Lung Adenomas Research Articles

Interaction between MYC and MCL1 in the Genesis and Outcome of Non–Small-Cell Lung Cancer

MYC exerts both positive and negative functions in cancer cells, such that its procancerous effects are unmasked only after its anticancer effects are blocked. Here we used multiple mouse models of lung adenocarcinoma to identify genetic events that can cooperate with MYC activation to promote the genesis of non-small-cell lung cancer (NSCLC), the most common form of lung cancer in humans. MYC overexpression targeted to pulmonary alveolar cells was sufficient to induce lung adenomas and carcinomas. Tumorigenesis was assisted by either spontaneous mutations in Kras or experimental introduction of activated RAS, but investigations revealed that additional events were required to circumvent apoptosis, one of the most significant negative functions exerted by MYC. We determined that overexpression of the antiapoptotic protein MCL1 was sufficient to circumvent apoptosis in this setting. Previous clinical studies have indicated that prognosis of human NSCLC is not associated with MCL1, despite its overexpression in many NSCLCs. In reexamining the prognostic value in this setting, we found that MCL1 overexpression does correlate with poor patient survival, but only when accompanied by MYC overexpression. Our findings therefore produce a convergence of mouse and human results that explain how MCL1 can block an important negative consequence of MYC overexpression in both experimental models and clinical cases of NSCLC.

High frequency of development of B cell lymphoproliferation and diffuse large B cell lymphoma in Dbl knock-in mice

Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho, Rac, and Cdc42 and to induce a transformed phenotype in murine fibroblasts. We previously reported that Dbl-null mice are viable and fertile but display defective dendrite elongation of distinct subpopulations of cortical neurons, suggesting a role of Dbl in controlling dendritic growth. To gain deeper insights into the role of Dbl in development and disease, we attempted a knock-in approach to create an endogenous allele that encodes a missense-mutation-mediated loss of function in the DH domain. We generated, by gene targeting technology, a mutant mouse strain by inserting a mutagenized human proto-Dbl cDNA clone expressing only the Dbl N terminus regulatory sequence at the starting codon of murine exon 1. Animals were monitored over a 21-month period, and necropsy specimens were collected for histological examination and immunohistochemistry analysis. Dbl knock-in mice are viable and did not manifest either decreased reproductive performances or gross developmental phenotype but revealed a reduced lifespan compared to wild-type (w.t.) mice and showed, with aging, a B cell lymphoproliferation that often has features of a frank diffuse large B cell lymphoma. Moreover, Dbl knock-in male mice displayed an increased incidence of lung adenoma compared to w.t. mice. These data indicate that Dbl is a tumor susceptibility gene in mice and that loss of function of Dbl DH domain by genetic missense mutations is responsible for induction of diffuse large B cell lymphoma.

Lung Tumorigenesis Suppressing Effects of a Commercial Kava Extract and Its Selected Compounds in A/J Mice

Lung cancer is the most deadly malignancy in the US. Chemoprevention is potentially a complementary approach to smoking cessation for lung cancer control. Recently, we reported that a commercially available form of kava extract significantly inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo(a)pyrene (BaP)-induced lung tumorigenesis in A/J mice at a dose of 10 mg per gram diet. In the present study, we examined the dose-dependent lung tumor inhibitory activities of kava and investigated potential active constituent(s). Mice treated with carcinogen alone contained 12.1±5.8 lung adenomas per mouse 22 weeks after final carcinogen administration. Mice that were fed diets containing kava at dosages of 1.25, 2.5, 5, and 10 mg/g of diet had 8.4±3.5, 6.6±3.5, 4.3±2.4, and 3.8±2.3 lung adenomas per mouse, respectively. This corresponds to a reduction of 31%, 46%, 65% and 69% in tumor multiplicity, which were all statistically significant (p < 0.05). Analyses of lung adenoma tissues derived from kava-treated animals revealed that kava significantly inhibited adenoma cell proliferation while it had no detectable effect on cell death, indicating that kava primarily suppressed lung tumorigenesis in A/J mice via inhibition of cell proliferation. Flavokawains A, B, and C, three chalcone-based components from kava, demonstrated greatly reduced chemopreventive efficacies even at concentrations much higher than their natural abundance, suggesting that they alone were unlikely to be responsible for kava's chemopreventive activity. Kava at all dosages and treatment regimens did not induce detectable adverse effects, particularly with respect to liver. Specifically, kava treatment showed no effect on liver integrity indicator enzymes or liver weight, indicating that kava may be potentially safe for long-term chemopreventive application.

