RIPA) and assayed for protein concentration. Twenty micrograms of proteins was resolved on 10% SDS-PAGE, transferred to nitrocellulose paper, probed with mouse monoclonal anti-HIF-1{alpha} Ab, and detected with HRP-conjugated goat anti-mouse Ab and ECL. For NF-{kappa}B-p65 phosphorylation, zymosan-elicited PMN (1 x 107) were treated with or without CXCL1 (1 µM) in PBS for 10 min at 37°C. Then, cells were washed, lysed, and immunoblotted with either anti-phospho-NF-{kappa}B-p65 or anti-NF-{kappa}B-p65. For βarr1 and βarr2, LLC and rat basophilic leukemia (RBL-2H3) cells (1 x 106) were lysed in RIPA and 20 µg of proteins was probed with mouse monoclonal anti-βarr1/2 Ab.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
βarr2 depletion enhanced tumorigenesis and metastasis

To determine the role of βarr2 in tumor development, 6–8 wk old male mice deficient in βarr2 (βarr2–/–) and their littermate controls (βarr2+/+) were injected heterotopically with LLC cells (5 x 105) into the dorsal skin. Tumor size was measured 4 wk after injection (Fig. 1A). As shown in Fig. 1B, tumor growth occurred 3.2-fold faster in βarr2–/– mice as compared with littermate controls (βarr2+/+). The time course of tumor growth in female mice showed no significant difference relative to male animals (Fig. 1C).


Figure 1
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  		FIGURE 1. βarr2 deficiency promotes heterotopic LLC tumor growth, lung metastasis, and mortality. βarr2+/+ and βarr2–/– mice (n = 14) were injected s.c. with LLC cells (1 x 105). Heterotopic tumor growth was measured weekly with a Thorpe caliper. Tumor volume was calculated using the formula: volume = (d1 x d2 [infi] x d3)0.5236, where the dn represents the three orthogonal diameter measurements. A–D, Shown are representative photographs of tumor dissected from βarr2+/+ (WT) and βarr2–/– (KO) mice (A), as well as heterotopic LLC tumor growth over 4 wk in male mice (B) and in female mice (C). For lung metastasis (D), LLC cells (3 x 105) were injected via the tail vein of βarr2+/+ and βarr2–/– mice (n = 8). The mice were euthanized by CO2 after 4 wk, and lungs were removed and inflated with Bouin’s fixative. Photographs are representative of LLC cell colonization and growth in lungs of βarr2+/+ and βarr2–/– mice. E, The number of metastatic nodules on the lungs was counted with the aid of a dissecting microscope. F, For survival rate, mice (n = 14) were injected with LLC cells via tail vein for lung metastasis as described above and observed daily up to 60 days mortality. βarr2–/– mice displayed decreased survival rate compared with βarr2+/+ mice. The experiments were repeated three times with similar results. *, p < 0.05; **, p < 0.005; ***, p < 0.0001.

 
We also determined the role of βarr2 deficiency in lung metastasis by assessing the number of metastatic nodules on the lungs. Colonization of the lung was significantly greater in βarr2–/– mice as compared with βarr2+/+ mice (Fig. 1D). The number of metastatic tumor nodules was ~5.66-fold higher in lungs from the βarr2–/– mice relative to wild-type animals (Fig. 1E). We next determined the effect of βarr2 deletion on the survival rate of mice inoculated with LLC cells. As shown in Fig. 1F, a significant reduction was observed in the median survival of βarr2–/– mice (33 days) relative to the βarr2+/+ mice (53 days). Morphometric analysis of the necrotic area was also performed to determine whether tumor necrosis was a factor in the effect of βarr2 deficiency in tumor growth. Tumors from βarr2–/– mice showed a slight but not significant decrease of necrotic areas as compared with βarr2+/+ animals (data not shown).

To determine whether the effect of βarr2 deficiency in tumor growth is due to the host, we measured the expression of βarr1 and βarr2 in LLC cells. As shown in Fig. 2, LLC cells (lane 2), as well as RBL-2H3 cells (lane 1) used as control, expressed both βarr1 and βarr2.


Figure 2
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  		FIGURE 2. Expression of βarr1 and βarr2 in LLC cells. RBL-2H3 and LLC (2 x 106 cells) were lysed in RIPA. Cell lysates were assayed for protein concentration, and 20 µg of proteins was resolved on 10% SDS-PAGE, transferred to nitrocellulose paper, and probed with mouse monoclonal anti-βarr1/2 Ab.

