**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 continued ERK activation. Of all the *BRAF* mutations, *BRAF-V600E* is the most frequent. (Mercer *et al*., 2003). Dankort *et al.* (2007) created BRaf(CA) (CA: constitutively active) mice to express normal BRaf prior to Cre-mediated recombination after which *BRaf(V600E)* was expressed at physiological levels. *BRaf(CA)* mice infected with an Adenovirus expressing Cre recombinase developed benign lung tumors that only rarely progressed to AdCA. The reason for this is the initial proliferation is halted by increased expression of senescence markers p53 and Ink4a/Arf. Consistent with the tumor suppressor function for Ink4a/Arf and p53, BRaf(V600E) expression combined with mutation of either locus led to lung cancer progression. Moreover, *BRaf(VE)*-induced lung tumors were prevented by pharmacological inhibition of MEK1/2.

**4.2. PI3K and lung cancer**

also maintenance.

**4.3. Rac and lung cancer**

*ras* (Kissil *et al*., 2007).

Another important pro-survival pathway that is interlinked with RAS is PI3K/Akt signaling pathway. Phosphoinositide-3-kinase (PI3K) consists of a regulatory (p85) and a catalytic (p110) subunit. The overexpression of both subunits was reported in lung carcinomas (Sa‐ muels & Velculescu 2004; Wojtalla *et al*., 2011). Furthermore, selective *PIK3CA* amplification was found in lung squamous cell carcinomas (Angulo *et al*., 2008). To investigate the onco‐ genic potential of PIK3CA, transgenic mice were generated with a *tet*-inducible expression of an activated p110α mutant, H1047R, and it was crossed with CCSP-*rt*TA mice to generate *CCSP-rtTA;tetO7;PIK3CA(H1047R)* compound mice. Upon dox treatment of animals for 14 weeks, double transgenic mice developed AdCAs, which subsequently disappeared after dox withdrawal for 3 weeks (Engelman *et al*., 2008). To identify the effect of loss of PI3K sig‐ naling in *Kras*-induced lung tumorigenesis, PI3K activity was completely eliminated in *p85* knockouts (*Pik3r2-/-*;*Pik3r1-/-*), and a dramatic decrease in the number of lung tumors was ob‐ served in *LSL KrasG12D*;*Pik3r2-/-*;*Pik3r1-/-* mice (Engelman *et al*., 2008). The clinical efficacy of NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor was also evaluated against p110α H1047R-induced mouse lung tumors. Application of this drug led to marked tumor regression. In contrast, NVP-BEZ235 barely had effect on mouse lung cancers driven by mutant *Kras*. However, a combination of NVP-BEZ235 and a MEK inhibi‐ tor ARRY-142886, had marked synergistic effect on tumor regression. These *in vivo* studies suggest that inhibitors of the PI3K-mTOR pathway when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers. Of note, Ras proteins directly interact with the p110α subunit of PI3K and introduction of specific mutations (T208D and K227A) in *PIK3CA* blocks this interaction (Gupta *et al*., 2007). To study the Ras-p110α interactions *in vivo* and its effects on tumorigenesis, these point mutations were introduced into the *Pik3ca* gene in the mice and these mice were crossed with *KrasLA2* alleles (Gupta *et al*., 2007). Inter‐ estingly, they were highly resistant to *Kras* induced lung tumor development, which suggest Ras-p110α interaction is required for Ras-driven tumorigenesis (Gupta *et al*., 2007). All these results emphasize the importance of PI3K signaling, not only in lung tumor induction, but

Genetically Engineered Mouse Models for Human Lung Cancer

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

39

Rac is a member of the Rho family of small GTPases, and it mediates the regulation of vari‐ ous important cellular processes including cell migration, proliferation and adhesion, all of which may contribute to tumorigenesis (Mack *et al*., 2011). The important role of Rac in Ras induced lung tumorigenesis was demonstrated in a mice model in which an oncogenic allele of *Kras* was activated by Cre-mediated recombination in the presence or absence of condi‐ tional deletion of *Rac1*. They showed that Rac1 function was required for tumorigenesis in lung carcinogenesis for mice with *Rac1* deletion had tumor regression and longer survival. These data showed a specific requirement for Rac1 function in cells expressing oncogenic *K-*

