Induced Lung Tumors Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Response and Resistance to NF-κB Inhibitors in Mouse Models of Lung Adenocarcinoma

Lung adenocarcinoma is a leading cause of cancer death worldwide. We recently showed that genetic inhibition of the NF-κB pathway affects both the initiation and the maintenance of lung cancer, identifying this pathway as a promising therapeutic target. In this investigation, we tested the efficacy of small-molecule NF-κB inhibitors in mouse models of lung cancer. In murine lung adenocarcinoma cell lines with high NF-κB activity, the proteasome inhibitor bortezomib efficiently reduced nuclear p65, repressed NF-κB target genes, and rapidly induced apoptosis. Bortezomib also induced lung tumor regression and prolonged survival in tumor-bearing Kras(LSL-G12D/wt);p53(flox/flox) mice but not in Kras(LSL-G12D/wt) mice. After repeated treatment, initially sensitive lung tumors became resistant to bortezomib. A second NF-κB inhibitor, Bay-117082, showed similar therapeutic efficacy and acquired resistance in mice. Our results using preclinical mouse models support the NF-κB pathway as a potential therapeutic target for a defined subset of lung adenocarcinoma. Using small-molecule compounds that inhibit NF-κB activity, we provide evidence that NF-κB inhibition has therapeutic efficacy in the treatment of lung cancer. Our results also illustrate the value of mouse models in validating new drug targets in vivo and indicate that acquired chemoresistance may later influence bortezomib treatment in lung cancer.

Aerosolized BC-819 Inhibits Primary but Not Secondary Lung Cancer Growth

Despite numerous efforts, drug based treatments for patients suffering from lung cancer remains poor. As a promising alternative, we investigated the therapeutic potential of BC-819 for the treatment of lung cancer in mouse tumor models. BC-819 is a novel plasmid DNA which encodes for the A-fragment of Diphtheria toxin and has previously been shown to successfully inhibit tumor growth in human clinical study of bladder carcinoma. In a first set of experiments, we examined in vitro efficacy of BC-819 in human lung cancer cell-lines NCI-H460, NCI-H358 and A549, which revealed >90% reduction of cell growth. In vivo efficacy was examined in an orthotopic mouse xenograft lung cancer model and in a lung metastasis model using luminescent A549-C8-luc adenocarcinoma cells. These cells resulted in peri- and intra-bronchiolar tumors upon intrabronchial application and parenchymal tumors upon intravenous injection, respectively. Mice suffering from these lung tumors were treated with BC-819, complexed to branched polyethylenimine (PEI) and aerosolized to the mice once per week for a period of 10 weeks. Using this regimen, growth of intrabronchially induced lung tumors was significantly inhibited (p = 0.01), whereas no effect could be observed in mice suffering from lung metastasis. In summary, we suggest that aerosolized PEI/BC-819 is capable of reducing growth only in tumors arising from the luminal part of the airways and are therefore directly accessible for inhaled BC-819.

SU‐E‐J‐119: Development of Voxel‐by‐Voxel Respiratory Lung Motion Simulation Model

Purpose: 1. To justify the respiratory induced lung tumor motion is correlated to volume change in lung 2.To show that such motion is a hysteresis type of motion, i.e. the inspiration and expiration paths the tumor follows are different. Methods: The model equation was generated based on the data obtained from the axial displacement versus volume plot of the lung by Verschakelen et al (1989). The time variation of the lung volume change was introduced using linear and non‐linear functions. After comparison with literature (Lu et al, 2006), we verified the non‐linear time variation is more reliable. The lung motion amplitude as a function of time was then plotted and fitted with sinusoidal function. The dynamic 3D lung motion was simulated as a function of time based on the model equation. Two independent simulations were performed and compared: 1. Independent tumor motion amplitude and lung volume change. 2. Lung tumor motion dependent on the lung volume change. Results: 1.The model function generated from the fitting curve is periodic function with even power. This agrees with the periodic nature of respiration induced lung motion and also with the fact that longer time is spent at the end of exhalation than at the end of inhalation, which is proven by other researchers too. 2.Based on the simulated dynamic lung motion, we observe that the lung tumor motion is well correlated to the lung volume change. Conclusions: Clinically, chest wall as well as diaphragm amplitude measurements are used to provide lung motion gating window. In this study, we observe that respiration induced motion of the lung tumor is correlated to the lung volume change, and this can give a good prediction of: 1. The 3D location of the lung tumor. 2. Density change of the lung.

