Lung cancer is a devastating disease with poor prognosis. The design of better therapies for lung cancer patients would be greatly aided by good mouse models that closely resemble the human disease. Unfortunately, current models for lung adenocarcinoma are inadequate due to the absence of metastases. In this study, we incorporated both K-ras and p53 missense mutations into the mouse genome and established a more faithful genetic model for human lung adenocarcinoma, the most common type of lung cancer. Mice with both mutations developed advanced lung adenocarcinomas that were highly aggressive and metastasized to multiple intrathoracic and extrathoracic sites in a pattern similar to that of human lung cancer. These mice also showed a gender difference in cancer-related death. Additionally, the presence of both mutations induced pleural mesotheliomas in 23% of these mice. This mouse model recapitulates the metastatic nature of human lung cancer and will be invaluable to further probe the molecular basis of metastatic lung cancer and for translational studies.
Lung cancer is the leading cause of cancer-related deaths. Although considerable effort has provided insight into the molecular events leading to the progression and metastasis of lung cancer, little improvement in treatment outcome has been achieved.
Many mouse models have been developed for lung cancer to test new treatment options. Xenograft models, in which human tumor cells are grafted into immune compromised mice, have been extensively used for preclinical testing. However, these models have intrinsic flaws, generally resulting in poor predicative power of the clinical efficacy of anticancer agents (Becher and Holland, 2006; Sausville and Burger, 2006). Susceptible mouse strains, including A/J and SWR, spontaneously develop lung tumors, with the sensitivity strongly associated with a polymorphism in intron 2 of the K-ras gene (You et al., 1992; Malkinson and You, 1994). These strains are also highly sensitive to the induction of lung tumors by chemical carcinogens. For example, a single dose of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice induces alveolar hyperplasias, lung adenomas, and adenocarcinomas 42 weeks after carcinogen treatment (Belinsky et al., 1993). Increased tumor induction by vinyl carbamate was demonstrated in transgenic mice carrying a mutant p53 with an Ala-to-Val mutation at codon 135 and a deletion of a K-ras allele (Wang et al., 2006a, 2006b). These and many other studies illustrate that the A/J model is a useful one to evaluate chemointerventive agents of lung tumors (Castonguay et al., 1991; Hecht et al., 1991). Unfortunately, lung tumors in A/J mice treated with carcinogens are generally not aggressive and do not metastasize.
To overcome these deficiencies, investigators have attempted to establish models that mimic the genetic signature of lung cancer. Two of the most common molecular changes identified in human lung cancer are mutations of the p53 and K-ras genes (Mitsudomi et al., 1992; Salgia and Skarin, 1998). Missense mutations in the p53 gene are found in more than 50% of human lung cancers, while p53 deletions are rare (Takahashi et al., 1989; Chiba et al., 1990; Greenblatt et al., 1994). The hot spot p53 mutations in lung cancer occur at amino acids 157, 158, 175, 245, 248, 249 and 273 (Toyooka et al., 2003; Vahakangas et al., 2001). Constitutive activation of the K-ras gene through mutations occurs in 30% of lung cancers, about 80% of which occur at codon 12 (Rodenhuis et al., 1988; Rodenhuis and Slebos, 1992).
Current genetic models of human lung adenocarcinoma are also limited by the absence or rare occurrence of metastasis (Meuwissen and Berns, 2005). In one model, mice inherit a latent mutant K-ras allele at the endogenous locus (K-rasLA1), which is spontaneously activated in vivo (Johnson et al., 2001). The activated K-rasLA1 allele expresses mutant K-ras with an aspartic acid at codon 12, inducing multifocal lung adenocarcinomas in heterozygous mice (K-rasLA1/+). The advantage of this model is that somatic activation of the K-ras proto-oncogene in mouse lung mimics mutational activation of the K-ras gene occurring in human lung cancer. The disadvantage, however, is that lung adenocarcinomas in this model, and in one with an accompanying deletion of p53, rarely metastasize.