In another study, Ji *et al* generated a lung-specific, *tet*-inducible, mice model in which the *CCSP-rtTA*;*tetO7-BRAFV600E* induced a development of lung AdCA with bronchioalveolar carcinoma type. The extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway was highly activated by the expression of *BRAF(V600E)* mutant. Upon dox withdrawal, the dein‐ duction of *BRAF*-mutant expression led to regression of lung tumors together with a marked decrease in phosphorylation of ERK1/2. Furthermore, the *in vivo* use of a specific MAPK/ERK kinase (MEK) inhibitor also induced lung tumor regression. All these results showed that both activated BRAF and KRAS signaling converge onto the same MAPK path‐ way, making this pathway a potential target for lung tumor intervention.

The significance of c-Raf was also investigated in *K-RasG12V*-driven NSCLCs. Ablation of c-Raf in *K-Ras+/G12V; c-Raf lox/lox* mice induced dramatic increase of survival rate and life span due to the decrease of tumor burden. This result suggests the essential role of c-Raf in mediating oncogenic Ras signaling in NSCLCs (Blasco *et al*, 2011).

Further investigation during *KrasG12D*-driven lung tumorigenesis showed the MAPK antago‐ nist Sprouty-2 (Spry-2) was upregulated. When *Spry-2* was knocked out in Cre/lox depend‐ ent *Spry-2flox/flox;LSL KrasG12D* mice, both tumor number and total tumor area were significantly increased. This clearly suggested a tumor suppressor activity for *Sprouty-2* dur‐ ing *Kras*-dependent lung tumorigenesis by involving in antagonism of Ras/MAPK signaling (Shaw *et al*., 2007).

By using *CCSP-rtTA;TetO-Cre;LSL-Kras(G12D)*mice Cho *et al.* (2011) established a dox-indu‐ cible, Kras(G12D)-driven lung AdCA to pursue the cellular origin and molecular processes involved in *Kras*-induced tumorigenesis. The EpCAM(+)MHCII(-) cells (bronchiolar origin) were more enriched with tumorigenic cells in generating secondary tumors than Ep‐ CAM(+)MHCII(+) cells (alveolar origin). In addition, secondary tumors derived from Ep‐ CAM(+)MHCII(-) cells showed diversity of tumor locations compared with those derived from EpCAM(+)MHCII(+) cells. Secondary tumors from EpCAM(+)MHCII(-) cells expressed differentiation marker, pro-SPC, consistent with the notion that cancer-initiating cells dis‐ play not only the abilities for self-renewal, but also the features of differentiation to generate tumors of heterogeneous phenotypes. High level of ERK1/2 activation and colony-forming ability as well as lack of Sprouty-2 expression were also observed in EpCAM(+)MHCII(-) cells. Their data suggested that bronchiolar Clara cells are the origin of tumorigenic cells for Kras(G12D)-induced lung cancer.

#### **4.2. PI3K and lung cancer**

continued ERK activation. Of all the *BRAF* mutations, *BRAF-V600E* is the most frequent. (Mercer *et al*., 2003). Dankort *et al.* (2007) created BRaf(CA) (CA: constitutively active) mice to express normal BRaf prior to Cre-mediated recombination after which *BRaf(V600E)* was expressed at physiological levels. *BRaf(CA)* mice infected with an Adenovirus expressing Cre recombinase developed benign lung tumors that only rarely progressed to AdCA. The reason for this is the initial proliferation is halted by increased expression of senescence markers p53 and Ink4a/Arf. Consistent with the tumor suppressor function for Ink4a/Arf and p53, BRaf(V600E) expression combined with mutation of either locus led to lung cancer progression. Moreover, *BRaf(VE)*-induced lung tumors were prevented by pharmacological

