Mouse Model Of Lung Adenocarcinoma Research Articles

Abstract 2255: Mechanisms governing lung adenocarcinoma metastasis

Abstract Lung cancer is the leading cause of cancer deaths in men and women in the United States. Most lung cancer patients are diagnosed at a stage when they already have inoperable metastases. Identifying the drivers of lung cancer metastatic potential and the underlying mechanisms by which this state is induced, reversed or maintained is a question of immense clinical importance. Understanding the metastatic process in humans is limited, as early tumors exist in undiagnosed patients. Genetically engineered mouse models of lung adenocarcinoma provide an opportunity to study lung tumor progression and metastasis in vivo. We have recently improved upon existing lung adenocarcinoma mouse models enabling us to alter genes in developing tumors in vivo using lentiviral vectors that co-deliver Cre-recombinase and a cDNA. Additionally, the introduction of a tdTomato Cre reporter allows us to identify and isolate cells from all stages of cancer progression including primary tumors, disseminating tumor cells, circulating tumor cells, and micro-and macro-metastases. We hypothesize that differential gene expression between primary tumors and their metastases will identify key determinants of lung cancer metastatic progression. To this end, we have used RNA-Seq to identify differentially expressed genes between tumors and their metastases and validated a subset of these genes by Fluidigm® microfluidics qPCR. Our validated genes include the lysyl hydroxylase Plod2 (a collagen modifier) and the transcription factor Arntl2, both recapitulating gene expression correlations observed in human metastatic lung adenocarcinoma. Our data demonstrate that knockdown of Plod2 decreases the metastatic properties of mouse metastatic cell lines, specifically migration and the ability to give rise to metastases in transplantation assays. Migration and colony morphology defects upon knockdown of Plod2 could be rescued by the addition of normal collagen. Further in vitro experiments using soft agar assays demonstrated that an additional candidate gene, the transcription factor Arntl2, is required for anchorage independent growth of mouse metastatic cell lines. These findings were extended to in vivo transplant experiments, demonstrating that Arntl2 is critical for metastatic seeding. This in vivo phenotype was rescued by expression of a hairpin insensitive Arntl2 cDNA, demonstrating the specific role of Arntl2 in metastatic progression. Current efforts are focused on confirming the role of Arntl2 in human lung adenocarcinoma and determining the molecular mechanism of how Arntl2 modulates cancer progression and metastasis using RNA-Seq. Together, these studies will increase our knowledge of factors that mediate human lung adenocarcinoma progression with the potential to identify new targets for intervention at distinct steps of the metastatic cascade. Citation Format: Jennifer J. Brady, Chen-Hua Chuang, Deborah R. Caswell, Monte M. Winslow. Mechanisms governing lung adenocarcinoma metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2255. doi:10.1158/1538-7445.AM2015-2255

Therapeutic effect of anti CEACAM6 monoclonal antibody against lung adenocarcinoma by enhancing anoikis sensitivity

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) plays a crucial role in tumorigenesis of lung cancer. However, the therapeutic potential for anti CEACAM6 monoclonal antibody (mAb) has only been limitedly explored. Here, we evaluate the therapeutic potential of naked anti CEACAM6 mAb against lung adenocarcinoma. Clone 8F5, recognizing B domain of CEACAM6, is established by immunizing A549 cells and screening for clones double positive for A549 and CEACAM6-Fc recombinant protein. We found that 85.7% of 70 resected lung adenocarcinoma tissue sections were positive for CEACAM6, whereas all squamous cell carcinoma examined were negative. A549 cells with high levels of CEACAM6 demonstrated more aggressive growth nature and showed increased paclitaxel chemosensitivity upon 8F5 binding. Treatment with 8F5 to A549 decreased cellular CEACAM6 expression and reversed anoikis resistance. 8F5 also decreased cellular status of Akt phosphorylation and increased apoptosis via caspase activation. In a mouse model of lung adenocarcinoma with xenotransplanted A549 cells, 8F5 treatment alone demonstrated 40% tumor growth inhibition. When combined with paclitaxel treatment, 8F5 markedly enhanced tumor growth inhibition, up to 80%. In summary, we demonstrate that anti CEACAM6 mAb is an effective therapeutic treatment for lung adenocarcinoma whose effect is further enhanced by combined treatment with paclitaxel.

