highexpressionofligandsforchemokinereceptorcxcr2in ... · neutrophil antigen p40, and factor viii;...

High Expression of Ligands for Chemokine Receptor CXCR2 in

Alveolar Epithelial Neoplasia Induced by Oncogenic Kras

Marie Wislez,1,8Nobukazu Fujimoto,

1Julie G. Izzo,

2Amy E. Hanna,

1Dianna D. Cody,

3

Robert R. Langley,4Hongli Tang,

1Marie D. Burdick,

6Mitsuo Sato,

7John D. Minna,

7

Li Mao,1Ignacio Wistuba,

1,5Robert M. Strieter,

6and Jonathan M. Kurie

1

Departments of 1Thoracic/Head and Neck Medical Oncology, 2Experimental Therapeutics, 3Imaging Physics, 4Cancer Biology, and5Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas; 6Division of Pulmonary and Critical Care Medicine,Department of Medicine, David Geffen School of Medicine and Lung Cancer Research Program, Jonsson Comprehensive Cancer Center,University of California, Los Angeles, California; 7Hamon Center for Therapeutic Oncology Research, University of Texas-SouthwesternMedical Center, Dallas, Texas; and 8Department of Pulmonology, Hopital Tenon, AP-HP, Universite Pierre et Marie Curie-ParisVI, Paris, France

Abstract

CXCL8, a ligand for the chemokine receptor CXCR2, wasrecently reported to be a transcriptional target of Rassignaling, but its role in Ras-induced tumorigenesis has notbeen fully defined. Here, we investigated the role of KC andMIP-2, the murine homologues of CXCL8, in KrasLA1 mice,which develop lung adenocarcinoma owing to somaticactivation of the KRAS oncogene. We first investigatedbiological evidence of CXCR2 ligands in KrasLA1 mice.Malignant progression of normal alveolar epithelial cells toadenocarcinoma in KrasLA1 mice was associated with en-hanced intralesional vascularity and neutrophilic inflamma-tion, which are hallmarks of chemoattraction by CXCR2ligands. In in vitro migration assays, supernatants ofbronchoalveolar lavage samples from KrasLA1 mice chemo-attracted murine endothelial cells, alveolar inflammatorycells, and the LKR-13 lung adenocarcinoma cell line derivedfrom KrasLA1 mice, an effect that was abrogated by pretreat-ment of the cells with a CXCR2-neutralizing antibody. CXCR2and its ligands were highly expressed in LKR-13 cells andpremalignant alveolar lesions in KrasLA1 mice. Treatment ofKrasLA1 mice with a CXCR2-neutralizing antibody inhibitedthe progression of premalignant alveolar lesions and inducedapoptosis of vascular endothelial cells within alveolar lesions.Whereas the proliferation of LKR-13 cells in vitro wasresistant to treatment with the antibody, LKR-13 cellsestablished as syngeneic tumors were sensitive, supporting arole for the tumor microenvironment in the activity of CXCR2.Thus, high expression of CXCR2 ligands may contribute to theexpansion of early alveolar neoplastic lesions induced byoncogenic KRAS . (Cancer Res 2006; 66(8): 4198-207)

Introduction

Non–small cell lung cancer (NSCLC) is highly invasive andfrequently metastatic at the time of diagnosis. Because metastatic

NSCLC is refractory to all known interventions, a logical approachto reducing mortality from NSCLC is to intervene before thedevelopment of invasive disease. Success in this effort will requirean understanding of the genetic and biochemical changes inNSCLC that confer invasive properties.

The biological features of NSCLC associated with advancedstages of disease include neutrophil infiltration and enhancedtumor vasculature (1–4). Neutrophils and vascular endothelial cellsare recruited to tumors by the class of CXC chemokines with anNH2-terminal Glu-Leu-Arg (ELR) motif (5–10). These chemokinesare also autocrine growth factors for certain types of cancer cells(11–13). There are three known receptors for these chemokines:CXCR1 and CXCR2, which are G-protein-coupled receptors, andDuffy antigen receptor. Endothelial cells, as well as other cell types,express CXCR2, thereby promoting angiogenesis in tumors thatexpress ELR-positive CXC chemokines (14–17).

CXCL8, also known as interleukin-8 (IL-8), is a CXCR2 ligandthat is present in freshly isolated specimens of human NSCLC (18).Two murine functional homologues of CXCL8 (KC and MIP-2)have been implicated as the dominant mediators of aberrantangiogenesis in a syngeneic murine Lewis lung cancer model andin a human NSCLC/severe combined immunodeficient mousechimera (5, 15). A recent study showed that CXCL8 is atranscriptional target of Ras signaling and is required for theinitiation of tumor-associated inflammation and neovasculariza-tion in xenograft models (19). Mutations in the proto-oncogeneKRAS occur in 30% to 50% of lung adenocarcinomas, the mostcommon subtype of NSCLC, and expression of mutant KRAS in thealveolar epithelium leads to the development of lung adenocarci-noma in mice (20–23). In addition to its role in the transformationof alveolar epithelial cells, the presence of KRAS mutations is apredictor of shorter survival in NSCLC patients (24).

