Mouse Lung Adenocarcinoma Research Articles

Co-Clinical Trials Demonstrate Superiority of Crizotinib to Chemotherapy in ALK-Rearranged Non–Small Cell Lung Cancer and Predict Strategies to Overcome Resistance

To extend the results of a phase III trial in patients with non-small cell lung cancer with adenocarcinomas harboring EML4-ALK fusion. We conducted a co-clinical trial in a mouse model comparing the ALK inhibitor crizotinib to the standard-of-care cytotoxic agents docetaxel or pemetrexed. Concordant with the clinical outcome in humans, crizotinib produced a substantially higher response rate compared with chemotherapy, associated with significantly longer progression-free survival. Overall survival was also prolonged in crizotinib- compared with chemotherapy-treated mice. Pemetrexed produced superior overall survival compared with docetaxel, suggesting that this agent may be the preferred chemotherapy in the ALK population. In addition, in the EML4-ALK-driven mouse lung adenocarcinoma model, HSP90 inhibition can overcome both primary and acquired crizotinib resistance. Furthermore, HSP90 inhibition, as well as the second-generation ALK inhibitor TAE684, demonstrated activity in newly developed lung adenocarcinoma models driven by crizotinib-insensitive EML4-ALK L1196M or F1174L. Our findings suggest that crizotinib is superior to standard chemotherapy in ALK inhibitor-naïve disease and support further clinical investigation of HSP90 inhibitors and second-generation ALK inhibitors in tumors with primary or acquired crizotinib resistance.

Multiple cells-of-origin of mutant K-Ras–induced mouse lung adenocarcinoma

Much controversy surrounds the cell-of-origin of mutant K-Ras (K-RasG12D)-induced lung adenocarcinoma. To shed light on this issue, we have used technology that enables us to conditionally target K-RasG12D expression in Surfactant Protein C (SPC)(+) alveolar type 2 cells and in Clara cell antigen 10 (CC10)(+) Clara cells by use of cell-type-restricted recombinant Adeno-Cre viruses. Experiments were performed both in the presence and absence of the tumor suppressor gene p53, enabling us to assess what effect the cell-of-origin and the introduced genetic lesions have on the phenotypic characteristics of the resulting adenocarcinomas. We conclude that both SPC-expressing alveolar type 2 cells and CC10-expressing Clara cells have the ability to initiate malignant transformation following the introduction of these genetic alterations. The lungs of K-Ras(lox-Stop-lox-G12D/+) and K-Ras(lox-Stop-lox-G12D/+);tumor suppressor gene Trp53(F/F) mice infected with Adeno5-SPC-Cre and Adeno5-CC10-Cre viruses displayed differences in their tumor spectrum, indicating distinct cellular routes of tumor initiation. Moreover, using a multicolor Cre reporter line, we demonstrate that the resulting tumors arise from a clonal expansion of switched cells. Taken together, these results indicate that there are multiple cellular paths to K-RasG12D-induced adenocarcinoma and that the initiating cell influences the histopathological phenotype of the tumors that arise.

Transdifferentiation of lung adenocarcinoma in mice with Lkb1 deficiency to squamous cell carcinoma

Lineage transition in adenocarcinoma (ADC) and squamous cell carcinoma (SCC) of non-small cell lung cancer, as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma, remains a fundamental yet unsolved question. Here we provide in vivo evidence showing the transdifferentiation of lung cancer from ADC to SCC in mice: Lkb1-deficient lung ADC progressively transdifferentiates into SCC, via a pathologically mixed mAd-SCC intermediate. We find that reduction of lysyl oxidase (Lox) in Lkb1-deficient lung ADC decreases collagen disposition and triggers extracellular matrix remodelling and upregulates p63 expression, a SCC lineage survival oncogene. Pharmacological Lox inhibition promotes the transdifferentiation, whereas ectopic Lox expression significantly inhibits this process. Notably, ADC and SCC show differential responses to Lox inhibition. Collectively, our findings demonstrate the de novo transdifferentiation of lung ADC to SCC in mice and provide mechanistic insight that may have important implications for lung cancer treatment.