Abstract A102: Chemopreventive efficacy and mechanistic studies of Targretin in the B[a]P-induced mouse lung tumor prevention model

Abstract Aspiration route has been demonstrated to be as effective as inhalation in administering agents to lung with associated reduction in cost for equipment and chemopreventive agent. The current study evaluated the chemopreventive efficacy and mechanism (s) of Targretin, a novel oral synthetic retinoid that selectively binds and activates retinoid X receptors, via oropharyngeal aspiration against the strain A/J mouse lung adenoma induced by benzo[a]pyrene (B[a]P). Lung adenomas were induced in 8-week old female strain A/J mice by three oral gavages of B[a]P (2 mg /0.2 ml cotton seed oil). The mice were allowed 4 weeks to develop dysplasia, then given one of three doses of Targretin (0.1, 0.3 and 1 mg/kg) for 16 weeks. At different time points, the expression of a series of biomarkers (e.g., Cox-2 and 5-Lox at 2 and 16 weeks, survivin, PCNA and apoptosis at 16 weeks) in lung was evaluated by Western Blot and immunohistochemistry. Tumor incidence and multiplicity in the lung were measured following 16-week aspiration of Targretin and compared with B[a] P only group. Targretin showed dose-related inhibition of B[a]P-induced tumor incidence (p&amp;lt;0.05) at 0.1 and 0.3 mg/kg following 16-week treatment with no effect on tumor multiplicity. Targretin also showed dose-dependent inhibition of 5-Lox (25-47%) at 2 and 16 weeks, and Cox-2 (27-38%) and survivin (39-48%) at 16 weeks. A dose-dependent induction of apoptosis (2.2-3.0-fold) and a marginal but dose-dependent inhibition of PCNA were also observed at 16 weeks. In conclusion, this study demonstrated that Targretin via aspiration showed chemopreventive efficacy against mouse lung adenoma with good correlation to early and late biomarkers of lung cancer progression. Also, it indicated that the aspiration route can be an excellent inexpensive alternative to inhalation for direct drug delivery to the rodent lung for efficacy testing of potential chemopreventive agents (Supported by NCI #N01-CN-53301). Citation Information: Cancer Prev Res 2010;3(12 Suppl):A102.

Synergism between 2,3,7,8-tetrachlorodibenzo-p-dioxin and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone on lung tumor incidence in mice

Although 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is classified as a human carcinogen, TCDD only induced oxidative DNA damages. In our present study, we combined TCDD with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to investigate their tumorigenic effects on lung tumor formation in A/J mice. Application of NNK at a tumorigenic dose (2mg/mouse) induced lung adenoma in both male and female A/J mice. Neither application of NNK at a non-tumorigenic dose (1mg/mouse) nor repeated application of TCDD alone increased tumor incidence. Following the single injection of NNK at a non-tumorigenic dose (1mg/mouse), repeated application of TCDD significantly increased the lung tumor incidence in female, but not in male, A/J mice 24 weeks later. Utilizing the real-time RT-PCR array, we found that P16 mRNA was significantly reduced in female lung, but not male lung, of NNK/TCDD co-treated A/J mice. With immunohistochemical staining, we confirmed that nuclear P16 protein was reduced in the lungs of NNK/TCDD co-treated female mice. These data suggest that P16 reduction at least partially contributed to synergistic effects of TCDD in lung tumorigenesis.

RETRACTED ARTICLE: Carboxypeptidase E: Elevated Expression Correlated with Tumor Growth and Metastasis in Pheochromocytomas and Other Cancers

Expression of carboxypeptidase E (CPE), a prohormone processing enzyme in different cancer types, was analyzed from data in the GEO profile database (http://www.ncbi.nlm.nih.gov/geo/) and experimentally in pheochromocytomas. Analysis of microarray data demonstrated that significantly elevated levels of CPE mRNA was found in many metastatic non-endocrine cancers: cervical, colon rectal, renal cancers, Ewing sarcomas (bone cancer), and various types of astrocytomas and oligodendrogliomas, whereas expression of CPE mRNA was virtually absent in their respective counterpart normal tissues. Moreover, there was higher CPE mRNA expression in cells from the metastatic tumor compared to those from the primary tumor in colorectal cancer. Elevated CPE mRNA expression was found in neuroendocrine tumors in lung and pituitary adenomas, although the significance is unclear since endocrine and neuroendocrine cells normally express CPE. However, studies of neuroendocrine tumors, pheochromocytomas, revealed expression of not only wild-type CPE, but a variant which was correlated with tumor behavior. Extremely high CPE mRNA copy numbers of the variant were found in very large or invasive tumors, both of which usually indicate poor prognosis. Thus, collectively the data suggest that CPE may play a role in promoting tumor growth and invasion. CPE could potentially serve as a diagnostic and prognostic biomarker for metastasis in different cancer types.