Abstract LB-464: Phenylbutyl isoselenocyanate (ISC-4) and phenylbutyl isothiocyanate (PBITC) inhibit 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced lung tumorigenesis in A/J mouse

Abstract Lung cancer is the leading cause of cancer deaths in both men and women with 90% of cases attributed to tobacco smoking. Evidence has shown that there is a 20–30 year latency period from tobacco exposure to invasive disease making chemopreventive approaches desirable. Our lab has previously shown the organoselenium compound ISC-4, an analog of naturally occurring established chemopreventive isothiocyanates, potently inhibits Phase I CYP450 enzymes at the protein level and induces Phase II enzyme activity via a transcriptional mechanism. In A/J mice treated with the tobacco procarcinogen NNK, dietary ISC-4 was able to reduce the number of O6-methyl guanine DNA adducts, which are critical for NNK induced carcinogenesis, in liver and lung. Based on these results we used an A/J mouse model of lung cancer to test the chemopreventive effects of ISC-4 against NNK induced lung tumorigenesis. As a positive control we included a group of high dose PBITC, the sulfur analog of ISC-4, which had never been tested in a chronic feeding model but shown to be very effective in pre-initiation assays at inhibiting NNK tumorigenesis. Based on bioavailability and MTD studies, A/J mice were fed ISC-4 in the AIN-76 diet at 0.19 μmol/g, 0.38 mol/g, and 0.57 μmol/g diet. PBITC, which is tolerated at higher dose, was given at 0.38 μmol/g and 3.0 mol/g diet. Mice (n=30/group) were fed control or experimental diet for two weeks and then injected with a single dose of 10 μmol NNK (IP). They were continued on diet for 22 weeks and then sacrificed. Lung tumors were counted and blood and tissues were collected for toxicity analyses. Mice treated with NNK plus dietary ISC-4 at 0.19 μmol/g, 0.38 μmol/g, and 0.57 μmol/g resulted in tumor multiplicity of 4.14 ± 0.35, 1.90 ± 0.28, and 2.13 ± 0.33 tumors/lung, respectively, compared to 13.9 ± 0.83 tumors/lung for mice treated with NNK on control diet, a reduction of 72–90% (p < 0.0001). Mice treated with 0.38 μmol/g and 3.0 μmol/g diet PBITC resulted in tumor multiplicity of 2.93 ± 0.40 and 0.80 ± 0.20 tumors/lung, respectively, a reduction of 82 and 98% (p < 0.0001). None of the treatment groups showed a decrease in body weight. Mice in all the groups, except 0.38 μmol/g PBITC and NNK control diet group, gained ∼10–20% less weight as compared to untreated control AIN-76 group. Blood analysis showed normal toxicity values for all groups except for the highest dose of ISC-4. HE staining of the liver and kidney samples showed no signs of toxicity except for two mice in the high dose of PBITC. In conclusion, both ISC-4 and PBITC showed great promise as chemopreventive agents for inhibition of NNK induced lung tumors. These results show that PBITC has a larger chemopreventive index, but at equimolar doses ISC-4 was more effective at tumor inhibition than PBITC (p=0.04). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-464. doi:10.1158/1538-7445.AM2011-LB-464

Abstract 1118: Role of the type I insulin-like growth factor receptor in lung tumorigenesis