The radiosensitizing effect of Paeonol on lung adenocarcinoma by augmentation of radiation-induced apoptosis and inhibition of the PI3K/Akt pathway

Purpose: To investigate the radiosensitizing effect and mechanism of action by the natural product Paeonol on lung adenocarcinoma both in vitro and in vivo.Materials and methods: Two lung adenocarcinoma cell lines (human lung adenocarcinoma cell line A549 and mouse Lewis lung carcinoma (LLC) cell line) were chosen for this research. In order to select the experimental concentrations of Paeonol, cytotoxicity was determined using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) assay. A clonogenic assay was performed to measure the radiosensitizing effects. Apoptosis was determined by the Tunel (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) assay and flow cytometry. Protein expression was analyzed by Western blotting. To test the radiosensitizing effect in vivo, a transplanted tumor model was established.Results: The MTT assay showed that Paeonol inhibited proliferation of cells. Paeonol concentration ranged from an IC5 (5% inhibiting concentration) to an IC20 and was used at non-toxic concentrations for subsequent experiments. The clonogenic assay showed that Paeonol enhanced the radiosensitivity of cells. Data from the Tunel assay and flow cytometry verified that Paeonol enhanced radiation-induced apoptosis. Paeonol inhibited the activation of the PI3K/AKT (Phosphatidylinositol 3-kinase/ Protein Kinase B) pathway and down-regulated the expression of COX-2 (Cyclooxygenase-2) and Survivin. Paeonol (1718 mg/kg) combined with 10 Gy irradiation inhibited the growth of a transplanted tumor model in vivo, resulting in the longest tumor growth time, tumor growth delay and the highest inhibition ratio when compared with the radiotherapy alone group.Conclusions: It is reported for the first time that Paeonol has a radiosensitizing effect on lung adenocarcinoma both in vitro and in vivo. This effect could be related to the augmentation of radiation-induced apoptosis and the inhibition of the PI3K/Akt signalling pathway and its downstream proteins: COX-2 and Survivin.

Estrogen and insulin‐like growth factor 1 synergistically promote the development of lung adenocarcinoma in mice

Estrogen receptor (ER) and insulin-like growth factor-1 receptor (IGF-1R) signaling are implicated in lung cancer progression. Based on their previous findings, the authors sought to investigate whether estrogen and IGF-1 act synergistically to promote lung adenocarcinoma (LADE) development in mice. LADE was induced with urethane in ovariectomized Kunming mice. Tumor-bearing mice were divided into seven groups: 17β-estradiol (E2), E2+fulvestrant (Ful; estrogen inhibitor), IGF-1, IGF-1+AG1024 (IGF-1 inhibitor), E2+IGF-1, E2+IGF-1+Ful+AG1024 and control groups. After 14 weeks, the mice were sacrificed, and then the tumor growth was determined. The expression of ERα/ERβ, IGF-1, IGF-1R and Ki67 was examined using tissue-microarray-immunohistochemistry, and IGF-1, p-ERβ, p-IGF-1R, p-MAPK and p-AKT levels were determined based on Western blot analysis. Fluorescence-quantitative polymerase chain reaction was used to detect the mRNA expression of ERβ, ERβ2 and IGF-1R. Tumors were found in 93.88% (46/49) of urethane-treated mice, and pathologically proven LADE was noted in 75.51% (37/49). In the E2+IGF-1 group, tumor growth was significantly higher than in the E2 group (p < 0.05), the IGF-1 group (p < 0.05) and control group (p < 0.05). Similarly, the expression of ERβ, p-ERβ, ERβ2, IGF-1, IGF-1R, p-IGF-1R, p-MAPK, p-AKT and Ki67 at the protein and/or mRNA levels was markedly higher in the ligand group than in the ligand + inhibitor groups (all p < 0.05). This study demonstrated for the first time that estrogen and IGF-1 act to synergistically promote the development of LADE in mice, and this may be related to the activation of the MAPK and AKT signaling pathways in which ERβ1, ERβ2 and IGF-1R play important roles.

Abstract 343: Transgenic C57/BL6 x Balb/c F1 mice expressing mitochondrial targeted catalase have less spontaneous lung cancer.