 
βarr2 depletion enhanced expression of CXCL1/KC, CXCL2/MIP-2, and VEGF and promoted angiogenesis

To determine whether the tumor-promoting effect of βarr2 depletion was due to an increased production of ELR+ chemokines and proangiogenic factors, we measured the levels of CXCL1, CXCL2, and VEGF in the homogenate of the LLC tumors from βarr2–/– and control mice. As shown in Fig. 3, expression of both CXCL1/KC (Fig. 3A) and CXCL2/MIP-2 (Fig. 3B) increased by ~2 and ~2.5-fold, respectively, in βarr2–/– mice relative to βarr2+/+ mice. VEGF expression also increased significantly (~3.4-fold) in βarr2–/– mice as compared with control mice (Fig. 3C). Because LLC cells were shown to express both ELR+ chemokine and VEGF (21), we also measured CXCL1 production in media from LLC cells cultured under both normoxic and hypoxic conditions. No significant difference was found in the levels of CXCL1 (~0.75 vs ~0.90 ng/ml for normoxic and hypoxic cells, respectively).


Figure 3
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  		FIGURE 3. Effect of βarr2 deficiency on production of proangiogenic factors. Levels of CXCL1/KC (A), CXCL2/MIP-2 (B), and VEGF (C) in the homogenate of heterotopic LLC tumors from βarr2+/+ and βarr2–/– mice (n = 8) were determined by specific double Ab sandwich ELISA as described in Materials and Methods. *, p < 0.05.

 
To further assess the role of βarr2 depletion in angiogenesis, tumors from βarr2–/– and control βarr2+/+ animals were stained with an Ab specific for factor VIII-related Ag for determination of microvessel density. Tumors from βarr2–/– mice (Fig. 4, B and C, filled bars) showed a statistically significant increase in the number of microvessel density as compared with tumors from control mice (Fig. 4, A and C, open bars).


Figure 4
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  		FIGURE 4. βarr2 deficiency promotes angiogenesis in LLC tumors. Heterotopic LLC tumors were dissected from βarr2+/+ and βarr2–/– mice (n = 10) after 4 wk, fixed in 10% neutral buffered formaldehyde, and embedded in paraffin. Tissue sections were immunostained with anti-factor VIII-related Ag Ab (A and B) and the number of microvessels in a 1.0- mm2 area were counted by light microscope with a grid eyepiece (C). *, p < 0.05.

 
βarr2 depletion enhanced infiltration of leukocyte subpopulations

We next analyzed the intratumor leukocyte infiltration of cell isolates from LLC tumors of βarr2–/– and βarr2+/+ mice. As shown in Fig. 5A–D, respectively, tumor-infiltrating NK+, CD3+, CD4+, and CD8a+ cells were decreased in βarr2–/– as compared with βarr2+/+ mice. No significant difference was found in tumor-infiltrating CD45+, PMNs, and CXCR2+ cells (Fig. 5E–G, respectively).


Figure 5
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  		FIGURE 5. FACS analysis of intratumor-infiltrating leukocyte subpopulations. Single-cell isolates from heterotopic LLC tumors from βarr2+/+ and βarr2–/– mice (n = 6) were stained for different leukocyte subpopulations: (A) NK, (B) CD3, (C) CD4, (D) CD8a, (E) CD45, (F) PMN and (G) CXCR2 were analyzed by a FACScan flow cytometer using CellQuest software. *, p < 0.05.

 
Inhibition of CXCR2 or NF-{kappa}B abrogated the effect of βarr2 depletion in LLC tumor development

Recent studies have suggested that CXCR2 promotes angiogenesis in non-small cell lung cancer and renal cell carcinoma (18, 22). To determine whether the effect of βarr2 is mediated via CXCR2, we used a CXCR2-neutralizing Ab to inhibit the receptor in the heterotopic model. Both βarr2–/– and βarr2+/+ mice treated with anti-mouse CXCR2 Ab showed a significant decreased in tumor size relative to nontreated animals (Fig. 6A). The inhibitory effect of anti-mouse CXCR2 treatment in βarr2–/– mice (~50%) was less effective than than in βarr2+/+ mice (~100%).


Figure 6
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  		FIGURE 6. Inhibition of heterotopic LLC tumor growth in mice treated with CXCR2-neutralizing Abs or NF-{kappa}B-inhibitor. βarr2+/+ and βarr2–/– mice were injected s.c. with LLC cells (1 x 105). A, For CXCR2 inhibition, mice were treated with neutralizing anti-mouse CXCR2 serum (n = 6) or goat preimmune serum (n = 6) daily. B, For NF-{kappa}B-inhibition, mice were treated with BAY 11–7085 5 mg/kg (n = 12) or vehicle (n = 12) three times a week. After 4 wk, mice were euthanized by CO2 and tumor size was measured with a Thorpe caliper (C–E). Levels of CXCL1/KC (C), CXCL2/MIP-2 (D), and VEGF (E) in tumor homogenates from BAY-treated and untreated βarr2+/+ and βarr2–/– mice were determined by ELISA as described in Materials and Methods. *, p < 0.05; **, p < 0.005.

