**4.6. Cyclin D1 and lung cancer**

The development of human lung carcinogenesis is very complex. Several oncogenes in‐ volved in this process have been identified, one of which is cyclin D1 (Meuwissen & Berns, 2005). Cyclin D1 is a crucial regulator in mammalian cell cycle, which drives cells to enter S phase by binding and activating CDK4/6. The cyclin D1/CDK4 complex phosphorylates the retinoblastoma protein (pRb), which releases E2F transcriptional factors from pRb con‐ straint. The E2Fs can then activate genes that are required for the cell to enter S phase (Sherr, 1996, 2004). Cyclin D1 overexpression results in deregulation of phosphorylation of pRB, which can cause loss of growth control. In fact, Cyclin D1 gene and protein products are fre‐ quently overexpressed in a wide rang of cancers. In NSCLC, the *CCND1* locus at 11q13 is amplified in up to 32% of cases, and its protein is expressed at high level in average of 45% of all cases (Gautschi *et al.*, 2007).

The ability of cyclin D1 to cause malignant transformation has been demonstrated in breast cancer transgenic mice model, in which *MMTV-Cyclin D1* transgenic mice developed mam‐ mary AdCA (Wang *et al.,* 1994). Just like in breast cancer, *CCND1* is often found amplified and overexpressed in NSCLC patients. It has been shown that cyclin D1 overexpression is a marker for an increased risk of upper aerodigestive tract premalignant lesions for progress‐ ing to cancer (Kim *et al*., 2011). A polymorphism, G/A870, has been identified in the *CCND1* gene and it results in an aberrantly spliced protein (Cyclin D1b) lacking the Thr-286 phos‐ phorylation site necessary for nuclear export (Diehl *et al*., 1997). It has been shown that the *MMTV-D1T286A* (analogous to Cyclin D1b in humans) mice developed mammary AdCAs at an increased rate relative to *MMTV-D1* mice. Even though cyclin D1b was detected in all NSCLC samples, and the G/A870 polymorphism in *CCND1* gene is predictive of the risk of lung malignancy (Gautschi *et al*., 2007), its impact on lung carcinogenesis has never been in‐ vestigated. Thus creation of mouse models for aberrant cyclin D1 expression in lung epithe‐ lial tissue is needed to test whether it is a key factor in the development of lung carcinogenesis.

Cancer chemoprevention uses dietary or pharmaceutical agents to suppress or prevent car‐ cinogenic progression to invasive cancer. In a recent study, it was shown that a combination of retinoid bexarotene and EGFR inhibitor erlotinib can suppress lung carcinogenesis in transgenic lung cancer cells as well as NSCLC patients in both early and advanced stages. Bexarotene can induce the proteasomal degradation of cyclin D1 and erlotinib can act as an inhibitor of EGFR which represses transcription of cyclin D1 (Kim *et al*., 2011). This finding implicates cyclin D1 as a chemopreventive target and the combination of bexarotene and er‐ lotinib is an attractive candidate for lung cancer chemoprevention (Dragnev *et al*., 2011). Be‐ fore using this regimen in clinical lung cancer chemoprevention, its activity should first be tested in clinically predictive cyclin D1 mouse lung cancer models.

#### **4.7. PTEN and lung cancer**

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‐

The development of human lung carcinogenesis is very complex. Several oncogenes in‐ volved in this process have been identified, one of which is cyclin D1 (Meuwissen & Berns, 2005). Cyclin D1 is a crucial regulator in mammalian cell cycle, which drives cells to enter S phase by binding and activating CDK4/6. The cyclin D1/CDK4 complex phosphorylates the retinoblastoma protein (pRb), which releases E2F transcriptional factors from pRb con‐ straint. The E2Fs can then activate genes that are required for the cell to enter S phase (Sherr, 1996, 2004). Cyclin D1 overexpression results in deregulation of phosphorylation of pRB, which can cause loss of growth control. In fact, Cyclin D1 gene and protein products are fre‐ quently overexpressed in a wide rang of cancers. In NSCLC, the *CCND1* locus at 11q13 is amplified in up to 32% of cases, and its protein is expressed at high level in average of 45%