Abstract Lung cancer is the leading cause of cancer related mortalities worldwide. The type I insulin-like growth factor receptor (IGF-IR) is frequently expressed at high levels in lung cancer. This receptor is involved in cell proliferation, apoptosis, migration and differentiation and is emerging as a potential target for molecular therapies. Furthermore, IGF-IR downregulation has been reported to sensitize cancer cells to agents that promote apoptosis. To investigate the importance of the IGF-IR in lung tumorigenesis, our lab generated doxycycline-inducible transgenic mice in which the IGF-IR was overexpressed in type II alveolar or Clara cells. IGF-IR overexpression was sufficient to induce lung tumor development in these mice indicating that this receptor has an important function in the initiation of lung tumorigenesis. To further investigate the role of IGF-IR in lung tumorigenesis, the levels and function of IGF-IR were evaluated in two human lung tumor cell lines (A549 and NCI-H358) and one murine lung tumor cell line (LA-4) as well as in human and murine normal bronchial epithelial cells. Western blotting revealed increased levels of IGF-IR in the lung cancer cell lines compared to the normal lung epithelial cells lines. The efficacy of the IGF-IR small-molecule inhibitor, BMS-754807, which is a drug currently in clinical trials, was also evaluated in the lung tumor cells. BMS-754807 inhibited A549, NCI-H358 and LA-4 cell survival in a dose-dependent manner with approximate IC50 concentrations of 1.70uM for A549, 1.25uM for NCI-H358 and 0.74uM for LA-4 cells. The effects of BMS-754807 on cell proliferation (Ki67 immunofluorescence), cell survival (JC-1 immunofluorescence) and migration (scratch wound and Boyden chamber assays) are currently being examined to determine which properties are affected by disruption of IGF-IR signaling. Similar studies will be performed following downregulation of IGF-IR expression using RNAi. To evaluate whether BMS-754807 could sensitize lung tumor cells to the cytotoxic effects of chemotherapeutic agents commonly employed in the treatment of human lung cancer, BMS-754807 was combined with cisplatin and with carboplatin. Neither the BMS-754807/cisplatin nor the BMS-754807/carboplatin combinations appeared to have a synergistic effect on cell survival. Combinations of BMS-754807 and lung cancer chemotherapeutic agents that do not target DNA are currently being evaluated. Additionally, key downstream molecules of the IGF-IR, including Akt and ERK1/2, are being studied as well as the transcription factor, KLF5, which is expressed at high levels in the lung tumors of the IGF-IR transgenic mice. Initial results support the importance of IGF-IR signaling in lung cancer. Further understanding of the function of IGF-IR in lung cancer will enable the development of more effective targeted therapies and improve existing therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1118. doi:10.1158/1538-7445.AM2011-1118

Differential Impacts of Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) in Epithelial IGF-Induced Lung Cancer Development

The IGF axis has been implicated in the risk of various cancers. We previously reported a potential role of tissue-derived IGF in lung tumor formation and progression. However, the role of IGF-binding protein (IGFBP)-3, a major IGFBP, on the activity of tissue-driven IGF in lung cancer development is largely unknown. Here, we show that IGF-I, but not IGF-II, protein levels in non-small-cell lung cancer (NSCLC) were significantly higher than those in normal and hyperplastic bronchial epithelium. We found that IGF-I and IGFBP-3 levels in NSCLC tissue specimens were significantly correlated with phosphorylated IGF-IR (pIGF-IR) expression. We investigated the impact of IGFBP-3 expression on the activity of tissue-driven IGF-I in lung cancer development using mice carrying lung-specific human IGF-I transgene (Tg), a germline-null mutation of IGFBP-3, or both. Compared with wild-type (BP3(+/+)) mice, mice carrying heterozygous (BP3(+/-)) or homozygous (BP3(-/-)) deletion of IGFBP-3 alleles exhibited decreases in circulating IGFBP-3 and IGF-I. Unexpectedly, IGF(Tg) mice with 50% of physiological IGFBP-3 (BP3(+/-); IGF(Tg)) showed higher levels of pIGF-IR/IR and a greater degree of spontaneous or tobacco carcinogen [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]-induced lung tumor development and progression than did the IGF(Tg) mice with normal (BP3(+/+;) IGF(Tg)) or homozygous deletion of IGFBP-3 (BP3(-/-); IGF(Tg)). These data show that IGF-I is overexpressed in NSCLC, leading to activation of IGF-IR, and that IGFBP-3, depending on its expression level, either inhibits or potentiates IGF-I actions in lung carcinogenesis.

Akt1 deletion prevents lung tumorigenesis by mutant K-ras

K-ras mutations are associated with smoking-induced lung cancer and poor clinical outcomes. In mice, K-ras mutations are sufficient to induce lung tumors, which require phosphoinoside-3-kinase (PI3K) and further downstream, mammalian target of rapamycin (mTOR) activation. However, the roles of individual Akt isoforms that link PI3K and mTOR are unknown. Here, we show that deletion of Akt1 but not Akt2 or Akt3 prevents lung tumorigenesis in a tobacco carcinogen-induced model and a genetic model. Akt1 deletion prevented tumor initiation as well as tumor progression, coincident with decreased Akt signaling in tumor tissues. In contrast, deletion of Akt3 increased tumor multiplicity in the carcinogen model and increased tumor size in the genetic model. Fibroblasts lacking Akt1 are resistant to transformation by mutant K-ras and stimulation by epidermal growth factor. Human lung cancer cells with mutant K-ras and diminished Akt1 levels fail to grow in vivo. These data suggest that Akt1 is the primary Akt isoform activated by mutant K-ras in lung tumors, and that Akt3 may oppose Akt1 in lung tumorigenesis and lung tumor progression. Given that Akt inhibitors in clinical development as cancer therapeutics are not isoform selective, these studies support specific targeting of Akt1 to mitigate the effects of mutant K-ras in lung cancer.

