**2. The first generation mouse models for lung cancer**

The first generation transgenic models for lung cancer were created by ectopic transgene ex‐ pression under control of lung-specific promoters. Thus transgenic expression was constitu‐ tive. Transgene expression was mainly found in specific subsets of lung epithelial cells. Lung *surfactant protein C* (*SPC*) promoter was used for constitutive gene expression in type II

alveolar cells whereas *Clara Cell Secretory Protein* (*CCSP*) promoter was used to target the non-ciliated secretory (Clara) cells that exist on the airways. In early studies, *SV40 Tag* (Sim‐ ian virus large T-antigen) that neutralizes the activity of both Rb and p53 was constitutively expressed under the control of *CCSP* (DeMayo *et al*., 1991; Sandmoller *et al*., 1994) or *SPC* promoters (Wikenheiser *et al*., 1992). Although each tumor originated from either Clara cells or type II alveolar cells, they both resulted in quite similar aggressive AdCAs without meta‐ stases (Wikenheiser *et al*., 1997). A similar strategy was used to express distinct oncogenes (such as *c-Raf* and *c-Myc* [Geick *et al*., 2001]) in the lung/bronchial epithelium, ending up with a milder phenotype, as both transgenic mice mainly developed adenomas, and a few progressed to AdCAs without any metastases.

The receptor tyrosine kinase RON (recepteur d'origine nantais) is a member of the MET pro‐ to-oncogene family, which is expressed by a variety of epithelial-derived tumors and cancer cell lines and has been implicated in the pathogenesis of lung adenocarcinomas (Chen *et al.*, 2002). To determine the oncogenic potential of RON, transgenic mice were generated using the lung *SPC* promoter to express human wild-type RON in type II cell phenotypes (Chen *et al.*, 2002). The mice were born normal without morphological alterations in the lung, howev‐ er, multiple adenomas 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 which transformed from pre-malignant adenomas to adenocarcinomas. Interestingly, Ras expression was dra‐ matically increased in the majority of tumors without mutation in the 'hot spots' of the *K-Ras* or *p53* genes suggesting that *SPC-RON* is a mouse lung tumor model with unique

Genetically Engineered Mouse Models for Human Lung Cancer

http://dx.doi.org/10.5772/53721

33

Many prominent genetic lesions found in human lung cancer clearly link the inactivation of well-known tumor suppressor genes (Sekido *et al*., 2003) to lung cancer development. Initial attempts to mimic some of these lesions implicated in lung cancer by using conventional knockout mice had limited success with respect to the onset of lung cancer. The main reason for this failure was that germ-line deletion of many essential tumor suppressor genes (such as the *retinoblastoma* gene (*Rb*) (Jacks *et al*., 1992) lead to embryonal lethality. Non-essential tumor suppressor gene (for embryonic survival) knockout mice often had a very broad tu‐ mor spectrum of which lung tumors formed only a minor fraction. Thus, *p53*, *p16Ink4a* and *p19Arf* (Meuwissen & Berns, 2005) null allele mice seldom develop lung AdCAs. However, introducing similar mutations into endogenous *p53* alleles, such as those prominently found in Li–Fraumeni patients, generated *p53R270H/+* and *p53R172H/+* which had a different tumor spec‐ trum compared with *p53+/-* mice (Olive *et al*., 2004), although their mean survival times were identical. Interestingly these mice, but especially *p53R270H/+* and *p53R270H/-* mice, gave rise to more malignant lung AdCAs, and even their metastases, which never occurred in *p53-/* mice. These results suggest that "humanized" *p53* mutations have a greater impact on lung

tumor progression than complete *p53* loss (Olive *et al*., 2004; Lang *et al*., 2004).

Targeting genes deleted early in human lung tumorigenesis, such as the complete cluster at chromosome 3p21.3, showed that heterozygous deletion for this 370 kb region showed no obvious predisposition for lung cancer development albeit homozygous deletion caused embryonal lethality (Smith *et al*., 2002). A more specific deletion of candidate tumor sup‐ pressor genes on chromosome 3 like *RassF1a*, *FHIT* and *VHL*, showed that 31% of *Rassf1a-/* mice produced spontaneous mainly lymphomas but also lung adenomas (Tommasi *et al*., 2005). Treatment of *Rassf1a-/-* mice with BP or urethane resulted in an even higher rate of lung tumors. No spontaneous lung tumors were observed in *Fhit-/-* or *Vhl+/-* mice, but 44% of *Fhit-/-;Vhl+/-* mice developed AdCAs by age 2 years. Again use of mutagens such as dimethyl‐ nitrosamine led to 100% adenoma and AdCA induction in *Fhit-/-;Vhl+/-* mice and even adeno‐ mas in 40% of *Fhit-/-* mice by age 20 months (Zanesi *et al*., 2005). This showed the usefulness of these knockout mice in recapitulating a pattern of early lung cancer development similar

biological characteristics (Chen *et al.*, 2002).

to human pattern.

