**3. The second generation models**

#### **3.1. K-rasLA and LSL K-ras models**

A different approach to address lung cancer onset was the use of knock-in alleles to activate oncogenes. One example of this is based on the somatic *K-ras* activation *via* an oncogenic *KrasG12D* knock-in allele (*KrasLA2*), which is expressed only after a spontaneous recombination event (Johnson *et al.,* 2001). In this way, sporadic KrasG12D expression occurred on an endoge‐ nous level, which in turn augments efficient development of lung AdCAs. However, these mice also developed other tumor lesions as K-RasG12D expression was not limited to the lung epithelial tissues.

ing and multiplicity of tumor initiation. Through the ability to synchronize tumor initiation in these mice, they could characterize the stages of tumor progression. Of particular signifi‐ cance, this system led to the identification of a new cell type contributing to the develop‐ ment of pulmonary AdCA (Jackson *et al*., 2001). By using this Cre-lox system, the same group later created conditional knock-in mice with mutations in *K-ras* combined with one of mutant *p53* alleles (Jackson *et al.*, 2005). *p53-*loss strongly promoted the progression of *Kras*induced lung AdCAs, yielding a mouse model that precisely recapitulates advanced human lung AdCA. The influence of *p53*-loss on malignant progression was observed as early as 6 weeks after tumor initiation. They also found that the contact mutant p53R270H behaved in a dominant-negative fashion to promote *K-ras*-driven lung AdCAs. Of note, a subset of mice also developed sinonasal adenocarcinomas, suggesting specific expression of *K-ras* in this tissue. In contrast to the lung tumors, expression of the point-mutant *p53* alleles strongly promoted the development of sinonasal AdCAs compared with simple loss-of-function,

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35

suggesting a tissue-specific gain-of-function of mutant p53 (Jackson *et al.*, 2005).

inflammatory cells, chemokines, and tumor progression (Ji *et al*., 2006).

**3.2. Doxycycline (dox)-inducible/de-inducible lung cancer models**

Since activating *K-ras* mutation models recapitulate the human lung tumor phenotypes well, closer analyses of early lung tumor initiating events were performed (Ji *et al*., 2006). A com‐ bination of both *CCSP-Cre* recombinase and *LSL KrasG12D* alleles (Jackson *et al.*, 2005) resulted in a progressive phenotype of cellular atypia, adenoma and finally AdCA. The activation of *K-ras* mutant allele in CC10-positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately AdCA. Surprisingly, *Kras* activation in the bron‐ chiolar epithelium was associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that in‐ creased significantly with age. Thus, *Kras* activation in the lung induces inflammatory che‐ mokines and provides an excellent means to study the complex interactions between

*In KrasLA* mice, oncogene can be induced, but it cannot be de-induced after lung carcinogene‐ sis. To improve this mouse model, a better method of replicating gene expression patterns of target oncogenes had to be taken into account. Furthermore, a general knock-in or knockout procedure only poorly represents genetic events that occur during sporadic lung cancer since genes are already deleted already *in utero* (Jonkers & Berns, 2002). Conditional regula‐ tion of the temporal-spatial expression of oncogenes or inactivation of tumor suppressor genes in somatic tissues of choice can more accurately mimic the *in vivo* situation leading to the onset of sporadic cancer (Jonkers & Berns, 2002; Lewandoski, 2001). This is why the sec‐ ond generation of mouse models for lung cancer makes use of a conditional bitransgenic tetinducible system (Lewandoski, 2001). Most often, the reverse tetracycline (tet)-controlled transactivator (*rt*TA) inducible system is used. The first transgene with the *rt*TA element be‐ hind a tissue-specific promoter causes the *rt*TA expression in a specific cell types, e.g. MMTV-*rt*TA, CCSP-*rt*TA. This transgene is then combined with a second transgene, consist‐