Based on recent evidence that CXCL8 plays a crucial role in theestablishment of Ras-induced tumors (19), we sought to determinewhether CXCR2 ligands contribute to malignant progression in arelevant model of KRAS-induced lung tumorigenesis. We investi-gated KrasLA1 mice, which develop lung adenocarcinoma throughsomatic activation of a KRAS allele carrying an activating mutationin codon 12 (G12D; ref. 23). Alveolar epithelial cells in this mousemodel recapitulate the series of morphologic stages through whichhuman atypical alveolar hyperplasia (AAH) evolves into adenocar-cinoma. We previously reported prominent infiltrates of macro-phages in premalignant alveolar lesions of KrasLA1 mice (25),supporting the presence of chemokines at early stages of lungtumorigenesis. We found neutrophils, vascular endothelial cells,and high expression of KC, MIP-2, and CXCR2 in premalignant

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

M. Wislez is a postdoctoral fellow of La Fondation de France and La Societe dePneumologie de Langue Francaise.

Requests for reprints: Jonathan M. Kurie, Department of Thoracic/Head and NeckMedical Oncology, The University of Texas M.D. Anderson Cancer Center, Unit 432,1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-6363; Fax: 713-796-8655; E-mail: [email protected].

I2006 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-3842

Cancer Res 2006; 66: (8). April 15, 2006 4198 www.aacrjournals.org

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alveolar lesions of KrasLA1 mice. CXCR2 inhibition blocked theexpansion of early alveolar neoplastic lesions, and the antitumoreffect of CXCR2 inhibition required the presence of the tumormicroenvironment. Thus, high expression of CXCR2 ligands maybe an important early event in alveolar neoplasia induced byoncogenic KRAS .

Materials and Methods

Animal experiments. Animal experiments were done in compliance

with the guidelines of The University of Texas M.D. Anderson Cancer

Center. KrasLA1 mice were provided by Dr. Tyler Jacks (MassachusettsInstitute of Technology, Cambridge, MA). For the CXCR2 neutralization

experiments, 16-week-old KrasLA1 mice were given 500 AL of CXCR2

immune serum (CIS; n = 13) or normal goat preimmune serum (NGS;

n = 14) by i.p. injection thrice a week for 3 weeks as previously described(15). Mice were subjected to respiratory-gated micro-computed tomogra-

phy (micro-CT) at the beginning and end of the treatment (26), and they

were killed by cervical dislocation within 6 hours of the final scan. During

the autopsy, visible lesions were counted on the surfaces of both lungs byinvestigators (M.W. and A.H.) blinded to the treatment group. The lungs

were then perfused with PBS and removed from the body. One lung was

kept at �80jC for protein extraction or immunohistochemistry of frozensections, and the other was fixed in 4% glutaraldehyde/paraformaldehyde

for 30 minutes followed by 10% formalin overnight before being embedded

in paraffin as previously described (25).

Syngeneic tumors were created with LKR-13, a lung adenocarcinoma cellline derived from a KrasLA1 mouse, in wild-type (129/sv) littermates of

KrasLA1 mice as described (25). Briefly, 107 LKR-13 cells in 100 AL of PBS

were s.c. injected into the flanks of the mice. When tumor volumes reached

50 mm3, mice were randomly assigned to receive 500 AL of CIS (n = 5) orNGS (n = 5) by i.p. injection thrice a week. Tumor diameters were measured

daily by an investigator (M.W.) who was blinded to the treatment group.

Tumor volumes were calculated as 0.5 � greatest diameter � (shortest

diameter)2. Treatment was continued until day 8, when one tumor reached1,500 mm3, at which time all mice were killed. Tumors (n = 19) were then

removed and either frozen at �80jC for protein extraction or fixed in 10%

formalin overnight before being embedded in paraffin.Cell lines. The LKR-13 and LKR-10 cell lines were derived by serial

passaging of minced lung adenocarcinoma tissues isolated from a KrasLA1

mouse. The cells were passaged in RPMI 1640 supplemented with 10% fetal

bovine serum on standard Falcon plasticware (Becton Dickinson, Bedford,MA) at 37jC in an atmosphere containing 5% CO2. To generate conditioned

medium, LKR-13 cells were plated in a 10-mm dish until 70% confluent,

washed in PBS, and exposed overnight to 4 mL of RPMI without serum. The

conditioned medium was recovered and centrifuged, and the supernatantwas frozen for later use.

Murine lung endothelial cells (MEC) were derived from the lungs of

transgenic mice homozygous for a temperature-sensitive mutant of SV40large T antigen (H-2Kb-tsA58 mice; refs. 27, 28). The cells were passaged in

DMEM supplemented with 10% fetal bovine serum on standard plasticware

at 33jC in an atmosphere containing 5% CO2. The expression of the SV40

large T antigen in these cells is thermolabile and can be inactivated byincubating the cells at 37jC for 48 hours before the beginning of

experiments (28). MECs were cultured at the nonpermissive temperature to

avoid the possibility of confounding effects of SV40 T-antigen expression.

Human bronchial epithelial cells (HBEC) were immortalized by theintroduction of genes encoding cyclin-dependent kinase-4 and human

telomerase reverse transcriptase (29). KRAS/HBECs were derived by stably

infecting parental HBECs with the retroviral vector pBabe-hyg-KRAS2-V12.

Immortalized HBECs and KRAS/HBECs were cultured with keratinocyteserum-free medium containing bovine pituitary extract and recombinant

epidermal growth factor (Life Technologies, Gaithersburg, MD). For

ELISAs, the cells were plated at 106 in 10-mm dishes. Conditionedmedium was recovered at 24, 48, and 72 hours; centrifuged; and frozen

for later use.

