**6. Clinical implications and future directions for mouse lung cancer models**

Xenograft models where manipulated human lung cancer cell lines are subcutaneously in‐ jected into nude mice have been extensively used for pre-clinical testing of novel drugs for lung cancer. The major issue for this approach is that lung cancer cell lines have already been adapted for long-term culture in a plastic dish with artificial medium and acquired stem-cell like phenotypes, and thus are not suitable for models of primary human lung can‐ cer obtained by surgical resection. The more preferred method, however, have been ortho‐ topical transplantation of human lung tumor cells in their lung cavity. To date, the results have shown that xenograft models do not accurately predict the clinical efficacy of anti-tu‐ mor drugs. Therefore, a question arises as to whether spontaneous and/or genetically-engi‐ neered mouse models for lung cancer would be more useful as tools for pre-clinical drug tests. It is obvious that there are differences in the lung anatomy and physiology between mice and humans, but some of the mouse models that we have described have a striking histological similarity, with an analogous genetic signature to that of human NSCLC. Impor‐ tantly, genetically-engineered mouse model-derived tumors develop in an innate immune environment and, therefore, have all the tumor-stromal interactions, such as angiogenesis and degradation of the tissue matrix.

(Contag *et al*., 2000; Hadjantonakis *et al*., 2003). In case of latter studies, transgenic expression of luciferase allows accurate longitudinal monitoring and good quantification of tumor bur‐ den as has been shown in the *LSL Kras* lung tumor model (Jackson *et al.*, 2001). These novel imaging techniques will greatly enhance the accuracy and reproducibility of mouse models.

Genetically Engineered Mouse Models for Human Lung Cancer

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

49

Transgenic lung cancer models created by Chen *et al*. (2002) can be applied to clinics by rais‐ ing Ron-specific antibodies. O'Toole *et al*. (2006) conducted an antibody phage display li‐ brary to generate a human IgG1 antibody IMC-41A10 that binds with high affinity to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein. They found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migra‐ tion, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, MAPK, and AKT in several cancer cell lines. In NCI-H292 lung cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent. This antibody should

Recent strategies showed the importance of aberrant promoter methylation in lung cancer development, such a *p16INK4a*, *Death-associated protein kinase 1,* and, *RAS association domain family 1A* (Shames *et al.*, 2006). Since chronic inflammations have been implicated in cancer pathogenesis (Shacter & Weitzman, 2002), altered methylation for lung surfactant proteins are good topics for future lung cancer studies; their signatures may serve as valuable mark‐ ers in lung cancer detection. The lung surfactant protein (*SP*) genes, *SP-A* and *SP-D* have been identified with high throughput approach that showed an altered methylation pattern in lung cancer compared to normal lung tissue (Vaid & Floros, 2009). However, *SP-A*-defi‐ cient mice were able to survive with no apparent pathology in a sterile environment (Korf‐ hagen et al., 1996), although their pulmonary immune responses were insufficient during immune challenge. *SP-D*-deficient mice, on the other hand, showed phenotypic abnormali‐ ties in alveolar macrophages and type II pneumocytes with increased lipid pools, indicating that *SP-D* has an important role in surfactant homeostasis (Botas et al., 1998). Paradoxically overexpression of *SP-A* and/or *SP-D* as a result of promoter hypomethylation has also been reported in lung cancer suggesting that it is critical to keep these protein levels within phys‐ iological ranges to prevent neoplastic transformation. Since the role of these lung surfactant proteins in lung carcinogenesis has never been studied *in vivo*, it will be worthwhile to cross lung surfactant-deficient mice with available transgenic/knockout strains to elucidate the

be tested *in vivo* using the *SPC-RON* mice with developing lung AdCAs.

roles of surfactant proteins in lung cancer initiation and development.

pre-doctoral fellowship BC100907. We thank K. Klein for editorial assistance.

K. Inoue has been supported by NIH/NCI 5R01CA106314, ACS RSG-07-207-01-MGO, and by WFUCCC Director's Challenge Award #20595. D. Maglic has been supported by DOD

**Acknowledgements**

We have described two models for NSCLC in which either the continuous oncogenic activi‐ ty of Kras (Fisher *et al*, 2001) or EGFR (Politi *et al*, 2006) are prerequisites of tumor mainte‐ nance since lung tumors underwent spontaneous regression with disappearance of the oncogene by dox withdrawal. This not only shows that tumor growth critically depends on the initiating active oncogenic pathways, but it also stresses the usefulness of these oncogen‐ ic pathways as therapeutic targets. Direct tumor intervention studies with tyrosine kinase inhibitors against EGFR mutations proved to be highly effective in several *hEGFR*-transgen‐ ic mouse models. TKIs such as gefitinib, erlotinib, and HKI-272 led to complete tumor re‐ gression (Politi *et al*., 2006; Ji *et al*., 2006a,b). In addition, treatment of lung cancer with humanized anti-hEGFR antibody (cetuximab) caused a significant tumor regression (Ji *et al*., 2006a). Further studies will be needed to investigate the signaling cascades that determine the sensitivity and resistance to EGFR-related tyrosine kinase interventions.