Abstract Human lung cancer is a major cause of morbidity and among the most frequent and deadly malignancies throughout the world. Discrepancies in classification and characterization approaches have resulted in confusions and difficulties in the comparative pathology of mouse and human lung tumors. Currently, there are very few mouse models of spontaneous lung cancer that are available to validate human lung carcinogenesis. Male C57/BL6 x Balb/c F1 (CB6 F1) mice have a high incidence of pulmonary adenocarcinoma that increases with increasing age. For example, 28 month old males have a 52 per cent incidence, while 32 month old males have close to 80 per cent incidence. In order to determine if these tumors were driven by mitochondrial reactive oxygen species (ROS), we crossed wild type Balb/c females with transgenic males on the C57/BL6 background expressing human catalase targeted to mitochondria (mCAT). The offspring were followed through the end of their lifespan when tissues were collected for histological evaluation. Of 65 males examined, we found that those expressing mCAT had a 29 per cent incidence of lung tumors, while wild type littermates had an incidence of 71 per cent (p ≤ 0.01). The presence of mCAT had a similar effect on the incidence of lung tumors in females although the overall incidence was less than in males. Histologically, lung tumors in wild type mice had higher grade malignancy than lung tumors in mice expressing mCAT suggesting that ROS generated by mitochondria might be playing a role in driving this type of cancer in mice. We conclude that spontaneous lung adenocarcinoma in old CB6 F1 mice could be a useful model to study the anti-tumor effect of mitochondrial-targeted anti-oxidants for intervention of human lung cancer. Citation Format: Christina Pettan-Brewer, John Morton, Piper Treuting, Warren Ladiges. Transgenic C57/BL6 x Balb/c F1 mice expressing mitochondrial targeted catalase have less spontaneous lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 343. doi:10.1158/1538-7445.AM2013-343 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 194: Delay in the progression of lung adenoma to adenocarcinoma in mice by oral consumption of pomegranate fruit extract.

Abstract Lung cancer has the highest United States and worldwide rate of cancer mortality, exceeding the mortality rates of colorectal, breast and prostate cancers combined. Tobacco smoking is well established as the major etiological risk factor for lung cancer, contributing to an increased risk in long-term smokers compared with non-smokers. The risk of the development of lung cancer in lifelong smokers is 20-40 times higher than that in non-smokers. Moreover, tobacco use accounts for 30% of overall lung cancer mortality. Given the large population at increased risk of developing lung cancer, the development of approaches to prevent smoking related lung cancer would have great clinical benefit. Pomegranate (Punica granatum, Punicaceae), is an edible fruit cultivated in Mediterranean countries, Afghanistan, India, China, Japan, Russia and the United States. Based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, pomegranate fruit extract (PFE) was found to contain anthocyanins (such as delphinidin, cyanidin and pelargonidin) and several hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) which account for 92% of the antioxidant activity of the whole fruit. We have earlier reported 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 effect of PFE consumption during progression of lung adenomas to adenocarcinomas was investigated in two mouse tumor protocols. NNK, a tobacco-specific N-nitrosamine, is believed to be one of the most promising candidates of lung carcinogen in humans. B(a)P is one of the most ubiquitous environmental polycyclic aromatic hydrocarbons present in automobile exhaust and cigarette smoke and it induces lung tumors in mice. Since majority of current and ex-smokers have small nodules, it is highly desirable to prevent additional growth of lung cancer after the appearance of nodules. To mimic this situation, PFE (0.2% PFE, w/v) was given to A/J mice in drinking water after the establishment of lung adenomas induced by NNK and B(a)P. Treatment with PFE significantly reduced the lung tumor multiplicity by 33% in mice treated with NNK and 29% in B(a)P-treated mice. PFE also caused significant reduction in the expression of COX-2, RAR-β, VEGF, CD-31 and angiopoietin-2 in lungs of mice treated with NNK and B(a)P. On administration of PFE, there was decrease in the multiplicity and incidence of lung adenomas and adenocarcinomas in mice treated with NNK and B(a)P. Thus, our results indicate that PFE has the potential to be developed as an agent against tobacco smoke-induced lung cancer and for improving the quality of life of early stage lung cancer patients and ex-smokers. Citation Format: Naghma Khan, Ruth Sullivan, Tien Hoang, Tejaswi Dittakavi, Hasan Mukhtar. Delay in the progression of lung adenoma to adenocarcinoma in mice by oral consumption of pomegranate fruit extract. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 194. doi:10.1158/1538-7445.AM2013-194