Antibodies. We purchased rabbit polyclonal antibodies against KC

(R&D Systems, Minneapolis, MN), factor VIII (DakoCytomation, Carpin-

teria, CA), prosurfactant protein C (SPC; Research Diagnostics, Concord,

MA), and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA); rat

monoclonal antibodies against F4/80 and the neutrophil antigen p40

(Serotec, Oxford, United Kingdom); and mouse monoclonal antibodies

against vascular endothelial growth factor (VEGF; Santa Cruz Biotechnol-

ogy, Santa Cruz, CA). For immunofluorescence staining, we used rabbit

antibodies against cleaved caspase-3 (FITC conjugated; Cell Signaling

Technology) and CD31 (PE conjugated; Research Diagnostics) and

secondary anti-rabbit and anti-rat antibodies (TRITC conjugated; Immu-

nology Consultants Laboratory, Newburg, OR). Nuclei were counterstained

with 4V,6-diamidino-2-phenylindole (DAPI) II (Vysis, Downers Grove, IL).

CIS and NGS were obtained as described previously (15). IgG fractions

were purified from the sera with a protein G column (IgG purification kit;

Pierce, Rockford, IL) and concentrated with a centrifugal filter (Millipore,

Bedford, MA).

Bronchoalveolar lavage for isolation of inflammatory cells andsupernatants. Briefly, KrasLA1 mice and wild-type littermates were killed by

cervical dislocation, and three 1-mL aliquots of PBS were injected directly

into the tracheae. The liquid was recovered by gentle aspiration and

centrifuged. The supernatant was recovered and frozen at �80jC for lateruse. The total cell count was done in a Neubauer chamber (Reichert,

Buffalo, NY), and differential cell counts were done on cytospin

preparations stained with H&E. The cell concentrations obtained bybronchoalveolar lavage varied between samples (1-8 � 104 cells and

10-40 � 104 cells for 129/sv and KrasLA1 mice, respectively). Cell viability

was z98%, as assessed by trypan blue exclusion. Alveolar cells were

suspended in RPMI 1640 serum-free medium containing penicillin G(100 IU/mL), streptomycin (100 Ag/mL), and amphotericin B (0.25 Ag/mL;

Life Technologies) for the in vitro migration experiments.

Micro-CT analysis. Mice were anesthetized, intubated by veterinary

personnel, and connected to a SAR-830/P small-animal ventilator (CWE,Ardmore, PA) operated using LabView software (National Instruments,

Austin, TX), which provided controlled repeated breath holds during CT

data acquisition (30). A single respiratory-gated, three-dimensional micro-CT image set was acquired for each mouse using 80 kVp, 405 AA, 400milliseconds per view, 720 views, and 0.5 degree incrementation per view.

This acquisition resulted in a set of contiguous axial DICOM-formatted

images through each mouse thorax, with voxels of dimensions 91 � 91 �91 Am3. Lesions were characterized on the initial and final scans by one

investigator (M.W.) who was blinded to the treatment group and autopsy

results. Lesions visualized on the CT images included solid or ‘‘ground-

glass’’ opacities or areas of consolidation resembling adenocarcinoma withbronchioloalveolar features in humans (31).

Tissue microarrays. Microarrays were constructed with cores from

formalin-fixed, paraffin-embedded blocks. One array was made withspecimens of normal lung tissue (n = 30), AAH (n = 40), adenoma

(n = 206), and adenocarcinoma (n = 11) from lungs of KrasLA1 mice

according to the histologic criteria established by Johnson et al. (23).

A second array comprised all lesions identified by histologic analysis fromthe mice treated with CIS or NGS (a total of 36 AAH and 103 adenomas).

Each murine lesion was sampled with a single core, 1 mm in diameter, as

the lesions were too small to permit multiple cores to be obtained.

Immunohistochemical analysis. Four-micrometer tissue sections weredeparaffinized, rehydrated, and washed with PBS as described (26).

Antigens were retrieved with 0.01 mol/L citrate buffer (pH 6; DakoCyto-

mation) for 30 minutes in a steamer. To detect the macrophage antigen

F4/80, slides were exposed to 0.025% trypsin-EDTA for 10 minutes.Samples were blocked for endogenous peroxidase activity in 3% hydrogen

peroxide/PBS, avidin/biotin solution (Zymed, San Francisco, CA), and

DAKO serum-free protein block (DakoCytomation) before being incubatedwith the primary antibody overnight at 4jC. Standard avidin/biotin

immunoperoxidase methods with diaminobenzidine as the chromogen

were used for detection. As negative controls to determine the specificity

of the immunostaining results, we omitted the primary antibody forcleaved caspase-3; stained a paraffin-embedded pellet of LKR-13 mouse

CXCR2 Ligands in KRAS-Induced Lung Cancer

www.aacrjournals.org 4199 Cancer Res 2006; 66: (8). April 15, 2006



lung adenocarcinoma cells for the macrophage antigen F4/80, theneutrophil antigen p40, and factor VIII; and used the isotype control for

CXCR2. For positive controls, we used normal lung tissue from KrasLA1

mice (F4/80, p40 neutrophil antigen, and factor VIII) and lymphoma tissue

from EA-myc mice (cleaved caspase-3).Staining was quantified independently by two investigators (M.W. and

I.W.) who were blinded to the treatment group. Lesions were scored based

on the frequency of stained cells within lesions. Tissues were visualized at�20 magnification for scoring of all antigens except cleaved caspase-3,which was visualized at �40 magnification.