Dmp1 (Dmtf1) is a Myb-like protein with tumor suppressive activity that had been isolated in a yeast two-hybrid screen with cyclin D2 bait (Hirai and Sherr, 1996; Inoue and Sherr, 1998; for review, Inoue *et al.*, 2007; Sugiyama *et al*., 2008a). The promoter is activated by oncogenic Ras-Raf signaling and induces cell-cycle arrest in an Arf, p53-dependent fashion (Inoue *et al.*, 1999; Sreeramaneni *et al.*, 2005). Both *Dmp1+/-* and *Dmp1-/-* mice are prone to spontaneous and carcino‐ gen-induced tumor development, indicating that it is haplo-insufficient for tumor suppres‐ sion, the mechanism of which have not been elucidated yet (Inoue *et al.*, 2000, 2001, 2007). The survival of *K-rasLA* mice was shortened by approximately 15 weeks in both *Dmp1+/-* and *Dmp1-/* backgrounds, the lung tumors of which showed significantly decreased frequency of *p53* mu‐ tations compared to *Dmp1+/+*. Approximately 40% of *K-rasLA* lung tumors from *Dmp1* wild-type mice lost one allele of the *Dmp1* gene, suggesting the primary involvement of *Dmp1* in *K-ras*-in‐ duced tumorigenesis (Mallakin *et al.*, 2007). Tumors from *Dmp1*-deficient mice showed more invasive and aggressive phenotypes than those from *Dmp1* wild-type mice. Loss of hetero‐ zygosity (LOH) of the h*DMP1* locus was detectable in approximately 35% of human lung carci‐ nomas, which was found in mutually exclusive fashion with LOH of *INK4a/ARF* or that of *p53*. Thus, *DMP1* is a novel tumor suppressor for both human and murine NSCLC (Mallakin *et al.*, 2007; Sugiyama *et al.*, 2008b).

Integration of gene expression data from a *KrasLA2* mouse model and *KRAS* mutated human lung tumors showed a significant overlap but also revealed a gene-expression signature for *K-ras* mutation in human lung cancer itself (Sweet-Cordero *et al*., 2005). By using *KrasLA2* knock-in mouse model and human lung cancer specimen, they compared gene expression patterns between these two species (Sweet-Cordero *et al*., 2005). They applied this method to the analysis of a model of *KrasLA2*-mediated lung cancer and found a good relationship to hu‐ man lung AdCA, thereby validating the usefulness of this transgenic model. Furthermore, integrating mouse and human data uncovered a gene-expression signature of *KRAS2* muta‐ tion in human lung cancer. They confirmed the importance of this signature by gene-expres‐ sion analysis of shRNA-mediated inhibition of oncogenic *KrasLA2* (Sweet-Cordero *et al*., 2005). However, one problem of *KrasLA* mice is that they develop tumors other than lung cancer (Mallakin *et al*., 2007). To overcome this issue, Jackson *et al.* (2001) developed a new model of lung AdCA in mice having a conditionally activatable allele of oncogenic *K-ras* (*LSL KrasG12D*)*.* They show that the use of a recombinant adenovirus expressing Cre recombi‐ nase (AdenoCre) to induce *KrasG12D* expression in the lungs of mice allows control of the tim‐ ing and multiplicity of tumor initiation. Through the ability to synchronize tumor initiation in these mice, they could characterize the stages of tumor progression. Of particular signifi‐ cance, this system led to the identification of a new cell type contributing to the develop‐ ment of pulmonary AdCA (Jackson *et al*., 2001). By using this Cre-lox system, the same group later created conditional knock-in mice with mutations in *K-ras* combined with one of mutant *p53* alleles (Jackson *et al.*, 2005). *p53-*loss strongly promoted the progression of *Kras*induced lung AdCAs, yielding a mouse model that precisely recapitulates advanced human lung AdCA. The influence of *p53*-loss on malignant progression was observed as early as 6 weeks after tumor initiation. They also found that the contact mutant p53R270H behaved in a dominant-negative fashion to promote *K-ras*-driven lung AdCAs. Of note, a subset of mice also developed sinonasal adenocarcinomas, suggesting specific expression of *K-ras* in this tissue. In contrast to the lung tumors, expression of the point-mutant *p53* alleles strongly promoted the development of sinonasal AdCAs compared with simple loss-of-function, suggesting a tissue-specific gain-of-function of mutant p53 (Jackson *et al.*, 2005).