The ability of cyclin D1 to cause malignant transformation has been demonstrated in breast cancer transgenic mice model, in which *MMTV-Cyclin D1* transgenic mice developed mam‐ mary AdCA (Wang *et al.,* 1994). Just like in breast cancer, *CCND1* is often found amplified and overexpressed in NSCLC patients. It has been shown that cyclin D1 overexpression is a marker for an increased risk of upper aerodigestive tract premalignant lesions for progress‐ ing to cancer (Kim *et al*., 2011). A polymorphism, G/A870, has been identified in the *CCND1* gene and it results in an aberrantly spliced protein (Cyclin D1b) lacking the Thr-286 phos‐ phorylation site necessary for nuclear export (Diehl *et al*., 1997). It has been shown that the *MMTV-D1T286A* (analogous to Cyclin D1b in humans) mice developed mammary AdCAs at an increased rate relative to *MMTV-D1* mice. Even though cyclin D1b was detected in all NSCLC samples, and the G/A870 polymorphism in *CCND1* gene is predictive of the risk of lung malignancy (Gautschi *et al*., 2007), its impact on lung carcinogenesis has never been in‐ vestigated. Thus creation of mouse models for aberrant cyclin D1 expression in lung epithe‐ lial tissue is needed to test whether it is a key factor in the development of lung

Cancer chemoprevention uses dietary or pharmaceutical agents to suppress or prevent car‐ cinogenic progression to invasive cancer. In a recent study, it was shown that a combination of retinoid bexarotene and EGFR inhibitor erlotinib can suppress lung carcinogenesis in transgenic lung cancer cells as well as NSCLC patients in both early and advanced stages. Bexarotene can induce the proteasomal degradation of cyclin D1 and erlotinib can act as an inhibitor of EGFR which represses transcription of cyclin D1 (Kim *et al*., 2011). This finding implicates cyclin D1 as a chemopreventive target and the combination of bexarotene and er‐ lotinib is an attractive candidate for lung cancer chemoprevention (Dragnev *et al*., 2011). Be‐

finitive conclusions (Pinder, 2011).

42 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

**4.6. Cyclin D1 and lung cancer**

of all cases (Gautschi *et al.*, 2007).

carcinogenesis.

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‐ ating with *Pten*-loss during NSCLC development.

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

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‐ morigenesis. It has been reported that LKB1 directly activates AMP-activated kinase and regulates apoptosis in response to energy stress (Shaw *et al*., 2004).

frequent down regulation of the *let-7* miRNA family as well as an upregulation of *miR-17-92* have been reported (Hayashita *et al*., 2005). *miR-17-92* encodes a cluster of seven miRNAs transcribed as single primary transcript. To date, functional analyses of *Dicer1* and *let-7* have been performed in the background *Kras*-induced NSCLC models. A conditional deletion of *Dicer1* in the background of *LSLKrasG12D;Dicer1flox/flox* mice let to a marked increase of tumor development (Kumar *et al*., 2007). However, since the 3′ UTR region of *Kras* transcripts has been shown to be a direct target of *let-7* (Johnson *et al*., 2005), it has become very tempting to increase *let-7* expression in *KrasG12D* lung tumors. *let-7* inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts *in vivo*. Intranasal application of both adenoviral (Esquela-Kerscher *et al*., 2008) and lentiviral (Ku‐ mar *et al*., 2008) *let-7* miRNA caused a significant decrease of *KrasG12D;p53-/-* lung tumors. These findings provide direct evidence that *let-7* acts as a tumor suppressor gene in the lung and indicate that this miRNA might be useful as a novel therapeutic agent in lung cancer.