Ehrhardt *et al.* (2001) created transgenic mouse models to study tumorigenesis of bronchio‐ lo-alveolar AdCAs derived from alveolar type II pneumocytes. Transgenic lines expressing c-*Myc* under the control of the *SPC* promoter developed multifocal bronchiolo-alveolar hy‐ perplasias, adenomas, AdCAs, whereas transgenic lines expressing a secretable form of the epidermal growth factor, TGFα, developed hyperplasias of the alveolar epithelium. Since the oncogenes c-Myc and TGFα are frequently overexpressed in human lung bronchiolo-al‐ veolar carcinomas, these mouse lines will be useful as those for human lung bronchiolo-al‐ veolar carcinomas (Ehrhardt *et al*., 2001).

Sunday *et al.* created a transgenic model for primary pulmonary neuroendocrine cell hyperpla‐ sia/neoplasia using *v-Ha-ras* driven by the *neuroendocrine* (NE)-specific calcitonin promoter (named *rascal*). All rascal transgenic mouse lineages developed hyperplasias of NE and non-NE cells, but mostly non-NE cells developed lung carcinomas (Sunday *et al*., 1999). Analyses of embryonic lung demonstrated *rascal* mRNA in undifferentiated epithelium, consistent with expression in a common pluripotent precursor cell. These observations indicate that *v*-*Ha-ras* can lead to both NE and non-NE hyperplasia/carcinoma *in vivo* (Sunday *et al*., 1999).

A strong correlation exists between *p53* mutations and lung malignancies, and LOH for *p53* has been reported in 40% of NSCLC with specific primers (Mallakin *et* al., 2007). Preceding this study, Morris *et al*. (1998) established a transgenic mouse model with disrupted p53 function in the epithelial cells of the peripheral lung. A dominant-negative mutant form of *p53* was expressed from the human *SPC* promoter. The dominant-negative p53 (dnp53) ex‐ pressed from the *SPC* promoter antagonized wild-type p53 functions in alveolar type II pneumocytes and some bronchiolar cells of the transgenic animals, and thereby promoted the development of carcinoma of the lung. This mouse model should prove useful to the study of lung carcinogenesis and to the identification of agents that contribute to neoplastic conversion in the lung. Another group later created *CCSP-dnp53* transgenic mice and report‐ ed significant increase in the incidence of spontaneous lung cancer in 18-month-old trans‐ genic mice (Tchon-Wong *et al*., 2002). In addition to the increased incidence of spontaneous lung tumor, these transgenic mice were more susceptible to the development of lung adeno‐ carcinoma after exposure to BaP. The risk of lung tumors was 25.3 times greater in BaPtreated mice adjusted for transgene expression. These results suggest that p53 function is important for protecting mice from both spontaneous and BaP-induced lung cancers.

The receptor tyrosine kinase RON (recepteur d'origine nantais) is a member of the MET pro‐ to-oncogene family, which is expressed by a variety of epithelial-derived tumors and cancer cell lines and has been implicated in the pathogenesis of lung adenocarcinomas (Chen *et al.*, 2002). To determine the oncogenic potential of RON, transgenic mice were generated using the lung *SPC* promoter to express human wild-type RON in type II cell phenotypes (Chen *et al.*, 2002). The mice were born normal without morphological alterations in the lung, howev‐ er, multiple adenomas 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 which transformed from pre-malignant adenomas to adenocarcinomas. Interestingly, Ras expression was dra‐ matically increased in the majority of tumors without mutation in the 'hot spots' of the *K-Ras* or *p53* genes suggesting that *SPC-RON* is a mouse lung tumor model with unique biological characteristics (Chen *et al.*, 2002).

alveolar cells whereas *Clara Cell Secretory Protein* (*CCSP*) promoter was used to target the non-ciliated secretory (Clara) cells that exist on the airways. In early studies, *SV40 Tag* (Sim‐ ian virus large T-antigen) that neutralizes the activity of both Rb and p53 was constitutively expressed under the control of *CCSP* (DeMayo *et al*., 1991; Sandmoller *et al*., 1994) or *SPC* promoters (Wikenheiser *et al*., 1992). Although each tumor originated from either Clara cells or type II alveolar cells, they both resulted in quite similar aggressive AdCAs without meta‐ stases (Wikenheiser *et al*., 1997). A similar strategy was used to express distinct oncogenes (such as *c-Raf* and *c-Myc* [Geick *et al*., 2001]) in the lung/bronchial epithelium, ending up with a milder phenotype, as both transgenic mice mainly developed adenomas, and a few

Ehrhardt *et al.* (2001) created transgenic mouse models to study tumorigenesis of bronchio‐ lo-alveolar AdCAs derived from alveolar type II pneumocytes. Transgenic lines expressing c-*Myc* under the control of the *SPC* promoter developed multifocal bronchiolo-alveolar hy‐ perplasias, adenomas, AdCAs, whereas transgenic lines expressing a secretable form of the epidermal growth factor, TGFα, developed hyperplasias of the alveolar epithelium. Since the oncogenes c-Myc and TGFα are frequently overexpressed in human lung bronchiolo-al‐ veolar carcinomas, these mouse lines will be useful as those for human lung bronchiolo-al‐