**3. The second generation models**

34 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

A different approach to address lung cancer onset was the use of knock-in alleles to activate oncogenes. One example of this is based on the somatic *K-ras* activation *via* an oncogenic *KrasG12D* knock-in allele (*KrasLA2*), which is expressed only after a spontaneous recombination event (Johnson *et al.,* 2001). In this way, sporadic KrasG12D expression occurred on an endoge‐ nous level, which in turn augments efficient development of lung AdCAs. However, these mice also developed other tumor lesions as K-RasG12D expression was not limited to the lung

Dmp1 (Dmtf1) is a Myb-like protein with tumor suppressive activity that had been isolated in a yeast two-hybrid screen with cyclin D2 bait (Hirai and Sherr, 1996; Inoue and Sherr, 1998; for review, Inoue *et al.*, 2007; Sugiyama *et al*., 2008a). The promoter is activated by oncogenic Ras-Raf signaling and induces cell-cycle arrest in an Arf, p53-dependent fashion (Inoue *et al.*, 1999; Sreeramaneni *et al.*, 2005). Both *Dmp1+/-* and *Dmp1-/-* mice are prone to spontaneous and carcino‐ gen-induced tumor development, indicating that it is haplo-insufficient for tumor suppres‐ sion, the mechanism of which have not been elucidated yet (Inoue *et al.*, 2000, 2001, 2007). The survival of *K-rasLA* mice was shortened by approximately 15 weeks in both *Dmp1+/-* and *Dmp1-/* backgrounds, the lung tumors of which showed significantly decreased frequency of *p53* mu‐ tations compared to *Dmp1+/+*. Approximately 40% of *K-rasLA* lung tumors from *Dmp1* wild-type mice lost one allele of the *Dmp1* gene, suggesting the primary involvement of *Dmp1* in *K-ras*-in‐ duced tumorigenesis (Mallakin *et al.*, 2007). Tumors from *Dmp1*-deficient mice showed more invasive and aggressive phenotypes than those from *Dmp1* wild-type mice. Loss of hetero‐ zygosity (LOH) of the h*DMP1* locus was detectable in approximately 35% of human lung carci‐ nomas, which was found in mutually exclusive fashion with LOH of *INK4a/ARF* or that of *p53*. Thus, *DMP1* is a novel tumor suppressor for both human and murine NSCLC (Mallakin *et al.*,

Integration of gene expression data from a *KrasLA2* mouse model and *KRAS* mutated human lung tumors showed a significant overlap but also revealed a gene-expression signature for *K-ras* mutation in human lung cancer itself (Sweet-Cordero *et al*., 2005). By using *KrasLA2* knock-in mouse model and human lung cancer specimen, they compared gene expression patterns between these two species (Sweet-Cordero *et al*., 2005). They applied this method to the analysis of a model of *KrasLA2*-mediated lung cancer and found a good relationship to hu‐ man lung AdCA, thereby validating the usefulness of this transgenic model. Furthermore, integrating mouse and human data uncovered a gene-expression signature of *KRAS2* muta‐ tion in human lung cancer. They confirmed the importance of this signature by gene-expres‐ sion analysis of shRNA-mediated inhibition of oncogenic *KrasLA2* (Sweet-Cordero *et al*., 2005). However, one problem of *KrasLA* mice is that they develop tumors other than lung cancer (Mallakin *et al*., 2007). To overcome this issue, Jackson *et al.* (2001) developed a new model of lung AdCA in mice having a conditionally activatable allele of oncogenic *K-ras* (*LSL KrasG12D*)*.* They show that the use of a recombinant adenovirus expressing Cre recombi‐ nase (AdenoCre) to induce *KrasG12D* expression in the lungs of mice allows control of the tim‐

**3.1. K-rasLA and LSL K-ras models**

epithelial tissues.

