Multiple pulmonary adenomas in the lung of transgenic mice overexpressing the RON receptor tyrosine kinase
Yi-Qing Chen1,
Yong-Qing Zhou2,
Lu-Hong Fu3,
Dong Wang1 and
Ming-Hai Wang1,4
1 Department of Medicine, University of Colorado School of Medicine, CU Cancer Center, and Denver Health Medical Center, Denver, CO 80204, USA,
2 Division of Neurosurgery, The First Affiliated Teaching Hospital, Zhejiang University School of Medicine, Hangzhou, Peoples Republic of China and
3 Division of Cancer Diagnosis and Treatment, Jinhua Guangfu Hospital, Jinhua, Zhejiang, Peoples Republic of China
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Abstract
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The receptor tyrosine kinase RON (recepteur dorigine nantais), a member of the MET proto-oncogene family, has been implicated in the pathogenesis of certain epithelial cancers including lung adenocarcinomas. To determine the oncogenic potential of RON, transgenic mice were generated using the surfactant protein C promoter to express human wild-type RON in the distal lung epithelial cells. The mice were born normal without morphological defects in the lung, however, multiple lung adenomas with distinct morphology and growth pattern were observed. Tumors appeared as a single mass in the lung around 2 months of age and gradually developed into multiple nodules throughout the lung. Most of the tumors were characterized as cuboidal epithelial cells with type II cell phenotypes. They grew along the alveolar walls and projected into the alveolar septa. A transition from pre-malignant adenomas to adenocarcinomas was observed. The RON transgene is highly expressed and constitutively activated in the tumors as evident by immunohistochemical staining and western blot analyses. Moreover, we found that Ras expression was dramatically increased in the majority of tumors. However, no mutation in the hot spots of the K-Ras or p53 gene was observed, although limited genomic instability occurs in individual tumors. Taken together, this is a mouse lung tumor model with unique biological characteristics. The model may provide an opportunity to study the role of RON in lung tumors and to elucidate the mechanisms underlying this distinct lung tumor.
Abbreviations: IHC, immunohistochemical; Inter-SSR-PCR, inter-simple sequence repeat; mAb, monoclonal antibody; MSP, macrophage-stimulating protein; RON, recepteur dorigine nantais; SPC, surfactant protein C; SSCP, single-stranded conformation polymorphism; wt, wild-type
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Introduction
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Lung adenocarcinoma belongs to a category of non-small cell lung cancers, which comprise
2540% of all lung cancers (1). Four histological subtypes are recognized, including acinar-, papillary-, mucous secreting- and bronchiolo-alveolar carcinomas (1,2). The biological aspects of lung adenocarcinoma are complex, and little is known about the precise gene alterations that underlie the earliest steps in the multiple stages of lung adenocarcinogenesis. However, multiple genetic changes, in particular chromosome regions (3p, 9p, 13q and 17p) or in specific candidate genes (Ras, p53, fragile histidine triad and p16), have been elucidated for established lung adenocarcinoma (3).
The RON (recepteur dorigine nantais) receptor tyrosine kinase (4), also known as stem cell-derived tyrosine kinase (STK) in mice (5), belongs to the MET proto-oncogene family (6). The RON gene is located in chromosome 3p21, a region frequently altered in certain types of lung cancers (4,7). Mature RON is a 180 kDa heterodimer composed of a 40 kDa extracellular chain and a 150 kDa transmembrane ß chain with intrinsic tyrosine kinase activity (4). RON is activated by growth factor macrophage-stimulating protein (MSP) (8,9), a serum protein with the structure similar to hepatocyte growth factor (10,11). MSP was originally identified by its activity in mouse peritoneal resident macrophage (10). Activated RON phosphorylates different intracellular proteins and activates a variety of signal pathways that regulate crucial epithelial cell functions, including growth, migration, differentiation and survival (1215).