Sunday *et al.* created a transgenic model for primary pulmonary neuroendocrine cell hyperpla‐ sia/neoplasia using *v-Ha-ras* driven by the *neuroendocrine* (NE)-specific calcitonin promoter (named *rascal*). All rascal transgenic mouse lineages developed hyperplasias of NE and non-NE cells, but mostly non-NE cells developed lung carcinomas (Sunday *et al*., 1999). Analyses of embryonic lung demonstrated *rascal* mRNA in undifferentiated epithelium, consistent with expression in a common pluripotent precursor cell. These observations indicate that *v*-*Ha-ras*

A strong correlation exists between *p53* mutations and lung malignancies, and LOH for *p53* has been reported in 40% of NSCLC with specific primers (Mallakin *et* al., 2007). Preceding this study, Morris *et al*. (1998) established a transgenic mouse model with disrupted p53 function in the epithelial cells of the peripheral lung. A dominant-negative mutant form of *p53* was expressed from the human *SPC* promoter. The dominant-negative p53 (dnp53) ex‐ pressed from the *SPC* promoter antagonized wild-type p53 functions in alveolar type II pneumocytes and some bronchiolar cells of the transgenic animals, and thereby promoted the development of carcinoma of the lung. This mouse model should prove useful to the study of lung carcinogenesis and to the identification of agents that contribute to neoplastic conversion in the lung. Another group later created *CCSP-dnp53* transgenic mice and report‐ ed significant increase in the incidence of spontaneous lung cancer in 18-month-old trans‐ genic mice (Tchon-Wong *et al*., 2002). In addition to the increased incidence of spontaneous lung tumor, these transgenic mice were more susceptible to the development of lung adeno‐ carcinoma after exposure to BaP. The risk of lung tumors was 25.3 times greater in BaPtreated mice adjusted for transgene expression. These results suggest that p53 function is

can lead to both NE and non-NE hyperplasia/carcinoma *in vivo* (Sunday *et al*., 1999).

important for protecting mice from both spontaneous and BaP-induced lung cancers.

progressed to AdCAs without any metastases.

32 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

veolar carcinomas (Ehrhardt *et al*., 2001).

Many prominent genetic lesions found in human lung cancer clearly link the inactivation of well-known tumor suppressor genes (Sekido *et al*., 2003) to lung cancer development. Initial attempts to mimic some of these lesions implicated in lung cancer by using conventional knockout mice had limited success with respect to the onset of lung cancer. The main reason for this failure was that germ-line deletion of many essential tumor suppressor genes (such as the *retinoblastoma* gene (*Rb*) (Jacks *et al*., 1992) lead to embryonal lethality. Non-essential tumor suppressor gene (for embryonic survival) knockout mice often had a very broad tu‐ mor spectrum of which lung tumors formed only a minor fraction. Thus, *p53*, *p16Ink4a* and *p19Arf* (Meuwissen & Berns, 2005) null allele mice seldom develop lung AdCAs. However, introducing similar mutations into endogenous *p53* alleles, such as those prominently found in Li–Fraumeni patients, generated *p53R270H/+* and *p53R172H/+* which had a different tumor spec‐ trum compared with *p53+/-* mice (Olive *et al*., 2004), although their mean survival times were identical. Interestingly these mice, but especially *p53R270H/+* and *p53R270H/-* mice, gave rise to more malignant lung AdCAs, and even their metastases, which never occurred in *p53-/* mice. These results suggest that "humanized" *p53* mutations have a greater impact on lung tumor progression than complete *p53* loss (Olive *et al*., 2004; Lang *et al*., 2004).

Targeting genes deleted early in human lung tumorigenesis, such as the complete cluster at chromosome 3p21.3, showed that heterozygous deletion for this 370 kb region showed no obvious predisposition for lung cancer development albeit homozygous deletion caused embryonal lethality (Smith *et al*., 2002). A more specific deletion of candidate tumor sup‐ pressor genes on chromosome 3 like *RassF1a*, *FHIT* and *VHL*, showed that 31% of *Rassf1a-/* mice produced spontaneous mainly lymphomas but also lung adenomas (Tommasi *et al*., 2005). Treatment of *Rassf1a-/-* mice with BP or urethane resulted in an even higher rate of lung tumors. No spontaneous lung tumors were observed in *Fhit-/-* or *Vhl+/-* mice, but 44% of *Fhit-/-;Vhl+/-* mice developed AdCAs by age 2 years. Again use of mutagens such as dimethyl‐ nitrosamine led to 100% adenoma and AdCA induction in *Fhit-/-;Vhl+/-* mice and even adeno‐ mas in 40% of *Fhit-/-* mice by age 20 months (Zanesi *et al*., 2005). This showed the usefulness of these knockout mice in recapitulating a pattern of early lung cancer development similar to human pattern.