Genetically Engineered Mouse Models for Human Lung Cancer

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

45

A large scale survey conducted by a different group to determine the miRNA signature of >500 lung, breast, stomach, prostate, colon, and pancreatic cancers and their normal adjacent tissue revealed that *miR-21* was the only miRNA up-regulated in all these tumors (Volinia *et al.*, 2006). Functional studies in cancer cell lines suggest that *miR-21* has oncogenic activity. Knockdown of *miR-21* in cultured glioblastoma cells activated caspases leading to apoptotic cell death, suggesting *miR-21* is an anti-apoptotic factor (Chan *et al.*, 2005). In MCF-7 cells, *miR-21* knock-down resulted in suppression of cell growth both *in vitro* and *in vivo* (Si *et al.*, 2007). Knock-down of *miR-21* in the breast cancer cells reduced invasion and metastasis (Zhu *et al.*, 2008). Targeted deletion of *miR-21* colon cancer cells resulted in tumorigenesis through compromising cell cycle progression and DNA damage-induced checkpoint func‐ tion by targeting *Cdc25a* (Wang *et al.*, 2009). *miR-21* expression is increased and predicts poor survival in NSCLC. Hatley *et al.* used transgenic mice with loss-of-function and gainof-function *miR-21* alleles combined with a model of NSCLC (*K-rasLA2*) to determine the role of *miR-21* in lung cancer (Hatley *et al*., 2010). They showed that overexpression of *miR-21* enhances lung tumorigenesis and that genetic deletion of *miR-21* protects against tumor for‐ mation. *miR-21* drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis (Hatley *et al*., 2010). These studies indi‐ cate that knocking-down of *miR-21* expression in cancer cells results in phenotypes impor‐

Hennessey *et al.* (2012) conducted Phase I/II biomarker study to examine the feasibility of using serum miRNA as biomarkers for NSCLC. Examination of miRNA expression levels in serum from a multi-institutional cohort of 50 subjects (30 NSCLC patients and 20 healthy controls) identified differentially expressed miRNAs. They found that 140 candidate miRNA pairs distinguished NSCLC from healthy controls with a sensitivity and specificity of at least 80% each. Several miRNA pairs involving miRNAs-106a, miR-15b, miR-27b, miR-142-3p, miR-26b, miR-182, 126#, let7g, let-7i (described above) and miR-30e-5p exhibited a negative predictive value and a positive predictive value of 100%. Notably, a combination of two dif‐ ferentially expressed miRNAs *miR-15b* and *miR-27b*, was able to discriminate NSCLC from

tant for tumor biology.

A large fraction of NSCLC cells have germ-line mutations and impaired expression of *LKB1.* LOH for *LKB1* has been reported in more than 50% in lung cancer (Makowski & Hayes, 2008) and thus *LKB1* inactivation is a common event for NSCLC (Sanchez-Cespedes *et al*., 2002, Sanchez-Cespedes, 2007). The highest numbers of mutations were found in AdCAs, es‐ pecially in those with *KRAS* mutations (Matsumoto *et al*., 2007; Sanchez-Cespedes, 2007). *LKB1* inactivation cooperates with *KRAS* activation, suggesting a role for LKB1 as an active repressor of the KRAS downstream pathway (Ji *et al*., 2007). *Lkb1flox/flox;LSLKrasG12D* mice showed a broad spectrum of NSCLCs: the majority of lung tumors were AdCAs, but SqCLCs and large cell carcinoma (LCLC) also occurred. Conversely, no SqCLC or LCLC was detected in *p53flox/flox;LSLKrasG12D* and *(Ink4a/Arf)flox/flox;LSLKrasG12D* mice. Furthermore, 61% of AdCA in *Lkb1flox/flox;LSLKrasG12D* mice developed metastases, but none found for SqCLC and LCLC. These results show that *LKB1*-loss permits squamous differentiation and facilitates metastases, but these two are independent events. AdCA from *Lkb1 flox/flox;LSLKrasG12D* mice had reduced pAMPK (phosphorylated, adenosyl monophosphate-activated protein kinase) and pACCA (phosphorylated, acetyl-CoA carboxylase α-subunit) levels and activated mTOR pathway. It is probable that *LKB1*-loss influences differentiation of NSCLC into sub‐ types by affecting discrete pathways (Shah *et al*., 2008). A large panel of human NSCLC showed *LKB1* mutations in AdCA (34%), SqCLC (19%), and LCC (16%) (Ji *et al.*, 2007). Si‐ multaneous mutations in *p53* and *LKB1* suggest non-overlapping roles in NSCLC. Moreover, reconstitution of LKB1 in human NSCLC cell lines showed anti-tumor effects independent of their *p53* or *INK4A/ARF* status (Ji *et al.*, 2007). Finally, loss of LKB1 expression in alveolar adenomatous hyperplasia, precursor lesion for AdCA, suggests an early role of *LKB1*-inacti‐ vation during AdCA development (Ghaffar *et al.*, 2003).