The LIM-domain only protein 4 contributes to lung epithelial cell proliferation but is not essential for tumor progression

BackgroundThe lung is constantly exposed to environmental challenges and must rapidly respond to external insults. Mechanisms involved in the repair of the damaged lung involve expansion of different epithelial cells to repopulate the injured cellular compartment. However, factors regulating cell proliferation following lung injury remain poorly understood. Here we studied the role of the transcriptional regulator Lmo4 during lung development, in the regulation of adult lung epithelial cell proliferation following lung damage and in the context of oncogenic transformation.MethodsTo study the role of Lmo4 in embryonic lung development, lung repair and tumorigenesis, we used conditional knock-out mice to delete Lmo4 in lung epithelial cells from the first stages of lung development. The role of Lmo4 in lung repair was evaluated using two experimental models of lung damage involving chemical and viral injury. The role of Lmo4 in lung tumorigenesis was measured using a mouse model of lung adenocarcinoma in which the oncogenic K-Ras protein has been knocked into the K-Ras locus. Overall survival difference between genotypes was tested by log rank test. Difference between means was tested using one-way ANOVA after assuring that assumptions of normality and equality of variance were satisfied.ResultsWe found that Lmo4 was not required for normal embryonic lung morphogenesis. In the adult lung, loss of Lmo4 reduced epithelial cell proliferation and delayed repair of the lung following naphthalene or flu-mediated injury, suggesting that Lmo4 participates in the regulation of epithelial cell expansion in response to cellular damage. In the context of K-RasG12D-driven lung tumor formation, Lmo4 loss did not alter overall survival but delayed initiation of lung hyperplasia in K-RasG12D mice sensitized by naphthalene injury. Finally, we evaluated the expression of LMO4 in tissue microarrays of early stage non-small cell lung cancer and observed that LMO4 is more highly expressed in lung squamous cell carcinoma compared to adenocarcinoma.ConclusionsTogether these results show that the transcriptional regulator Lmo4 participates in the regulation of lung epithelial cell proliferation in the context of injury and oncogenic transformation but that Lmo4 depletion is not sufficient to prevent lung repair or tumour formation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0228-0) contains supplementary material, which is available to authorized users.

Isolation of epithelial, endothelial, and immune cells from lungs of transgenic mice with oncogene-induced lung adenocarcinomas.

Genetically engineered mouse models of lung adenocarcinoma have proven invaluable for understanding mechanisms of tumorigenesis, therapy response, and drug resistance. However, mechanistic studies focused on studying these processes in tumor-bearing mouse lungs are confounded by the fact that, in most cases, relevant signaling pathways are analyzed in whole-lung preparations, which are composed of a heterogeneous mixture of cells. Given our increasing knowledge about the roles played by different subpopulations of cells in the development of lung adenocarcinoma, separating the major cellular compartments of the tumor microenvironment is recommended to allow for a precise analysis of relevant pathways in each isolated cell type. In this study, we optimized magnetic- and fluorescence-based isolation protocols to segregate lung epithelial (CD326/epithelial cell adhesion molecule-positive), endothelial (CD31-positive), and immune (CD45-positive) cells, with high purity, from the lungs of transgenic mice with mutant epidermal growth factor receptor-induced lung adenocarcinomas. This approach, which can potentially be extended to additional lung adenocarcinoma models, enables delineation of the molecular features of individual cell types that can be used to gain insight into their roles in lung adenocarcinoma initiation, progression, and response to therapy.

Therapeutic efficacy of the F8-IL2 immunocytokine in a metastatic mouse model of lung adenocarcinoma.

Antibody-cytokine fusion proteins (immunocytokines) represent a novel class of armed antibodies in oncology. In particular, IL2- and TNF-based immunocytokines targeting the EDB domain of fibronectin and the A1 domain of tenascin-C have demonstrated promising anti-tumor activity and are currently investigated in Phase I and Phase II clinical trials. To advance the development of immunocytokines for NSCLC, we here report on the therapeutic efficacy of F8-IL2, an immunocytokine directed against the alternatively spliced EDA domain of fibronectin in a fully immunocompetent, orthotopic model of NSCLC, and the characterization of the target antigen expression in human NSCLC specimens. We evaluated the therapeutic efficacy of the F8-IL2 immunocytokine utilizing a K-ras mutant, p53 deficient metastatic mouse model of NSCLC derived from the latest generation of genetically engineered and conditional tumor models. In parallel, we assessed the presence of the EDA domain of fibronectin by immunofluorescence in lung biopsies obtained from patients with NSCLC. The EDA domain of fibronectin was broadly expressed in lung metastases obtained from our model. Treatment with F8-IL2 induced substantial local changes within immune effector cell populations and demonstrated promising therapeutic efficacy as monotherapy. The target of F8-IL2, the EDA domain of fibronectin, was present in all human lung adenocarcinoma specimens tested. Both the therapeutic efficacy in a metastatic mouse model of NSCLC and the extensive presence of the EDA domain of fibronectin in human NSCLC biopsies support the rational development of therapies based on the F8-IL2 immunocytokine for the treatment of NSCLC.

Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma.

Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma.

Characterization of the cell of origin and propagation potential of the fibroblast growth factor 9-induced mouse model of lung adenocarcinoma.

Fibroblast growth factor 9 (FGF9) is essential for lung development and is highly expressed in a subset of human lung adenocarcinomas. We recently described a mouse model in which FGF9 expression in the lung epithelium caused proliferation of the airway epithelium at the terminal bronchioles and led to rapid development of adenocarcinoma. Here, we used this model to characterize the effects of prolonged FGF9 induction on the proximal and distal lung epithelia, and examined the propagation potential of FGF9-induced lung tumours. We showed that prolonged FGF9 over-expression in the lung resulted in the development of adenocarcinomas arising from both alveolar type II and airway secretory cells in the lung parenchyma and airways, respectively. We found that tumour cells harboured tumour-propagating cells that were able to form secondary tumours in recipient mice, regardless of FGF9 expression. However, the highest degree of tumour propagation was observed when unfractionated tumour cells were co-administered with autologous, tumour-associated mesenchymal cells. Although the initiation of lung adenocarcinomas was dependent on activation of the FGF9-FGF receptor 3 (FGFR3) signalling axis, maintenance and propagation of the tumour was independent of this signalling. Activation of an alternative FGF-FGFR axis and the interaction with tumour stromal cells is likely to be responsible for the development of this independence. This study demonstrates the complex role of FGF-FGFR signalling in the initiation, growth and propagation of lung cancer. Our findings suggest that analysing the expressions of FGF-FGFRs in human lung cancer will be a useful tool for guiding customized therapy.

Fulvestrant-mediated inhibition of estrogen receptor signaling slows lung cancer progression.

Estrogens are key signaling molecules that regulate various physiological processes such as cell growth, development, and differentiation. They also play a major role in many pathological conditions, such as hormone-dependent cancer. The importance of inhibiting estrogen receptor signaling in diseases of estrogen target tissues, such as breast cancer, is well documented. However, the role of estrogen signaling in diseases of nontarget tissues, such as lung cancer, is not well characterized. The aim of the current study is to examine the expression of estrogen receptor β (ERβ) and the roles of estradiol (E2) and fulvestrant on the progression of lung cancer. Tissue microarray (TMA) and immunohistochemistry (IHC) analyses were used to detect the expression of aromatase, ERα, and ERβ in 198 patients. We performed analyses to determine if there was any correlation among these three proteins. A mouse model of urethane-induced lung adenocarcinoma was used in the study. Mice were divided into three treatment groups: blank control, E2 alone, and E2 + fulvestrant (ERβ antagonist). Western blot analysis and fluorescence quantitative PCR (FQ-PCR) were used to measure expression of ERβ protein and mRNA levels, respectively. ERβ, but not ERα, was overexpressed in NSCLC samples. Lung cancer progression in mice treated with E2 was significantly increased compared to either the control group or the E2 + fulvestrant group. Mice in the E2 treatment group had significantly increased expression of ERβ at both the mRNA and protein levels compared to mice treated with E2 + fulvestrant or control. Our data suggest that ERβ promotes lung cancer progression in mice and that this progression can be inhibited with fulvestrant. These findings may help elucidate the role of ERβ in lung cancer and suggest that estrogen receptor antagonists, such as fulvestrant, may be therapeutically beneficial for the treatment of the disease.