Likewise, mouse models with p53 mutations have recently been described. One particular mutation generated at the endogenous p53 allele contains an arginine-to-histidine substitution at codon 172, which corresponds to the hot spot mutation at 175 in human p53 (Liu et al., 2000; Lang et al., 2004; Olive et al., 2004). The first mouse model with this mutation expresses low levels of mutant p53 because of an intronic deletion of a single nucleotide (p53R172HΔg) (Liu et al., 2000). Strikingly, however, p53R172HΔg/+ mice develop osteosarcomas and carcinomas that metastasize at a frequency of 69 and 40%, respectively, a phenotype not observed in p53+/− mice. This metastatic phenotype was confirmed in the latest models expressing mutant p53 at appropriate levels (Lang et al., 2004; Olive et al., 2004). These models demonstrate that mutant p53 has a gain of function in addition to loss of function in vivo, and reproduce the metastatic nature of human cancers. Unfortunately, however, mice expressing mutant p53 rarely develop lung adenocarcinomas.
Since mice inheriting a p53 missense mutation develop a wide range of tumors that metastasize, we postulated that K-ras mutation could initiate lung adenocarcinoma, while p53 mutation could exacerbate the phenotype by promoting dissemination of cancer cells. In an effort to develop a more faithful mouse model, one with similar molecular changes and metastatic behavior to human lung cancer, we generated mice with both p53 and K-ras missense mutations. We found that the combination of these mutations resulted in lung adenocarcinomas with a high incidence of metastases and gender differences in cancer-related death. This new mouse model thus most closely simulates human metastatic lung cancer and provides an immunocompetent system to test novel therapeutic agents in vivo.
Lung cancer in p53R172HΔg/+ K-rasLA1/+ mice
The p53R172HΔg/+ mice carrying a missense mutation in one p53 allele (Liu et al., 2000) were crossed with K-rasLA1/+ mice carrying a latent mutant K-ras allele (Johnson et al., 2001) to generate K-rasLA1/+, p53R172HΔg/+ and p53R172HΔg/+ K-rasLA1/+ mice. All genotypes produced from the above cross were included in the cohort study and were in a 129Sv background. The p53R172HΔg/+ K-rasLA1/+ mice are sometimes referred to as double mutant mice for simplicity. For comparison, p53+/− K-rasLA1/+ mice were also generated and included in this study.
At necropsy, p53R172HΔg/+ K-rasLA1/+ mice had substantial lung tumor burden (Figure 1Aa). Histologically, the lesions were atypical adenomatous hyperplasia (AAH), adenomas and adenocarcinomas. AAH, the precursor lesion of human lung adenocarcinoma (Kitamura et al., 1999), was identified in multifocal and diffuse patterns contiguous with well-defined or ill-defined adenomas and adenocarcinomas (Figure 1Ba). Lung adenomas and adenocarcinomas were frequently juxtaposed and merging (Figure 1Bb). The majority of double mutant mice had multiple large or diffuse adenocarcinomas, which had papillary/glandular phenotypes or were poorly differentiated (Figure 1Aa and Bc, d). Lung adenocarcinoma cells usually had enlarged vesicular nuclei with prominent nucleoli, similar to human lung adenocarcinoma cells. Surfactant protein C (SPC), which is frequently expressed in human lung adenocarcinoma, was detected in murine lung adenocarcinomas by immunohistochemistry (Figure 1Be). Papillary hyperplasia of bronchial epithelial cells was also observed in double mutant mice (Figure 1Bf). These hyperplastic cells are positive for CC10, a Clara cell marker, suggesting an alternative origin of lung adenocarcinoma in this model (Figure 1Bg).
Strikingly, lung adenocarcinomas in this model were highly invasive and metastatic (Figure 1Ab–e and Bh, i). Metastatic lesions in the lymph node and liver stained positive for SPC (Figure 1Bh and i, respectively).