Increased expression of FoxM1 transcription factor in respiratory epithelium inhibits lung sacculation and causes Clara cell hyperplasia

Foxm1 is a member of the Forkhead Box (Fox) family of transcription factors. Foxm1 (previously called Foxm1b, HFH-11B, Trident, Win, or MPP2) is expressed in multiple cell types and plays important roles in cellular proliferation, differentiation and tumorigenesis. Genetic deletion of Foxm1 from mouse respiratory epithelium during initial stages of lung development inhibits lung maturation and causes respiratory failure after birth. However, the role of Foxm1 during postnatal lung morphogenesis remains unknown. In the present study, Foxm1 expression was detected in epithelial cells of conducting and peripheral airways and changing dynamically with lung maturation. To discern the biological role of Foxm1 in the prenatal and postnatal lung, a novel transgenic mouse line that expresses a constitutively active form of FoxM1 (FoxM1 N-terminal deletion mutant or FoxM1-ΔN) under the control of lung epithelial-specific SPC promoter was produced. Expression of the FoxM1-ΔN transgene during embryogenesis caused epithelial hyperplasia, inhibited lung sacculation and expression of the type II epithelial marker, pro-SPC. Expression of FoxM1-ΔN mutant during the postnatal period did not influence alveologenesis but caused focal airway hyperplasia and increased proliferation of Clara cells. Likewise, expression of FoxM1-ΔN mutant in conducting airways with Scgb1a1 promoter was sufficient to induce Clara cell hyperplasia. Furthermore, FoxM1-ΔN cooperated with activated K-Ras to induce lung tumor growth in vivo. Increased activity of Foxm1 altered lung sacculation, induced proliferation in the respiratory epithelium and accelerated lung tumor growth, indicating that precise regulation of Foxm1 is critical for normal lung morphogenesis and development of lung cancer.

Requirement of the NF-κB Subunit p65/RelA for K-Ras–Induced Lung Tumorigenesis

K-Ras-induced lung cancer is a very common disease, for which there are currently no effective therapies. Because therapy directly targeting the activity of oncogenic Ras has been unsuccessful, a different approach for novel therapy design is to identify critical Ras downstream oncogenic targets. Given that oncogenic Ras proteins activate the transcription factor NF-kappaB, and the importance of NF-kappaB in oncogenesis, we hypothesized that NF-kappaB would be an important K-Ras target in lung cancer. To address this hypothesis, we generated a NF-kappaB-EGFP reporter mouse model of K-Ras-induced lung cancer and determined that K-Ras activates NF-kappaB in lung tumors in situ. Furthermore, a mouse model was generated where activation of oncogenic K-Ras in lung cells was coupled with inactivation of the NF-kappaB subunit p65/RelA. In this model, deletion of p65/RelA reduces the number of K-Ras-induced lung tumors both in the presence and in the absence of the tumor suppressor p53. Lung tumors with loss of p65/RelA have higher numbers of apoptotic cells, reduced spread, and lower grade. Using lung cell lines expressing oncogenic K-Ras, we show that NF-kappaB is activated in these cells in a K-Ras-dependent manner and that NF-kappaB activation by K-Ras requires inhibitor of kappaB kinase beta (IKKbeta) kinase activity. Taken together, these results show the importance of the NF-kappaB subunit p65/RelA in K-Ras-induced lung transformation and identify IKKbeta as a potential therapeutic target for K-Ras-induced lung cancer.

The function of IGF-IR in NNK-mediated lung tumorigenesis

The type I insulin-like growth factor receptor (IGF-IR) is associated with many different types of cancer and it has been found to be involved in many aspects of the malignant phenotype, such as mitogenesis, survival, transformation and metastasis. This receptor has been observed to be overexpressed in the majority of lung cancer cell lines and human lung tumor biopsies. Two doxycycline-inducible transgenic mouse models in which the human IGF-IR was overexpressed in either the Clara cells or the type II alveolar cells of the lung were used in this study to examine the interaction between the nicotine derivative, nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the IGF-IR. NNK was injected into both of the transgenic mouse models that overexpress human IGF-IR in the lung tissue in order to determine whether IGF-IR overexpression would affect NNK-induced tumorigenesis. No significant differences in the overall tumor burden were found between mice overexpressing the IGF-IR transgene that were treated with NNK and those that were not, however NNK-treated mice expressing high levels of IGF-IR transgene developed larger tumors than mice expressing high levels of IGF-IR transgene that did not receive NNK injections. In addition, endogenous murine IGF-IR was found to be expressed at high levels in the tumors that developed in the wild type, NNK injected mice suggesting that NNK induces lung tumors through inducing endogenous IGF-IR expression.

