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Inhibition of Oncogenic K-ras Signaling by Aerosolized Gene Delivery in a Mouse Model of Human Lung Cancer¹

Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [H-Y. L., Y-A. S., J. I. L., K. A. H., L. M., J. M. K.]; Molecular Cardiology Research Institute, New England Medical Center, Boston, Massachusetts 02111 [T. F.]; Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030 [B. E. G.]; and Howard Hughes Medical Institute, Center for Cancer Research, and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 [T. J.]

	ABSTRACT

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Purpose: Transfer of growth-suppressive genes to lung tumors has therapeutic potential, but effective delivery techniques have not been developed. Here, we investigated gene delivery to lung tumors by aerosolization of adenoviral vectors incorporated into calcium phosphate precipitates.

Experimental Design: To investigate the efficacy of this delivery method in normal and neoplastic lung, an adenoviral vector expressing ß-galactosidase was administered by jet nebulization to K-ras^LA1 mice, which develop lung adenocarcinomas through activation of a latent allele carrying mutant K-ras(G12D). Furthermore, we investigated whether aerosolized delivery of Ad-MKK4 (KR), an adenoviral vector expressing dominant-negative mutant mitogen-activated protein kinase kinase 4(MKK4), can block Ras-dependent signaling in K-ras^LA1 mice.

Results: After a single administration, ß-galactosidase was detected in lung tissue for up to 21 days, and expression was much greater in tumors than in normal lung tissue. MKK4 was activated in the lungs of K-ras^LA1 mice, and aerosolized treatment with Ad-MKK4 (KR) decreased c-Jun-NH₂-terminal kinase but not extracellular signal- regulated kinase activity, providing evidence that MKK4 was selectively inhibited.

Conclusions: These findings demonstrate a novel approach to targeting oncogenic pathways in lung tumors by aerosolized gene delivery.

	INTRODUCTION

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Lung cancer remains the primary cause of cancer-related death in the United States, and its incidence is increasing in developing countries (1) . Progress has been made in the treatment of other malignancies by targeting the protein products of mutated proto-oncogenes (2) , and similar efforts have begun in the treatment of lung cancer patients. Interest has focused on the proto-oncogene K-ras, which is mutated in 30–50% of lung adenocarcinomas, the most common histological subtype of non-small cell lung cancer (3) . K-ras has been inhibited through the development of small molecules that block posttranslational modifications required for K-ras activation and by gene therapeutic strategies that decrease its expression (4 , 5) .

Gene therapies have shown promise in the replacement of wild-type p53in mouse lung tumors after intratracheal delivery of vector (6, 7, 8, 9) . Gene therapies for the treatment of lung cancer patients have been delivered through intratumoral, intrabronchial, and intrapleural injections (10) . These approaches have achieved limited success for several reasons. First, tumors are not always easily accessible to direct injection, increasing the morbidity associated with this procedure. Second, intratumoral injections do not consistently deliver adenovirus to the entire tumor volume, leaving portions of the tumor untreated. Third, gene transfer to lung tumor cells is inhibited by soluble factors in malignant pleural effusions (11) , reducing the efficacy of treating tumor cells in this tissue compartment.

Aerosolized delivery is a potentially more efficacious approach for gene delivery. It can reach a large surface area of the bronchial epithelium, does not carry the risks associated with intrathoracic injections, and avoids the toxicities associated with systemic administration. Despite these advantages, the efficacy of aerosolized gene transfer has been limited by the vector systems used. Cationic lipids and polymers encapsulating DNA have demonstrated high transfection efficiency in normal HBE³ cells and lung tumor cells (6 , 12) , but they are associated with significant inflammatory reactions in the bronchial epithelium (13 , 14) . Aerosolized delivery of adenoviral and retroviral vectors has achieved gene transfer to lung cancers in animal models, but this approach is limited by variable viral receptor expression in lung cancer cells, and administration of viral vectors to the lung induces antiviral immune responses that reduce gene transfer (15, 16, 17) . In addition to their limited transfection efficiencies, lipid and viral vectors are fragile and can be degraded during the jet nebulization process (18) .