The RON gene is normally transcribed in the lungs as evident in northern blot analysis (4). Immunohistochemical (IHC) staining confirmed that RON expression is restricted to bronchial epithelial cells, and not alveolar cells including type II cells (16). The role of RON in the pathogenesis of lung tumors is unknown. Previous studies have established that unlike its mouse counterpart STK, which has tumorigenic activities (17), overexpression of human wild-type (wt)RON in rodent fibroblasts does not cause cell transformation in vitro and tumor formation in vivo (13,18,19). However, recent studies have shown that RON expression is altered in epithelial carcinomas including those from lung, colon and breast (2022), indicating that overexpression of this receptor could play a role in the progression of certain epithelial cancers. It has been shown that RON expression is increased in the lung after administration of chemical carcinogens (22). Altered RON expression has also been found in several lung cancer cell lines (23) and in primary lung adenocarcinomas such as bronchioloalveolar carcinoma (our unpublished data). Moreover, two reports have shown that oncogenic RON can be experimentally created by point mutations, such as D1232V or M1254T, in the kinase domain of RON (18,19). Recently, a mutation in the RON gene has been identified in a primary human lung adenocarcinoma sample (24). Thus, RON may be involved in the progression of certain lung tumors in vivo.
The present work is to study the oncogenic potential of human wtRON in vivo using transgenic mice as a model. Targeted expression of RON in the lung was achieved by using a lung-specific transcription element, the human surfactant protein C (SPC) promoter (25). It was discovered that forced expression of RON causes the formation of multiple lung adenomas and adenocarcinomas with unique cell morphology and growth pattern. Tumors displayed type II cell phenotype with increased Ras expression and genomic instability. These results suggest that overexpression of human wtRON could initiate oncogenic programs and play an important role in the pathogenesis of certain types of lung cancers.
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Materials and methods
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Cell lines and reagents
Mouse lung epithelial cell line CCL-196 was from ATCC (Rockville, MD). The 3.7SPC/SV40 vector containing a 3.7 kb human SPC promoter fragment (25) was provided by Dr J.A.Whitsett (University of Cincinnati, Cincinnati, OH). Mouse monoclonal antibody (mAb) (clone ID2) and rabbit IgG antibodies to human RON were used as described (21). Mouse mAb 4G10 to phosphotyrosine was from Upstate Biotechnology (Lake Placid, NY). Rabbit anti-rat proSPC serum and goat anti-rabbit CC10 serum (26) were provided by Dr G.Singh (Department of Veterans Affairs Medical Center, Pittsburgh, PA).
Techniques used to establish SPCp-RON transgenic mice
A human RON cDNA (4) was inserted into the vector 3.7SPC/SV40 between the SPC promoter and the 0.6 kb SV40 sT intro-poly A sequence. The generation of transgenic mice was conducted at the Transgenic Core Facility at Wayne State University (Detroit, MI). The purified transgene fragment was injected into the pronuclei of fertilized eggs isolated from B6C3/F1 hybrid mice (C57BL/6xC3H, Taconic). Eggs surviving microinjection were implanted into the oviducts of pseudopregnant foster mothers (CD1, Charles River Laboratories) following previously described procedures (27,28).
Southern blot analysis
Genomic DNA was isolated from tails of transgenic mice and digested with restriction enzyme BamHI. DNA was separated on a 1% agarose gel and transferred to a nylon membrane. Hybridization was conducted with 32P-labeled RON cDNA as described (28).
Tissue processing and histological examination
All procedures performed on mice were approved by the institutional review board. The lungs were inflated with PBS, fixed with 4% paraformaldehyde in solution, and then processed. Fresh lung tissue was also obtained before lung fixation and used for RNA isolation and protein extraction. Histological examination was conducted by Dr W.Gunning (Medical College of Ohio, Toledo, OH) with hematoxylineosin (H&E) staining of formalin-fixed, paraffin-embedded sections.
Immunoprecipitation and western blotting
These methods were performed as described previously (12,21). The mAb ID2 was used for immunoprecipitation of human RON. Rabbit IgG to RON was used as primary antibody in western blotting followed by goat anti-rabbit IgG conjugated with horseradish peroxidase. The reaction was developed with enhanced chemiluminescent reagents and recorded on film.