Pleural mesothelioma, which originates from mesothelial cells of the pleura, was also observed in p53R172HΔg/+ K-rasLA1/+ mice. Grossly, multiple lesions appeared on the pleura and multiple enlarged lymph nodes were observed in the mediastinum (Figure 2A). Microscopically, mesothelial cells were observed proliferating in a papillary pattern into pleural space (Figure 2Ba). Early-stage pleural mesotheliomas were localized and clearly identified on top of hyperplastic lung tissue and adenoma (Figure 2Bb and c). Late-stage pleural mesotheliomas invaded lung parenchyma (Figure 2Bd). The majority of pleural mesotheliomas in this model were biphasic, composed of both epithelioid and sarcomatoid cancer cells with pronounced pleomorphism and marked nuclear atypia. Sarcomatoid lesions were associated with spindle cell fibrous components, resembling human fibrous mesothelioma (Figure 2Be). Highly aggressive mesotheliomas extended into surrounding tissues and metastasized to mediastinal lymph nodes, liver, pancreas, ovary, and other sites (Figure 2Bf and data not shown). Pleural mesotheliomas in this model were positive for at least one marker, Calretinin or Cytokeratin 6 (Figure 2Bg, h and Table 1), and negative for SPC (Figure 1Be) and for periodic acid-Schiff (PAS) staining (data not shown). These data further verified the histological diagnosis of mesothelioma. There are no reports of pleural mesothelioma in previously published K-rasLA1/+ mice probably due to the low frequency of this tumor type (Johnson et al., 2001). We also did not observe mesotheliomas in p53R172HΔg/+ or p53R172H/+ mice (Liu et al., 2000; Lang et al., 2004).
Tumor spectrum in p53R172HΔg/+ K-rasLA1/+ mice
All p53R172HΔg/+ K-rasLA1/+ mice developed lung tumors. The major type of malignancy was lung adenocarcinoma, which developed in 52 of 56 double mutant mice (Table 2). Pleural mesothelioma was identified in 13 of these 56 mice. Six out of these 13 mice also had peritoneal mesotheliomas. Grossly, anatomical extension of thoracic lesions into the abdominal cavity through the diaphragm was noted in three of the six mice with peritoneal mesotheliomas. In addition to lung tumors, lymphoma infiltration in lung appeared in 6/56 (10.7%) of double mutant mice. Mice with both lung adenocarcinomas and lung lymphomas did not develop any metastases, probably because they died at a younger age than mice with only lung adenocarcinomas (median survival of 256 days compared to 317 days for those without lymphomas; P=0.0053).
For comparison, 48 K-rasLA1/+ mice were also analysed. They developed a tumor spectrum similar to that of double mutant mice (Table 2). Pleural mesotheliomas were identified in 4/48 K-rasLA1/+ mice, but no mesotheliomas were identified in the peritoneum. The incidence of pleural mesotheliomas in K-rasLA1/+ mice was lower than that in double mutant mice (χ2, P<0.05). As previously reported, p53R172HΔg/+ mice develop lymphomas, sarcomas, and carcinomas, a few of which are lung adenocarcinomas (Liu et al., 2000) (Table 2).
High metastatic potential of lung adenocarcinoma in p53R172HΔg/+ K-rasLA1/+ mice
Lung adenocarcinomas in p53R172HΔg/+ K-rasLA1/+ mice were much more aggressive and metastatic than those in K-rasLA1/+ mice. At 3–4 months of age, double mutant mice developed AAH, lung adenomas, and small, focal adenocarcinomas with no metastasis (data not shown). Metastases were identified at 7–14 months of age in dissected double mutant mice. Histologically, metastases were found in 19 of 52 p53R172HΔg/+ K-rasLA1/+ mice that had developed lung adenocarcinomas (36.5%), while metastases were found in the two of 44 K-rasLA1/+ mice that had developed lung adenocarcinomas (4.5%), which was significantly different (χ2, P<0.001). Lung adenocarcinomas were widely disseminated to multiple intrathoracic and extrathoracic sites in double mutant mice (Table 3). Seeding was restricted to the mediastinum in the two K-rasLA1/+ mice with metastases (Table 3). The average number of sites of metastases was 2.3 per p53R172HΔg/+ K-rasLA1/+ mouse. Mediastinal structures were the major metastatic sites (mediastinal lymph nodes/adipose tissue and heart). Extrathoracic sites of metastases included liver, adrenal glands, body wall, kidneys, and mesentery/lymph nodes. Previously published data show that p53+/− K-rasLA1/+ mice develop lung adenocarcinomas with more malignant features, but no metastasis (Johnson et al., 2001). We also analyzed five p53+/− K-rasLA1/+ mice and found that they developed lung tumors but no metastasis, as reported previously (data not shown). Thus, the metastatic burden was the most remarkable difference among p53R172HΔg/+ K-rasLA1/+, p53+/− K-rasLA1/+ and K-rasLA1/+ mice. Notably, not all enlarged nodules seen in the mediastinum at necropsy were metastases. These were reactive lymph nodes, an observation consistent with that in human lung cancer (Kerret al., 1992).