Abstract 1587: Targeted deletion of Fra-1 blocks oncogenic K-ras induced lung tumor development and progression in mice

Abstract Background: Fra-1 (Fosl1) is a dimeric partner of AP-1 transcription factor and regulates gene expression in a context-dependent (cell-type and stimulus-specific) manner through the TPA response element (AP-1 site). We have previously shown that Fra-1 is strongly activated in lung epithelial cells by cigarette smoke, a major determinant of lung cancer, as well as tumor promoting mitogenic and pro-inflammatory stimuli. Ectopic expression of Fra-1 promotes human lung cancer cell progression in vitro, and tumor development in nude mice. We hypothesized that Fra-1, an effector of ERK signaling, is required for oncogenic K-ras promoted lung tumor development in vivo. Methods: To test our hypothesis and to better understand the role of Fra-1 in lung tumor development and progression in vivo, we have generated transgenic mice bearing floxed allele Fra-1 (Fra-1FF) and LSL K-rasG12D by cross-breeding Fra1F/F and LSL K-rasG12D mice (NCI), which carry a latent point-mutant allele of K-ras (K-rasG12D). Fra1wt/wt +LSL-K-rasG12D mice (abbreviated as Kras+/−) and Fra1F/F+ LSL-K-rasG12D mice (Fra1FF- K-rasG12D) were instilled intratracheally with adenovirus Cre (2.5 ×107 pfu) and then were allowed to recover for an additional 13 weeks. Mice were sacrificed, lungs were fixed, and lung tumor induction was analyzed by histopathology. Results: As anticipated, activation of K-ras oncogene by Ade-Cre instillation induced lung tumor development in Kras+/− mice bearing Fra-1 wild type alleles. Hyperplastic lesions with papillary structures and adenomas were clearly visible in these mice. Strikingly, the lung tumor formation in Fra-1 floxed mice bearing LSL-K-rasG12D allele (Fra1F/F+ LSL-K-rasG12D mice) infected with Adeno-cre virus was markedly reduced. In all animals, with exception of few hyperplastic lesions, no adenomas were detected in mice lacking Fra-1. Conclusions: The present data suggest that Fra-1 is critical for oncogenic K-ras promoted lung tumor development in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1587.

Abstract 3471: Reciprocal roles of cytochromes P4501A1 and 1A2 in polycyclic aromatic hydrocarbon (PAH)-mediated tumorigenesis in mice: Implications for lung cancer in humans

Abstract Humans are constantly exposed to environmental carcinogenic polycyclic aromatic hydrocarbons (PAHs) through cigarette smoke, diesel exhausts, charcoal-broiled meats, etc. Cytochrome P4501A (CYP1A) enzymes play important roles in the activation of PAHs such as 3-methylcholanthrene (MC) to carcinogenic DNA-binding metabolites. The resulting DNA adducts may lead to mutations of tumor suppressor genes such as p53, thereby leading to tumorigenesis. In this study, we tested the hypothesis that CYP1A1 and 1A2 have reciprocal roles in PAH-mediated tumorigenesis. Eight week-old female wild type (WT) (A/J) mice or mice lacking the gene for CYP1A1 or CYP1A2 on the A/J background were treated with a single dose of MC (40 µmol/kg), benzo[a]pyrene (BP), or vehicle (corn oil), and liver and lung tumors were studied after 28 weeks. While 100% of WT or Cyp1a2-null mice exposed to MC showed lung tumors after 28 weeks, about 80% of the Cyp1a1-null mice showed lung tumors. However, there were striking differences in the Cyp1a1-null and Cyp1a2-null in regard to lung tumor multiplicities and sizes of tumors. The WT mice treated with MC had about 15 lung tumors/animal. On the other hand, the Cyp1a2-null mice displayed about 40 lung tumors/animal, while the Cyp1a1-null mice showed about 2-3 tumors/mouse. Also, the tumor sizes were much larger in the Cyp1a2-null mice, compared to the much smaller sizes in the Cyp1a1-null mice. Although BP was less potent in inducing lung tumors in the WT mice compared to MC, it was similar to MC In that tumors were suppressed in the Cyp1a1-null mice and augmented in the Cyp1a2-null mice. Immunohistochemical studies showed that CYP1A1 was overexpressed in the tumorous portions of the lung in the Cyp1a2-null or WT mice, compared to non-tumorous regions, These results suggest that CYP1A1 contributes to the formation of tumorigenesis by PAHs, while CYP1A2 plays an important role in the protection against carcinogenesis mediated by PAHs, presumably through it role in PAH detoxification. In conclusion, our results strongly suggest that CYP1A1 and 1A2 could be novel candidates for cancer prevention and therapy though either inhibition of CYP1A1 or induction of CYP1A2. Thus, CYP1a1-null and Cyp1a2-null mice could be developed as novel animal models for future cancer prevention and/or therapeutic intervention in humans exposed to environmental PAHs. (Supported by NIH grant ES009132.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3471.