In this study, we investigated a novel aerosolized treatment strategy. We delivered recombinant adenoviral vectors incorporated into calcium phosphate precipitates, a modification that enhances adenoviral gene delivery in airway epithelia, which lack the receptor activity to bind adenovirus fiber protein (19 , 20) . Gene transfer was achieved in lung tumors that arise in K-ras^LA1 mice, which express mutant K-rasthrough stochastic activation of a latent allele. Aerosolized delivery of an adenovirus expressing dominant-negative mutant MKK4inhibited Ras-dependent signaling in the lungs of K-ras^LA1 mice. These findings provide evidence that aerosolized delivery of calcium phosphate-precipitated adenoviral vectors is an effective means of gene transfer to lung tumors that should be considered for therapeutic trials in lung cancer patients.

	MATERIALS AND METHODS

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Animals, Cells, and Materials.
We conducted studies on K-ras^LA1 mice, which carry a latent K-ras allele with two copies of exon 1, one wild type and the other mutant (G12D; Ref. 21 ). The latent allele is activated stochastically in cells through homologous recombination, which results in the deletion of the wild-type copy of exon 1 and expression of an oncogenic form of the K-ras gene. Multifocal lung adenocarcinomas develop spontaneously in 100% of the mice. Mutant K-rasmRNA and protein are detectable in tumors but not in normal lung tissue. Tumors are first detectable at 2–3 weeks, grow to essentially fill the thoracic cavity, and cause pulmonary insufficiency to which the mice succumb at 8–10 months of age (21) . The spontaneously immortalized HBE cell line HB56B (22) was grown in keratinocyte serum-free medium (Life Technologies, Inc., Gaithersburg, MD) on standard plasticware (Falcon; Becton-Dickinson, Bedford, MA) at 37°C with a pCO₂ of 5%. We purchased rabbit polyclonal antibody against human ERK1 and ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA), goat polyclonal antibody against human JNK1 (Santa Cruz Biotechnology), and rabbit polyclonal antibody against human phospho-MKK4 (Thr261; New England Biolabs, Beverly, MA).

Preparation and Administration of Calcium Phosphate-precipitated Adenovirus.
Particles (5 x 10⁹) of adenovirus expressing ß-galactosidase (Ad-ßGal), dominant-negative mutant MKK4(Ad-MKK4-KR; Ref. 23 ), or parental adenovirus (Ad5CMV) were incubated in Eagle’s Minimal Essential Medium (Sigma Chemical Co., St. Louis, MO) containing 5.8 mM Ca²⁺ and 0.86 mM P_i for 30 min. Adenoviruses were administered unmodified or as calcium phosphate precipitates by continuous aerosolization using an Aerotech II nebulizer (CIS-USA, Inc., Bedford, MA) flowing at 10 liters of dry, compressed air/min over a 30-min period into an airtight box housing K-ras^LA1 mice. Three days after a single administration, the mice were sacrificed. The lungs were perfused with 1x PBS, fixed with 10% formaldehyde, and stained with X-Gal as described previously (19) . Lungs were then paraffin-embedded, cut into 5-µm sections, and stained with H&E.

Western Analysis.
Lung tissues were collected, frozen in dry ice, and kept at -70°C until use. Lung tissues were ground into powder in liquid nitrogen and suspended in 500 µl of 1x PBS containing 1 mM phenylmethylsulfonyl fluoride. Samples were centrifuged at 15,000 x g for 20 min. The pellet was suspended in 400–800 µl of cold lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 0.2 mM EGTA, 1% NP40, 10% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 20 mM sodium fluoride, 5 mM sodium orthovanadate, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 2 mg/ml pepstatin, and 1 mM benzamidine] and left on wet ice for 20 min. Samples were vortexed vigorously for 15 s and clarified by centrifugation at 15,000 x g for 20 min. Whole cell lysates of H441 cells were isolated by incubation in lysis buffer for 20 min on ice followed by centrifugation at 15,000 x g for 20 min. Protein concentration was determined with the Protein Assay Kit (Bio-Rad, Hercules, CA). Protein was separated by electrophoresis on a SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA), and immunoblotted overnight at 4°C with primary antibodies, and antibody binding was detected using the enhanced chemiluminescence kit (Amersham, Inc., Arlington Heights, IL) according to the manufacturer’s directions.