2007; Sugiyama *et al.*, 2008b).

Since activating *K-ras* mutation models recapitulate the human lung tumor phenotypes well, closer analyses of early lung tumor initiating events were performed (Ji *et al*., 2006). A com‐ bination of both *CCSP-Cre* recombinase and *LSL KrasG12D* alleles (Jackson *et al.*, 2005) resulted in a progressive phenotype of cellular atypia, adenoma and finally AdCA. The activation of *K-ras* mutant allele in CC10-positive cells resulted in a progressive phenotype characterized by cellular atypia, adenoma and ultimately AdCA. Surprisingly, *Kras* activation in the bron‐ chiolar epithelium was associated with a robust inflammatory response characterized by an abundant infiltration of alveolar macrophages and neutrophils. These mice displayed early mortality in the setting of this pulmonary inflammatory response. Bronchoalveolar lavage fluid from these mutant mice contained the MIP-2, KC, MCP-1 and LIX chemokines that in‐ creased significantly with age. Thus, *Kras* activation in the lung induces inflammatory che‐ mokines and provides an excellent means to study the complex interactions between inflammatory cells, chemokines, and tumor progression (Ji *et al*., 2006).

#### **3.2. Doxycycline (dox)-inducible/de-inducible lung cancer models**

*In KrasLA* mice, oncogene can be induced, but it cannot be de-induced after lung carcinogene‐ sis. To improve this mouse model, a better method of replicating gene expression patterns of target oncogenes had to be taken into account. Furthermore, a general knock-in or knockout procedure only poorly represents genetic events that occur during sporadic lung cancer since genes are already deleted already *in utero* (Jonkers & Berns, 2002). Conditional regula‐ tion of the temporal-spatial expression of oncogenes or inactivation of tumor suppressor genes in somatic tissues of choice can more accurately mimic the *in vivo* situation leading to the onset of sporadic cancer (Jonkers & Berns, 2002; Lewandoski, 2001). This is why the sec‐ ond generation of mouse models for lung cancer makes use of a conditional bitransgenic tetinducible system (Lewandoski, 2001). Most often, the reverse tetracycline (tet)-controlled transactivator (*rt*TA) inducible system is used. The first transgene with the *rt*TA element be‐ hind a tissue-specific promoter causes the *rt*TA expression in a specific cell types, e.g. MMTV-*rt*TA, CCSP-*rt*TA. This transgene is then combined with a second transgene, consist‐ ing of a target gene behind a tet-responsive promoter (*tetO7*) vector, e.g. pTRE-Tight (2nd generation vector from Clontech). The presence of tet/dox ensures stable interaction of the *rt*TA element with the *tetO7* promoter, which, in turn, expresses the target gene upon expo‐ sure to tet or dox.

human lung cancers, by introducing somatic mutations in a limited number of differentiated cells of choice whereby other cells of the fully developed lung remained normal. In short, mutations of targeted regions, flanked by loxP (also known as being "floxed") or **f**lippase **r**ecombination **t**arget (Frt) sequence sites, were introduced through deletion by their respec‐ tive site-specific recombinases Cre or Flp. Thus, in the case of tumor suppressor genes, con‐ ditional hypomorphic mutations (i.e., lower than normal function of the protein) or null allele, several coding or non-coding exons are floxed and can, therefore, be deleted by its corresponding recombinase. Conversely, floxed transcription stops (Lox-Stop-Lox or LSL) in front of oncogene or knock-in alleles can control their respective conditional activation (Jack‐

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37

son *et al.*, 2001) as in the case of *LSL KRasG12D* mice described in the previous section.

it resembles human lung cancer events.