Abstract 2741: NF-κB activation by IL-1α and p62 feedforward loops is required for Kras-induced pancreatic ductal adenocarcinoma.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States with a less than six month mean survival period and less than five percent of an overall five-year survival rate. Despite developments in the management of pancreatic cancer, the survival rate has remained unchanged in the past 50 years. Thus, a better understanding of the mechanism of pancreatic cancer inception and development is the key to identifying prevention strategies and therapeutic targets for increasing patient survival rates, which is one of the greatest challenges in pancreatic cancer research. The mutational activation of Kras is an early event in PDAC development and has been detected in 90% of PDAC and 50% of pancreatic intraepithelial neoplasia (PanIN). The genetic mutation of oncogenic Kras in the pancreas induces chronic pancreatitis and pancreatic cancer. However, the key signaling pathway of the downstream Kras remains unidentified. Here, we report that pancreas-specific targeted IKK2/β inactivation inhibited NF-κB activation and PDAC development in KrasG12D and KrasG12D; Ink4a/ArfF/F mice, demonstrating a mechanistic link between IKK2/β/NF-kB signaling pathway and KrasG12D signaling pathways in PDAC inception. Our findings reveal that KrasG12D-activated AP-1 induces IL-1α, which, in turn, activates NF-κB and its target genes IL-1α and p62, to initiate IL-1α/p62 feedforward loops for inducing and sustaining NF-κB activity. Furthermore, IL-1α overexpression correlates with Kras mutation, NF-κB activity, and poor survival in PDAC patients. Knock-down of IL-1α in mouse PDAC cell lines and lung adenocarcinoma cell lines significantly reduces tumorigenesis in orthotopic xenograft mouse and subcutaneous xenograft mouse models. Therefore, our findings demonstrate the new mechanism by which IKK2/β/NF-κB is activated by KrasG12D through dual feedforward loops of IL-1α/p62 to promote development of PDAC. This suggests that the interruption of the dual feedforward loops of IL-1α/p62 could be the therapeutic targets for inhibiting oncogenic Kras signaling in PDAC. Citation Format: Jianhua Ling, Ya'an Kang, Paul Chiao. NF-κB activation by IL-1α and p62 feedforward loops is required for Kras-induced pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2741. doi:10.1158/1538-7445.AM2013-2741

The effect of EMD 87580, an Na+/H+ isoform 1 inhibitor, on the rate of cell proliferation in mouse lung adenocarcinoma cells

BackgroundDysregulated pH is an emerging hallmark that maybe permissive for cancer progression. The Na+/H+ exchanger (NHE1) is one of the most notable pH regulating transporters and as such warrants further investigation for its role in non small cell lung cancer (NSCLC).MethodsNHE1 protein expression and the rate of cell proliferation were examined in cultured mouse lung adenocarcinoma cells ±10μM NHE1 inhibitor (EMD 87580) or Etoposide for 24 or 48 hours using western blotting and the MTT assay, respectively.ResultsWestern blotting demonstrated an increase in NHE1 protein expression in mouse lung adenocarcinoma cells. In the presence of EMD 87580 (N), NHE1 protein expression showed a trend towards reduction vs. control group (C) (100% C vs. 61.5% N). Treatment of mouse lung adenocarcinoma cells with EMD 87580 for 24 hours did not alter the rate of cell proliferation. Similarly, Etoposide, an anticancer topoisomerase inhibitor, had no effect on the rate of cell proliferation following 24 hours of treatment (92.8±6.3%). However, 48 hours of treatment with EMD 87580 revealed a trend towards reduction in the rate of cell proliferation in mouse lung adenocarcinoma cells (100±11.3% C vs. 77.4±2.6% N). The rate of cell proliferation was significantly less in mouse lung adenocarcinoma cells treated with Etoposide (34.9±7.3%).ConclusionTaken together, these data demonstrate that NHE1 protein was over expressed in the mouse lung adenocarcinoma cells. NHE1 inhibition regresses cell proliferation with prolonged treatment. It is evident the tumor progression and metastasis require NHE1.

Abstract A60: Foxf1 transcription factor drives progression of benign lung adenomas to malignant adenocarcinomas