Immune Complex Kinase Assay.
Immune complex kinase assays were performed as described previously (23) on lysates from mouse lung tissues and HB56B cells. HB56B cells were transiently transfected for 1 h with expression vectors (5 µg) containing K-ras(G12D) or empty vector using LipofectAMINE (Life Technologies, Inc.) followed by infection for 48 h with calcium phosphate-precipitated Ad-MKK4 (KR) (24) or Ad5CMV (10⁴ particles). Cell lysates were prepared and subjected to immunoprecipitation (100 µg/sample) using antibodies (1 µg) that recognize JNK1 or ERK1/2 (Santa Cruz Biotechnology) by rotation at 4°C for at least 2 h. Protein A/G-agarose beads (20 µl; Santa Cruz Biotechnology) were added and incubated at 4°C for 1–2 h. The beads were washed three times with lysis buffer and once with kinase buffer [20 mM HEPES (pH 7.5), 20 mM ß-glycerol phosphate, 10 mM p-nitrophenyl phosphate, 10 mM MgCl₂, 1 mM DTT, and 50 mM sodium vanadate]. Kinase assays were performed by incubating the beads with 30 µl of kinase buffer containing 20 µM cold ATP, 5 µCi of [-³²P]ATP (2000 cpm/pmol), and 1 µg of GST-cJun or myelin basic protein as a substrate for JNK and ERK, respectively. The kinase reaction was performed at 30°C for 20 min. The samples were suspended in Laemmli buffer, boiled for 5 min, and analyzed by SDS-PAGE. The gel was dried and autoradiographed.

	RESULTS

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Calcium Phosphate Enhanced Aerosolized Adenovirus Delivery to K-ras^LA1 Mice.
K-ras^LA1 mice were treated with unmodified Ad-ßGal or Ad-ßGal incorporated into calcium phosphate precipitates (Ad-ßGal + Ca) by continuous aerosolization into an airtight box. Three days after a single administration, the mice were sacrificed, and the lungs were fixed in formalin and stained with X-Gal. Whole lung preparations from mice treated with Ad-ßGal + Ca demonstrated intense staining in tumors and less prominent staining in normal lung parenchyma (Fig. 1) . Lungs from mice treated with unmodified Ad-ßGal demonstrated no visible staining (Fig. 1) . X-Gal staining was detected in lung tumors of mice sacrificed up to 21 days after treatment (data not shown). We performed histological analysis to investigate X-Gal expression in tumor and normal lung tissues. X-Gal staining was detected in lung tumor cells and detected less frequently in normal bronchial epithelial cells of mice treated with Ad-ßGal + Ca but was not detected in lung tissues from mice treated with unmodified Ad-ßGal (Fig. 1) . These findings indicate that calcium phosphate precipitation was required for adenoviral gene expression in tumors.

View larger version (84K): [in this window] [in a new window]   Fig. 1. Aerosol delivery of Ad-ßGal to the lungs of K-rasLA1 mice. K-rasLA1 mice (14 weeks old) were subjected to no treatment (Untreated) or to aerosolized treatment with unmodified Ad-ßGal (Ad-ßGal) or calcium phosphate-precipitated Ad-ßGal (Ad-ßGal + Ca). Three days after treatment, the mice were sacrificed, and the lungs were perfused with 1x PBS, fixed with 10% formalin, and stained with X-Gal overnight at 37°C. Whole lung preparations are illustrated. These lungs were serially sectioned, counterstained with H&E, and subjected to histological analysis. Representative sections of tumor and normal bronchiole are illustrated. Magnifications are indicated in the bottom left corner of each image.

View larger version (66K): [in this window] [in a new window]   Fig. 2. MKK4 was activated in lungs of K-rasLA1 mice. A, top panels, Western analysis was performed on whole lung extracts prepared from K-rasLA1 mice (Mutant) and wild-type (WT) mice, using antibodies that recognize phospho-MKK4 (Thr261) (pMKK4) and pan-MKK4 (MKK4) to control for relative MKK4 levels. A, bottom panels, in vitro kinase assays of JNK activity were performed on whole lung extracts using GST-cJun as substrate. JNK1 Western analysis was performed on these extracts to control for relative JNK1 levels. B, top right panel, immunohistochemical analysis of MKK4 expression was performed on lung tissue derived from K-rasLA1 mice using antibodies against phospho-MKK4 (Thr261). Illustrated is MKK4 staining of a lung tumor (x10). B, bottom panel, high magnification (x40) of the bracketed area in the top right panel. Cells staining positively are brown. B, top left panels, specificity of phospho-MKK4 antibody for activated MKK4 was examined in H441 non-small cell lung cancer cells that were serum-starved (-) or serum-stimulated (+), trypsinized, cytospun onto slides, and stained with phospho-MKK4 (Thr261) antibody (illustrated at x40 magnification).