The same group conducted a mouse trial that mirrors a human clinical trial in patients with KRAS-mutant lung cancers (Chen *et al*., 2012). They demonstrated that simultaneous loss of either *p53* or *Lkb1*, strikingly weakened the response of *Kra*s-mutant cancers to single thera‐ py by docetaxel. Addition of selumetinib provided substantial benefit for mice with lung cancer caused by *Kras* and *Kras* and *p53* mutations, but not in mice with *Kras* and *Lkb1* muta‐ tions (Chen *et al*., 2012). Thus synchronous 'clinical' trials performed in mice, not only will be useful to anticipate the results of ongoing human clinical trials, but also to generate clini‐ cally-relevant hypotheses that will affect the analysis and design of human studies.

#### **4.9. miRNAs and lung cancer**

Not only might genetic mutations in oncogenes and tumor suppressor genes affect their tar‐ get gene expression during lung tumorigenesis, but also microRNAs (miRNAs) can also per‐ form similar roles. microRNAs are evolutionarily conserved, endogenous, non-protein coding, 20–23 nucleotide, single-stranded RNAs that negatively regulate gene expression in a sequence-specific manner. In order to become active, small interfering RNA (siRNA) must undergo catalytic cleavage by the RNase DICER1. In human lung cancer, increased activities of DICER1 and variant regulations of miRNA clusters have been observed. For the latter, a frequent down regulation of the *let-7* miRNA family as well as an upregulation of *miR-17-92* have been reported (Hayashita *et al*., 2005). *miR-17-92* encodes a cluster of seven miRNAs transcribed as single primary transcript. To date, functional analyses of *Dicer1* and *let-7* have been performed in the background *Kras*-induced NSCLC models. A conditional deletion of *Dicer1* in the background of *LSLKrasG12D;Dicer1flox/flox* mice let to a marked increase of tumor development (Kumar *et al*., 2007). However, since the 3′ UTR region of *Kras* transcripts has been shown to be a direct target of *let-7* (Johnson *et al*., 2005), it has become very tempting to increase *let-7* expression in *KrasG12D* lung tumors. *let-7* inhibits the growth of multiple human lung cancer cell lines in culture, as well as the growth of lung cancer cell xenografts *in vivo*. Intranasal application of both adenoviral (Esquela-Kerscher *et al*., 2008) and lentiviral (Ku‐ mar *et al*., 2008) *let-7* miRNA caused a significant decrease of *KrasG12D;p53-/-* lung tumors. These findings provide direct evidence that *let-7* acts as a tumor suppressor gene in the lung and indicate that this miRNA might be useful as a novel therapeutic agent in lung cancer.

morigenesis. It has been reported that LKB1 directly activates AMP-activated kinase and