In another study, Ji *et al* generated a lung-specific, *tet*-inducible, mice model in which the *CCSP-rtTA*;*tetO7-BRAFV600E* induced a development of lung AdCA with bronchioalveolar carcinoma type. The extracellular signal-regulated kinase (ERK)-1/2 (MAPK) pathway was highly activated by the expression of *BRAF(V600E)* mutant. Upon dox withdrawal, the dein‐ duction of *BRAF*-mutant expression led to regression of lung tumors together with a marked decrease in phosphorylation of ERK1/2. Furthermore, the *in vivo* use of a specific MAPK/ERK kinase (MEK) inhibitor also induced lung tumor regression. All these results showed that both activated BRAF and KRAS signaling converge onto the same MAPK path‐

The significance of c-Raf was also investigated in *K-RasG12V*-driven NSCLCs. Ablation of c-Raf in *K-Ras+/G12V; c-Raf lox/lox* mice induced dramatic increase of survival rate and life span due to the decrease of tumor burden. This result suggests the essential role of c-Raf in mediating

Further investigation during *KrasG12D*-driven lung tumorigenesis showed the MAPK antago‐ nist Sprouty-2 (Spry-2) was upregulated. When *Spry-2* was knocked out in Cre/lox depend‐ ent *Spry-2flox/flox;LSL KrasG12D* mice, both tumor number and total tumor area were significantly increased. This clearly suggested a tumor suppressor activity for *Sprouty-2* dur‐ ing *Kras*-dependent lung tumorigenesis by involving in antagonism of Ras/MAPK signaling

By using *CCSP-rtTA;TetO-Cre;LSL-Kras(G12D)*mice Cho *et al.* (2011) established a dox-indu‐ cible, Kras(G12D)-driven lung AdCA to pursue the cellular origin and molecular processes involved in *Kras*-induced tumorigenesis. The EpCAM(+)MHCII(-) cells (bronchiolar origin) were more enriched with tumorigenic cells in generating secondary tumors than Ep‐ CAM(+)MHCII(+) cells (alveolar origin). In addition, secondary tumors derived from Ep‐ CAM(+)MHCII(-) cells showed diversity of tumor locations compared with those derived from EpCAM(+)MHCII(+) cells. Secondary tumors from EpCAM(+)MHCII(-) cells expressed differentiation marker, pro-SPC, consistent with the notion that cancer-initiating cells dis‐ play not only the abilities for self-renewal, but also the features of differentiation to generate tumors of heterogeneous phenotypes. High level of ERK1/2 activation and colony-forming ability as well as lack of Sprouty-2 expression were also observed in EpCAM(+)MHCII(-) cells. Their data suggested that bronchiolar Clara cells are the origin of tumorigenic cells for

way, making this pathway a potential target for lung tumor intervention.

oncogenic Ras signaling in NSCLCs (Blasco *et al*, 2011).

38 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

inhibition of MEK1/2.

(Shaw *et al*., 2007).

Kras(G12D)-induced lung cancer.

Another important pro-survival pathway that is interlinked with RAS is PI3K/Akt signaling pathway. Phosphoinositide-3-kinase (PI3K) consists of a regulatory (p85) and a catalytic (p110) subunit. The overexpression of both subunits was reported in lung carcinomas (Sa‐ muels & Velculescu 2004; Wojtalla *et al*., 2011). Furthermore, selective *PIK3CA* amplification was found in lung squamous cell carcinomas (Angulo *et al*., 2008). To investigate the onco‐ genic potential of PIK3CA, transgenic mice were generated with a *tet*-inducible expression of an activated p110α mutant, H1047R, and it was crossed with CCSP-*rt*TA mice to generate *CCSP-rtTA;tetO7;PIK3CA(H1047R)* compound mice. Upon dox treatment of animals for 14 weeks, double transgenic mice developed AdCAs, which subsequently disappeared after dox withdrawal for 3 weeks (Engelman *et al*., 2008). To identify the effect of loss of PI3K sig‐ naling in *Kras*-induced lung tumorigenesis, PI3K activity was completely eliminated in *p85* knockouts (*Pik3r2-/-*;*Pik3r1-/-*), and a dramatic decrease in the number of lung tumors was ob‐ served in *LSL KrasG12D*;*Pik3r2-/-*;*Pik3r1-/-* mice (Engelman *et al*., 2008). The clinical efficacy of NVP-BEZ235, a dual pan-PI3K and mammalian target of rapamycin (mTOR) inhibitor was also evaluated against p110α H1047R-induced mouse lung tumors. Application of this drug led to marked tumor regression. In contrast, NVP-BEZ235 barely had effect on mouse lung cancers driven by mutant *Kras*. However, a combination of NVP-BEZ235 and a MEK inhibi‐ tor ARRY-142886, had marked synergistic effect on tumor regression. These *in vivo* studies suggest that inhibitors of the PI3K-mTOR pathway when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers. Of note, Ras proteins directly interact with the p110α subunit of PI3K and introduction of specific mutations (T208D and K227A) in *PIK3CA* blocks this interaction (Gupta *et al*., 2007). To study the Ras-p110α interactions *in vivo* and its effects on tumorigenesis, these point mutations were introduced into the *Pik3ca* gene in the mice and these mice were crossed with *KrasLA2* alleles (Gupta *et al*., 2007). Inter‐ estingly, they were highly resistant to *Kras* induced lung tumor development, which suggest Ras-p110α interaction is required for Ras-driven tumorigenesis (Gupta *et al*., 2007). All these results emphasize the importance of PI3K signaling, not only in lung tumor induction, but also maintenance.