Aggressive mammary carcinoma progression in Nrf2 knockout mice treated with 7,12-dimethylbenz[a]anthracene

BackgroundActivation of nuclear factor erythroid 2-related factor (Nrf2), which belongs to the basic leucine zipper transcription factor family, is a strategy for cancer chemopreventive phytochemicals. It is an important regulator of genes induced by oxidative stress, such as glutathione S-transferases, heme oxygenase-1 and peroxiredoxin 1, by activating the antioxidant response element (ARE). We hypothesized that (1) the citrus coumarin auraptene may suppress premalignant mammary lesions via activation of Nrf2/ARE, and (2) that Nrf2 knockout (KO) mice would be more susceptible to mammary carcinogenesis.MethodsPremalignant lesions and mammary carcinomas were induced by medroxyprogesterone acetate and 7,12-dimethylbenz[a]anthracene treatment. The 10-week pre-malignant study was performed in which 8 groups of 10 each female wild-type (WT) and KO mice were fed either control diet or diets containing auraptene (500 ppm). A carcinogenesis study was also conducted in KO vs. WT mice (n = 30-34). Comparisons between groups were evaluated using ANOVA and Kaplan-Meier Survival statistics, and the Mann-Whitney U-test.ResultsAll mice treated with carcinogen exhibited premalignant lesions but there were no differences by genotype or diet. In the KO mice, there was a dramatic increase in mammary carcinoma growth rate, size, and weight. Although there was no difference in overall survival, the KO mice had significantly lower mammary tumor-free survival. Also, in the KO mammary carcinomas, the active forms of NF-κB and β-catenin were increased ~2-fold whereas no differences in oxidized proteins were observed. Many other tumors were observed, including lymphomas. Interestingly, the incidences of lung adenomas in the KO mice were significantly higher than in the WT mice.ConclusionsWe report, for the first time, that there was no apparent difference in the formation of premalignant lesions, but rather, the KO mice exhibited rapid, aggressive mammary carcinoma progression.

Spontaneous development of lung adenocarcinoma in the CADM1 gene-deficient mice

Aberration of cell adhesion is a critical step in the development and progression of human tumors. A tumor suppressor gene, CADM1(Cell adhesion molecule 1)/TSLC1(Tumor suppressor in lung cancer 1), was originally identified in non-small cell lung cancer (NSCLC) by its tumor suppressor activity in nude mice. CADM1 encodes an immunoglobulin superfamily cell adhesion molecule which is expressed in the brain, testis, lung and various other epithelial tissues. In normal polarized epithelia, CADM1 is expressed along the lateral membrane and associates in the cytoplasm with a member of 4.1-family proteins carrying actin-binding activity. On the other hand, CADM1 is inactivated by loss of the chromosomal fragment and/or methylation of the gene promoter in 30-60% of various human cancers, including NSCLC. To understand the physiological roles of CADM1, we have generated Cadm1/Tslc1 gene-deficient mice and have found that Cadm1-/- male mice are infertile due to the disruption of cell adhesion between the immature spermatocytes and Sertoli cells. Here, we demonstrate that more than 30% of Cadm1-/- mice developed lung adenomas or adenocarcinomas spontaneously at 15 months of age. In the tumors, normal alveolar structure was completely replaced by the tumor cells with atypical nuclei. Lung tumors also developed in Cadm1+/- mice at 18 months of age through the second hit of the Cadm1 gene, indicating that CADM1 cascade is critical to lung tumor suppression. Immunohistochemical study revealed that the membrane localization of 4.1N and CADM4, another member of CADM family proteins expressed in the lung, was abrogated in the tumors but not in the normal epithelia. These results suggest that the disruption of the CADM-4.1 cascade of cell adhesion is prerequisite to lung tumor formation.