**4. Specific oncogenes in mouse lung cancer models**

**4.1. Kras downstream effectors and lung cancer − Roles of Raf**

Since *Kras* mutations are very common (20-25%) in NSCLC, the understanding of the precise signaling cascade of the Kras pathway is very important (Ji *et al*., 2007). One of the best char‐ acterized Ras pathways is Ras/Raf/MEK/ERK. In fact, *BRAF* gene mutations have been found in a variety of human cancers including NSCLC (Davies *et al*., 2002; Ji *et al*., 2007). On‐ cogenic mutations of *BRAF* render constitutively phosphorylation of the protein, resulting in

The determining factor of this conditional approach is the control of temporal-spatial Cre or FRT recombinase expression. For that purpose, several *Cre* transgenic lines have been gener‐ ated, with or without *tet*-inducible promoters (Perl *et al.*, 2002). Apart from this, Cre-mediat‐ ed recombination can also be achieved through the administration of an engineered Adeno-Cre virus *via* nasal or tracheal inhalation (Meuwissen *et al.*, 2001; Jackson *et al.*, 2001). An advantage of the latter method is that a limited amount of adult lung cells can be targeted in a very concise, localized, and timely fashion. Efficacy of this method was tested with condi‐ tional alleles of *KRasG12D* and *KRasG12V* (Jackson *et al.*, 2001; Guerra *et al.*, 2003). Infection of adult lungs with Adeno-Cre virus rapidly resulted in the onset of adenomatous alveolar hy‐ perplasia, followed by the development of adenomas and AdCAs at 3-4 months post-infec‐ tion. Although a latency of 8 months was also observed (Guerra *et al.*, 2003), no metastases could be found in any of the models. Most probably a single *K-ras* activation is not enough to allow the AdCAs to progress into a higher state of malignancy as would be required for fully metastasizing lesions. However, these straightforward experiments disclosed the im‐ portant role of *K-ras* in human lung cancer onset and progression (Guerra *et al.*, 2003). An‐ other important aspect of this model was that lung tumor multiplicity could be controlled by the dose of *Adeno-Cre* virus infecting only a subset of lung epithelial cells. This, together with a controlled time-point of *Adeno-Cre* application, mimics sporadic character of human lung cancer development. However, one has to be careful to note that variability of the *Ade‐ no-Cre* virus delivery and infection (especially with the intranasal method) might lead to in‐ consistent experimental results. Nevertheless this versatile method remains powerful in that

Therefore, on/off target gene expression is possible depending on administration or with‐ drawal of tet/dox (Gossen *et al.,* 1992). Both *SPC-rtTA* and *CCSP-rtTA* transgenes (Perl *et al.,* 2002) have been used for directing dox-responsive *rt*TA to either alveolar type II or Clara cells. Although both of these promoters have been used to create lung cancer models of mice, CCSP-*rt*TA has more widely been used than SPC-*rt*TA since the *CCSP* promoter is ac‐ tive in both Clara cells and alveolar type II cells while the *SPC* promoter is active only in alveolar type II cells (Floyd *et al.*, 2005). Several transgenic mice such as *CCSP-rtTA;tetO7- FGF-7* and *CCSP-rtTA;tetO7-KrasG12D* have been successfully created to induce lung lesions in response to antibiotics (Tichelaar *et al*., 2000; Fisher *et al*., 2001). Induction of FGF-7 caused initial epithelial cell hyperplasia followed by adenomatous hyperplasia after dox applica‐ tion. All hyperplasia disappeared after withdrawal of dox (Tichelaar *et al*., 2000). However, mouse KrasG12D induction caused epithelial cell hyperplasia, adenomatous hyperplasia and, after 2 months dox application, multiple adenomas and AdCAs. Again, no lesion was de‐ tected after 1 month of dox withdrawal (Fisher *et al*., 2001). When the *CCSP-rtTA;tetO7 KrasG12D* alleles were combined with conventional *p53* or *Ink4a/Arf*-null alleles, AdCAs with a more malignant phenotype appeared after 1 month dox treatment, thus showing a synergy of mutant *K-ras* and *p53* or *Ink4a/Arf* deficiencies. However, even in these compound *tet*-in‐ ducible mouse models, all lesions disappeared after dox withdrawal. This finding demon‐ strated the importance of mutant *K-ras* as a "driving" oncogene not only at tumor onset, but also during maintenance of AdCA in these mice (Fisher *et al*., 2001).