#### **4.3. Rac and lung cancer**

Rac is a member of the Rho family of small GTPases, and it mediates the regulation of vari‐ ous important cellular processes including cell migration, proliferation and adhesion, all of which may contribute to tumorigenesis (Mack *et al*., 2011). The important role of Rac in Ras induced lung tumorigenesis was demonstrated in a mice model in which an oncogenic allele of *Kras* was activated by Cre-mediated recombination in the presence or absence of condi‐ tional deletion of *Rac1*. They showed that Rac1 function was required for tumorigenesis in lung carcinogenesis for mice with *Rac1* deletion had tumor regression and longer survival. These data showed a specific requirement for Rac1 function in cells expressing oncogenic *Kras* (Kissil *et al*., 2007).

#### **4.4. Receptor-type protein tyrosine kinase and lung cancer − Roles of EGFR**

**4.5. HER2 and lung cancer**

(Swanton *et al*., 2006).

The c-*ERBB2* gene is located on chromosome 17q11.2-12 and encodes **H**uman **E**pidermal Growth Factor **R**eceptor **2** (HER2) (Hu *et al*., 2011). This is a transmembrane glycoprotein re‐ ceptor p185HER2, which has been targeted by the humanized monoclonal antibody trastuzu‐ mab (Herceptin). *HER2* is amplified and overexpressed in approximately 25% of breast cancer patients and is associated with an aggressive clinical course and poor prognosis. HER2 pro‐ tein overexpression without gene amplification happens in some cases, possibly due to pro‐ moter activation and/or protein stabilization. HER2 overexpression stimulates cell growth in *p53*-mutated cells while it inhibits cell proliferation in those with wild-type *p53*. The molecular mechanisms for these differential responses have recently been clarified: the *Dmp1* promoter was activated by HER2/neu through the PI3K-Akt-NF-κB pathway, which in turn stimulated *Arf* transcription and p53 activation to prevent tumorigenesis. Conversely HER2 simply stim‐

Genetically Engineered Mouse Models for Human Lung Cancer

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

41

HER2 receptor overexpression has been reported in 11% to 32% of NSCLC tumors, with gene amplification found in 2%-23% of cases (Hirsch *et al*., 2009; Swanton *et al*., 2006). Highlevel ERBB2 amplification occurs in a small fraction of lung cancers with a strong propensity to high-grade adenocarcinomas (Grob *et al.*, 2012). The frequency of *HER2* amplification in NSCLC and the widespread availability of HER2 fluorescence *in situ* hybridization analysis may justify a study of trastuzumab monotherapy in NSCLC cases. However, sensitivity to HER2-directed therapies is complex and involves expression not only of HER2, but also of other EGFR family members (HER1, HER2, and HER4), their ligands, and molecules that in‐ fluence pathway activity (Swanton *et al*., 2006). The role played by HER2 as a heterodimeri‐ zation partner for other EGFR family members makes HER2 an attractive target regardless of receptor overexpression in lung cancer. However, targeted therapies in patients overex‐ pressing HER2 have proven less successful in clinical trials for NSCLC. One reason to ex‐ plain the failure is intratumoral heterogeneity of *ERBB2* amplification, which was found in 4 of 10 cases (Grob *et al*., 2012). Of note, this heterogeneity is rare in breast cancer that re‐ sponds relatively well to anti-HER2 therapy. Laboratory data indicate that forced expression of HER2 in a NSCLC line increases sensitivity to gefitinib. They speculated that this may re‐ sult from the gefitinib-mediated inhibition of HER2/HER3 heterodimerization and HER3 phosphorylation. It might thus be expected that combinatorial approaches, such as EGFR in‐ hibition (by gefitinib) together with HER2 dimerization blockade (by pertuzumab) may be even more effective. Preclinical data indicate this may be the case, with the combination of erlotinib and pertuzumab promoting more than additive antitumor activity in the NSCLC