 
Since NF-{kappa}B regulates CXCL1 and CXCL8 expression (23, 24), we next determined the effect of NF-{kappa}B inhibition on ELR+ chemokines and tumor growth. βarr2–/– and βarr2+/+ mice were treated with an NF-{kappa}B inhibitor, BAY 11–7085 (5 mg/kg), three times a week for 4 wk. As shown in Fig. 6B, tumor size was significantly reduced (~65%) in the BAY 11–7085-treated βarr2–/– mice as compared with vehicle-treated mice. Tumor size was also reduced (~35%) by BAY 11–7085 in βarr2+/+ animals, although not to a statistically significant extent (Fig. 6B, open bars). NF-{kappa}B inhibition diminished significantly CXCL1/KC (Fig. 6C), CXCL2/MIP-2 (Fig. 6D), and VEGF (Fig. 6E) expression in both βarr2–/– and βarr2+/+ animals.

βarr2 depletion increased NF-{kappa}B activity in neutrophils

To assess the effect of βarr2 inhibition in NF-{kappa}B activation, we measured p65 phosphorylation in neutrophils stimulated with CXCL1. To this end, zymosan-elicited peritoneal neutrophils were collected from βarr2–/– mice and control littermates βarr2+/+ and treated with or without CXCL1 (1 µM). As shown in Fig. 7, cells from βarr2–/– mice displayed a significant increase in CXCL1-induced p65 phosphorylation as compared with βarr2+/+ animals.


Figure 7
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  		FIGURE 7. βarr2 deficiency promotes p65 phosphorylation. A, Mouse peritoneal macrophages were treated with 1 µM CXCL1/KC for 10 min at 37°C. Cell lysates were immunoprecipitated with anti-NF-{kappa}B-p65 and immunoblotted with anti-phospho-p65. The experiments were repeated three times with similar results. B, Band density from radiogram was calculated by Image-Pro Plus software (Media Cybernetics) and is represented as relative p65 expression (arbitrary units). Data shown are averages of three experiments. **, p < 0.005.

 
Effect of βarr2 depletion on HIF-1{alpha} expression

HIF-1{alpha} has been shown to induce expression of proangiogenic chemokine as well as the chemokine receptors CXCR1 and CXCR2 (25, 26, 27, 28). We next determined whether increased chemokine production observed in βarr2–/– mice correlated with an increase in HIF-1{alpha} expression. As shown in Fig. 8, tumor from βarr2–/– showed decreased HIF-1{alpha} expression relative to βarr2+/+. No expression of HIF-1{alpha} was detected in PMN from either βarr2–/– or βarr2+/+ mice.


Figure 8
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  		FIGURE 8. Expression of HIF-1{alpha} in LLC tumors. A, Tumors and zymosan-elicited peritoneal PMN from βarr2+/+ and βarr2–/– mice were lysed in RIPA, and 20 µg of protein was analyzed in 10% SDS-PAGE and probed with monoclonal anti-HIF-1{alpha} Ab. B, Band density from radiogram was calculated by Image-Pro Plus software (Media Cybernetics) and is represented as relative HIF-1{alpha} expression (arbitrary units). Data shown are averages of three experiments. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Arrestins are known to promote receptor desensitization and internalization by interacting with the phosphorylated form of GPCRs, thereby preventing their coupling with G protein (13). Several studies have demonstrated that in addition to GPCR desensitization, arrestins also modulate a variety of intracellular functions, including activation of MAPK, tyrosine kinase, and growth factors (12, 29, 30, 31, 32). Recent studies in our laboratory have shown that βarr2 suppression in mice promoted angiogenesis and wound closure (14). The data presented in this work further demonstrate that βarr2 plays a crucial role in tumor growth, invasion, and metastasis. First, heterotopically implanted LLC tumors in βarr2–/– mice showed a 2-fold increase in tumor growth compared with wild-type mice (Fig. 1, A–C). Second, in the tail vein metastasis assay, the number of metastatic nodules significantly increased in the lung of βarr2–/– mice (Fig. 1, D and E). Third, survival rate of βarr2–/– mice decreased very significantly relative to wild-type animals (Fig. 1F). Finally, tumors from βarr2–/– mice showed significantly greater microvessel density as compared with βarr2+/+ animals (Fig. 4).