Abstract 99: Wnt signaling promotes tumor propagation in a mouse model of lung adenocarcinoma

Abstract The considerable variability within tissue microenvironments as well as the multiclonality of cancers leads to heterogeneity within tumors, increasing the probablility of cellular states that promote resistance to therapy and eventually lead to reconstitution of the tumor by treatment-resistant cancer cells, which in some case have properties of normal tissue stem cells. Wnt signals are important in the maintenance of stem cells in various epithelial tissues, including in lung development and regeneration. We hypothesized that Wnt signals would also contribute to hierarchial organization of KrasG12D and KrasG12D; tp53Δ/Δ mouse lung adenocarcinomas. Indeed, rare cells with nuclear βeta-catenin were detected in mouse tumors in vivo. Stimulating KP mouse lung adenocarcinoma cells with recombinant Wnt3a promoted their spheroid-forming ability in a 3-dimensional Matrigel culture system, whereas RNAi or small-molecule inhibitors targeting Porcupine, an enzyme required for post-translational palmitoylation of Wnt, suppressed tumor initiation in vitro and in vivo. Interestingly, either the stimulation or inhibition of the Wnt signaling pathway had no effect on growth of KP lung cancer cells in standard 2-dimensional cell culture, suggesting that Wnt signaling qualitatively modulates the cellular phenotype instead of controlling cell proliferation. We propose that targeting the Wnt signaling pathway can stimulate re-differentiation of lung adenocarcinoma cells, which may render them more sensitive to conventional anti-tumor therapies. Citation Format: Tuomas Tammela, Natanya Kerper, Wen Xue, Nikhil Joshi, Pekka Katajisto, Tyler Jacks. Wnt signaling promotes tumor propagation in a mouse model of lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 99. doi:10.1158/1538-7445.AM2014-99

Abstract 70: Mouse models of human lung adenocarcinoma: Identification of potential tumor suppressors by functional in vivo shRNA screening

Abstract Lung cancer is one of the leading human cancer malignancies, and histopathologically divided into Non small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Lung adenocarcinoma is a major form of NSCLC. We have developed a lentiviral-vector-mediated mouse model, which enables generation of lung adenocarcinoma from less than 100 alveolar epithelial cells, and investigated the role of IKK2 and NF-κB in lung-cancer development. IKK2 depletion in tumour cells significantly attenuated tumour proliferation and significantly prolonged mouse survival. We identified Timp1, one of the NF-κB target genes, as a key mediator for tumour growth by activating ERK signaling pathway. Knockdown of either Ikbkb or Timp1 by short hairpin RNAs reduced tumour growth in both xenograft and lentiviral models. Further, by utilizing lentiviral mediated mouse models, we performed a genome wide in vivo shRNA screen to findout tumor suppressors or tumor suppressor like genes that will cooperate with KRasG12D expression. The screen identified several novel molecules as potential tumor suppressor properties, which will cooperate with KRasG12D. Several of these molecules were also altered in human patient cohorts of The Cancer Genome Atlas. Ephrin receptor A2, EphA2, is one of the molecule knockdown of which cooperated with KRasG12D expression to generate lung adenocarcinomas comparable to knockdown of potent tumorsuppressor P53 thus validating our screen. The mechanisms underlying EPhA2 mediated tumorsuppression are being studied. Overall, our study identified novel potential tumorsuppressors. Citation Format: Narayana Yeddula, Yifeng Xia, Inder Verma. Mouse models of human lung adenocarcinoma: Identification of potential tumor suppressors by functional in vivo shRNA screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 70. doi:10.1158/1538-7445.AM2014-70

Abstract 5343: Continuing expression of SHP2E76K is required to maintain lung tumors induced by the active SHP2E76K mutant in transgenic mice