While HER2 is overexpressed in about 20% of lung cancers, mutations in HER2 also occur in about 2-3% of cases. HER2 mutations typically occur in adenocarcinomas and are more fre‐ quent in women and never-smokers (Pinder, 2011). Mutations in HER2 lead to constitutive activation of the HER2 receptor, similar to the situation with EGFR. In good contrast to what we experienced in breast cancer, early clinical trials of Herceptin combined with chemother‐ apy in lung cancer patients with HER2 overexpression did not show a benefit for patients. However, there are case reports of lung cancer with HER2 mutations who have responded

ulate cell proliferation in cells that lack *Dmp1*, *Arf*, or *p53* (Taneja *et. al.*, 2010).

#### *4.4.1. EGFR and lung cancer*

Epidermal growth factor (EGF) receptor family is one type of RTKs, on which the tyrosine residues phosphorylation lead to activation of downstream TK signaling that contributes to cell proliferation, motility and invasion (Stella *et al*., 2012). The activation mutations on *EGFR* gene are found in about 10-20% of advanced NSCLC cases and its protein overexpres‐ sion is found in more than 60% of all lung cancers (Lynch *et al*., 2004; Soria, *et al.*, 2012). Lynch *et al.* reported that EGFR mutation correlated with clinical responsiveness to the tyro‐ sine kinase inhibitor gefitinib (2004). Since these mutations lead to increased growth factor signaling with susceptibility to the inhibitor, screening for such mutations in lung cancers will identify patients who will have a response to gefitinib. To study a specific oncogenic potential of *EGFR* mutant, the variant III (vIII) deletion, Ji *et al*. (2006a) produced *Tet-op-EGFRvIII*; *CCSP-rtTA* mice, in which the EGFRvIII expression was induced in lung type II pneumocytes upon dox administration. Mice developed atypical adenomatous hyperplasia after 6-8 weeks of dox induction and progressed to lung adenocarcinomas after 16 weeks with high activation of AKT and ERK signaling pathways. De-induction of EGFRvIII result‐ ed in significant tumor regression, supporting the requirement of continuous EGFRvIII ex‐ pression in lung tumorigenesis. Furthermore, by using an EGFR/ERB2 inhibitor HKI-272, they found tumor volume in *EGFRvIII* ; *CCSP-rtTA; Ink4a/Arf*-/- mice was dramatically de‐ creased, suggesting a therapeutic strategy for lung cancers with *EGFRvIII* mutation by an ir‐ reversible EGFR inhibitor (Ji *et al*., 2006a). Politi *et al.* (2006) also studied the role of EGFR mutations in the initiation and maintenance of lung cancer, and developed transgenic mice that express an exon 19 deletion mutant (EGFR(ΔL747-S752)) or the L858R mutant (EGFR(L858R)) in type II pneumocytes under the control of dox, and reported that expres‐ sion of either EGFR mutant lead to the development of lung AdCa. Ji *et al*. (2006b) later cre‐ ated bitransgenic mice with inducible expression in type II pneumocytes of two common hEGFR mutants (hEGFRDEL and hEGFRL858R) seen in human lung cancer. Both bitransgenic lines developed lung AdCa with hEGFR mutant expression, confirming their oncogenic po‐ tential. Maintenance of transformed phenotypes of these lung cancers was dependent on sustained expression of the EGFR mutants. Treatment with small molecule inhibitors (erloti‐ nib or HKI-272) as well as a humanized anti-hEGFR antibody (cetuximab) led to dramatic tumor regression (Ji *et al*., 2006b). Thus persistent EGFR signaling is required for tumor maintenance in human lung AdCas expressing EGFR mutants. Li *et al.* (2007) generated an‐ other dox-inducible lung cancer mice model harboring both erlotinib sensitizing and resis‐ tence mutations L858R and T790M (*EGFR TL*). They found that specific expression of *EGFR TL* in lung compartments led to the development of typical bronchioloalveolar carcinoma af‐ ter 4-5 weeks and peripheral adenocarcinoma after 7-9 weeks. Treatment of *EGFR TL*-driven tumors is most effective when using combined regimen of HKI-272 and rapamycin, suggest‐ ing that this combination therapy may benefit pateints harboring erlotinib resistence EGFR mutation (Li *et al*., 2007).