IHC staining
Lung sections were treated with 15% acetic acid to block endogenous alkaline phosphatase and then blocked with 1% BAS in Tris buffer (10 mm Tris pH 7.4, with 150 mM NaCl, 1% BSA and 0.1% Tween 20). Antibodies to human RON, proSPC, CC10 or Ras were used as primary antibody followed by a second antibody conjugated with alkaline phosphatase. The substrate for the reaction was Fast Red. H322 cells grown on a glass slide were used as positive controls. Lung sections incubated with pre-immunized rabbit serum served as the negative control. Slides were examined under a microscope and photographed. Overexpression was assessed using a semi-quantitative scoring system (29), utilizing the sum of a proportion score (05) and an intensity score (03). The combined score ranges from 0 to 8 with six or more considered overexpression (29).
Mutational analysis of the K-ras and p53 genes by single-stranded conformation polymorphism and DNA sequencing
Genomic DNA was isolated from individual lung tumor nodules according to previously described methods (28). Primers used for mutational analysis, which cover the K-Ras (codes 12 and 61) or the p53 (exons 5 and 8) gene, were designed according to previously published sequence data (30,31). PCR-based single-stranded conformation polymorphism (SSCP) was performed as described previously (21). Double-stranded DNA sequencing was performed at the University of Colorado DNA Sequence Core facility.
Inter-simple sequence repeat-PCR for genomic instability
The appearance of genomic instability in individual lung tumors was determined by the inter-simple sequence repeat (Inter-SSR)-PCR techniques (32,33). Genomic DNA was prepared from individual lung tumors. Oligonucleotide primers (CA)8 RG or (CA)8 RY (R = a 50:50 mix of purines A and G; Y = a 50:50 mix of pyrimidines T and C) were synthesized. The primers were 5'-end-labeled with [
-32P]ATP (6000 Ci/mmmol, NEC) using 5'-end-labeling kit (Promega). PCR was carried out in 20 ml reaction mixture consisting of 1 mM primer (a 1:5 mixture of labeled and unlabeled primers), 50 ng DNA, and 0.3 U Taq polymerase in PCR buffer (Life Technologies). After initial denaturation for 5 min at 94°C, 30 PCR cycles were performed at 30 s at 94°C, 45 s at 52°C and 2 min at 72°C, followed by a final 7 min extension at 72°C. After PCR, 5 ml of each sample was analyzed on a non-denaturing 8% polyacrylamide gel buffered with 1x TBE. Gels were cast in a Sequi-Gen 0.04x38x50 cm sequencing gel apparatus (Life Technologies). Electrophoresis was carried out at 14 V/cm for 1013 h. Gels were dried and exposed to film for 210 h at room temperature.
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Results
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Generation of transgenic mice that specifically express human wtRON in the lung
To determine if the transgenic expression of human wtRON in the mouse lung has any oncogenic effect, the SPCp-RON transgene vector was constructed (Figure 1A
) and transgenic mice were produced. The integration of the SPCp-RON transgene into the genome was determined by Southern blot analysis using mouse-tail DNA. Results are shown in Figure 1B
. Five founder mice, nos 35, 36, 39, 40 and 71, were found among 19 mice to harbor the transgene with varying copy number (Figure 1B
and Table I
). Founder mouse no. 39 died accidentally at the age of 4 months. The remaining founder mice (line nos 35, 36, 40 and 71) were bred with B6C3/F1 hybrid mice to produce offspring (F1). The F1 mice with the integrated transgene were determined by PCR analysis using tail DNA as templates (data not shown). Germline transmission was identified in line nos 35, 36 and 71. Founder mouse no. 40 failed to pass the transgene to offspring, probably due to the mosaic effect on the founder mouse, with the germline cells being free of the transgene (34,35). Founder mice nos 35, 36 and 71 and their offspring were used for further studies.


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Fig. 1. Generation of SPCp-RON transgenic mice that specifically express human RON in the lungs. (A) Schematic representation of the SPCp-RON transgene construct. The human RON cDNA was inserted into SalI and EcoRI sites in the vector 3.7SPC/SV40 (34). Digestion of the construct with restriction enzymes NdeI and SmaI results in an 8.6 kb fragment, which was used for microinjection. (B) Southern blot analysis of human RON cDNA integrated into the genomic DNA of founder mice. RON cDNA was used as positive control (data not shown). Hybridization was performed as described in the Materials and methods.