Abstract The SHP2 protein tyrosine phosphatase is activated by growth factor receptor oncogenes or by gain-of-function (GOF) missense mutations such as SHP2E76K in human cancer. To study the oncogenic function of GOF SHP2 mutants and the dependency of SHP2 mutant-induced tumors, we established transgenic mice containing the tetO-SHP2E76K transgene. tetO-SHP2E76K mice were bred to CCSP-rtTA mice to direct expression of the transgene in type II pneumocytes to assess the role of the SHP2 mutant in lung adenocarcinoma. When the CCSP-rtTA/tetO-SHP2E76K bitransgenic mice were induced with doxycycline (dox), they developed papillary adenomas that progressed to adenocarcinoma in 6-9 months. This result demonstrates the oncogenic activity of the GOF SHP2 mutant in our mouse model of lung adenocarcinoma. Consistent with previous studies in cultured cells, lung tissues from dox-induced CCSP-rtTA/tetO-SHP2E76K have elevated pGab1, pErk, pSrc and c-Myc. Using magnetic resonance imaging (MRI), we were able to detect lung tumors in the dox-induced CCSP-rtTA/tetO-SHP2E76K bitransgenic mice. To determine if the SHP2E76K-induced lung tumors require continuing expression of the SHP2 mutant for tumor maintenance, dox was withdrawn from the CCSP-rtTA/tetO-SHP2E76K mice after detection of lung tumors by MRI. When these mice were examined again by MRI one month after dox withdrawal, the lung tumors were no longer detectable. Histological evaluation of lung tissues revealed residual hyperplastic lesions as well as evidence of necrosis, while biochemical analysis showed that pGab1, pErk, pSrc and Myc returned to basal levels. Thus, discontinuation of SHP2E76K expression resulted in tumor regression. These data indicate that continuing expression of SHP2E76K is required for maintenance of the SHP2 mutant-induced lung tumors. Citation Format: Valentina E. Schneeberger, Noreen Luetteke, Yuan Ren, Parastou Foroutan, Gary V. Martinez, Eric B. Haura, Domenico Coppola, Jie Wu. Continuing expression of SHP2E76K is required to maintain lung tumors induced by the active SHP2E76K mutant in transgenic mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2014-5343

Abstract 2485: Secretome signature associated with TITF1/NKX2-1-negative non-small cell lung cancer

Abstract Background: TITF1/NKX2-1 is a master transcription factor of peripheral airway cells and a known lineage-specific oncogene in lung cancer cells, whereas it also suppresses tumor progression and is associated with favorable prognosis of lung adenocarcinoma. In our previous study, a protein signature for TITF1 was found in plasmas of mouse models of lung adenocarcinoma, leading to the identification of concordant circulating protein signatures in human non-small cell lung cancer (NSCLC). Following study demonstrated one of TITF1 signature proteins, pro-SFTPB, as a predictive biomarker for NSCLC. To identify additional biomarkers which would complement the performance of pro-SFTPB, we studied secretome from forty NSCLC cell lines in terms of TITF1 expression. Materials and Methods: Forty NSCLC cell lines were cultured according to the standard SILAC protocol. The same batch of cells was used for analysis of whole cell extract and conditioned media. Samples were fractionated and subjected to mass spectrometric analysis. Gene expression data for the same set of NSCLC cell lines were obtained using the Illumina Human WG-6 v3.0 Expression BeadChips. Results: NSCLC cell lines were divided into two groups by median of TITF1 gene expression (TITF1-positive and negative). In secretome analysis, overall 10,399 proteins were identified and semi-quantified with using total number of spectral counts. Interestingly, top abundant secretome proteins in TITF1-negative NSCLC cell lines contain many cytokine/chemokines (IL6, CXCL1, CXCL2 and CXCL5) and growth factors (CTGF, PDGFA, PDGFC, and VEGFC). Among top abundant secretome proteins, gene expression of SRGN was most significantly and inversely correlated with TITF1 gene expression. SRGN is a proteoglycan and physiologically associated with some proteases and cytokine secretion in hematopoietic cells. In addition recent studies have indicated that SRGN is also associated with aggressive phenotype in some cancers, thus considered as a promising biomarker candidate for TITF1-negative NSCLC. Then we determined whether SRGN would be directly regulated by TITF1. Suppression of TITF1 by siRNA or TNF alpha treatment increased SRGN expression, suggesting potential transcriptional regulation of SRGN by TITF1. Finally we studied functional roles of SRGN in TITF1-negative cell lines. Knockdown of SRGN by siRNA did not affect cell proliferation. However two chemokines, CXCL1 and CXCL5, which are abundant in secretome of TITF1-negative cell lines, were decreased by SRGN knockdown. In addition, macrophage migration was also decreased in the conditioned media of SRGN knockdown cells compared with control cells. Conclusion: Our findings suggest functional relevance of SRGN in tumor microenvironment and tumor aggressiveness in TITF1/NKX2-1-negative NSCLC. Validation study for plasma levels of CXCL1 and CXCL5 in NSCLC subjects is now on-going. Citation Format: Ayumu Taguchi, Muge Celiktas, Dhillon Dilsher, Qing Zhang, Chee-Hong Wong, Alice Chin, Adi Gazdar, Samir Hanash. Secretome signature associated with TITF1/NKX2-1-negative non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2485. doi:10.1158/1538-7445.AM2014-2485

Ghrelin relieves cancer cachexia associated with the development of lung adenocarcinoma in mice