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States with current 5-year survival only 8—12%. Patients with non-small cell lung cancer (NSCLC) develop metastases prior to clinical symptoms, which reduces successful treatment outcomes. The new therapeutic approaches targeting progression and metastasis of NSCLC are needed. Foxf1 transcription factor is expressed in mesenhyme-derived cells, such as fibroblasts, smooth muscle cells and endothelial cells, but absent from epithelial-derived cells, such as alveolar and airway epithelial cells, and benign lung adenoma cells. In present study, we used immunohistochemistry to demonstrate that Foxf1 expression is up-regulated in the invasive front of human lung adenocarcinomas. Foxf1 expression is also induced in mouse lung adenocarcinomas, caused by either activation of K-Ras G12D transgene or transplantation of Lewis Lung Carcinoma (LLC) cells. In vitro, shRNA-mediated depletion of Foxf1 from LLC cells decreased migration, invasion and colony formation of LLC cells on soft agar. Depletion of Foxf1 reduced mRNA and protein levels of Twist1, and prevented TGFbeta-induced EMT. In vivo, Foxf1-depleted LLC cells failed to form lung metastasis in the transplantation model of lung cancer. Furthermore, we generated transgenic mice with Doxycycline-inducible expression of Foxf1 under control of epithelial-specific SPC-promoter (epFoxf1 mice). Lung adenomas in these mice were induced by urethane. Activation of the Foxf1 transgene in pre-existing lung adenomas promoted their progression towards adenocarcinomas that invaded adjacent bronchiolar lumens and pleural surfaces. Transgenic expression of Foxf1 in tumor cells resulted in the loss of cell polarity, decreased E-cadherin, and increased expression of aSMA, Twist1 and other EMT associated genes. Foxf1 directly bound to promoter region of Twist1, implicating the Foxf1 in the regulation of the Twist1 gene. The present study provides the first genetic evidence that aberrant expression of Foxf1 induces EMT in lung tumors and promotes the progression of lung adenomas to invasive adenocarcinomas, suggesting that Foxf1 is a promising target for anti-cancer therapy. Citation Format: Xinjian Wang, David Balli, Tien Le, Vladimir V. Kalinichenko, Tatiana V. Kalin. Foxf1 transcription factor drives progression of benign lung adenomas to malignant adenocarcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A60.

Ganetespib (STA-9090), a Nongeldanamycin HSP90 Inhibitor, Has Potent Antitumor Activity in In Vitro and In Vivo Models of Non–Small Cell Lung Cancer

We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC(50) values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20-3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t(1/2) 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.

A Cross-Platform Comparison of Genome-Wide Expression Changes of Laser Microdissected Lung Tissue of C-Raf Transgenic Mice Using 3′IVT and Exon Array

Microarrays are widely used to study genome-wide gene expression changes in different conditions most notably disease, growth, or to investigate the effects of drugs on entire genomes. While the number and gene probe sequences to investigate individual gene expression changes differs amongst manufactures, the design for all of the probes is biased towards the 3′ region. With the advent of exon arrays, transcripts of any known or predicted exon can be investigated to facilitate the study of genome-wide alternative splicing events. Thus, the use of exon arrays provides unprecedented opportunities in gene expression studies. However, it remains a major challenge to directly compare gene expression data derived from oligonucleotide to exon arrays. In the present study, genome-wide expression profiling of Laser Micro-dissected Pressure Catapulted (LMPC) samples of c-Raf mouse lung adenocarcinoma, dysplasia, unaltered transgenic and non-transgenic tissues was performed using the Affymetrix GeneChip Mouse Genome 430 2.0 Array and whole genome Mouse Exon 1.0 ST Array. Based on individual group comparisons 52 to 83% of regulated genes were similar in direction, but fold changes of regulated genes disagreed when data amongst the two platforms were compared. Furthermore, for 27 regulated genes opposite direction of gene expression was observed when the two platforms were compared pointing to the need to assess alternative splicing events at the 3′ end. Taken collectively, exon arrays can be performed even with laser microdissected samples but fold change gene expression changes differ considerably between 3′IVT array and exon arrays with alternative splicing events contributing to apparent differences in gene expression changes.

Poly (lactide-co-glycolide) encapsulated extract of Phytolacca decandra demonstrates better intervention against induced lung adenocarcinoma in mice and on A549 cells

We tested relative efficacy of the extract of Phytolacca decandra (PD) and its PLGA nano-encapsulated form (NPD) in mice intoxicated with benzo[a]pyrene (BaP) (25mg/kgb.w.) plus sodium-arsenite (SA) (10mg/kg b.w.) and on A549 lung cancer cells in vitro. We characterized nanoparticles by physico-chemical and morphological studies using dynamic light scattering, scanning electron and atomic force microscopies. We also conducted FTIR and 1H NMR studies to determine if NPD had a co-polymeric nature and analyzed drug–DNA interaction through circular dichroism spectra (CD) and melting temperature profiles (Tm) taking calf thymus DNA as target. An oral dose of 0.3mg/kgb.w. for NPD and 30mg/kgb.w. for PD in mice showed chemopreventive effects in regard to DNA fragmentation, comet tail length and toxicity biomarkers like ROS generation, NFκβ, p53, PARP, CYP1A1 and caspase 3. NPD showed greater effects than that by PD. Results of in vivo studies showed similar effects on A549 in regard to cell viability, DAPI and PI staining, Comet tail length, DNA fragmentation. To further confirm the biological molecule present in PD we analyzed its chromatographic fraction through mass spectroscopy, NMR and FT-IR studies and characterized it to be a tri-terpenoid, a derivative of betulinic acid with a molecular formula C30H46O2. Thus, overall results suggest that nano-encapsulation of PD (NPD) increases drug bioavailability and thereby has a better chemo-preventive action against lung cancer in vivo and on A549 cells in vitro than that of PD.