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Specific expression of RON in type II cells in the lung of transgenic mice
To determine if the transgene is specifically expressed in the lung, various organs were collected from SPCp-RON mice. RON was immunoprecipitated from tissue lysates with mAb ID2 followed by western blot analysis. Results are shown in Figure 2A
. RON was detected in lung homogenates, but not in other organs or tissues. The size of the detected band is identical to the mature RON ß chain expressed by lung epithelial CCL-196 cells, indicating that RON is correctly processed in the lung. The level of wtRON in the transgeinc lung was significantly higher than that in CCL-196 cells. We also performed RTPCR to confirm the above results. RON cDNA fragment was amplified only from RNA isolated from the lung tissues but not from other organs (data not shown). Similar results were also found in F1 mice derived from line nos 35, 36 and 71 (data not shown).


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Fig. 2. Specific expression of RON in the lungs of SPCp-RON transgenic mice. (A) Various tissues were harvested from F1 mice from three lines of transgenic mice. Immunoprecipitation was carried out using tissue homogenates (1 mg protein/sample) with mAb ID2. Western blotting was performed using rabbit IgG antibody to RON. Lung epithelial CCL-196 cells were used as the positive control (first lane). The data showed here are from a F1 mouse of the line 71. (B) Lung sections from three 2-month-old transgenic mice were used in IHC staining with rabbit IgG to human RON (top panel) or pre-immunized rabbit IgG (bottom panel) as detailed in the Materials and methods (400x magnification).
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To determine transgene-expressing cells in the lung, IHC staining was performed on lung sections prepared from three 2-month-old transgenic mice with antibodies specific to human RON. Results are shown in Figure 2B
. Numerous alveolar cells were stained positively for RON. No positive staining was observed in bronchial epithelial cells (data not shown). Histological analysis indicated that the RON-positive cells have morphological features similar to type II cells. It was estimated that >90% of type II cells express the RON transgene. These results, together with those in Figure 2A
, suggest that the SPC promoter directs human wtRON expression specifically in type II cells in SPCp-RON transgenic mice.
Development and histological features of multiple lung adenomas in SPCp-RON mice
Between 6 and 14 months of age, depending on individual lines of transgenic mice, all founder mice and the majority of F1 mice (92 of 102 in line no. 71, 74 of 79 in line no. 35, and 47 of 51 in line no. 36) showed signs of respiratory distress and weight loss. Lungs were removed from killed mice and examined. It was found that multiple tumors with uniform morphology were observed in the lung, although the overall structures of the lung were still intact (Figure 3A
).

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Fig. 3. Histological features of lung tumors in SPCp-RON transgenic mice. (A) Multiple lung tumors in SPCp-RON mice. The lung of a F1 mouse (6 months old from line no. 71) was collected for histological analysis. The section shown here is a lobe of the lung stained with H&E. (B) Morphological features of lung adenoma. The enlarged tumor was from the same F1 mouse shown in (A) (H&E staining, 400x magnification). (C) Lung adenomas with increased cellular atypia and increased mitotic index. Lung tumors were from a 20-month-old F1 mouse (line no. 71, 400x magnification).
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Detailed histological examination revealed that most of the tumors were located in peripheral portions of the lung. The majority of tumors appear as solid-alveolar adenomas, suggesting a pre-malignant state. No papillary growth morphology was observed. Tumors at early stages displayed a distinctive cuboidal morphology with round to oval nuclei and very low mitotic index as mitotic figures were infrequent (Figure 3B
). In a few cases, tumors showed a slight degree of cellular atypia. At later stages, tumors had significantly more cellular atypia with a high mitotic index (Figure 3C
). Some tumors showed distinct features of large cell undifferentiated carcinoma with the pallor of the cytoplasm present in the tumor as well as the tremendous pleomorphism of cellular size and nuclear morphology (Figure 3C
). The tumors appear to be expanding and compressing adjacent tissue. Evidence of vascular invasion and metastasis was not observed. We examined other organs and tissues and did not find any tumor formation. Also, no tumors were found in the lung of littermate controls (more than 120 mice examined within 818 months). These results suggest that tumor formation is not a randomly occurring event in SPCp-RON mice. A summary of tumors found in three lines of transgenic mice is shown in Table I
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Growth patterns of lung tumors in SPCp-RON mice
To characterize lung tumors in more detail, we first determined when tumors appear in the lung and how long it takes for tumors to become multiples in the lung. To this end, the lungs from F1 mice (line no. 71) were collected at different months after mice were born. A single tumor mass was found in the lung in three individual mice as early as 2 months of age (Figure 4A
). No tumors were found in newborn or 1-month-old mice (three mice were examined in each age group). Around 46 months of age, several tumor nodules were observed. Up to 8 or 14 months, numerous tumor masses were seen (Figure 3A
). At these stages, some mice showed signs of respiratory distress. These results suggest that the tumors are most probably initiated 2 months after birth. The progression of tumors from a single mass to multiple nodules appears to be a slow process taking
610 months. Because many type II cells express the RON transgene, these multiple tumors probably originate from individual type II cells within several months. However, the erogenous spreading of tumors that are loosened from alveolar walls may also play a role.