Cancer cachexia is a multifactorial, critical illness syndrome characterized by an ongoing loss of skeletal muscle and adipose tissue. The reductions in body weight and skeletal muscle mass are important prognostic indicators for cancer patients that are refractory to current therapies. Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced in the stomach, stimulates food intake and growth hormone secretion, suppresses inflammation, and prevents muscle catabolism. We investigated the pharmacological potential of ghrelin in the treatment of cancer cachexia by using urethane-treated, bronchioalveolar epithelium-specific Pten-deficient mice that developed lung adenocarcinomas. Ghrelin or phosphate-buffered saline was given to mice daily for four weeks beginning at five months after urethane injection, which corresponded to the time point of lung adenocarcinoma formation. Ghrelin inhibited the inductions of C-reactive protein, tumor necrosis factor-α, interleukin-1β, and interleukin-6, mitigated the reduction of food intake and fat mass, and consequently ameliorated body weight loss in the mouse model of lung adenocarcinoma. We also demonstrated that skeletal muscle mass and muscle contraction force in both fast-twitch muscle and slow-twitch muscle were retained in ghrelin-treated mice in conjunction with an upregulation of local insulin-like growth factor 1/Akt signaling. In addition, ghrelin administration reduced the expressions of phosphorylated-p38 mitogen-activated protein kinase, phosphorylated-nuclear factor-kappa B, Forkhead box protein O1, muscle RING-finger protein-1, and F-Box protein 32 in the lysates of skeletal muscle in the tumor-bearing state. Our results indicate that ghrelin administration exerts a protective effect against cancer cachexia by ameliorating skeletal muscle wasting and regulating systemic inflammation.

Exercise-induced lung cancer regression: mechanistic findings from a mouse model.

It has been demonstrated that regular exercise improves the quality of life in patients undergoing treatment for lung cancer and has been associated with reductions in cancer-specific mortality in patients with colon and breast cancer. The direct effects of cardiovascular exercise on lung cancer tumor biology, however, remain unknown. The authors evaluated the effects of cardiovascular exercise in a mouse model of lung adenocarcinoma. Luciferase-tagged A549 lung adenocarcinoma cells were injected through the tail vein of nude male mice. Then, the mice underwent weekly bioluminescent imaging until lung tumors were clearly identified. After lung tumors were identified, the mice were randomized to daily wheel running versus no wheel running, and they were imaged weekly. After 4 weeks, all mice were killed, and the lung tumors were harvested. Western blot and immunohistochemical analyses were conducted on tumor tissues to identify potential differences in protein expression levels in exercising mice versus sedentary mice. Lung tumors in exercising mice grew significantly more slowly relative to sedentary mice. There was no change in the development of metastatic lesions between the 2 groups. Protein analysis by Western blot or immunohistochemical analysis demonstrated increased p53 protein levels in exercising mice relative to sedentary mice as well as increased mediators of apoptosis, including Bax and active caspase 3, in tumor tissues. In both groups of mice, no normal tissue toxicity was observed in other organs. Daily cardiovascular exercise appears to mitigate the growth of lung adenocarcinoma tumors, possibly by activation of the p53 tumor suppressor function and increased apoptosis.

Obligate progression precedes lung adenocarcinoma dissemination.

Despite its clinical importance, very little is known about the natural history and molecular underpinnings of lung cancer dissemination and metastasis. Here, we used a genetically engineered mouse model of metastatic lung adenocarcinoma in which cancer cells are fluorescently marked to determine whether dissemination is an inherent ability or a major acquired phenotype during lung adenocarcinoma metastasis. We find very little evidence for dissemination from oncogenic KRAS-driven hyperplasias or most adenocarcinomas. p53 loss is insufficient to drive dissemination but rather enables rare cancer cells in a small fraction of primary adenocarcinomas to gain alterations that drive dissemination. Molecular characterization of disseminated tumor cells indicates that downregulation of the transcription factor Nkx2-1 precedes dissemination. Finally, we show that metastatic primary tumors possess a highly proliferative subpopulation of cells with characteristics matching those of disseminating cells. We propose that dissemination is a major hurdle during the natural course of lung adenocarcinoma metastasis. Because of its aggressively metastatic nature, lung cancer is the top cancer killer of both men and women in the United States. We show that, unlike in other cancer types, lung cancer dissemination is a major initial barrier to metastasis. Our findings provide insight into the effect of p53 deficiency and downregulation of Nkx2-1 during lung adenocarcinoma progression.