## **4.5. HER2 and lung cancer**

**4.4. Receptor-type protein tyrosine kinase and lung cancer − Roles of EGFR**

40 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

Epidermal growth factor (EGF) receptor family is one type of RTKs, on which the tyrosine residues phosphorylation lead to activation of downstream TK signaling that contributes to cell proliferation, motility and invasion (Stella *et al*., 2012). The activation mutations on *EGFR* gene are found in about 10-20% of advanced NSCLC cases and its protein overexpres‐ sion is found in more than 60% of all lung cancers (Lynch *et al*., 2004; Soria, *et al.*, 2012). Lynch *et al.* reported that EGFR mutation correlated with clinical responsiveness to the tyro‐ sine kinase inhibitor gefitinib (2004). Since these mutations lead to increased growth factor signaling with susceptibility to the inhibitor, screening for such mutations in lung cancers will identify patients who will have a response to gefitinib. To study a specific oncogenic potential of *EGFR* mutant, the variant III (vIII) deletion, Ji *et al*. (2006a) produced *Tet-op-EGFRvIII*; *CCSP-rtTA* mice, in which the EGFRvIII expression was induced in lung type II pneumocytes upon dox administration. Mice developed atypical adenomatous hyperplasia after 6-8 weeks of dox induction and progressed to lung adenocarcinomas after 16 weeks with high activation of AKT and ERK signaling pathways. De-induction of EGFRvIII result‐ ed in significant tumor regression, supporting the requirement of continuous EGFRvIII ex‐ pression in lung tumorigenesis. Furthermore, by using an EGFR/ERB2 inhibitor HKI-272, they found tumor volume in *EGFRvIII* ; *CCSP-rtTA; Ink4a/Arf*-/- mice was dramatically de‐ creased, suggesting a therapeutic strategy for lung cancers with *EGFRvIII* mutation by an ir‐ reversible EGFR inhibitor (Ji *et al*., 2006a). Politi *et al.* (2006) also studied the role of EGFR mutations in the initiation and maintenance of lung cancer, and developed transgenic mice that express an exon 19 deletion mutant (EGFR(ΔL747-S752)) or the L858R mutant (EGFR(L858R)) in type II pneumocytes under the control of dox, and reported that expres‐ sion of either EGFR mutant lead to the development of lung AdCa. Ji *et al*. (2006b) later cre‐ ated bitransgenic mice with inducible expression in type II pneumocytes of two common hEGFR mutants (hEGFRDEL and hEGFRL858R) seen in human lung cancer. Both bitransgenic lines developed lung AdCa with hEGFR mutant expression, confirming their oncogenic po‐ tential. Maintenance of transformed phenotypes of these lung cancers was dependent on sustained expression of the EGFR mutants. Treatment with small molecule inhibitors (erloti‐ nib or HKI-272) as well as a humanized anti-hEGFR antibody (cetuximab) led to dramatic tumor regression (Ji *et al*., 2006b). Thus persistent EGFR signaling is required for tumor maintenance in human lung AdCas expressing EGFR mutants. Li *et al.* (2007) generated an‐ other dox-inducible lung cancer mice model harboring both erlotinib sensitizing and resis‐ tence mutations L858R and T790M (*EGFR TL*). They found that specific expression of *EGFR TL* in lung compartments led to the development of typical bronchioloalveolar carcinoma af‐ ter 4-5 weeks and peripheral adenocarcinoma after 7-9 weeks. Treatment of *EGFR TL*-driven tumors is most effective when using combined regimen of HKI-272 and rapamycin, suggest‐ ing that this combination therapy may benefit pateints harboring erlotinib resistence EGFR

*4.4.1. EGFR and lung cancer*

mutation (Li *et al*., 2007).