TIP30 loss enhances cytoplasmic and nuclear EGFR signaling and promotes lung adenocarcinogenesis in mice

Lung adenocarcinoma, the most common type of human non-small cell lung cancer (NSCLC), frequently overexpresses EGFR. However, the mechanisms underlying EGFR overexpression are not completely understood. Recent studies have identified that decreased expression of TIP30 is associated with the metastasis of human NSCLCs, but a causative relationship between TIP30 deficiency and NSCLC development remains unclear. We show here that Tip30 deletion leads to spontaneous development of lung adenomas and adenocarcinomas in mice. Lung tumor development was preceded by aberrant expansion of bronchioalveolar stem/progenitor and alveolar type II cells, as well as increased expression of EGFR and its downstream signaling factors in the lung of Tip30−/− mice. Moreover, TIP30 knockdown in human lung adenocarcinoma cells resulted in prolonged EGFR activity in early endosomes, delayed EGFR degradation, increased EGFR nuclear localization, leading to up-regulated pAKT and pERK1/2 expression. Importantly, in human lung adenocarcinomas, low TIP30 expression correlates with prolonged patient overall and post-progression survival times. Together, these results suggest that TIP30 functions as a tumor suppressor to inhibit EGFR cytoplasmic and nuclear signaling and suppress adenocarcinogenesis in the lung and highlight the potential of therapeutic strategies aiming at inhibiting EGFR signaling for patients with low TIP30 expression lung adenocarcinoma.

A novel immunocompetent murine tumor model for the evaluation of RCAd-enhanced RDAd transduction efficacy

Low gene transfer rate in tumors, high dose-induced acute inflammatory response, and lack of an immunocompetent preclinical animal model to accurately reflect the therapeutic efficacy are prominent reasons for the lack of clinical success of adenoviral (Ad) vectors. In this study, we tested whether human replication-competent adenovirus (RCAd) can replicate in T739 mouse bladder transitional tumor cells (BTT) and lung adenocarcinoma cells (LA795), and whether RCAd can enhance the transduction rate and transgene expression of human replication defective adenoviruses (RDAd) in these tumor cells in vitro and in vivo. We demonstrated that human RCAd exhibited good infectability and cytopathologic effects in mouse BTT and LA795 cells, which was comparable to that in A549 and NCIH460 human tumor cells. In contrast, no infectability and cytopathologic effects were observed in other three mouse tumor cells such as 4T1, B16, and Lewis cells. The combined use of RCAd with RDAd significantly enhanced RDAd transduction efficiency in BTT and LA795 tumor cells in vitro and in vivo. When BTT and LA795 cells were co-infected with RDAd Ad-EGFP and RCAd, a large amount of E1a expression and 2-3 orders of increases in Ad-EGFP genomic DNA were observed. In contrast, the expression of the late gene Hexon is very low, which may explain ineffective packaging of viral particles. In conclusion, our study provided a novel immunocompetent animal model which is useful for evaluating RCAd infectability, cytopathy, and replication. The combined use of RCAd and RDAd provided a new solution for cancer gene therapy.