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Fig. 4. Growth patterns of lung tumors in SPCp-RON mice. (A) Detection of single lung tumor in F1 mice at 2 months of age. Individual lungs from three F1 mice, derived from line nos 35 (ii), 36 (iii) or 71 (iv), respectively, were collected at 2 months of age (H&E staining, 100x magnification). A normal lung section from a littermate at the same age was used as control (i). (B) Maturation stages of tumors in the lung. The lung was collected from a F1 mouse (8 months old, line no. 71). Tumor progression at different stages: (i) an early tumor mass (hyperplasia) growing in alveoli; (ii) another relatively early tumor mass (hyperplasia) in alveoli; (iii) a partially developed tumor mass; (iv) a fully-grown tumor mass. (H&E stain, 100x magnification.) This growth pattern is different from those seen in spontaneous or chemically induced lung adenomas in strain A mice, which have solid, papillary, or mixed growth patterns (42,43).
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To determine the patterns of tumor growth, individual tumors at different maturation stages were studied. Results are shown in Figure 4B
. The tumors appeared to originate from alveolar and grow along alveolar walls. The spaces of the alveolar septa were still present when tumor growth was at early stages (Figure 4B, i and ii
). However, tumors gradually grew into the solid-shape mass occupying the alveolar septa at later stages (Figure 4B, iii and iv
). These results demonstrate the sequential maturation stages of the tumors in the lung.
Specific expression and activation of RON in lung tumors
To determine if RON is expressed in tumors, IHC staining was performed on lung sections using rabbit IgG antibodies specific to human RON. Results are shown in Figure 5A
. Immunoreactive RON was strongly detected in tumors (Figure 5A, i
). The RON staining is displayed mostly as cytoplasm/membrane granular patterns. By analyzing the immunoreactivity, it seems that the cells at the edges of the tumor have more intensive staining than those in the center. No positive staining was found in control tumor sections (Figure 5A, ii
), in which pre-immunized rabbit serum was used. Similar results were also found in lung sections derived from other lines of transgenic mice (data not shown).


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Fig. 5. Specific expression and activation of RON in lung tumors. (A) Detection of RON expression by IHC staining. Lung sections from a F1 mice (line no. 71) were stained with rabbit anti-RON IgG followed by DAKO LSAB Staining Kit (i). Normal rabbit IgG was used as controls (ii). (B) Constitutive phosphorylation of RON in vivo. Individual tumor nodules were isolated from the lungs of a F1 mouse (line no. 71). The mAb ID2 immunoprecipitation of RON was done with tissue homogenates (3 mg/sample) followed by western blotting. The phosphorylated RON protein was detected with mAb 4G10 (iii). Human wtRON was also detected with rabbit IgG to RON using the same amount of tumor lysates (iv). NL, lung tissues from littermate control; TM, mixed lung tumors. All others are individual tumor nodules.
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To determine if RON is constitutively active in vivo, tissue lysates were prepared from individual lung tumor nodules. Normal lung lysates prepared from littermate control mice were used as controls. RON was immunoprecipitated with mAb ID2 and detected in western blotting using mAb 4G10 to phosphotyrosine. As shown in Figure 5B, iii
, RON is autophosphorylated in individual tumor nodules with variable levels. The high levels of tyrosine phosphorylation were observed in tumor nodules T12, T13 and T14. Also, RON was highly expressed in these individual lung tumors (Figure 5B, iv
). RON was not detected in lung lysates derived from control mice. The data, together with results shown in Figure 5A
, suggest that RON is highly expressed and constitutively activated in lung tumors of transgenic mice.