Low dosage 5-fluorouracil increases the transfection efficiency of Ad/VEGF-A in mouse lung carcinoma cell line LA795 and inhibits tumor growth

Adenovirus vectors were widely used in gene therapy for tumors. We used adenovirus vector to transfer small interfering RNA (siRNA) against vascular epithelium growth factor A (VEGF-A) molecules to mouse lung adenoma LA795 cells and used low dose of chemotherapeutic drugs to further elevate the infection efficiency of adenovirus vector in and therapeutic effect of RNAi on tumor cells. LA795 cells were infected by Ad/EGFP and treated with different dosages of gemcitabin, epirubicin, cisplatin, or 5-fluorouracil (5-FU). Cells were observed under fluorescence microscope continuously using green fluorescent protein (GFP) as the reporter gene. The percentage of GFP-positive cells and fluorescent intensity were tested by flow cytometry to determine optimum concentrations of drugs. Ad/siVEGF-A containing VEGF-A siRNA was constructed. Real-time PCR and ELISA were applied to measure the expression level of VEGF-A after LA795 cells were infected by Ad/siVEGF-A and treated with 5-FU. The combination of Ad/siVEGF-A and 5-FU was also applied in treating subcutaneous tumor in mice. Low dose of 5-FU elevated the Ad/EGFP infection in LA795 cells significantly, and also enhanced the effect of Ad/siVEGF-A in down-regulating VEGF-A mRNA and protein levels in tumor cells. When used in tumor in vivo, the combination strategy repressed tumor growth effectively. Low dose of 5-FU can enhance the capability of adenovirus infecting tumor cells and promote the efficiency of gene therapy by adenovirus.

Carcinogenic Interactions between a Single Inhalation of239PuO2and Chronic Exposure to Cigarette Smoke in Rats

Rats were exposed once by inhalation to plutonium-239 dioxide ((239)PuO(2)), resulting in chronic alpha-particle irradiation of the lung, and exposed chronically to cigarette smoke to examine carcinogenic interactions between the two exposures. F344 rats were exposed to (239)PuO(2) to achieve an initial lung burden of 0.5 kBq and then exposed 6 h/day, 5 days/week to cigarette smoke at 100 or 250 mg particulate matter/m(3) for up to 30 months. Exposure to cigarette smoke increased the cumulative radiation dose to lung by slowing the clearance of (239)PuO(2). (239)PuO(2) alone did not affect survival, but the higher cigarette smoke exposure shortened survival in females. Combined exposure to (239)PuO(2) and cigarette smoke acted synergistically to shorten survival in both genders. The combined effects of cigarette smoke and (239)PuO(2) were approximately additive for lung hyperplasia and adenomas but were strongly synergistic for carcinomas. Differences between observed incidences and incidences predicted by survival-adjusted models accounting for increased radiation dose revealed a substantial component of synergy for carcinomas above that attributable to the radiation dose effect. The synergy for malignant lung tumors is consistent with findings from uranium miners and nuclear weapons production workers. These results bolster confidence in the epidemiological findings and have implications for risk assessment.

Abstract SY02-03: Studying tumor evolution in mouse models of cancer

Abstract Like other solid tumors, lung adenocarcinoma progresses through multiple stages including tumor initiation, expansion in the lung, and metastasis of the primary tumor to distant organs. Dissection of this process requires quantitative and tractable models that recapitulate the human disease at the genetic and histological level. We have employed autochthonous mouse models of lung adenocarcinoma that recapitulate tumor initiation, progression and metastasis in the human disease both genetically, and pathologically. We have established oncogenic Kras as the initiating event in lung adenoma and amplification of the oncogenic signaling is associated with progression to adenocarcinoma. The p53 tumor suppressor limits tumor progression in this model. Restoration of the Trp53 locus has pinpointed the adenoma-adenocarcinoma transition as the stage in which p53 function is critical for tumor suppression. Expression of oncogenic Kras in the absence of p53 leads to the development of distant metastases after long latency. Identification of the lentiviral integration site in each tumor allows the clonal relationship between metastases and the primary tumors to be unequivocally established. Gene expression and DNA copy number analyses of metastasis-derived cell lines and primary lung tumors have uncovered a metastasis associated gene expression signature and recurrent genomic alterations. Integration of these datasets, other genome-wide analyses and human tumor data are providing insights into the molecular mechanism and biology of lung cancer progression and metastasis. Citation Format: Tyler Jacks. Studying tumor evolution in mouse models of cancer [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY02-03