The role of tumor-infiltrating lymphocytes in tumor progression is still not fully understood. Tumor infiltrates from βarr2–/– mice showed a significant decrease in NK+ cells, CD3+, CD4+, and CD8+ T lymphocytes relative to βarr2+/+ mice (Fig. 5). Whether this decrease in T cell infiltration contributes to the significant difference in tumor development and metastasis in βarr2–/– mice is unclear. However, elevated levels of T lymphocytes in tumor infiltrates have been associated with better prognosis due to their antitumor immune responses (33, 34). Impaired CD3+ and CD4+ T lymphocyte infiltration into the lung was also shown in a mouse model of allergic asthma, being deficient in βarr2 expression (35). This phenomenon was correlated with a decrease in production of Th2 cytokines that are necessary for the maturation and migration of T cells (36). Therefore, it is possible that the decrease in infiltration of T cells into tumor observed in this study is also due to the defective secretion of cytokines by βarr2–/– animals (35). Surprisingly, despite the significant increase in CXCR2 chemokine production, PMN infiltration into tumor was not affected in βarr2–/– as compared with βarr2+/+ mice (Fig. 5F). Similar results were shown in CXCR2–/– mice as well as in mice pretreated with anti-CXCR2 Ab (18). These data likely indicate that PMN infiltration into tumors is mediated via a CXCR2-independent mechanism.

Several studies have indicated that the ELR+ chemokines activate the CXCR2 receptor to mediate angiogenesis (6, 7). CXCR2-deficient mice as well as mice treated with CXCR2-neutralizing Ab displayed impaired angiogenesis, diminished healing in excisional and chemical burn wounds, and tumor development (18, 22, 37, 38). Elevated expression levels of CXCR2 ligands were observed in several tumor cells, including melanoma and lung and colon carcinoma cells, and they were shown to be associated with enhanced angiogenesis and leukocyte infiltration (39, 40, 41, 42, 43). These studies suggest that CXCR2 and CXCR2 ligands play a crucial role in tumor development as well as tumor-associated inflammation. The data herein also support this contention. First, both CXCL1/KC and CXCL2/MIP-2 expression levels are significantly increased in tumor from βarr2–/– mice relative to control mice (Fig. 3, A and B). Second, pretreatment of βarr2–/– animals with the CXCR2-neutralizing Ab blocked the effect of βarr2 deficiency and reduced tumor size to those of wild-type animals (Fig. 6A). Previous studies by our group and others have shown that CXCR2 internalizes rapidly (~95% in 2–5 min) upon activation by ligand (11). Leukocytes from βarr2–/– mice showed decreased CXCR2 internalization but increased CXCR2-mediated cellular responses (i.e., GTPase activity, chemotaxis, intracellular Ca2+ mobilization, and superoxide production) and angiogenesis relative to control animals (14). Thus, it could be that the increase in tumor development and metastasis is due to an increase in CXCR2-mediated cellular activities due to both an increase in ELR+ chemokine production and receptor resistance to desensitization and internalization.

Treatment of βarr2–/– animals with the pharmacological inhibitor of NF-{kappa}B BAY 11–7085 reduced tumor size in both βarr2–/– (~65%) and βarr2+/+ (~30%) animals (Fig. 6B) and suppressed CXCL1, CXCL2, and VEGF production (Fig. 6C–E). Inhibition of NF-{kappa}B in squamous cell carcinoma and human ovarian cancer cells has also been shown to decrease ELR+ chemokine and VEGF production and to suppress angiogenesis (44, 45). NF-{kappa}B is present in the cytosol in an inactive form as a complex bound with the inhibitory protein I{kappa}-B (46). Phosphorylation of I{kappa}-B by the I{kappa}-B kinase promotes its dissociation from the NF-{kappa}B complex following its degradation (47). BAY 11–7085 prevents phosphorylation and degradation of I{kappa}B, thereby inhibiting NF-{kappa}B activation (48, 49). Gao et al. have shown that βarr2 complexes with I{kappa}-B to inhibit nuclear translocation of NF-{kappa}B (50). Thus, it could be that depletion of βarr2 promotes phosphorylation and degradation of I{kappa}B and thereby NF-{kappa}B nuclear translocation and gene transcription. This may lead to the increased production of ELR+ chemokine (i.e., CXCL1/KC, CXCL2/MIP-2) and VEGF, thereby enhancing angiogenesis, tumor progression, and metastasis (44, 45). Supporting this contention is that PMN from βarr2–/– mice showed a significant increase in p65 phosphorylation relative to βarr2+/+ animals (Fig. 7). Suppression of βarr2 expression with small interfering RNA has also been shown to enhance NF-{kappa}B activity (50, 51).