Expression of the type II cell marker by lung adenomas
It has been shown previously that the SPC promoter functions in type II and Clara cells (25). The specific cellular marker for type II cells is SPC, which is produced only by type II cells (36,37). In contrast, non-ciliated bronchiolar Clara cells express CC10 (26), which serves as the cellular marker for Clara cells. To determine if lung tumors express the type II or Clara cell marker, IHC staining was performed using antibodies specific to proSPC or CC10. The results are shown in Figure 6
. Positive staining of the bronchial epithelial cells with the anti-CC10 antibody was observed (Figure 6A
). No tumor cells were stained positively with CC10. The results of detecting SPC are shown in Figure 6B
. Positive staining of tumor cells with anti-SPC antibody was documented. As a control, type II cells from littermates also expressed SPC (data not shown). These results, together with those from Figure 6A
, suggest that lung tumors do not express the Clara cell marker CC10, but display type II cell phenotypes.

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Fig. 6. Determination of specific cellular markers expressed by tumors. IHC staining was performed as described in Figure 5A . (A) CC10 is expressed in bronchiole epithelial cells but not in tumors. The lung tumor sections from F1 mice from line no. 71 were incubated with goat IgG against rabbit CC10 (cross reacts with mouse CC10) (100x magnification). Normal goat IgG was used as negative controls (data not shown). (B) SPC is expressed in lung adenomas. The rabbit IgG antibody to rat proSPC was used as primary antibody (400x magnification). Lung sections from F1 mice were used in this experiment. Normal rabbit IgG was used as negative controls (data not shown).
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Increased expression of the Ras protein in lung tumors
To determine if Ras expression is altered in lung tumors, IHC staining was performed in 25-lung tumor sections derived from 25 mice. Adjacent normal lung tissues were used as controls. By using the Allreds scoring method (29), Ras expression in normal alveolar cells scored 4 (16 cases) and 5 (nine cases) with the mean score of 4.36. In contrast, overexpression of Ras was observed in tumor nodules. The IHC score ranged from 5 (four cases), 6 (seven cases), 7 (10 cases) to 8 (four cases). The mean score was 6.56, which is significantly higher than that from normal alveolar cells (P < 0.05). Results shown in Figure 7
are the representative staining of Ras expressed in tumors with moderate (Figure 7A
) or high levels (Figure 7B
). Results in Figure 7C
are lung sections incubated with control rabbit IgG for negative controls. No meaningful staining was observed.

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Fig. 7. Increased expression of the Ras protein in lung tumors derived from SPCp-RON mice. IHC staining was performed as described in the Materials and methods. Rabbit IgG to mouse Ras was used as primary antibody. Normal rabbit IgG was used as the negative control. The levels of Ras expression were assessed according to a previously published method (29). The moderate (A) or high levels (B) of Ras expression with the negative control (C) are shown (100x magnification). Similar results were found in other lung tumor sections.
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Genomic instability in lung tumors from RON transgenic mice
To determine if RON-mediated tumor formation is accompanied with genomic alteration, Inter-SSR-PCR (32,33) was used to determine the presence of genomic instability in individual tumors. This method simultaneously samples numerous diverse regions of the genome, specifically those regions of the genome present between inverted abundant repetitive elements (32,33). Representative results are shown in Figure 8
. In these four sets of samples (normal versus tumors), changes in the amplified DNA patterns were observed in tumor samples with the appearance of a new band or bands with reduced or increased intensity. These results suggest that genomic instability occurs in individual lung tumors in RON transgenic mice.

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Fig. 8. Increased genetic instability in individual lung tumors isolated from SPCp-RON transgenic mice. Genomic DNA was isolated either from individual lung tumor (T) nodules or from normal (N) lung tissues of the same mouse. Inter-SSR-PCR was performed as described in the Materials and methods. Samples were repeated three times to ensure the accuracy of the results. Representative results from four sets of samples are shown here. Similar findings were also found in >30-paired samples from different transgenic mice.