The status of the wild-type p53 allele was examined in lung adenocarcinomas and metastases of p53R172HΔg/+ K-rasLA1/+ mice by quantitative real-time PCR using specific TaqMan probes to differentiate wild-type and mutant alleles. Loss of heterozygosity (LOH) was detected in 81.8% (9/11) of end-stage lung adenocarcinomas and 71.4% (10/14) of macroscopic metastases (Table 4).
Since the K-rasLA1 allele is a latent allele that is spontaneously activated in somatic cells, the expression of mutant K-ras was analysed in lung cancers and metastases at mRNA and protein levels (Figure 3). Mutant K-rasG12D having a glycine-to-aspartic acid substitution at codon 12 was expressed in lung adenocarcinomas isolated from both double mutant and K-rasLA1/+ mice, and in metastases and mesotheliomas isolated from double mutant mice. Moreover, histologically normal lung tissue adjacent to lung adenocarcinoma isolated from double mutant mice and normal lung tissue isolated from wild-type mice did not show the expression of mutant K-ras by either protein or mRNA analysis. Thus, both p53 (inherited) and K-ras (acquired) mutations were present in lung cancers and metastases.
Survival of p53R172HΔg/+K-rasLA1/+ mice
Mice of the various genotypes were monitored daily and killed if they showed signs of respiratory distress, lethargy, decreased body weight, or abdominal distension. Double mutant mice had significantly reduced survival when compared to other littermates (P<0.0001, Figure 4a). Double mutant mice developed much more aggressive lung cancers and higher tumor burden than K-rasLA1/+ mice, leading to shorter lifespans. The median survival for double mutant mice was 266 days compared to 373 days for K-rasLA1/+ mice. The p53+/− K-rasLA1/+ mice showed a very similar survival curve to published data, and indicated no difference in survival as compared to p53R172HΔg/+ K-rasLA1/+ mice (Figure 4a). However, a significant difference in cumulative cancer-related death was observed between male and female double mutant mice (P=0.0071, Figure 4b). This gender difference in cancer-related death was not observed for K-rasLA1/+ and p53R172HΔg/+ littermates (Supplementary Figure 1A-B). The cumulative deaths for female and male wild-type littermates were also similar to each other (Supplementary Figure 1C).
Current mouse models for lung cancer develop lung adenocarcinomas, but do not metastasize (Nikitin et al., 2004; Meuwissen and Berns, 2005). The deletion of a p53 allele enhances lung tumorigenesis induced by mutant K-ras, but again does not promote the development of metastasis (Johnson et al., 2001; Jackson et al., 2005). In this study, we generated mice with both p53 and K-ras missense mutations to test the suitability of such a model for metastatic lung adenocarcinoma.
In this model, primary lung cancer disseminated widely through both hematogenous and lymphatic pathways to multiple sites strikingly similar to those in human lung cancer (Tamura et al., 1992; Quint et al., 1996; Sadikot et al., 1997). Surprisingly, no metastasis of lung cancer was found in bone and brain. Additional genetic changes may be needed to produce tumors that can metastasize to these organs.