Immunofluorescence staining. For dual-fluorescence staining, frozen

sections 4 Am thick were fixed in acetone and incubated with a

fluorochrome-coupled primary antibody or with a primary antibody

followed by a fluorochrome-coupled secondary antibody. Immunofluores-

cence-generated signals were visualized with a Zeiss Axioplan epifluor-

escence microscope (Nikon, Melville, NY) equipped with an oil immersion

objective and single bandpass filters for FITC, Texas red, and DAPI.

Digitized images of each fluorochrome were captured individually using a

high-resolution image analysis system (MetaMorph, Universal Imaging,

Downington, PA) with a cooled charge-coupled device camera (C4742-95-

12; Hamamatsu Photonics, Hamamatsu, Japan).ELISA. To measure concentrations of KC, MIP-2, VEGF, and CXCL8 in

lung homogenates, bronchoalveolar lavage supernatants, and conditioned

medium samples, ELISA was done according to the manufacturer’s

instructions (R&D Systems). Results were expressed as the mean

concentration (pg/mL) F SE normalized to the amount of total protein.Western blot analysis. Lysates from cell lines and tissue samples were

separated by SDS/PAGE and transferred onto a polyvinylidene fluoride

nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes

were immunoblotted overnight at 4jC with primary antibodies in TBScontaining 5% nonfat dry milk. Antibody binding was detected with an

enhanced chemiluminescence kit according to the manufacturer’s instruc-

tions (Amersham, Arlington Heights, IL).

Cytokine array. Cytokine antibody arrays (Array 3.1, Ray Biotech,

Norcross, GA) were treated for 30 minutes at room temperature with

blocking buffer and then incubated overnight at 4jC with lysates (100 Ag)from KrasLA1 mouse–derived lung tissues. Protein binding was detected

using the detection buffers as recommended by the manufacturer. The

membrane has controls for the detection reaction (immobilized antigen/

antibody complexes) and antibody specificity (antibodies against unrelated

peptides or the absence of antibodies). Antibodies against murine peptides

are arranged in duplicate on the array.

Proliferation assay. LKR-13 cells and MECs were plated at 1,000 per

well in 96-well tissue culture plates and incubated for 24 hours before

being treated with different concentrations of recombinant KC and MIP-2(0-25 ng/mL; R&D Systems) or CXCR2 antibodies from CIS or IgG from

NGS (1:50 to 1:2,000). Proliferation was quantified after 3 and 6 days by

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Migration assay. Cells (1 � 105) in 750 AL of RPMI with 0.1% bovineserum albumin (Sigma, St Louis, MO) were plated in the upper chambers

of wells containing a polyethylene terephthalate filter with 8-Am pores (BD

BioCoat, San Jose, CA). For the experiments on MECs and LKR-13 cells

(Fig. 2A and C-H), four wells per condition (with or without chemo-attractant) were used in each experiment, and the experiments were

repeated thrice. The data shown are an average of the three experiments

(12 data points per condition). For experiments on primary inflammatorycells derived from lavage samples (Fig. 2B), each experiment included two

lavage samples (one sample from two KrasLA1 mice). Cells recovered from

each lavage sample were split into two wells, which were treated with or

without chemoattractant (two wells per condition, a total of four wells).This experiment was repeated thrice, and the mean values were calculated

from six data points per condition. In Fig. 2G , primary inflammatory cells

were recovered from lavage samples from five KrasLA1 mice. Cells from

each mouse were split into four wells, two wells per condition (IgG- orCXCR2-neutralizing antibody). The two values per condition were

averaged. The results from each mouse were presented separately, and

the results from all five mice were combined to determine statistical

significance. For these experiments, RPMI alone, RPMI with recombinantKC and MIP-2 (0-25 ng/mL), LKR-13 cell-conditioned medium, or

bronchoalveolar lavage supernatant (1:2 in RPMI) was used as a

chemoattractant in the lower chamber. To measure sensitivity to

CXCR2-neutralizing antibodies, the cells were exposed to CXCR2-neutralizing antibodies or IgG (1:50 to 1:2,000) purified from CIS and

NGS, respectively, during the assay. After 24 hours of incubation at 37jC,the cells on the upper surface of the membrane were removed with a

cotton swab, leaving the cells on the lower surface, which were stainedwith Wright stain in methanol and examined by microscopy at � 20

magnification. Five fields were counted per filter.

Statistical analysis. Immunostaining scores, lung lesion numbers, and

results from the proliferation and migration assays were consideredcontinuous variables. The Kruskal-Wallis test was used for three-group

comparisons and the Mann-Whitney nonparametric test for two-group

comparisons. Spearman’s r coefficient was used for correlative studiesbetween quantitative variables. Ps V 0.05 were considered significant. Data

were processed with StatView and Survival Tools 5.0 software (Abacus

Concepts, Berkeley, CA).

Results

Chemokine expression in the lungs of KrasLA1 mice. We firstinvestigated chemokine expression in lung extracts derived fromKrasLA1 mice using an array of antibodies against a panel of 62murine chemokines. We examined lung tissues from mice at anearly stage of lung tumorigenesis (5 months old) when AAH andadenomas but no adenocarcinomas were present to identifychemokines that might play a role in tumorigenesis. The chemo-kines expressed most prominently were cutaneous T-cell–attractingchemokine/C-C chemokine-27, CXCL-16, insulin-like growthfactor binding protein-3, IL-1a, lipopolysaccharide-induced CXC-chemokine, IL-12/p70, lymphotactin, monocyte chemoattractantprotein-1 (MCP-1), MCP-5, macrophage colony stimulating factor,MIP-2, MIP-3a, platelet factor-4, P-selectin, thymus-derivedchemotactic agent-3, and vascular cell adhesion molecule-1(Supplementary Fig. S1). Thus, a variety of chemokines werepresent in lung tissues of KrasLA1 mice before the development oflung adenocarcinoma.