A large fraction of NSCLC cells have germ-line mutations and impaired expression of *LKB1.* LOH for *LKB1* has been reported in more than 50% in lung cancer (Makowski & Hayes, 2008) and thus *LKB1* inactivation is a common event for NSCLC (Sanchez-Cespedes *et al*., 2002, Sanchez-Cespedes, 2007). The highest numbers of mutations were found in AdCAs, es‐ pecially in those with *KRAS* mutations (Matsumoto *et al*., 2007; Sanchez-Cespedes, 2007). *LKB1* inactivation cooperates with *KRAS* activation, suggesting a role for LKB1 as an active repressor of the KRAS downstream pathway (Ji *et al*., 2007). *Lkb1flox/flox;LSLKrasG12D* mice showed a broad spectrum of NSCLCs: the majority of lung tumors were AdCAs, but SqCLCs and large cell carcinoma (LCLC) also occurred. Conversely, no SqCLC or LCLC was detected in *p53flox/flox;LSLKrasG12D* and *(Ink4a/Arf)flox/flox;LSLKrasG12D* mice. Furthermore, 61% of AdCA in *Lkb1flox/flox;LSLKrasG12D* mice developed metastases, but none found for SqCLC and LCLC. These results show that *LKB1*-loss permits squamous differentiation and facilitates metastases, but these two are independent events. AdCA from *Lkb1 flox/flox;LSLKrasG12D* mice had reduced pAMPK (phosphorylated, adenosyl monophosphate-activated protein kinase) and pACCA (phosphorylated, acetyl-CoA carboxylase α-subunit) levels and activated mTOR pathway. It is probable that *LKB1*-loss influences differentiation of NSCLC into sub‐ types by affecting discrete pathways (Shah *et al*., 2008). A large panel of human NSCLC showed *LKB1* mutations in AdCA (34%), SqCLC (19%), and LCC (16%) (Ji *et al.*, 2007). Si‐ multaneous mutations in *p53* and *LKB1* suggest non-overlapping roles in NSCLC. Moreover, reconstitution of LKB1 in human NSCLC cell lines showed anti-tumor effects independent of their *p53* or *INK4A/ARF* status (Ji *et al.*, 2007). Finally, loss of LKB1 expression in alveolar adenomatous hyperplasia, precursor lesion for AdCA, suggests an early role of *LKB1*-inacti‐

The same group conducted a mouse trial that mirrors a human clinical trial in patients with KRAS-mutant lung cancers (Chen *et al*., 2012). They demonstrated that simultaneous loss of either *p53* or *Lkb1*, strikingly weakened the response of *Kra*s-mutant cancers to single thera‐ py by docetaxel. Addition of selumetinib provided substantial benefit for mice with lung cancer caused by *Kras* and *Kras* and *p53* mutations, but not in mice with *Kras* and *Lkb1* muta‐ tions (Chen *et al*., 2012). Thus synchronous 'clinical' trials performed in mice, not only will be useful to anticipate the results of ongoing human clinical trials, but also to generate clini‐

Not only might genetic mutations in oncogenes and tumor suppressor genes affect their tar‐ get gene expression during lung tumorigenesis, but also microRNAs (miRNAs) can also per‐ form similar roles. microRNAs are evolutionarily conserved, endogenous, non-protein coding, 20–23 nucleotide, single-stranded RNAs that negatively regulate gene expression in a sequence-specific manner. In order to become active, small interfering RNA (siRNA) must undergo catalytic cleavage by the RNase DICER1. In human lung cancer, increased activities of DICER1 and variant regulations of miRNA clusters have been observed. For the latter, a

cally-relevant hypotheses that will affect the analysis and design of human studies.

regulates apoptosis in response to energy stress (Shaw *et al*., 2004).

44 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

vation during AdCA development (Ghaffar *et al.*, 2003).

**4.9. miRNAs and lung cancer**

A large scale survey conducted by a different group to determine the miRNA signature of >500 lung, breast, stomach, prostate, colon, and pancreatic cancers and their normal adjacent tissue revealed that *miR-21* was the only miRNA up-regulated in all these tumors (Volinia *et al.*, 2006). Functional studies in cancer cell lines suggest that *miR-21* has oncogenic activity. Knockdown of *miR-21* in cultured glioblastoma cells activated caspases leading to apoptotic cell death, suggesting *miR-21* is an anti-apoptotic factor (Chan *et al.*, 2005). In MCF-7 cells, *miR-21* knock-down resulted in suppression of cell growth both *in vitro* and *in vivo* (Si *et al.*, 2007). Knock-down of *miR-21* in the breast cancer cells reduced invasion and metastasis (Zhu *et al.*, 2008). Targeted deletion of *miR-21* colon cancer cells resulted in tumorigenesis through compromising cell cycle progression and DNA damage-induced checkpoint func‐ tion by targeting *Cdc25a* (Wang *et al.*, 2009). *miR-21* expression is increased and predicts poor survival in NSCLC. Hatley *et al.* used transgenic mice with loss-of-function and gainof-function *miR-21* alleles combined with a model of NSCLC (*K-rasLA2*) to determine the role of *miR-21* in lung cancer (Hatley *et al*., 2010). They showed that overexpression of *miR-21* enhances lung tumorigenesis and that genetic deletion of *miR-21* protects against tumor for‐ mation. *miR-21* drives tumorigenesis through inhibition of negative regulators of the Ras/MEK/ERK pathway and inhibition of apoptosis (Hatley *et al*., 2010). These studies indi‐ cate that knocking-down of *miR-21* expression in cancer cells results in phenotypes impor‐ tant for tumor biology.