Other models for early, benign lung tumor lesions have been created by using a bitransgenic *tet*-inducible human *KrasG12C* allele that can be expressed in both Clara and/or alveolar type II cells (Tichelaar *et al*., 2000; Floyd *et al*., 2005). Expression of human KrasG12C caused multi‐ ple, small lung tumors over a 12-month time period. Although tumor multiplicity increased upon continued *K-ras* expression, most lung lesions were hyperplasias or well-differentiated adenomas (Floyd *et al*., 2005). This is in good contrast to the more severe phenotypes ob‐ served in other transgenic mouse models in which different mutant *K-ras* alleles were ex‐ pressed in the lung. Expression of K-rasG12C was associated with a 2-fold increase in the activation of the Ras and Ral signaling pathways and increased phosphorylation of Ras downstream effectors, including Erk, p90 ribosomal S6 kinase, ribosomal S6 protein, p38 and MAPKAPK-2. In contrast, expression of K-rasG12C had no effect on the activation of the JNK and Akt signaling pathways explaining low tumor induction by human *KrasG12C*. This observation was in strong contrast to the effects of the previously described mouse *KrasG12D* models (Fisher et al., 2001).

## **3.3. Cre/loxP or Flp/Frt models**

The *Cre/loxP* or *Flp/FRT* system (Jonkers & Berns, 2002; Lewandoski, 2001; Dutt *et al*., 2006) provided excellent tools for reproducing more complicated lung tumor genetics found in human lung cancers, by introducing somatic mutations in a limited number of differentiated cells of choice whereby other cells of the fully developed lung remained normal. In short, mutations of targeted regions, flanked by loxP (also known as being "floxed") or **f**lippase **r**ecombination **t**arget (Frt) sequence sites, were introduced through deletion by their respec‐ tive site-specific recombinases Cre or Flp. Thus, in the case of tumor suppressor genes, con‐ ditional hypomorphic mutations (i.e., lower than normal function of the protein) or null allele, several coding or non-coding exons are floxed and can, therefore, be deleted by its corresponding recombinase. Conversely, floxed transcription stops (Lox-Stop-Lox or LSL) in front of oncogene or knock-in alleles can control their respective conditional activation (Jack‐ son *et al.*, 2001) as in the case of *LSL KRasG12D* mice described in the previous section.

ing of a target gene behind a tet-responsive promoter (*tetO7*) vector, e.g. pTRE-Tight (2nd generation vector from Clontech). The presence of tet/dox ensures stable interaction of the *rt*TA element with the *tetO7* promoter, which, in turn, expresses the target gene upon expo‐

Therefore, on/off target gene expression is possible depending on administration or with‐ drawal of tet/dox (Gossen *et al.,* 1992). Both *SPC-rtTA* and *CCSP-rtTA* transgenes (Perl *et al.,* 2002) have been used for directing dox-responsive *rt*TA to either alveolar type II or Clara cells. Although both of these promoters have been used to create lung cancer models of mice, CCSP-*rt*TA has more widely been used than SPC-*rt*TA since the *CCSP* promoter is ac‐ tive in both Clara cells and alveolar type II cells while the *SPC* promoter is active only in alveolar type II cells (Floyd *et al.*, 2005). Several transgenic mice such as *CCSP-rtTA;tetO7- FGF-7* and *CCSP-rtTA;tetO7-KrasG12D* have been successfully created to induce lung lesions in response to antibiotics (Tichelaar *et al*., 2000; Fisher *et al*., 2001). Induction of FGF-7 caused initial epithelial cell hyperplasia followed by adenomatous hyperplasia after dox applica‐ tion. All hyperplasia disappeared after withdrawal of dox (Tichelaar *et al*., 2000). However, mouse KrasG12D induction caused epithelial cell hyperplasia, adenomatous hyperplasia and, after 2 months dox application, multiple adenomas and AdCAs. Again, no lesion was de‐ tected after 1 month of dox withdrawal (Fisher *et al*., 2001). When the *CCSP-rtTA;tetO7 KrasG12D* alleles were combined with conventional *p53* or *Ink4a/Arf*-null alleles, AdCAs with a more malignant phenotype appeared after 1 month dox treatment, thus showing a synergy of mutant *K-ras* and *p53* or *Ink4a/Arf* deficiencies. However, even in these compound *tet*-in‐ ducible mouse models, all lesions disappeared after dox withdrawal. This finding demon‐ strated the importance of mutant *K-ras* as a "driving" oncogene not only at tumor onset, but