Biological evidence of CXCR2 ligands in the lungs of KrasLA1

mice. Given the recent report that CXCL-8 is a transcriptional targetof mutant KRAS (19), we further examined the role of the MIP-2 andKC in KrasLA1 mice. We investigated biological evidence of theseligands in KrasLA1 mice by examining premalignant alveolar lesionsfor neutrophils and vascular endothelial cells, which are recruitedby these CXCR2 ligands. A tissue microarray was constructedfor immunohistochemical analysis with punch biopsy samples ofnormal tissue, AAH, adenoma, and adenocarcinoma from lungs ofKrasLA1 mice. We examined the expression of a 40-kDa cell surfaceantigen expressed specifically in neutrophils (32) and factor VIII,which is expressed in endothelial cells. Staining for neutrophils(Fig. 1A) and endothelial cells (Fig. 1B) in neoplastic lesionsincreasedwith histologic progression fromAAH to adenocarcinoma.Cytospin preparations of bronchoalveolar lavage samples revealedthat macrophages were the predominant inflammatory cell in thelavage samples from KrasLA1 mice and 129/sv wild-type littermates(Table 1). Neutrophil counts were significantly higher in bronchoal-veolar lavage samples from KrasLA1 mice than in those from wild-type littermates (Table 1). Thus, malignant progression wasassociated with neutrophil inflammation and angiogenesis.

We next investigated biological evidence of CXCR2 ligands inbronchoalveolar lavage samples from KrasLA1 mice and inconditioned media samples from LKR-13 lung adenocarcinoma

Cancer Research




cells derived from KrasLA1 mice. The ability of these samples tochemoattract MECs, primary cultures of alveolar inflammatorycells, and LKR-13 cells was measured in vitro , and the specific role ofCXCR2 was examined by preincubation of the cells with a CXCR2-neutralizing antibody or IgG as a control (purified from CIS andNGS, respectively) to block cellular migration upon exposure tochemoattractants. Incubation with LKR-13 cell–conditioned medi-um recruited MECs (Fig. 2A) and alveolar inflammatory cells(Fig. 2B), whereas incubation with bronchoalveolar lavage super-natants recruited MECs (Fig. 2C) and LKR-13 cells (Fig. 2D).Preincubation with CXCR2-neutralizing antibody blocked therecruitment of MECs (Fig. 2E) and LKR-13 cells (Fig. 2F) by

bronchoalveolar lavage supernatants and the recruitment ofalveolar inflammatory cells (Fig. 2G) by LKR-13 cell–conditionedmedia samples, showing biological evidence of CXCR2 ligands inKrasLA1 mouse–derived lung tissues that was required for the re-cruitment of these cells. Preincubation with the CXCR2-neutralizingantibody did not block the recruitment of MECs by LKR-13 cell–conditioned medium (Fig. 2H), suggesting that LKR-13 cells secretefactors other than CXCR2 ligands that recruit MECs. Thus, lungtissues derived from KrasLA1 mice expressed soluble, biologicallyactive mediators that recruited inflammatory cells, vascularendothelial cells, and adenocarcinoma cells, and this recruitmentwas inhibited, in part, by a CXCR2-neutralizing antibody.

Table 1. Inflammatory cells in bronchoalveolar lavage samples

Mice No. cells/AL (% F SE total cells), mean F SE

Total cells Alveolar macrophages Neutrophils Lymphocytes

Wild type (n = 3) 106.6 F 52.3 99.3 F 47.7 (94.3 F 1.2) 1.7 F 1.3 (2.7 F 1.6) 5.5 F 5.3 (2.9 F 2.1)

KrasLA1 (n = 4) 180.0 F 58.4 136.4 F 41.7 (79.6 F 2.6) 25.7 F 9.3 (18.3 F 1.4) 9.3 F 4.2 (4.6 F 1.4)

P* 0.7 0.4 (0.03) 0.03 (0.03) 0.28 (0.7)

*Statistical analysis was done using the Mann-Whitney nonparametric test.

Figure 1. Malignant progression inKrasLA1 mice is associated with infiltrationof neutrophils and endothelial cells.Immunohistochemical analysis of p40, aneutrophil-specific protein (A), and factorVIII, an endothelial cell-specific protein (B),was done on lung tissues from KrasLA1

mice. Representative sections ofnormal tissue (N ), AAH, adenoma (Ad ),and adenocarcinoma (ADC ) at �20magnification. Columns, mean stainingscores for each histologic category;bars, SE.