Abstract 2869: Chemopreventive efficacy and mechanism of licofelone in mouse lung tumor model

Abstract Our previous study comparing inhalation and aspiration methods of administration of an agent to lung airway epithelia indicated that aspiration can be an equally effective route of exposure while reducing the need for inhalation equipment and amount of chemopreventive agent required. The current study evaluated the chemopreventive efficacy and mechanism of Licofelone, a dual inhibitor of cyclooxygenase-2 (Cox-2) and 5-lipoxygenase (5-Lox), against the strain A/J mouse lung adenoma induced by benzo[a]pyrene (B[a]P). The Licofelone was administered to the lung via oropharyngeal aspiration. Lung adenomas were induced in 8-wk old female strain A/J mice by three oral gavages of B[a]P (2 mg /0.2 ml cotton seed oil). The mice were left for 4 weeks to develop dysplasia, then given one of three doses of Licofelone (0.03, 0.1 and 0.3 mg/kg) for 16 weeks. At different time points, the expression of a series of biomarkers (e.g., Cox-2 and 5-Lox at 2 weeks, survivin, PCNA and apoptosis at 16 weeks) in lung was evaluated by Western Blot and immunohistochemistry. Tumor incidence and multiplicity in the lung were measured following 16-week aspiration of Licofelone and compared with B[a] P only group. The data showed that Licofelone inhibited Cox-2 (26-42%) and 5-Lox (43-61%) in a dose-dependent manner at 2 weeks. Both survivin (16-25%) and PCNA (41-61%) were also inhibited in a dose-dependent manner. There was a 2.4 fold induction of apoptosis at 0.3 mg/kg when compared to the B[a] P only group. Licofelone showed significant dose-related inhibition of B[a]P-induced tumor incidence (p = &amp;lt;0.05) and multiplicity at 0.03 and 0.1 mg/kg following 16-week treatment. In conclusion, this study demonstrated that Licofelone administered by aspiration showed chemopreventive efficacy against mouse lung adenoma with good correlation to early and late biomarkers of lung cancer progression. Also, it showed that the aspiration route can be an excellent alternative to inhalation for direct delivery of drugs to the rodent lung. (Supported by NCI #N01-CN-53301). 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 2869.

Abstract 4995: Loss of the lung-specific tumor suppressor Gprc5a causes persistent activation of Stat3 leading to increased survival signaling in normal and malignant lung epithelial cells

Abstract The G protein-coupled receptor, family C, group 5, member A (GPRC5A), a retinoic acid induced gene expressed primarily in human and mouse lung tissue. Our previous studies demonstrated that mice with a deletion of the Gprc5a gene develop spontaneous lung adenomas and adenocarcinomas. Since Gprc5a is expressed preferentially in epithelial cells in lung tissues, we isolated epithelial cells from tracheas of Gprc5a+/+ and Gprc5a−/− mice and used them for an analysis of phenotypic and genotypic differences that might explain the mechanisms of Gprc5a actions. We found that normal Gprc5a−/− cells can survive better than Gprc5a+/+ cells in basal medium deprived of supplemented growth factors. Furthermore, the Gprc5a−/− cells but not the Gprc5a+/+ cells were able to form colonies in semi-solid medium. To explore possible mechanisms that underlay the distinct phenotypes of the above cells, we compared and contrasted the survival signaling pathway mediated by the Signal Transducer and Activator of Transcription 3 (STAT3), which is triggered by certain cytokines and growth factors to regulate the expression of genes implicated in cell growth differentiation, survival, inflammation and transformation. Increased Stat3 activity by phosphorylation has been identified in a variety of tumors including non-small cell lung cancers (NSCLCs) and it has been linked to inflammation and tumor progression. The constitutive activity (phosphorylation of tyrosine 705) of Stat3 and the expression of several Stat3 regulated genes including survivin, Bcl-XL, and Mcl1 at both the mRNA and protein levels was higher in the Gprc5a−/− cells compared to normal Gprc5a+/+ cells. Stat3 activation was triggered by autocrine Leukemia inhibitory factor (Lif), a member of the interleukine-6 (IL-6) family of cytokines, as revealed after adding neutralizing antibodies against different Stat3 activating cytokines to the growth medium. Although Gprc5a+/+ cells produced and secreted sufficient amounts of Lif into their conditioned medium, their response to Lif was transient, whereas the Gprc5a−/− cells showed a prolonged response evidenced by a persistent Stat3 activation. Lung cancer cells isolated from an adenocarcinorma of Gprc5a−/− mice exhibited Lif-mediated Stat3 activation similar to the normal Gprc5a−/− cells. Blocking Stat3 activation using AG490, an inhibitor of Stat3 signaling, or using a dominant negative Stat3 (mutant Y705F) in the Gprc5a−/− normal and cancer cells increased apoptosis and inhibited anchorage-independent growth. These results demonstrate that Stat3 activation may be important for the early stages of lung tumorigenesis in Gprc5a−/− mice and by implication that the tumor suppressive effects of Gprc5a are mediated, at least in part, by suppression of Stat3 signaling. Supported in part by the Samuel Waxman Cancer Research Foundation. 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 4995.