Abstract 903: The IKKα kinase is a potential therapeutic target in K-Ras-induced lung cancer

Abstract Lung cancer is the leading cause of cancer deaths in the world. In lung cancer, K-Ras oncogenic mutations are widespread, and compounds used to target the biological activity of the Ras proteins failed in clinical trials. Therefore, it is imperative to identify novel therapeutic targets that reduce K-Ras-induced lung tumorigenesis. We have previously shown that K-Ras-induced lung tumorigenesis is potentiated by the subunit p65 of the transcription factor NF-κB; and that K-Ras-induced NF-κB activation in the lung requires IKKα. Therefore, we hypothesized the following: (1) the IKKα kinase promotes K-Ras-induced oncogenesis; and (2) pharmacological inhibition of IKKα activity will be beneficial therapeutically. We used genetic and/or pharmacological approaches to inactivate IKKα in primary lung epithelial cells transformed by K-Ras, as well as K-Ras-positive lung cancer cell lines, and determined that IKKα inhibiton reduces oncogenic properties in vitro. In addition we used a highly specific IKKα inhibitor (CmpdA, Bayer) to treat a K-Ras-induced lung cancer mouse model combined with loss of the tumor suppressor p53 (LSL-K-RasG12D/p53fl/fl). We chose this model for the following reasons: (1) K-Ras activation coupled to p53 loss leads to aggressive lung adenocarcinomas in mice that better resemble human lung tumors; (2) lung cancer patients harboring K-Ras oncogenic mutations frequently display p53 inactivation; and (3) it has been recently demonstrated that p53 loss can enhance NF-κB activation by K-Ras in lung tumors. To induce concomitant K-Ras activation and p53 loss in murine lung epithelial cells, we infected the lungs of LSL-K-RasG12D/p53fl/fl mice by intranasal administration of a Cre-expressing adenovirus (AdCre). Two treatment regimens were used to treat infected animals: CmpdA was either administered daily for 2 weeks immediately following AdCre infection or daily for 4 weeks starting 8 weeks post-infection. In either case, mice treated with CmpdA presented smaller and lower grade tumors than mice treated with placebo. These results show that IKKα promotes lung cancer growth and progression both in vitro and in vivo. In addition, we have shown that pharmacological administration of an IKKα inhibitor slows lung tumor growth and progression in vivo, supporting our hypothesis that IKKα inhibition therapy will have clinical benefits in lung cancer. This research is supported by a FAMRI YCSA Award and a FAPESP Young Investigator Award to DB and a NIH grant (CA73756) to AB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 903. doi:1538-7445.AM2012-903

Abstract 1711: Expression patterns of the G protein-coupled receptor, GPRC5A, in human malignant pleural mesothelioma

Abstract The G protein-coupled receptor family C group 5 member A (hGPRC5A, mGprc5a) was previously shown to function as a lung-specific tumor suppressor gene evidenced by the development of lung adenomas and adenocarcinomas in mice with knockout of the gene. Moreover, we have demonstrated that the immunohistochemical expression of GPRC5A protein was significantly lower in non-small cell lung cancer (NSCLC) resected specimens compared to normal bronchial epithelia from healthy individuals. The latter findings prompted us to examine the expression patterns of GPRC5A in malignant pleural mesothelioma (MPM) because, like NSCLC, this malignancy develops in the thoracic cavity. Analysis of two publicly available MPM microarray datasets revealed that GPRC5A mRNA expression is significantly higher in MPMs compared to matched normal pleura (5.5 fold higher, p=0.002; 6.57 fold higher, p=0.04). GPRC5A immunohistochemical protein expression was semi-quantitatively assessed in formalin-fixed paraffin embedded (FFPE) MPM surgical specimens (n=73) comprised of 41 epitheloid, 14 biphasic and 18 sarcomatoid type MPMs. GPRC5A protein expression was also assessed in a subset of the MPM specimens (n=51) that were reconstructed as high-density spiral tissue microarrays (TMA) developed by J. Fukuoka and colleagues (Sakura-Finetek, Tokyo, Japan) comprised of 35 epitheloid, 9 biphasic and 7 sarcomatoid type MPMs. ANOVA analysis demonstrated significant differences in GPRC5A immunohistochemical expression among MPMs based on the histological subtype (p&amp;lt;10-6) with relative highest expression in the epitheloid tumors (mean, 98.04 ± 54.55; min, 0; max, 200), intermediate expression in biphasic-type tumors (mean, 53.57 ± 38.55; min, 0, max, 140) and lowest expression in sarcomatoid-type MPMs (mean, 24.44 ± 21.48; min, 0; max, 70). The patterns of GPRC5A expression based on histological type of surgical MPM specimens were corroborated using the spiral TMA technology with relative highest expression in the epitheloid tumors lowest expression in sarcomatoid-type MPMs. Importantly, GPRC5A immunohistochemical protein expression was positively significantly correlated between the surgical and spiral TMA specimens (R=0.71, p&amp;lt;10-6). We then sought to examine the association of GPRC5A expression with overall survival in MPM. GPRC5A expression was not associated with overall survival when all histological-type MPMs were included. Interestingly, patients with non-epitheloid type MPMs with relatively higher GPRC5A expression exhibited significantly worse overall survival compared to MPM patients with similar histology and lower expression (p=0.02 of the log-rank test). These findings suggest that GPRC5A may function as an oncogene in MPMs and warrant further studies to probe the function of this receptor in mesotheliomas. Supported by DoD PROSPECT grant W81XWH-07-1-0306. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1711. doi:1538-7445.AM2012-1711