High expression of KC and MIP-2 in KrasLA1 mice. Weexamined expression of KC and MIP-2, the functional homologuesof CXCL8 in mice, in lung tissues derived from KrasLA1 mice andtheir wild-type littermates by immunohistochemical analysis andELISA. KC was detected by immunohistochemical analysis inepithelial cells, macrophages, and endothelial cells within alveolarepithelial lesions of KrasLA1 mice (Fig. 3A), whereas MIP-2 was notdetectable by immunohistochemical analysis (data not shown).ELISA done on lung homogenates and supernatants frombronchoalveolar lavage samples showed that KrasLA1 mice hadhigher expression of KC (Fig. 3B) and MIP-2 (Fig. 3C) than didtheir wild-type littermates. The concentrations of KC and MIP-2were highly correlated in lung homogenates (q = 0.92) andbronchoalveolar lavage fluid (q = 0.89; Fig. 3D), suggestingcoordinated regulation of KC and MIP-2 expression in alveolarneoplastic lesions. In contrast, the expression of anotherangiogenic factor (VEGF) was not increased in the lungs ofKrasLA1 mice (Fig. 3E and F), suggesting a specific role for CXCR2ligands as angiogenic factors in early neoplastic lesions.

To extend the analysis to human lung cells, we investigatedwhether the expression of CXCL8 in HBECs, which are humanbronchial epithelial cells that have been immortalized by theintroduction of genes encoding cyclin-dependent kinase-4 andhuman telomerase reverse transcriptase (29), is increased by

oncogenic KRAS. KRAS/HBECs, which have been transfected witha retroviral vector expressing mutant KRAS, acquire enhancedanchorage-independent growth and increased saturation density.9

CXCR2 expression was similar in HBECs and KRAS/HBECs(Fig. 4A), whereas CXCL8 expression, as measured by ELISA ofconditioned media, was higher in KRAS/HBECs than in HBECs(Fig. 4B). Thus, the introduction of mutant KRAS was sufficient toconfer high CXCL8 expression in HBECs.

CXCR2 neutralization inhibits alveolar epithelial neoplasiain KrasLA1 mice. We examined the role of CXCR2 in KrasLA1 lungtumorigenesis by passively immunizing mice with CIS or NGS (as acontrol). Beforehand, we examined CXCR2 expression in prema-lignant alveolar lesions by immunohistochemical analysis andfound that CXCR2 was expressed in epithelial cells, inflammatorycells, and endothelial cells (Fig. 5A), indicating that multiple celltypes were potential targets of CXCR2 neutralization. KrasLA1 micewere treated with CIS or NGS by i.p. injection thrice a week for 3weeks, beginning at 4 months of age. After treatment, we countedthe lesions on lung pleural surfaces at the time of autopsy. Themice were also subjected to micro-CT at the beginning and

Figure 2. CXCR2 neutralization inhibits the migration ofepithelial cells, endothelial cells, and inflammatory cells in vitro .Experiments were done, and the results were calculated, asdescribed in Materials and Methods. A and B, migration ofMECs (A ) and alveolar inflammatory cells (B) in the presence(+) or absence (�) of LKR-13 cell–conditioned medium (CM ).C and D, migration of MECs (C ) and LKR-13 cells (D ) usingbronchoalveolar lavage supernatants (BAL ) from wild-type(WT ) or KrasLA1 mice as a chemoattractant. E and F,migration of MECs (E) and LKR-13 cells (F ) preincubated withCXCR2-neutralizing antibody or IgG purified from CIS andNGS, respectively, before incubation with bronchoalveolarlavage. G, effect of CXCR2 neutralization on the migration offive samples of alveolar inflammatory cells in the presence ofLKR-13 cell conditioned media. H, migration of MECspreincubated with CXCR2-neutralizing antibody or IgG beforeincubation with LKR-13 conditioned medium.

9 J.D. Minna, unpublished data.

Cancer Research




completion of treatment to examine changes in lesion numbers(examples are shown in Fig. 5B). The autopsies and micro-CT scansrevealed that relative to the effects of NGS, treatment with CISdecreased the mean number of lesions (Fig. 5C). Histologic analysisof lung tissue sections showed that the CIS-treated mice had feweradenomas per mouse (mean F SE: 3.3 F 0.7 versus 6.0 F 0.8;P = 0.01) and more AAH lesions (2.3 F 0.4 versus 0.9 F 0.2;P = 0.01) than did the NGS-treated mice. Together, these datasuggest that CXCR2 neutralization decreased the number oflung adenocarcinoma precursors and inhibited the progressionof AAH to more advanced stages of alveolar malignancy.

CXCR2 neutralization induces apoptosis of vascular endo-thelial cells. Based on the decrease in number of alveolar lesionsobserved after CIS treatment, we examined whether CXCR2inhibition induces apoptosis. Western blot analysis showed thatcaspase-3 cleavage was higher in whole-lung extracts from micetreated with CIS than in those from mice treated with NGS (n = 6mice per group; Fig. 6A), suggesting the presence of apoptotic cells.We also examined caspase-3 cleavage specifically in alveolarlesions. A tissue microarray for immunohistochemical analysis wasconstructed with punch biopsy samples of normal tissue, AAH, andadenoma from lungs of KrasLA1 mice treated with CIS or NGS.Staining for cleaved caspase-3 was greater in alveolar lesions from

mice treated with CIS than in those from mice treated with NGS(Fig. 6B).

We next investigated which cell types underwent apoptosis byperforming dual immunofluorescence staining for cleaved caspase-3 and CD31 (endothelial cells), F4/80 (macrophages), p40(neutrophils), or SPC (type II alveolar cells). We chose SPC as anepithelial cell marker because it is expressed in lung adenocarci-nomas in KrasLA1 mice (22). We observed coexpression of cleavedcaspase-3 with CD31 (Fig. 6C) but not with F4/80, p40, or SPC(data not shown), suggesting that CXCR2 inhibition inducedapoptosis of endothelial cells but not inflammatory cells ortransformed alveolar epithelial cells.