Other models for early, benign lung tumor lesions have been created by using a bitransgenic *tet*-inducible human *KrasG12C* allele that can be expressed in both Clara and/or alveolar type II cells (Tichelaar *et al*., 2000; Floyd *et al*., 2005). Expression of human KrasG12C caused multi‐ ple, small lung tumors over a 12-month time period. Although tumor multiplicity increased upon continued *K-ras* expression, most lung lesions were hyperplasias or well-differentiated adenomas (Floyd *et al*., 2005). This is in good contrast to the more severe phenotypes ob‐ served in other transgenic mouse models in which different mutant *K-ras* alleles were ex‐ pressed in the lung. Expression of K-rasG12C was associated with a 2-fold increase in the activation of the Ras and Ral signaling pathways and increased phosphorylation of Ras downstream effectors, including Erk, p90 ribosomal S6 kinase, ribosomal S6 protein, p38 and MAPKAPK-2. In contrast, expression of K-rasG12C had no effect on the activation of the JNK and Akt signaling pathways explaining low tumor induction by human *KrasG12C*. This observation was in strong contrast to the effects of the previously described mouse *KrasG12D*

The *Cre/loxP* or *Flp/FRT* system (Jonkers & Berns, 2002; Lewandoski, 2001; Dutt *et al*., 2006) provided excellent tools for reproducing more complicated lung tumor genetics found in

also during maintenance of AdCA in these mice (Fisher *et al*., 2001).

sure to tet or dox.

36 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

models (Fisher et al., 2001).

**3.3. Cre/loxP or Flp/Frt models**

The determining factor of this conditional approach is the control of temporal-spatial Cre or FRT recombinase expression. For that purpose, several *Cre* transgenic lines have been gener‐ ated, with or without *tet*-inducible promoters (Perl *et al.*, 2002). Apart from this, Cre-mediat‐ ed recombination can also be achieved through the administration of an engineered Adeno-Cre virus *via* nasal or tracheal inhalation (Meuwissen *et al.*, 2001; Jackson *et al.*, 2001). An advantage of the latter method is that a limited amount of adult lung cells can be targeted in a very concise, localized, and timely fashion. Efficacy of this method was tested with condi‐ tional alleles of *KRasG12D* and *KRasG12V* (Jackson *et al.*, 2001; Guerra *et al.*, 2003). Infection of adult lungs with Adeno-Cre virus rapidly resulted in the onset of adenomatous alveolar hy‐ perplasia, followed by the development of adenomas and AdCAs at 3-4 months post-infec‐ tion. Although a latency of 8 months was also observed (Guerra *et al.*, 2003), no metastases could be found in any of the models. Most probably a single *K-ras* activation is not enough to allow the AdCAs to progress into a higher state of malignancy as would be required for fully metastasizing lesions. However, these straightforward experiments disclosed the im‐ portant role of *K-ras* in human lung cancer onset and progression (Guerra *et al.*, 2003). An‐ other important aspect of this model was that lung tumor multiplicity could be controlled by the dose of *Adeno-Cre* virus infecting only a subset of lung epithelial cells. This, together with a controlled time-point of *Adeno-Cre* application, mimics sporadic character of human lung cancer development. However, one has to be careful to note that variability of the *Ade‐ no-Cre* virus delivery and infection (especially with the intranasal method) might lead to in‐ consistent experimental results. Nevertheless this versatile method remains powerful in that it resembles human lung cancer events.