The c-*ERBB2* gene is located on chromosome 17q11.2-12 and encodes **H**uman **E**pidermal Growth Factor **R**eceptor **2** (HER2) (Hu *et al*., 2011). This is a transmembrane glycoprotein re‐ ceptor p185HER2, which has been targeted by the humanized monoclonal antibody trastuzu‐ mab (Herceptin). *HER2* is amplified and overexpressed in approximately 25% of breast cancer patients and is associated with an aggressive clinical course and poor prognosis. HER2 pro‐ tein overexpression without gene amplification happens in some cases, possibly due to pro‐ moter activation and/or protein stabilization. HER2 overexpression stimulates cell growth in *p53*-mutated cells while it inhibits cell proliferation in those with wild-type *p53*. The molecular mechanisms for these differential responses have recently been clarified: the *Dmp1* promoter was activated by HER2/neu through the PI3K-Akt-NF-κB pathway, which in turn stimulated *Arf* transcription and p53 activation to prevent tumorigenesis. Conversely HER2 simply stim‐ ulate cell proliferation in cells that lack *Dmp1*, *Arf*, or *p53* (Taneja *et. al.*, 2010).

HER2 receptor overexpression has been reported in 11% to 32% of NSCLC tumors, with gene amplification found in 2%-23% of cases (Hirsch *et al*., 2009; Swanton *et al*., 2006). Highlevel ERBB2 amplification occurs in a small fraction of lung cancers with a strong propensity to high-grade adenocarcinomas (Grob *et al.*, 2012). The frequency of *HER2* amplification in NSCLC and the widespread availability of HER2 fluorescence *in situ* hybridization analysis may justify a study of trastuzumab monotherapy in NSCLC cases. However, sensitivity to HER2-directed therapies is complex and involves expression not only of HER2, but also of other EGFR family members (HER1, HER2, and HER4), their ligands, and molecules that in‐ fluence pathway activity (Swanton *et al*., 2006). The role played by HER2 as a heterodimeri‐ zation partner for other EGFR family members makes HER2 an attractive target regardless of receptor overexpression in lung cancer. However, targeted therapies in patients overex‐ pressing HER2 have proven less successful in clinical trials for NSCLC. One reason to ex‐ plain the failure is intratumoral heterogeneity of *ERBB2* amplification, which was found in 4 of 10 cases (Grob *et al*., 2012). Of note, this heterogeneity is rare in breast cancer that re‐ sponds relatively well to anti-HER2 therapy. Laboratory data indicate that forced expression of HER2 in a NSCLC line increases sensitivity to gefitinib. They speculated that this may re‐ sult from the gefitinib-mediated inhibition of HER2/HER3 heterodimerization and HER3 phosphorylation. It might thus be expected that combinatorial approaches, such as EGFR in‐ hibition (by gefitinib) together with HER2 dimerization blockade (by pertuzumab) may be even more effective. Preclinical data indicate this may be the case, with the combination of erlotinib and pertuzumab promoting more than additive antitumor activity in the NSCLC (Swanton *et al*., 2006).