Sensitivity to CXCR2 inhibition requires the tumor micro-environment. From the above findings, we could not exclude thepossibility that CXCR2 neutralization inhibited lung tumorigenesisthrough effects on the tumor microenvironment, epithelial cells, orboth. To investigate this question, we compared the in vitro andin vivo sensitivity of LKR-13 cells to CXCR2 neutralization. Despitetheir high expression of CXCR2 and KC (Fig. 7A), LKR-13 cellsshowed no change in proliferation after 5 days of treatment in vitrowith CXCR2-neutralizing antibody (data not shown). To investigateeffects of CXCR2 neutralization on LKR-13 cells in vivo , weestablished LKR-13 cells as syngeneic tumors in wild-type

Figure 3. Alveolar lesions in KrasLA1 micehave high expression of KC and MIP-2.A, immunohistochemical analysis of KC intumor cells (left ; magnification, �20) andmacrophages (right ; magnification, �40) ofan adenoma from a KrasLA1 mouse.Arrows, positively stained cells. ELISA wasused to detect KC (B), MIP-2 (C ), andVEGF (E) in lung homogenates andbronchoalveolar lavage supernatants(BAL) from KrasLA1 mice (n = 9) andwild-type (WT ) littermates (n = 6). Theconcentrations of cytokines werenormalized to the total protein content ofeach sample. Each ELISA was done induplicate. Columns, means; bars, SE.D, KC and MIP-2 concentrations werecorrelated in each sample (.) of lunghomogenate and bronchoalveolar lavagesupernatant. F, immunohistochemicalanalysis of VEGF (magnification, 20�)shows staining in normal bronchial cells(left) and faint staining in tumor cells(right ). Arrows, positively stained cells.





littermates. Once established as syngeneic tumors, LKR-13 cellsrecruited neutrophils and endothelial cells (Fig. 7B). Treatment ofthe mice with immune sera (CIS or NGS) every other day revealedthat after 8 days, tumor volumes were lower in CIS-treated micethan in NGS-treated control mice (P < 0.05; Fig. 7C). Thus, thetumor microenvironment was required for the antitumor effect ofCXCR2 inhibition on LKR-13 cells.

Discussion

This study provides evidence that alveolar epithelial cellstransformed by oncogenic KRAS have high expression of CXCR2ligands, which recruit inflammatory and endothelial cells, creatinga milieu that promotes the progression of early neoplasia. Our

finding that oncogenic KRAS triggers tumor-host interactions thatare crucial to the promotion of tumor growth provides a rationaleto target CXCR2 or its ligand, CXCL8, in the treatment ofoncogenic KRAS-induced lung malignancy. If such a treatmentapproach is found to be efficacious, the implications areconsiderable given that there is currently no effective treatmentfor this subset of NSCLC.

Introduction of mutant RAS into cells is sufficient to confertransformed properties in vitro (19). In addition to these cell-autonomous effects, Ras promotes tumorigenesis in vivo byeliciting a stromal response, an effect mediated, in part, throughCXCR2 ligands (19), which we showed here to have relevance tolung cancer. Mutant RAS increases the expression of CXCL8through activation of Rac- and Akt-dependent pathways (19). Wepreviously showed that Rac- and Akt-dependent pathways areactivated in premalignant alveolar lesions of KrasLA1 mice (33, 34),and that these pathways cooperate to maintain the survival of lungcancer cells (35). Rac- and Akt-dependent pathways promote cellsurvival by regulating the expression of BCL2 family members,caspase-9, FOXO proteins, and GSK-3, among others (36–38). Thus,oncogenic RAS induces cellular transformation through cell-autonomous and stroma-dependent processes, both of which areactivated through Rac- and Akt-dependent pathways, whichcooperate to modulate the expression of a diverse set ofdownstream mediators.

Findings reported here on the role of CXCR2 ligands ininflammation and neovascularization corroborate evidence fromother pulmonary diseases in which these processes are prominentfactors. In bronchiolitis obliterans syndrome, which occurs afterlung transplantation, and in the adult respiratory distress

Figure 4. Mutant KRAS increases CXCL8 expression in HBECs. A, Westernblot analysis of HBECs and KRAS /HBECs using specific antibodies againstCXCR2 and h-actin. B, ELISA for CXCL8 in conditioned medium collected fromHBECs and KRAS /HBECs at various times after seeding. Normalized to totalprotein content.

Figure 5. CXCR2 neutralization inhibits malignant progression inKrasLA1 mice. A, immunohistochemical analysis of CXCR2 intumor cells (top ; magnification, �20) and endothelial cells (bottom ;magnification, �40) in a lung adenoma from a KrasLA1 mouse.Arrows, positively stained cells. B, representative micro-CT axialimages of one KrasLA1 mouse at the beginning (top ) and end(bottom ) of treatment with CXCR2-neutralizing antibody. Thelesions, which appear as peripheral nodules in the right and leftlower lobes (arrows ), disappeared with treatment; the other lesionsdecreased moderately or were stable. C, mean numbers of lesionsper mouse were determined for each group [mice treated withCXCR2 antibody (CIS) or NGS)] by counting visible lesions on thepleural surfaces at autopsy (left ). Changes in lesion numbers overtime were determined from micro-CT scans done at the beginningand end of treatment (right ). Columns, means; bars, SE.