View larger version (22K): [in this window] [in a new window]   Fig. 3. MKK4 (KR) blocked JNK activation by mutant K-ras. HB56B cells were transiently transfected with a plasmid containing K-ras (G12D) or empty vector (-) and infected for 48 h with Ad-MKK4 (KR) or empty virus (-). Cell extracts were prepared and subjected to in vitro kinase assays (KA) using GST-cJun as substrate and to Western analysis (W) using anti-JNK1 antibodies to control for relative JNK1 levels.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Aerosolized delivery of Ad-MKK4 (KR) selectively inhibited JNK activity in the lungs of K-rasLA1 mice. K-rasLA1 mice were subjected to a single aerosolized treatment with calcium phosphate-precipitated Ad-MKK4 (KR), empty virus (EV), or no treatment (-). Three days later, the mice were sacrificed, and whole lung extracts were subjected to in vitro kinase assays (KA) using GST-cJun and myelin basic protein (MBP) as substrates for JNK and ERK, respectively. The extracts were also subjected to Western analysis (W) of JNK and ERK using anti-JNK1 and ERK1/2 antibodies to control for relative JNK and ERK levels.

	DISCUSSION

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Previous studies have shown that MKK4 expression and activity are altered in human tumor cells and support a role for MKK4 as both a promoter and a suppressor of human tumorigenesis. The MKK4 gene is deleted or mutated in a subgroup of pancreatic, biliary, and breast carcinomas, and reintroduction of MKK4inhibits the metastatic ability of certain tumor cells, demonstrating that MKK4has tumor suppressor activity (26, 27, 28) . Potentially mediating this effect, Ras pathway activation induces the expression of p53 and p16^INK4a, which induce premature cellular senescence, and inactivation of p53 or p16 prevents Ras-induced growth arrest (29) . In contrast to these studies, MKK4 is known to be a downstream mediator of Rac1, and Rac1 activation contributes to Ras-induced cellular transformation (25) , indicating that MKK4 plays a role in cellular transformation. Supporting the latter hypothesis, we found that MKK4 activity was increased in the lungs of K-ras^LA1 mice, and phospho-MKK4 was detected mostly in lung tumor cells. This supports in vitro studies on lung cancer cells demonstrating that MKK4-dependent pathways play a dominant role in mutant Ras-induced colony formation (30 , 31) . Thus, MKK4 and its downstream mediators play apparently contradictory roles in the regulation of cellular growth and transformation that may depend on the presence of cell type-specific factors or the activity of tumor suppressor pathways that inhibit the mitogenic and transforming effects of MKK4.

We found that aerosolized delivery of calcium phosphate-precipitated Ad-MKK4 (KR) demonstrated selectivity at the molecular level, inhibiting the activity of JNK but not ERK in the lungs of K-ras^LA1 mice. Based on our findings, this technique has several potentially useful applications. First, it can be used in animal models of human lung cancer to investigate, in an in vivo setting, the role of specific intracellular pathways in lung tumorigenesis. Second, it can be implemented in the clinical setting for the delivery of genes with anticancer activity to lung cancer patients. Similar to previous reports (19) , short-term treatments of K-ras^LA1 mice caused no histological evidence of inflammation to the airway epithelium (data not shown), suggesting that calcium phosphate may be less toxic to normal tissues than other materials used for adenoviral encapsulation (13 , 14) . In future studies, it will be important to investigate the antitumor efficacy of this approach and issues related to chronic adenoviral administration, such as toxicities to normal bronchial tissues and the development of antiadenoviral immune responses that reduce gene transfer efficiency.

	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 Supported in part by American Cancer Society Grant RPG-98-189-CNE and NIH Grants R01CA80686, P50 CA70907 (Lung Cancer Spore), and U01CA84306 (Mouse Models of Human Cancer Consortium). T. J. is a Howard Hughes Medical Institute Investigator, and J. M. K. is a Sidney Kimmel Scholar.

² To whom requests for reprints should be addressed, at Box 432, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Fax: (713) 796-8655;E-mail: jmkurie{at}mdanderson.org.

³ The abbreviations used are: HBE, human bronchial epithelial; ERK, extracellular signal-regulated kinase; JNK, c-Jun-NH₂-terminal kinase; MKK4, mitogen-activated protein kinase kinase-4; X-Gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; GST, glutathione S-transferase.

Received 2/25/02; revised 5/29/02; accepted 6/ 6/02.

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