Abstract 257: R4 knock-out mice showed lung hyperplasia and susceptible to urethane-induced lung carcinogenesis

Abstract Lung cancer is responsible for 1.3 million deaths worldwide annually. There have been several reports on candidate genes driving lung cancers including ras. However still it is needed to find new genes which can cause lung cancers. Recently we developed R4 knock-out (KO) mice which showed larger relative lung weight and volume than age-matched wild-type (WT) mice. Lung hyperplasia was found with increased alveolar thickness in 6, 18 and 40 week-old R4 KO mice. Also, this finding was observed in E16.5 and 18.5 day KO mice. Cell cycle markers including PCNA and cyclinD1 were highly expressed in the lung of R4 KO mice than WT mice. PCNA positive cells were mostly type 2 pneumocytes. To determine the susceptibility of lung cancer in R4 KO mice, we induced lung cancer by the injection of 1g/kg urethane weekly for a total of eight doses. Twenty weeks after the initial injection, mice were sacrificed, and the lungs were fixed, and the number of tumors on the lung surface was assessed. Also lung tumors were scanned by micro-CT at the end of the lung carcinogenesis protocol for measure the tumor diameter. Surface lung tumor number is significantly increased in KO mice. And tumor size is also significantly larger in KO mice than WT. Histological findings showed more severe well differentiated lung adenoma in urethane-injected R4 KO mice. We conclude that R4 KO mice is susceptible to tumorigenesis in the urethane model and the functional loss of R4 gene may cause lung cancer. Therefore, R4 gene has a possibility as a potential target for chemoprevention of lung cancer. 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 257.

Abstract 3227: Lung-targeted expression of B-RAF V600E in alveolar type II cells causes emphysema and delayed adenoma in mice

Abstract Classically oncogenes are found associated with neoplastic diseases. More recently a connection with developmental syndromes has been reported. Here we describe a link between the most prevalent mutant form of B-RAF oncogene (B-RAF V600E) with Chronic Obstructive Pulmonary Disease (COPD) that often precede lung cancer in men. By use of targeted expression of this mutant B-RAF in type II pneumocytes of mice we have developed an animal model for these devastating diseases that are epidemic worldwide. Constitutive expression of B-RAF V600E led to emphysema as early as late embryonic development that is persistent throughout adult life when it is often accompanied by chronic inflammation. Infiltrating immune cells include localized macrophages, mast cells, and leukocytes as in the case of human COPD. Inflammatory cell infiltration does not precede emphysema formation but is a prelude to tumor development. Consistently, ablation of RAG1 alleles did not eliminate emphysema or late tumor development. The oncogene involved in tumor formation is also B-RAF V600E because the most frequent mutations of spontaneous lung adenomas in mice were absent. These data support a model where the regenerative process triggered by continuous emphysema formation is the driving force that converts B-RAF V600E from a latent to an active oncogene in this target cell type. Our data suggest that B-RAF V600E is not a complete oncogene in type II cells and requires promoting conditions that can be provided by emphysema. 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 3227.

Abstract 951: Inhibition of lung tumorigenesis by oral consumption of pomegranate fruit extract