Cancer Research




syndrome caused by ventilator-induced lung injury, lung expres-sion of CXCR2 ligands parallels the inflammatory response andlung injury (17, 39). Neutralization of CXCR2 is sufficient toattenuate neutrophil infiltration and lung injury in murine modelsof these diseases (17, 39). In the heterotopic and orthotopic Lewislung cancer models, tumor growth is associated with enhancedneovascularization, neutrophil inflammation, and expression ofCXCR2 ligands, and CXCR2 neutralization decreases tumor sizeand increases tumor necrosis (15). Similar to our findings withLKR-13 cells, the CXCR2-neutralizing antibody is efficaciousagainst Lewis lung cancer cells grown as heterotopic tumors buthas no effect on their proliferation in vitro , indicating that thetumor microenvironment is required for tumor sensitivity. Thus,high expression of CXCR2 ligands contributes to inflammationand neovascularization in a broad spectrum of pulmonarydiseases.

Our observation that CXCR2 ligands contribute to theestablishment and growth of tumor cells in a syngeneic hostraises the possibility that they can also promote the progression ofearly neoplasia toward invasive disease. In KrasLA1 mice, in whichthe expansion of hyperplastic alveolar epithelial cells intoadenomas precedes the development of adenocarcinomas (23),we found that malignant progression was associated withenhanced neutrophil inflammation and neovascularization andhigh expression of CXCR2 ligands. CXCR2 neutralization reducedthe numbers of early lesions, inhibited the progression of AAH into

adenomas, and induced apoptosis of vascular endothelial cells.These findings suggest that high expression of CXCR2 ligands andthe accompanying neovascularization are required for earlyalveolar neoplasia. The angiogenic activity of CXCR2 ligands hasbeen reported to be neutrophil-dependent in the Matrigel spongeangiogenesis assay (40). Although we have not excluded a role forneutrophils in angiogenesis in KrasLA1 mice, CXCR2 neutralizationdid not decrease the numbers of neutrophils in premalignantalveolar lesions by immunohistochemical analysis of lung tissuesections (data not shown), raising the possibility that CXCR2ligands promote angiogenesis through direct effects on vascularendothelial cells. Based on the response of this early neoplasticdisease model to CXCR2 inhibition, strategies to target CXCR2 mayfind application in treating early lung cancers in humans.

Oncogenic KRAS has been reported to increase the expression ofVEGF in a variety of tumor types, including lung cancer (41, 42).Based on these and other findings, clinical strategies haveused neutralizing antibodies to VEGF and VEGF receptor, VEGFligand traps, and small-molecule inhibitors of VEGF receptortyrosine kinase activity to block neovascularization in patientswith malignancies. However, a recent study found high CXCL8expression in oncogenic KRAS-transformed colon cancer cellsdeficient in hypoxia-inducible factor-1 and VEGF, and CXCL8was sufficient to promote angiogenesis in that setting (43).Thus, CXCL8 expression contributes to angiogenesis in KRAS-transformed cells and increases in response to VEGF blockade,

Figure 6. CXCR2 inhibition induces apoptosis ofvascular endothelial cells in alveolar lesions.A, Western blot analysis of whole-lung extracts fromKrasLA1 mice treated with NGS or CXCR2 antibody(CIS; n = 6 mice per group) using antibodiesspecific for cleaved caspase-3 (CC3 ) or h-actin.B, representative images of immunohistochemicalstaining for cleaved caspase-3 in KrasLA1 mice treatedwith NGS or CIS. Columns, mean staining scorescalculated from the scores of all alveolar lesions fromeach treatment group; bars, SE. C, representativeimages of immunofluorescent staining (magnification,�63) for DAPI, cleaved caspase-3, and CD31 in a lungsection from a KrasLA1 mouse treated with CXCR2antibody. Arrows, a cell that coexpressed cleavedcaspase-3 and CD31.





raising the possibility that treatment of KRAS-mutant tumors withantiangiogenic strategies targeting both CXCL8 and VEGFmay be more effective than VEGF blockade alone. Based onthese preclinical studies, efforts are warranted to investigateCXCR2 as a therapeutic target for the prevention and treatmentof NSCLC.

Acknowledgments

Received 10/24/2005; revised 1/30/2006; accepted 2/9/2006.Grant support: NIH grants R01 CA105155, P50 CA70907, P30 CA16672, CA87879,

and P50 CA90388.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Figure 7. CXCR2 inhibition shrinks LKR-13 syngeneictumors. A, left, Western blot analysis of LKR-10 andLKR-13 cells using specific antibodies against CXCR2and h-actin. Middle, immunohistochemical analysis ofCXCR2 in LKR-13 cells. Right, ELISA for MIP-2 andKC in conditioned medium from LKR-13 cells. B,immunohistochemical analysis of LKR-13 syngeneictumors to identify infiltrating endothelial cells andneutrophils using specific antibodies against factor VIII(left ) and p40 (right ; magnification, �40). C, changesin mean volumes of LKR-13 syngeneic tumors overtime in mice treated with CXCR2 antibody (CIS) orNGS. Tumors were measured daily. Points, meantumor volumes; bars, SE. *, P < 0.05.

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2006;66:4198-4207. Cancer Res Marie Wislez, Nobukazu Fujimoto, Julie G. Izzo, et al. in Alveolar Epithelial Neoplasia Induced by Oncogenic KrasHigh Expression of Ligands for Chemokine Receptor CXCR2

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