While HER2 is overexpressed in about 20% of lung cancers, mutations in HER2 also occur in about 2-3% of cases. HER2 mutations typically occur in adenocarcinomas and are more fre‐ quent in women and never-smokers (Pinder, 2011). Mutations in HER2 lead to constitutive activation of the HER2 receptor, similar to the situation with EGFR. In good contrast to what we experienced in breast cancer, early clinical trials of Herceptin combined with chemother‐ apy in lung cancer patients with HER2 overexpression did not show a benefit for patients. However, there are case reports of lung cancer with HER2 mutations who have responded well to treatment with Herceptin plus chemotherapy. For instance, BIBW2992 (a small mole‐ cule inhibitor of EGFR and HER2) has shown evidence of activity in lung cancer patients with HER2 mutations. Most of the patients described had cancers that had shown resistance to chemotherapy and/or EGFR inhibitors. More patients with SCLC should be screened for HER2 mutations since the number of patients described to date is too small to draw any de‐ finitive conclusions (Pinder, 2011).

fore using this regimen in clinical lung cancer chemoprevention, its activity should first be

Genetically Engineered Mouse Models for Human Lung Cancer

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

43

Since expression of phosphatase and tensin homologue deleted from chromosome 10 (PTEN; reviewed in Inoue *et al.*, 2012) is often down regulated in NSCLC, several mice mod‐ els have been generated in which *Pten* was inactivated in the bronchial epithelium (Yanagi *et al*., 2007; Iwanaga *et al*., 2008). *PTEN* is a tumor suppressor gene that acts by blocking the PI3K dependent activation of serine-threonine kinase Akt (Inoue *et al.*, 2012). Since *Pten-/* mice are embryonic lethal, one had to make use of floxed *Pten* alleles (*Ptenflox/flox*), combined with *CCSP-Cre* transgene, targeting *Pten* deletion into bronchial epithelial cells. However, these *Ptenflox/flox;CCSP-Cre* mice did not show any aberrant pulmonary development or phe‐ notypic abnormalities even when mice were followed for more than 12 months (Iwanaga *et al*., 2008). This changed dramatically when the *Ptenflox/flox;CCSP-Cre* alleles were crossed with *LSLKrasG12D*. Lung tumor development was markedly accelerated compared in *Pten-/-*;*KrasG12D* mice to that of single *LSLKrasG12D* mice. *Pten*-deficient, *Kras* mutant tumors were often of the more advanced AdCA with higher vascularity (Iwanaga *et al*., 2008), suggesting that *Pten*loss cooperates with *Kras* mutations in NSCLC. Contrary to these results were the findings of another study in which *Pten*-inactivation was targeted in bronchioalveolar epithelium with *SPC-rtTA;tetO7-Cre* (Yanagi *et al*., 2007). When dox was applied *in utero* at E10-16 dur‐ ing embryogenesis, most mice died post-natally from hypoxia. Their lungs showed an im‐ paired alveolar epithelial cell differentiation with an overall lung epithelial cell hyperplasia. The few surviving mice developed spontaneous lung AdCAs. Post-natal dox application during P21-27 resulted in a mild bronchiolar and alveolar cell hyperplasia and increased cell size but no lethality. A majority of these animals developed AdCAs in comparison to WT controls. Prior addition of urethane induced an even higher amount of AdCAs. Interesting‐ ly, most *Pten-/-* AdCAs (33%), with or without urethane addition, showed spontaneous *Kras* mutations. The latter observation again indicates the importance of Kras activity in cooper‐

Mutations in liver kinase B1 (*LKB1*) are found in Peutz–Jeghers syndrome (PJS) patients and are characterized by intestinal polyps (hamartoma) and increased incidence of epithelial tu‐ mors, such as hamartomatous polyps in the gastrointestinal tract, as well as breast, colorec‐ tal, and thyroid cancers (Giardiello *et al.*, 2000). It is a serine threonine kinase also known as *STK11* (Sanchez-Cespedes *et al*., 2002). LKB1 is a primary upstream kinase of adenine mono‐ phosphate-activated protein kinase (AMPK), a necessary element in cell metabolism that is required for maintaining energy homeostasis. It is now clear that LKB1 exerts its growth suppressing effects by activating a group of other ~14 kinases, creating a group of AMPK and AMPK-related kinases. Activation of AMPK by LKB1 suppresses cell growth and prolif‐ eration when energy and nutrient levels are low. The *LKB1* gene has been implicated in the regulation of multiple biological processes, signaling pathways (Wei *et al*., 2005), and tu‐

tested in clinically predictive cyclin D1 mouse lung cancer models.

ating with *Pten*-loss during NSCLC development.

**4.8. LKB1 and lung cancer − A novel player**

**4.7. PTEN and lung cancer**