Several studies have indicated that HIF-1{alpha} plays a significant role in tumor growth by promoting both ELR+ chemokine production as well as CXCR1 and CXCR2 expression (25, 26, 27, 28). The data herein, however, clearly indicate that the effect of βarr2 is independent of HIF-1{alpha} activation. This contention is supported by the following observations. First, HIF-1{alpha} expression decreased in tumors from βarr2–/– mice relative to control βarr2+/+ mice. Second, tumors from βarr2–/– mice showed no significant increase in CXCR2 expression (Fig. 5G and data not shown). Mizukami et al. (52) have also shown that in colon cancer cells deficient in HIF-1{alpha} expression, CXCL8 production increased by 2-fold relative to control cells. Pharmacological inhibition of NF-{kappa}B, however, blocked CXCL8 expression and reduced tumor growth (52). These data mirror the ones obtained in this study and further indicate that alternative pathways can be activated to promote angiogenesis and tumor growth (53).

LLC cells express both βarr2 and CXCR2. CXCR2-deficient mice, however, displayed decreased tumor progression and metastasis (Fig. 6A and Ref. 18), suggesting that the CXCR2 expressed in LLC cells plays no significant role in tumor development. Keane et al. (18) have also shown that in LLC tumors, the cells expressing CXCR2 are predominantly the vascular endothelial cells. A question that remains unanswered is the role of βarr2 expressed in LLC cells in tumor progression and metastasis of this study. Attempts to generate a stable LLC cell line deficient in βarr2 expression with small interfering RNA have failed. However, immunostaining of tumor sections with anti-factor VIII-related Ag that is specific for endothelial cells showed a significant increase in microvessel density in βarr2–/– mice relative to βarr2+/+ mice. These data further suggest that the increase in tumor progression and metastasis exhibited by βarr2–/– mice is due to increased CXCR2/NF-{kappa}B activity of the host endothelial cells.

Upon activation of the EP4 receptor, βarr1 has been shown to complex with c-Src to transactivate the epidermal growth factor receptor to modulate NF-{kappa}B activation and promote tumor progression and metastasis (54). Witherow et al. (51), using transfected HeLa cells, have shown that both βarr1 and βarr2 interact with I{kappa}B{alpha} to regulate NF-{kappa}B activity. Since CXCR2 has been shown to couple to both βarr1 and βarr2 to regulate signal transduction (12), an unanswered question is whether βarr1 plays a role in CXCR2-mediated tumor progression metastasis. Thus far, βarr1 knockout can only be identified by Southern blotting (55). The large number of animals required to carry out these experiments makes this question difficult to address at this time. CXCR2, however, internalizes via a clathrin/AP2-dependent mechanism (10). Both clathrin and AP2 have far greater affinity for βarr2 than for βarr1 (56, 57). Therefore, it is unlikely that CXCR2 interacts with βarr1 to modulate NF-{kappa}B activity.

In summary, the data herein demonstrate that arrestins play a feedback regulatory role in tumor development, invasion, and metastasis. These processes are mediated via the modulation of both CXCR2 and NF-{kappa}B activity by βarr2. These data suggest that arrestins can be targeted for therapeutic intervention against tumor development and metastasis.


   Acknowledgments
 
We are grateful to Dr. Robert J. Lefkowitz (Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC) for providing βarr2–/– mice and for critical reading of this manuscript. We thank Kimberly M. Malloy (JLC Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC) for mouse genotyping and technical support. We also thank Dr. Martin Oppermann (Department of Cellular and Molecular Immunology, George-August-Universität, Göttingen, Germany) for the generous gift of βarr1 and βarr2 Abs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflicts 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 This work was supported by National Institutes of Health Grants AI38910 and 056-CA92077, the National Center on Minority Health and Health Disparities (P20 MD00175), and the U.S. Army Medical Research and Materiel Command (07-1-0418). Back

2 Address correspondence and reprint requests to Dr. Ricardo M. Richardson, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 1801 Fayetteville Street, Durham, NC 27707. E-mail address: mrrichardson{at}nccu.edu Back

3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; βarr2, β-arrestin-2; CXCL8, IL-8; CXCR2, IL-8 receptor B; ELR, Glu-Leu-Arg; HIF-1{alpha}, hypoxia-inducible factor-1{alpha}; KC or CXCL1, keratinocyte-derived chemokine; LLC, Lewis lung cancer; MIP-2 or CXCL2, macrophage inflammatory protein-2; RBL, rat basophilic leukemia; RIPA, radioimmune precipitation assay; VEGF, vascular endothelial growth factor. Back

Received for publication August 28, 2007. Accepted for publication February 10, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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