Other mouse models for NSCLC have also been used for targeted therapies. First, dox-in‐ duced overexpression of the PI3K p110α catalytic subunit PIK3CA, mutated in its kinase do‐ main (H1047R) in *CCSP-rtTA;tetO7-PIK3CA(H1047R)* mice, induces adenocarcinomas (Engelman *et al.*, 2008). Treatment of these lung tumors with NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor, caused a marked lung tumor regres‐ sion. Interestingly, when this single agent NVP6-BEZ235 was tested on lung tumors in *CCSP-rtTA;tetO7-KrasG12D* mice, no regression was observed. However, when NVP-BEZ235 was combined with MEK inhibitor ARRY-142886, significant regression of *KrasG12D* tumors occurred (Engelman *et al*., 2008). Thus, two major RAS downstream effector pathways need‐ ed to be inactivated to get an irreversible regression in Ras mutated NSCLC.

Although *K-RAS* is mutated in ~30% of human NSCLC, direct targeting of RAS has been un‐ successful for lung cancer therapy. Many small molecules against Ras functions have been tested and farnesyl transferase inhibitors are the most marked examples of these failed at‐ tempts (Mahgoub *et al*., 1999; Omer *et al*., 2000). Recent results with lung cancer mouse mod‐ els strongly suggest that KRAS4A, and not KRAS4B is driving the onset of NSCLC. An explanation for this failure can thus be attributed to the fact that only KRAS4B is farnesylat‐ ed, but not its isoform KRAS4A. Although we still have to study if KRAS4A is important in the pathogenesis of human NSCLC, we can imagine the importance of *Kras* mouse models in testing functional inhibitiors for KRAS4A (To *et al*., 2008).

The use of optimized, genetically-modified mouse models for lung cancer for therapy re‐ search necessitates sophisticated non-invasive tools to follow tumor development and re‐ sponse to therapy *in vivo*. Measurement of tumor size as a function of time is the most obvious way of doing this and existing techniques such as computed-tomography imaging or magnetic resonance imaging for small animals are now in use (Engelman *et al*., 2008; Po‐ liti *et al*., 2006). However, these techniques are time-consuming and expensive, making them less suitable for large number of animals. Other techniques, such as fluorescence imaging and bioluminescence, can be used for measuring gene expression or tumor growth *in vivo* (Contag *et al*., 2000; Hadjantonakis *et al*., 2003). In case of latter studies, transgenic expression of luciferase allows accurate longitudinal monitoring and good quantification of tumor bur‐ den as has been shown in the *LSL Kras* lung tumor model (Jackson *et al.*, 2001). These novel imaging techniques will greatly enhance the accuracy and reproducibility of mouse models.

Transgenic lung cancer models created by Chen *et al*. (2002) can be applied to clinics by rais‐ ing Ron-specific antibodies. O'Toole *et al*. (2006) conducted an antibody phage display li‐ brary to generate a human IgG1 antibody IMC-41A10 that binds with high affinity to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein. They found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migra‐ tion, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, MAPK, and AKT in several cancer cell lines. In NCI-H292 lung cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent. This antibody should be tested *in vivo* using the *SPC-RON* mice with developing lung AdCAs.

Recent strategies showed the importance of aberrant promoter methylation in lung cancer development, such a *p16INK4a*, *Death-associated protein kinase 1,* and, *RAS association domain family 1A* (Shames *et al.*, 2006). Since chronic inflammations have been implicated in cancer pathogenesis (Shacter & Weitzman, 2002), altered methylation for lung surfactant proteins are good topics for future lung cancer studies; their signatures may serve as valuable mark‐ ers in lung cancer detection. The lung surfactant protein (*SP*) genes, *SP-A* and *SP-D* have been identified with high throughput approach that showed an altered methylation pattern in lung cancer compared to normal lung tissue (Vaid & Floros, 2009). However, *SP-A*-defi‐ cient mice were able to survive with no apparent pathology in a sterile environment (Korf‐ hagen et al., 1996), although their pulmonary immune responses were insufficient during immune challenge. *SP-D*-deficient mice, on the other hand, showed phenotypic abnormali‐ ties in alveolar macrophages and type II pneumocytes with increased lipid pools, indicating that *SP-D* has an important role in surfactant homeostasis (Botas et al., 1998). Paradoxically overexpression of *SP-A* and/or *SP-D* as a result of promoter hypomethylation has also been reported in lung cancer suggesting that it is critical to keep these protein levels within phys‐ iological ranges to prevent neoplastic transformation. Since the role of these lung surfactant proteins in lung carcinogenesis has never been studied *in vivo*, it will be worthwhile to cross lung surfactant-deficient mice with available transgenic/knockout strains to elucidate the roles of surfactant proteins in lung cancer initiation and development.