In vivo MRI for effective non-invasive detection and follow-up of an orthotopic mouse model of lung cancer.

One of the main reasons for the dismal prognosis of lung cancer is related to the late diagnosis of this pathology. In this study, we evaluated the potential of optimized lung MRI techniques as a completely non-invasive approach for non-small-cell lung cancer (NSCLC) MRI in vivo detection and follow-up in a mouse model of lung adenocarcinoma expressing the luciferase gene. Bioluminescent lung tumour cells were orthotopically implanted in immuno-deficient mice. Ultra-short echo-time (UTE) MRI free-breathing acquisitions were compared with standard gradient-echo lung MRI (FLASH) using both respiratory-gated and free-breathing protocols. The MRI findings were validated against bioluminescence imaging (BLI) and gold-standard histopathology analysis. Adenocarcinoma-like pathological tissue was successfully identified in all the mice with gated-FLASH and non-gated UTE MRI, and good tumour co-localization was found between MRI, BLI and histological analyses. An excellent or good correlation was found between the measured bioluminescent signal and the total tumour volumes quantified with UTE MRI or gated-FLASH MRI, respectively. No significant correlation was found when the tumours were segmented on non-gated MR FLASH images. MRI was shown to be a powerful imaging tool able to detect, quantify and longitudinally monitor the development of sub-millimetric NSCLCs. To our knowledge, this is the first study which proves the feasibility of a completely non-invasive MRI quantitative detection of lung adenocarcinoma in freely breathing mice. The absence of ionizing radiation and the high-resolution of MRI, along with the complete non-invasiveness and good reproducibility of the proposed non-gated protocol, make this imaging tool ideal for direct translational applications.

Gene-targeted radiation therapy mediated by radiation-sensitive promoter in lung adenocarcinoma and the feasibility of micro-PET/CT in evaluation of therapeutic effectiveness in small animals

To explore the combined anti-tumor effect of radiation therapy and gene-targeted suppression of tumor neovasculature in lung adenocarcinoma in vivo, and to explore the feasibility of micro-PET/CT in dynamic evaluation of treatment effectiveness. Thirty 5-6-week old male BALB/c nude mice were used in this study. The mouse models of xenotransplanted human lung adenocarcinoma were divided into 5 groups at random, six mice in each group: the control group, radiation treatment alone group and three groups of recombinant baculovirus plus radiation treatment (intratumoral injection, tail vein injection, and intramuscular injection). The tumor volume was measured every 2 days. Growth delay time (GD) and growth inhibition rate was calculated. FDG metabolism was evaluated by micro-PET-CT before and after treatment. The expressions of VEGF, CD31 and Ki-67 were detected by immunohistochemistry (IHC). The tumor growth delay was >12 days, and the tumor inhibition rate was >45% in the recombinant baculovirus combined with radiotherapy groups, significantly higher than that of the radiotherapy alone group (P < 0.05). Immunohistochemical analysis showed that the expressions of VEGF, CD31 and Ki-67 were significantly lower than that in other groups (P < 0.05). The micro-PET-CT assessment showed that the FDG-metabolism in the recombinant baculovirus combined with radiotherapy groups was significantly reduced (P < 0.05), and the SUVmax (FDG metabolism) of transplanted tumors after treatment was also markedly decreased in comparison with that of the control group. The tumor volume after treatment was significantly correlated with SUVmax in the recombinant baculovirus intratumoral injection + radiotherapy group(r = 0.976), recombinant baculovirus intravenous injection + radiotherapy group (r = 0.954), recombinant baculovirus intramuscular injection + radiotherapy group (r = 0.929), and radiotherapy alone group (r = 0.871, P < 0.05). The recombinant baculovirus containing Egr1 promoter and K5 gene combined with radiotherapy enhances the suppressing effect on the growth of lung adenocarcinoma in the tumor-bearing nude mice. The inducibility of Egr1 promoter by radiation allows the targeting and controllability of treatment. Micro-PET-CT results have a good correlation with the treatment effectiveness. Therefore, it can be used in real-time evaluation of tumor metabolic function in vivo.

Abstract A21: microRNA-200 regulates tumor cell PD-L1 expression to control lung cancer metastasis.