Interestingly, a significant discrepancy in cumulative cancer death was observed between female and male p53R172HΔg/+ K-rasLA1/+ mice, but not in littermates with either single mutation. This gender difference in survival is supported by a study showing a higher number of pulmonary lesions induced by a tobacco-specific carcinogen NNK in A/J females as opposed to males (Belinsky et al., 2003). In humans, the observation that female smokers are more susceptible to lung cancer than male smokers is supported by many case–control studies, but opposed by several prospective cohort studies (Zang and Wynder, 1996; Payne, 2001; Bach et al., 2003; Bain et al., 2004; Olak and Colson, 2004). Therefore, a gender difference in susceptibility to human lung cancer is still controversial. Nevertheless, the survival of female and male double mutant mice showed a statistically significant difference and will be an important model to probe this difference.
Pleural mesothelioma is an uncommon, but highly aggressive human malignancy that is invariably fatal because it is detected so late (Jaurand, 2005). In this study, 23% of p53R172HΔg/+ K-rasLA1/+ mice also developed pleural mesotheliomas. Four lines of evidence supported the diagnosis of this tumor in this model. The first is the restricted gross and microscopic localization to the pleural mesothelial lining and the distinct phenotypic differences from intra-pulmonary epithelial lesions. The second is positive staining of Calretinin and/or Cytokeratin 6, the two commonly used markers for human mesothelioma (Ordonez, 2003). Specifically, Calretinin was detected in 80% of murine mesotheliomas examined, corresponding to the frequency of 80% in human mesotheliomas (Abutaily et al., 2002). An antibody against Cytokeratin 5 and 6 is positive in 63% of human mesotheliomas (Abutaily et al., 2002). The cytokeratin 6 antibody detected staining in 40% of murine mesotheliomas (a murine-specific Cytokeratin 5 antibody is not available). Of note, these markers stain normal mesothelial cells and do not allow us to distinguish benign from malignant cells. None of lung adenocarcinomas examined in this study was positive for either marker. The third is negative PAS staining, which if positive, rules out mesothelioma. The fourth is that SPC staining was negative in mesothelioma, but positive in the adjacent lung adenocarcinoma. Taken together, histological features, immunohistochemical data, and PAS staining all support the diagnosis of mesothelioma in this study.
Thus, this new lung cancer model more faithfully simulates human metastatic lung cancer and promises to be invaluable in testing novel preventive or therapeutic modalities prior to clinical studies.
Materials and methods
Tumor samples and pathological analysis
The K-rasLA1/+ mice were crossed with p53R172HΔg/+ to generate K-rasLA1/+, p53R172HΔg/+, p53R172HΔg/+ K-rasLA1/+ and wild-type mice, and with p53+/− to generate p53+/− K-rasLA1/+ mice. The background of these mice was greater than 90% 129Sv. Genotypes were determined as previously described (Liu et al., 2000; Johnson et al., 2001). Mice were housed in sterilized plastic cages with hardwood bedding and dust covers in a HEPA-filtered, specific pathogen-free room (24±1°C, 45% humidity, 14/10 h light/dark cycle). The number of mice per cage was three to five. All mice were given sterilized NIH-31 mouse/rat diet (No. 7017, Harlan), and water ad libitum. Mice were monitored daily for signs of illness or obvious tumor burden and moribund mice were killed. Tumors and tissues were fixed with 10% buffered formalin, paraffin embedded, and sectioned at 4 μm. Sections (a single section for each tissue per mouse) were stained with hematoxylin and eosin (H&E) for histological evaluation. The diagnosis of a metastatic lesion was based on histological features and the gross finding that the lesion was not in direct contact with a lung tumor. A metastatic diagnosis of lung adenocarcinoma on parietal pleura was possible only if parietal and visceral pleurae were not adhering to each other.
Revese transcription–PCR and quantitative real-time TaqMan PCR
Total RNA was extracted from tumors or normal lung by using Trizol (Invitrogen, Carlsbad, CA, USA) and purified by an RNeasy kit (Qiagen, Gaithersburg, MD, USA). Reverse transcriptase reactions were performed using the First-Strand cDNA Synthesis Kit (Amersham Bioscience, Piscataway, NJ, USA). Primers for wild-type and mutant K-ras alleles were K-ras12G: IndexTermTTGTGGTGGTTGGAGGTGG, K-ras12D: IndexTermCTTGTGGTGGTTGGAGGTGA, and K-rasEx3R1: IndexTermCTGTCTTGTCTTTGCTGAGGTC. Specific amplification was confirmed by sequencing PCR products.