Hennessey *et al.* (2012) conducted Phase I/II biomarker study to examine the feasibility of using serum miRNA as biomarkers for NSCLC. Examination of miRNA expression levels in serum from a multi-institutional cohort of 50 subjects (30 NSCLC patients and 20 healthy controls) identified differentially expressed miRNAs. They found that 140 candidate miRNA pairs distinguished NSCLC from healthy controls with a sensitivity and specificity of at least 80% each. Several miRNA pairs involving miRNAs-106a, miR-15b, miR-27b, miR-142-3p, miR-26b, miR-182, 126#, let7g, let-7i (described above) and miR-30e-5p exhibited a negative predictive value and a positive predictive value of 100%. Notably, a combination of two dif‐ ferentially expressed miRNAs *miR-15b* and *miR-27b*, was able to discriminate NSCLC from healthy volunteers with high sensitivity, specificity (Hennessey *et al.,* 2012). Upon further testing on additional 130 subjects, this miRNA pair predicted NSCLC with a specificity of 84%, sensitivity of 100%. These data provide evidence that serum miRNAs have the poten‐ tial to be sensitive, cost-effective biomarkers for the early detection of NSCLC.

sets of genes are responsible for SqCLC and AdCA. Their model can be used in determining genetic modifiers that contribute to susceptibility or resistance to SqCLC development.

Genetically Engineered Mouse Models for Human Lung Cancer

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

47

The other group tried to induce SqCLC through constitutive expression of human K14 by creating *CC10-hK14* mice (Dakir *et al*., 2008). Although hK14 is highly expressed in bronchial epithelium, only precursor lesions varying from hyperplasia to squamous metaplasia were observed (Dakir *et al*., 2008). Clearly, the increased K14 expression and onset of squamous cell metaplasia alone was not sufficient to generate fully developed SqCLC. As far as trans‐ genic/knockout mice models are concerned, only the *LSLKrasG12D;Lkb1flox/flox* somatic mouse model has been able to generate advanced SqCLC. By using a somatically activatable mu‐ tant *Kras*-driven model of mouse lung cancer (*K-rasLA*), Ji *et al.* (2007) compared the role of Lkb1 to other tumor suppressors in lung cancer. Although *Kras* mutation cooperated with loss of *p53* or *Ink4a/Arf* in this system, the strongest cooperation was seen with homozygous inactivation of *Lkb1*. *Lkb1*-deficient tumors demonstrated shorter latency, an expanded histo‐ logical spectrum (adeno-, squamous, and large-cell carcinoma) and more frequent metasta‐ sis as compared to tumors lacking *p53* or *Ink4a/Arf*. Interestingly up to 60% of *Lkb1* deficient lung tumors had squamous or mixed squamous histology (Ji *et al*., 2007), which has not been reported in other mouse lung cancer models. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of *Lkb1*, confirming its haplo-insufficiency. Consistent with these findings, inactivation of *LKB1* was found in 34% and 19% of 144 human lung adeno‐ carcinomas and squamous cell carcinomas, respectively. They also identified a variety of metastasis-promoting genes, such as *NEDD9*, *VEGFC* and *CD24*, as targets of LKB1 repres‐ sion in lung cancer. These studies established LKB1 as a critical barrier to prevent lung carci‐

nogenesis, controlling initiation, differentiation and metastasis (Ji *et al*., 2007).

**models**

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

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