Abstract Lung cancer is the most common cause of cancer mortality worldwide and continues to be a major health problem, exceeding the combined mortality rates of colorectal, breast and prostate cancers. It has proven difficult to control lung cancer with conventional therapeutic and surgical approaches, and generally the prognosis is poor with an overall five-year survival rate of 10-14% in the USA. A competent approach to reduce the mortality from lung cancer is the early detection of premalignant lesions as the survival of early stage lung cancer patients is much better as compared to patients with advanced cancers. The current mainstays of lung cancer therapy are surgical resection, radiation ablation and systemic chemotherapy with considerable toxicity. However, it appears unlikely that these therapies can cause striking improvements in near future. Thus, effective approaches to the prevention of lung cancer need to be developed, especially efforts targeted to the progression of preneoplastic and benign neoplastic lesions to more malignant lung tumors. The pomegranate (Punica granatum; Punicaceae) fruit has been used for centuries in ancient cultures for its medicinal purposes and we have been defining the chemopreventive effects of pomegranate fruit extract (PFE). Based on MALDI-TOF MS analysis, PFE was found to contain anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose). We have earlier shown that PFE inhibits prosurvival signaling pathways in human lung carcinoma A549 cells and tumor growth in athymic nude mice (Carcinogenesis 2007;28:163-73) and A/J mice (Cancer Res 2007;67:3475-82). In this study, the chemopreventive activity of PFE during progression of lung adenomas was investigated in two mouse tumor protocols. Female A/J mice were treated with 0.2% PFE in drinking water (w/v, equivalent of human consumption of juice derived from two fruits) till the termination of the experiment. One week after treatment with PFE, mice were given a single i.p. injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at dose of 100 mg/kg body weight or urethane at dose of 1 mg/g body weight. Mice were sacrificed at 16 weeks after NNK treatment and 20 weeks after treated with urethane. PFE administration significantly reduced the lung tumor multiplicity by 47% in mice treated with NNK and 52% in urethane-treated mice. Markers of cell proliferation, apoptosis and angiogenesis were studied by immunohistochemical staining. Treatment with PFE caused significant reduction in Ki-67, proliferating cell nuclear antigen (PCNA), caspase-3, CD-31 and vascular endothelial growth factor (VEGF) in lungs of mice treated with NNK and urethane. Thus, our results show that PFE can act as a chemopreventive agent in smokers and ex-smokers with early lung lesions. 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 951.

Abstract 4992: Spontaneous development of lung adenocarcinoma in the Cadm1 gene-deficient mice

Abstract Aberration of cell adhesion is a critical step in the development and progression of human tumors. A tumor suppressor gene, CADM1(Cell adhesion molecule 1)/TSLC1(Tumor suppressor in lung cancer 1), was originally identified in non-small cell lung cancer (NSCLC) by its tumor suppressor activity in nude mice. CADM1 encodes an immunoglobulin superfamily cell adhesion molecule which is expressed in the brain, testis, lung and various other epithelial tissues. In normal polarized epithelia, CADM1 is expressed along the lateral membrane and associates in the cytoplasm with a member of 4.1-family proteins carrying actin-binding activity and a member of the membrane scaffold proteins, MAGuKs, carrying PDZ-domains. On the other hand, CADM1 is inactivated by loss of the chromosomal fragment and/or methylation of the gene promoter in 30-60% of various human cancers, including NSCLC. Here, we demonstrate that CADM1 expressing cell lines, HEK293 and Caco-2, lose their epithelia-like cell structure and show flat morphology with immature cell adhesion when the CADM1 function is abrogated by RNAi. CADM1-binding proteins, 4.1B and MPP2, as well as E-cadherin and ZO-1, are mislocalized from the membrane in HEK293 cells. Loss of CADM1 is also correlated with the transformed phenotype of cancer cells, suggesting that CADM1 is involved in the formation of epithelia-like cell structure with 4.1B and MPPs, while loss of its function could cause morphological transformation of cancer cells. To further understand the physiological roles of CADM1, we have generated Cadm1/Tslc1 gene-deficient mice and have found that Cadm1−/− male mice are infertile due to the disruption of cell adhesion between the immature spermatocytes and Sertoli cells. Here, we also demonstrate that more than 30% of Cadm1−/− mice developed lung adenomas or adenocarcinomas spontaneously at 15 months of age. In the tumors, normal alveolar structure was completely replaced by the tumor cells with atypical nuclei. Lung tumors also developed in Cadm1+/− mice at 18 months of age through the second hit of the Cadm1 gene by the promoter methylation or loss of the gene fragment, indicating that CADM1 cascade is critical to lung tumor suppression. Immunohistochemical study revealed that the membrane localization of 4.1N and CADM4, another member of CADM family proteins expressed in the lung, was abrogated in the tumors but not in the normal epithelia, suggesting that the disruption of the CADM-4.1 cascade of cell adhesion is prerequisite to lung tumor formation. Furthermore, several oncogenic signal transduction cascades were activated in these tumors. These results suggest that CADM1 is involved in lung tumor suppression through the maintenance of cell adhesion machinery as well as suppression of the growth factor cascades. 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 4992.