Abstract 4205: The Jackson Laboratory Repository: new mouse models of cancer

Abstract The Jackson Laboratory Repository serves as a centralized facility to import, develop, preserve, and distribute mouse models to the scientific community. More than 400 strains are added annually to one of the largest collection of characterized mouse strains available. New models of increased tumor incidence at the Repository include three transgenic strains for studies of prostate tumorogenesis that use the rat probasin promoter, (Pbsn-ERG*) develops prostate neoplasia resulting from the expression of the ERG oncogene in prostate epithelial cells; a companion strain that expresses hepsin and GFP in prostate epithelial cells (Pbsn-Hpn,-GFP); a transgenic (Pbsn-IGF1*) that develops prostate hyperplasia; a B cell lymphoma transgenic model expressing the human T-cell leukemia/lymphoma 1 gene (Cd79b-TCL1A); a mouse with a targeted mutation in the kinase Plk3 that develops spontaneous tumors in multiple organs (lung, kidney, liver); a mouse with a targeted mutation in Xpc that exhibits increased susceptibility to radiation induced skin cancer and acetylaminofluorene-induced lung and liver cancers; and a targeted mutation in the tumor suppressor, Tnk1, that develops high rates of spontaneous tumors (lymphomas and carcinomas). Other recently added mutants allow cancer research in different areas ranging from an amino acid substitution (S10A) in Cdkn1b (p27) that reduces susceptibility to induced lung tumors to new research tools, which include a tetracycline (tetO) regulated Erbb2 (TRE-Neu) transgenic strain that develops hyperplasia in tissues where Erbb2 is expressed when combined with a tTA/rtTA strain, and a transgenic mouse, which expresses human ERBB2 (HER2) in mammary gland and brain, displays tolerance to ERBB2 antigen and may be useful in testing ERBB2-based vaccines and immunotherapies. Equally important are the Cre recombinase-expressing strains used in conjunction with these mutants. Most of the existing Cre strains have not been fully characterized and ‘ectopic’ expression of Cre recombinase can confound analysis of experimental results. The Repository has undertaken a project to comprehensively characterize Cre recombinase activity in embryonic and adult mouse tissues. Annotated images are being published on a dedicated website and submitted to MGI as part of a collaboration to develop a Cre stain database (http://www.creportal.org/). This information will allow users to make informed judgments about the suitability of a particular line for their experiments. The Repository maintains a searchable on-line resource (www.jaxmice.jax.org/query). Researchers wishing to have strains considered for inclusion in the Repository are encouraged to submit their strains at: www.jax.org/grc/. The Jackson Laboratory Repository is supported by the NCRR, The Howard Hughes Medical Institute, The Ellison Medical Foundation and donations from private charitable foundations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4205.

The Kras Mutational Spectra of Chemically Induced Lung Tumors in Different Inbred Mice Mimics the Spectra of KRAS Mutations in Adenocarcinomas in Smokers versus Nonsmokers

Human lung cancer patients exhibit different KRAS mutations depending on smoking status. In a mouse model of human cancer, A/J and BALB/cBy mice treated with the tobacco carcinogen, 3-methylcholanthrene (MCA), followed by butylated hydroxytoluene (BHT)-elicited chronic inflammation develop a high multiplicity of lung tumors. DNA was isolated from MCA-induced lung tumors in A/J and BALB/cByJ mice. Kras codon 12 sequences from these tumors were compared to those in human lung tumors from smokers and never-smokers. The distribution of Kras codon 12 mutations in MCA-induced A/J lung tumors is strikingly similar to those found in adenocarcinomas from human smokers. In contrast, codon 12 mutations in BALB/cBy mice contain predominantly G --> D mutations, which is the most common mutation in never smokers. A single lung carcinogen induces different tumor initiating mutations in different strains of mice. This may be useful for investigating the role of specific KRAS mutations in adenocarcinoma pathogenesis in smokers versus never smokers, identifying mechanisms that select for certain KRAS mutations and developing new drugs that specifically target cells with different KRAS mutations.