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Mutational analysis of K-Ras and p53 genes in individual lung tumor nodules
Previous reports have demonstrated high frequencies of point mutations in the K-Ras or p53 gene in mouse lung tumor samples (38). To determine if K-Ras or p53 mutations occur in lung tumors from RON transgenic mice, PCR-based SSCP analysis was performed using genomic DNA isolated from >40 tumor nodules from either young (612 months) or old (1320 months) transgenic mice. Several hot spots in the K-Ras gene (code 12 or 61) or in the p53 gene (exons 5 or 8) were analyzed by SSCP and DNA sequencing analyses. To our surprise, no mutations were identified in these regions in all tumors sampled. These results suggest that the formation of lung tumors is not associated with the abnormalities in the K-Ras or p53 gene.
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Discussion
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The main findings in this report are the expression of human wtRON in distal lung epithelial cells of mice results in the formation of multiple lung adenomas. Tumors develop slowly with signs of progression towards malignancy with pathological features of large cell undifferentiated carcinomas. Tumors are characterized as cuboidal epithelial cells that line up along alveolar septa and project into the alveolar spaces in numerous solid formations. RON is highly expressed and constitutively activated in lung tumors. Tumors express proSPC, but not Clara cell product CC10, indicating that tumors display type II cell phenotypes. By Inter-SSR-PCR analysis, we found that individual tumor nodules display the altered patterns of genomic fingerprinting, suggesting that genomic instability occurs in these lung adenomas. Although increased Ras expression was found in the majority of lung tumor nodules, no point mutations were observed in the hot spots of the K-Ras or p53 gene, demonstrating that overexpression of human wtRON has the ability to initiate cell transforming and tumorigenic program in vivo, leading to lung tumor formation and progression. To our knowledge, this is the first evidence showing that overexpression of human wtRON in vivo results in tumor formation and progression.
Targeted expression of transgenes in distal lung epithelial cells by human SPC promoter has been reported previously (34,39,40). By analyzing RON transgene expression in our SPCp-RON mice, we demonstrated that human wtRON is specifically expressed in the mouse lung type II pneumocytes. As shown in western blot analysis, human wtRON is only detected in the homogenate of the transgenic lung, and not in other organs or tissues (Figure 2A
). RTPCR analysis also confirmed that the RON transgene is transcribed in the transgenic lungs. Moreover, as evident in IHC staining (Figure 2B
), RON is expressed in alveolar cells of the transgenic lung with type II cell morphology. Thus, specific expression of the RON transgene in the lung directed by the SPC promoter was achieved. It is interesting to note that despite its high level of expression, RON has no effects on lung development. All three lines of transgenic mice and their offspring grew normally without any respiratory and structural defects. Thus, increased RON expression has no negative effects on the development, morphology and function of the lung.
There are several characteristics of RON-mediated lung tumors in our transgenic mice. First, tumors in SPCp-RON mice have a unique cell morphology and growth pattern. They originate from alveoli, express the type II cell marker SPC, grow along the alveolar septa, and project into the alveolar spaces. Only solid-alveolar growth morphology was observed. We had examined >100 lung tumor samples and did not see any tumors with papillary growth patterns. This single growth pattern is different from those seen in other transgenic lung tumors (34,40,41) or in spontaneous or chemically induced lung tumors in strain A mice (42,43). As shown in SPCp-SV40T transgenic mice (34), lung tumors were adenocarcinomas with a mixed growth pattern: lepidic, papillary and solid (34), indicating that tumors display phenotypes of several subtypes of adenocarcinomas. Tumors also displayed mixed cellular markers including CC10 and SPC, suggesting heterogeneity of tumors from bronchiolar and alveolar origins (34). Spontaneous or chemically induced mouse lung tumors usually have solid, papillary or mixed histological growth patterns (37,42,43). Thus, lung tumors in SPCp-RON grow in a way that is different from lung tumors observed in other transgenic mice (34,40,41) and strain A mice (42,43). Secondly, the progression of lung tumors in SPCp-RON mice is a slow process. Tumors were initiated in the lung as a few masses