Abstract Background: Tumor cell regulation of the recruitment and activity of diverse inflammatory cells creates an immunosuppressive microenvironment that favors both tumor growth and metastasis. However, the mechanistic basis of how tumor cells trigger intra-tumoral immunosuppression have not been fully defined. Here we show that metastasis-prone lung adenocarcinoma cells suppress the proliferation and activity of intra-tumoral CD8+ T cells through a PD-L1 dependent mechanism and that PD-L1 expression is regulated by microRNA-200 (miR-200), a known mediator of tumor cell EMT. Results: Analysis of patient tumors from The Cancer Genome Atlas (TCGA) (230 adenocarcinomas) and a large MDACC cohort of resected tumors (N&amp;gt;150) demonstrated that tumors with a mesenchymal gene expression signature had repression of miR-200 family expression and higher levels of PD-L1 expression. These findings were confirmed experimentally in human and murine NSCLC cell lines. In vivo experiments with a Kras/p53 mutant mouse model of lung adenocarcinoma showed that decreased miR-200 led to increased expression of PD-L1 levels on tumor cells, causing profound CD8+ T cell suppression. Upon suppression of CD8+ T cell activity tumors acquired a growth advantage and metastatic capability. Confirmation of these results with the LLC model in PD-L1 knockout mice also demonstrated that PD-L1 expression on the tumor cells, rather than other cells in the tumor microenvironment, was critical to defining tumor immunity and metastastic ability. The metastases could be suppressed by genetic and pharmacologic blockade of PD-L1. Conclusion: Our findings demonstrate a novel biological role for miR-200 in cancer cells by simultaneous control of the cell-intrinsic EMT program and the cell-non-autonomous PD-L1-mediated immune evasion. These findings support further investigation into the mechanistic effects of miR-200 on metastasis and the use of specific EMT-related biomarkers to select immunomodulatory therapies. Citation Format: Don L. Gibbons, Limo Chen, Sangeeta Goswami, Young-Ho Ahn, Lauren A. Byers, Lixia Diao, Jing Wang, Angelica Cortez, Jonathon Roybal, James Welsh, Xuejun Zhang, David Dwyer, Xiaohui Yi, Chen Lieping, Ignacio Wistuba, John V. Heymach, Jonathan M. Kurie, F. Xiao-Feng Qin. microRNA-200 regulates tumor cell PD-L1 expression to control lung cancer metastasis. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A21.

Glycyrrhizin Suppresses Lung Adenocarcinoma Cell Growth Through Inhibition of Thromboxane Synthase

Background/Aims: The effects of glycyrrhizin treatment in lung cancer remain undetermined, despite extensive studies of the anti-tumor activities of glycyrrhizin. Methods: Lung adenocarcinoma A549 and NCI-H23 cell lines were used in this study. Cell growth was examined by MTS assays, while apoptosis and cell cycle were determined by flow cytometric analysis. Both real-time PCR and western blotting were used to examine the expression levels of thromboxane synthase (TxAS), and TxAS activity was measured using EIA detection of the biosynthesis of TxA2. TxAS was overexpressed in NCI-H23 cells by transfection with TxAS cDNA, while TxAS was inhibited by transfection with TxAS siRNA in A549 cells. For the mouse model of lung adenocarcinoma, the effects of glycyrrhizin on tumor growth were analyzed by western blot evaluation of TxAS, PTEN and survivin. TxAS activity was determined by EIA assay. Results: Glycyrrhizin suppressed cell growth in A549 cells, but not in NCI-H23 cells, by induction of apoptosis. TxAS was overexpressed in A549 cells, but the TxAS levels in NCI-H23 cells were minimal. Moreover, TxAS expression and activity were suppressed by glycyrrhizin. Glycyrrhizin had no additive effects with TxAS siRNA knockdown in suppressing A549 cell growth, whereas it completely suppressed cell growth of NCI-H23 cells transfected with TxAS cDNA. These results were further confirmed by the in vivo study. Conclusion: Our study suggests that the anti-tumor effect of glycyrrhizin in lung adenocarcinoma is, at least in part, TxAS-dependent. Therefore, glycyrrhizin is a promising anti-cancer agent for the treatment of lung adenocarcinoma.

Upregulation of the microRNA cluster at the Dlk1-Dio3 locus in lung adenocarcinoma

Mice in which lung epithelial cells can be induced to express an oncogenic KrasG12D develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of KrasG12D tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known SNPs revealed not only a change in expression of Cd22 but also that its expression became allele-specific in tumors. The most salient finding however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of ~53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was dramatically and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured KrasG12D tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.