Runx3 is required for the differentiation of lung epithelial cells and suppression of lung cancer

Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molecular abnormalities. K-ras mutations are associated with the initiation of lung adenocarcinomas, but K-ras-independent mechanisms may also initiate lung tumors. Here, we find that the runt-related transcription factor Runx3 is essential for normal murine lung development and is a tumor suppressor that prevents lung adenocarcinoma. Runx3-/- mice, which die soon after birth, exhibit alveolar hyperplasia. Importantly, Runx3-/- bronchioli exhibit impaired differentiation, as evidenced by the accumulation of epithelial cells containing specific markers for both alveolar (that is SP-B) and bronchiolar (that is CC10) lineages. Runx3-/- epithelial cells also express Bmi1, which supports self-renewal of stem cells. Lung adenomas spontaneously develop in aging Runx3+/- mice ( approximately 18 months after birth) and invariably exhibit reduced levels of Runx3. As K-ras mutations are very rare in these adenomas, Runx3+/- mice provide an animal model for lung tumorigenesis that recapitulates the preneoplastic stage of human lung adenocarcinoma development, which is independent of K-Ras mutation. We conclude that Runx3 is essential for lung epithelial cell differentiation, and that downregulation of Runx3 is causally linked to the preneoplastic stage of lung adenocarcinoma.

The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients

DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in many human diseases, including cancer. The tobacco-specific carcinogen NNK also induces DNA methylation. However, the role of DNMT1-mediated methylation in tobacco carcinogenesis remains unclear. Here we used human and mouse lung cancer samples and cell lines to determine a mechanism whereby NNK induced DNMT1 expression and activity. We determined that in a human lung cell line, glycogen synthase kinase 3beta (GSK3beta) phosphorylated DNMT1 to recruit beta-transducin repeat-containing protein (betaTrCP), resulting in DNMT1 degradation, and that NNK activated AKT, inhibiting GSK3beta function and thereby attenuating DNMT1 degradation. NNK also induced betaTrCP translocation to the cytoplasm via the heterogeneous nuclear ribonucleoprotein U (hnRNP-U) shuttling protein, resulting in DNMT1 nuclear accumulation and hypermethylation of the promoters of tumor suppressor genes. Fluorescence immunohistochemistry (IHC) of lung adenomas from NNK-treated mice and tumors from lung cancer patients that were smokers were characterized by disruption of the DNMT1/betaTrCP interaction and DNMT1 nuclear accumulation. Importantly, DNMT1 overexpression in lung cancer patients who smoked continuously correlated with poor prognosis. We believe that the NNK-induced DNMT1 accumulation and subsequent hypermethylation of the promoter of tumor suppressor genes may lead to tumorigenesis and poor prognosis and provide an important link between tobacco smoking and lung cancer. Furthermore, this mechanism may also be involved in other smoking-related human diseases.

Abstract A70: Kava as a lung cancer chemopreventive agent

Abstract Lung cancer is a serious malignancy with high incidence and death. Chemoprevention is a complementary approach to smoking cessation to help control lung cancer. Recently, we reported that kava inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo(a)pyrene (BaP)-induced lung tumorigenesis in A/J mice. In the present study, we examined the dose-dependent lung tumor inhibitory activities of kava and attempted to identify the active constituent(s). Carcinogen-treated mice, dosed eight times with a mixture of NNK plus BaP (2 µmol each) administered by gavage, had 12.2 ± 5.7 lung adenomas per mouse after 26 weeks. Carcinogen-treated mice given diets containing kava at dosages of 1.25, 2.5, 5, and 10 mg/g had 8.4 ± 3.5, 6.6 ± 3.5, 4.3 ± 2.4, and 3.8 ± 2.3 lung adenomas per mouse, corresponding to reductions of 31%, 46%, 64%, and 69%, respectively. All reductions were significant (P &amp;lt; 0.05). Flavokawains A, B, and C from kava demonstrated either no or much weaker chemopreventive efficacies, suggesting that they are unlikely to be responsible for the chemopreventive activity of kava. Western blot analyses of lung adenoma tissues and lung cancer cells reveal that kava modulates the NF-kB signaling pathway, a potential mechanism responsible for kava-induced lung cancer chemoprevention. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A70.