23 months after the mice were born. Multiple nodules were observed only at 612 months and lasted nearly 1224 months. As the RON transgene is expressed in numerous type II cells, it is conceivable that tumors were derived from individual type II cells in different sites. Mice that died due to respiratory failure usually lived to 2 years. This pattern of tumor progression is different from those found in SV40- or other oncogene-expressing transgenic mice (34,40,41,44). In these mice, lung tumors developed rapidly within months and mice died
46 months of age (34,40,41), mostly before transgenic descendants could be obtained. Thus, lung tumors in RON transgenic mice progress in a relatively natural course, which is relatively similar to those found in spontaneous or chemically induced mouse lung tumors (42,43). Thirdly, the majority of lung tumors in SPCp-RON mice are characterized as adenomas with a slight degree of atypia. This observation suggests that the expression of the RON transgene has oncogenic potential but is not sufficient to initiate a full oncogenic program leading to malignant adenocarcinomas. From analyzing lung tumor specimens from different ages of mice, we noticed that most tumors maintain the pre-malignant state for a longer period even in the later stages of their growth. As evident in histological examination (Figure 3
), tumors usually have a very low mitotic index as mitotic figures are infrequent. Cellular atypia was also infrequent in the majority of tumors found in mice aged from 6 to 12 months. No evidence of vascular invasion or metastasis was documented in >100 mice bearing lung tumors. Almost all mice that died during the period of our observation succumbed to respiratory failure caused mainly by tumors that expanded and compressed adjacent tissues, not by metastasis. Significant cellular atypia with increased mitotic index was only seen in a relatively small portion of tumors at later stages. Interestingly, these tumors displayed pathological features of large cell undifferentiated carcinomas.
The mechanisms by which RON expression results in lung adenoma in transgenic mice are unknown. As RON has the ability to activate numerous signaling pathways including Ras, PI-3 kinase, MAP kinase and JNK (1215), it is probable that constitutive RON activation facilitates the uncontrolled growth of distal lung epithelial cells towards formation of lung adenomas. As shown in Figure 4A
, lung tumors were not found in newborn mice but only in mice at 23 months of age, suggesting that a 6090 day period is required for tumors to develop. However, the progression from lung adenoma to adenocarcinoma might require a close collaboration between RON and other oncogenes such as Ras. In other words, a second hit with additional genomic or cellular changes is required for the complete progression towards lung adenocarcinoma. As demonstrated in Figure 7
, increased Ras expression and genomic instability were observed in individual lung tumors. These changes could provide opportunities for alterations in some oncogenes or tumor suppressor genes in RON-expressing type II cells. As observed in our studies, lung tumors in young mice (38 months old) are usually adenomas with some atypical cells showing early progression towards adenocarcinomas. In contrast, tumors in relatively old mice (1024 months) show increased malignant behavior, indicating that additional changes are needed for adenomas to develop into adenocarcinomas. It has been reported that K-Ras or p53 gene is frequently mutated in mouse lung tumors produced spontaneously (42) or induced either by chemical carcinogens or by transgenic expression of oncogenes (38,43). Nevertheless, mutations in several hot spots in the K-Ras or p53 gene was not detected, suggesting that the mechanism(s) by which RON-mediated lung tumor formation might be different from those of commonly observed lung tumors (38,42,43). Thus, it will be important in the future to study molecular and cellular mechanisms involved in the initiation and progression of lung tumors in SPCp-RON transgenic mice.
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Notes
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4 To whom correspondence should be addressed Email: ming-hai.wang{at}uchsc.edu 
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Acknowledgments
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We wish to thank Drs J.A.Whitsett (University of Cincinnati, Cincinnati, OH) for 3.7SPC/SV40 vector, W.Gunning (Medical College of Ohio, Toledo, OH) for histological examination of tumors, and J.H.Fisher (University of Colorado School of Medicine, Denver, CO) for critical reading of the manuscript. We are indebted to Dr Y.-S.Ho (Wayne State University, Detroit, MI) for his kind help in generation of transgenic mice. Ms J.Larsens assistance in editing the manuscript was greatly appreciated. This work was supported in part by National Institutes of Health Grant RO1 CA91980 and A143516 to M.H.W.
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Received June 5, 2002;
revised July 26, 2002;
accepted August 6, 2002.