Specific probes were used to differentiate mutant and wild-type p53 alleles in tumors by real-time PCR. Primers and probes were p53 forward 5′-IndexTermTCTACAAGAAGTCACAGCACATGAC-3′, p53 reverse 5′-IndexTermCCTTCCACCCGGATAAGATGC-3′, wild-type probe 5′-TET-IndexTermAGGTCGTGAGACACTGCC-3′, mutant probe 5′-FAM-IndexTermTCGTGAGACGCTGCCC-3′. The GSC (goosecoid homeobox protein gene) forward 5′-IndexTermCGGCACCGCACCATCT, GSC reverse 5′-IndexTermTCGTCTCCTGGAAGAGGTTCC, and GSC probe 5′-VIC-IndexTermCCGATGAGCAGCTCG-3′ were used for normalization. PCR reactions were performed in 384-well reaction plate using ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Each sample was measured in duplicate. The relative level of p53 gene was determined by ΔΔCt based on the formula ΔΔCt=(sample Ct [p53]−sample Ct [GSC])−(control Ct [p53]−control Ct [GSC]). All tumor samples used the same reference sample for calculation, non-tumor DNA with wild-type p53. Analysis of diluted samples showed 2ΔΔCt values of ⩽0.15, 0.15–0.6 or >0.6 indicating 0, 1, or 2 copies of wild-type p53 alleles, respectively (Hill et al., 2005).
Immunohistochemistry and PAS staining
Immunohistochemical analysis was performed on tissue sections using the Vectastain ABC kit (Vector, Burlingame, CA, USA). Antibodies used were Calretinin (Zymed, South San Francisco, CA, USA, 18-0291, 1:3000 dilution), SPC (Chemicon, Temecula, CA, USA, AB3786, 1:1000 dilution) and a murine-specific Cytokeratin 6 antibody (gift from Dennis R Roop). Sections were counterstained with nuclear fast red or hematoxylin (Vector). Immunoflourescence analysis was performed using a CC10 antibody (Santa Cruz SC-9772, Santa Cruz, CA, USA, 1:50 dilution).
Sections were also stained with PAS staining system (Sigma, St Louis, MO, USA). Normal mouse liver sections were used as positive controls. For a negative control, liver samples were treated with diastase (Sigma) to remove immunoreactivity.
Immunoprecipitation-western blot analysis
Tumor samples were homogenized in 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.5% NP40, and proteinase inhibitors (Roche, Mannheim, Germany). Agarose-conjugated anti-ras antibody Ab-1 (Calbiochem, San Diego, CA, USA, OP01A) was added according to the manufacturer’s recommendation. Blots were probed with an anti-pan-Ras12D antibody specific to mutant Ras with an aspartic acid at codon 12 (Oncogene, PC10). The same membrane was stripped and probed with an anti-K-Ras antibody (Santa Cruz, SC30, 1:150). The secondary antibody was horseradish peroxidase-conjugated and signals were detected with a chemiluminescence kit (Amersham Pharmacia, Piscataway, NJ, USA).
Survival curves were plotted by the Kaplan–Meier method. The statistical significance between different survival data was determined by the logrank test (GraphPad Prism 4). Tabular data between groups were compared by the χ2 test.
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This study was supported by a grant from the Department of Defense, DAMD17-01-1-0689 and the Cancer Center Support Grant CA16672 from the NIH. We thank Tyler Jacks for the K-rasLA1/+ mice.
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Zheng, S., El-Naggar, A., Kim, E. et al. A genetic mouse model for metastatic lung cancer with gender differences in survival. Oncogene 26, 6896–6904 (2007). https://doi.org/10.1038/sj.onc.1210493
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