Effect of Dietary Polyphenon E and EGCG on Lung Tumorigenesis in A/J Mice

To compare the chemopreventive efficacy of Polyphenon E (Poly E), (-)-epigallocatechin-3-gallate (EGCG) and Polyphenon E without EGCG (Poly E-EGCG) on the development of benzo(a)pyrene (B(a)P)-induced lung tumors in A/J mice. Female A/J mice were given a single intraperitoneal injection of B(a)P (100 mg/kg body weight). One week after B(a)P injection, animals received AIN-76A purified powder diet containing 0.975% (wt/wt) EGCG, 0.525% (wt/wt) Poly E-EGCG or 1.5% (wt/wt) Poly E for 24 weeks or control diet with no additives. Poly E treatment significantly decreased tumor multiplicity by 52% and tumor load by 64%, while EGCG and Poly E-EGCG did not significantly inhibit lung tumor multiplicity. EGCG was more stable in a complex mixture (Poly E) than as a pure compound. EGCG was ineffective when administered by diet likely due to its instability. Thus, EGCG's efficacy on mice lung tumorigenesis requires the presence of other tea catechins.

Abstract A14: A forward genetic screen for genes that cooperate with PTEN in lung cancer development

Abstract Phosphatase and Tensin Homolog (Pten) negatively regulates the PI3K signaling pathway and functions as a tumor suppressor in many forms of cancer. The role of Pten in lung cancer is not well understood, although protein expression is frequently lost. Evidence from a mouse model has suggested that loss of Pten is not sufficient to induce lung tumor formation, but will cooperate with activated Kras to decrease latency and increase severity of lung tumors. In order to better understand the role of Pten in lung cancer we performed a forward genetic screen in mice aimed at identifying mutations that cooperate with Pten during lung cancer development. To target both loss of Pten and random mutations to lung tissue we generated mice containing a conditional Sleeping Beauty (SB) transposase allele (Rosa26-Stopfl/fl-SB11) and a conditional Pten allele (Ptenfl/fl). When crossed to mice expressing Cre recombinase under control of the lung-specific surfactant protein C (SPC) promoter, the SB11 gene is expressed while Pten is rendered inactive in lung tissue. In these Pten null cells, mutations are created by SB11 mediated transposition of a mutagenic transposon, T2/Onc, engineered to disrupt tumor suppressor genes or overexpress oncogenes. We have performed IHC for the SB transposase on lung tissue of mice harboring both Rosa26-Stopfl/fl-SB11 and SPC-Cre alleles and find the transposase is expressed in approximately 20% of lung epithelial cells. Experimental mice harboring all four transgenes (Ptenfl/fl, Rosa26-Stopfl/fl-SB11, SPC-Cre, and T2/Onc), along with two control groups (Ptenfl/fl, Rosa26-Stopfl/fl-SB11, and SPC-Cre or Ptenfl/fl, Rosa26-Stopfl/fl-SB11, and T2/Onc), were aged and monitored for morbidity. Median survival of the experimental and control group containing SPC-Cre (lung tissue is Pten null) was approximately 300 days, while median survival of the control group containing T2/Onc (lungs are wild-type for Pten) was 500 days (p < 0.0001). Upon necropsy, 29% of experimental animals, 12% of the Pten null control group, and 3% of the Pten wild-type control group harbored macroscopic lung tumors. Using linker-mediated PCR and high-throughput sequencing we sequenced and mapped over 10,000 transposon insertions in 31 lung tumors from 18 mice. Statistical analysis of these insertions identified 44 common insertion sites which indicate potential driver mutations for lung cancer. Candidate genes identified include known cancer genes (e.g., Map2k4 and Lpp), genes dysregulated in human lung tumors (e.g., Rlf and Car8) and genes not previously associated with lung cancer (e.g., Mllt10 and Ppp3ca). We will be testing the effect of knocking-down or over-expressing these candidate cancer genes in cell lines and mouse models to determine their role in tumor formation and development. The results of this study will be used to identify new diagnostic markers and therapeutic targets for treating lung cancer. Citation Information: Cancer Res 2009;69(23 Suppl):A14.

HIF2α cooperates with RAS to promote lung tumorigenesis in mice

Members of the hypoxia-inducible factor (HIF) family of transcription factors regulate the cellular response to hypoxia. In non-small cell lung cancer (NSCLC), high HIF2alpha levels correlate with decreased overall survival, and inhibition of either the protein encoded by the canonical HIF target gene VEGF or VEGFR2 improves clinical outcomes. However, whether HIF2alpha is causal in imparting this poor prognosis is unknown. Here, we generated mice that conditionally express both a nondegradable variant of HIF2alpha and a mutant form of Kras (KrasG12D) that induces lung tumors. Mice expressing both Hif2a and KrasG12D in the lungs developed larger tumors and had an increased tumor burden and decreased survival compared with mice expressing only KrasG12D. Additionally, tumors expressing both KrasG12D and Hif2a were more invasive, demonstrated features of epithelial- mesenchymal transition (EMT), and exhibited increased angiogenesis associated with mobilization of circulating endothelial progenitor cells. These results implicate HIF2alpha causally in the pathogenesis of lung cancer in mice, demonstrate in vivo that HIF2alpha can promote expression of markers of EMT, and define HIF2alpha as a promoter of tumor growth and progression in a solid tumor other than renal cell carcinoma. They further suggest a possible causal relationship between HIF2alpha and prognosis in patients with NSCLC.

Tea polyphenols inhibit cyclooxygenase-2 expression and block activation of nuclear factor-kappa B and Akt in diethylnitrosoamine induced lung tumors in Swiss mice

Due to lack of validated screening methods and hence poor prognosis, treatment of lung cancer has not still improved up to the expectations. Therefore, risk of lung cancer needs to be minimized by efficient preventive measures. Tea (Camellia sinensis) and its bioactive polyphenols have been associated with prevention of human cancer for several organs. Thus, intake of tea polyphenols seems to be a viable mean to control lung cancer burden. In the present study, we studied the chemopreventive effects of green tea polyphenols (GTP) and black tea polyphenols (BTP) against diethylnitrosoamine (DEN) induced lung tumors in Swiss albino mice. Chemopreventive potential of tea polyphenols, was recorded as evident by, low incidence of alveologenic tumors in lungs of animals at tested doses (0.1% and 0.2% of both GTP and BTP) when compared with DEN (20 mg/kg b wt) treated animals. As a mechanism of cancer chemoprevention cellular signaling pathways were also targeted. GTP and BTP treatment inhibited the expression of Akt, cyclooxygenase-2 and inactivated nuclear factor-kappa B via blocking phosphorylation and subsequent degradation of IkappaB alpha. Thus, the study suggests that polyphenolic constituents of both cultivars of tea, i.e. green and black, have chemopreventive effects in DEN induced lung tumorigenesis in Swiss albino mice.

Effect of gefitinib on N-nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induced lung tumorigenesis in A/J mice

Gefitinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). N-Nitrosamine-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent carcinogen found in tobacco smoke, induces lung tumors in A/J mice. NNK induces cellular transformation resulting in the over-expression of EGFR. Accordingly, EGFR may be a target for cancer prevention. In this study, we investigated the effect of gefitinib on NNK-induced tumorigenesis and the carcinogenicity of gefitinib in A/J mice. A total of 180 four-week-old female A/J mice were randomly divided into six groups: group 1 (controls), treated with deionized water; group 2, treated with 5 mg/kg p.o. gefitinib; group 3, treated with 50 mg/kg p.o. gefitinib (to test the carcinogenicity of gefitinib); group 4 (controls for NNK treatment), treated with deionized water; group 5, treated with 5 mg/kg p.o. gefitinib; and group 6, treated with 50 mg/kg p.o. gefitinib and injected with NNK once at 8 weeks of age to test the chemopreventive activity of gefitinib. Gefitinib was given once a day, 5 days a week by gavage, beginning at 4 weeks of age and continuing for 26 weeks. All mice were sacrificed at 30 weeks of age. The multiplicities of the NNK-induced lung tumors were significantly suppressed in a dose-dependent manner. Gefitinib had no effect on body weight at a low dose. The administration of gefitinib alone for 26 weeks did not induce tumorigenesis; instead, it significantly suppressed the incidence of spontaneous tumors in the mice, in contrast with other anti-cancer agents. Gefitinib did not induce lung fibrosis when compared with control mice by Azan–Mallory staining. Our results suggest that gefitinib has a weak but significant chemopreventive effect with no carcinogenicity or pulmonary toxicity in A/J mice.