Abstract 1941: Development of novel NSAID conjugated molecules for lung cancer therapy

Abstract Background: Many promising chemopreventive Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) such as Flurbiprofen have acquired anti-cyclooxygenase 2 (COX2) effects with anti-cancer activities. Recently several phosphorylated-NSAID compounds were shown to improve the therapeutic potency over the parental NSAID molecules (JPET 111.183533, 2011) by 10 to 30 folds. Previously, we also reported several novel and potent molecules by the incorporation of NSAIDs such as aspirin, indomethancin, ketorolac, ketoprofen, and sulindac into the aclyting N-terminal of our lead mimetic molecules B317 (F5C-OC2Y-Atmp) (AACR 45: 476, 2004). Here we report a new series of highly potent molecules by replacing the N-terminal F5C with a flurbiprofen (Fbp). The resultant molecules effectively inhibit many cancer cell lines in vitro and in vivo, with efficacy much better than the unmodified parental NSAID molecules or those phosphorylated NSAID analogs. Methods: Standard MTT assays were used to access growth inhibitory effects of the parental and NSAID conjugated molecules. Effects of drugs on cell cycle and apoptosis were analyzed by flow cytometry. In vivo efficacy was evaluated in subcutaneous lung xenografts in athymic nude mice. Results: 32 lung cancer cell lines including 24 NSCLC and 8 SCLC cell lines were treated with various concentrations of some selected conjugated drugs for 4 days. The best compound had an average IC 50 value of 0.5 uM, with a range of 0.1 to 2 uM, while parental Flurbiprofen had IC50 value of 500 uM, with a range of 200 uM to 800uM. Cell cycle analysis showed that exposure of lung cancer cells to these new conjugates resulted in a significant shift of cells from S and G2 phases into G1 arrest, resulting in high percentage of apoptotic cell deaths in a dose- and time-dependent manner. Finally, our in vivo efficacy studies demonstrated that new NSAID conjugates at 5 mg/kg by daily ip injections significantly suppressed growth of subcutaneous xenografts of human lung adenocarcinoma in athymic nude mice without causing weight loss. Conclusion: Our new NSAID conjugate is a novel class of therapeutic agent with anti-growth effects against lung cancer cells in vitro and in vivo, with a 1000 fold increase in potency than the parental Flurbiprofen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1941. doi:1538-7445.AM2012-1941

Abstract 1173: AKT-mTOR pathway mediates mutant p53 gain-of-function by inhibiting autophagy

Abstract Nearly half of human cancers harbor p53 mutations, which are predominantly through missense mutations that result in accumulation of mutant p53 proteins in cancer cells. Persistent evidence supports that the mutant p53 proteins not only lose tumor suppressor activity, but also acquire gain-of-function abilities to promote carcinogenesis, metastasis, tumor recurrence and chemoresistance. Indeed, knock-in mouse models bearing the mutant p53R172H (equivalent to human R175H mutation) at physiological level in vivo display gain-of-function phenotypes, such as enhanced cell transformation potentials and tumor metastasis. However, the molecular mechanism of mutated p53R172H conferring gain-of-function remains poorly understood. In this study, utilizing mouse embryonic fibroblasts (MEFs) and lung adenocarcinoma cells (93-1) derived from p53R172H knock-in mice, we observed that AKT was preferentially activated in p53R172H mutant cells over p53-deficient cells. This activation of AKT allows p53R172H mutant cells to bypass contact inhibition, grow in an anchorage-independent manner and form tumors after subcutaneous injection into SCID mice. In contrast, expression of dominant negative AKT or shRNA-mediated AKT knockdown in p53R172H mutant cells inhibited colony formation in soft-agar and suppressed tumor growth in mouse xenograft tumor model. To directly dissect the essential role of AKT in p53R172H mutant-initiated tumorigenesis in vivo, we bred AKT1 knockout mice to p53R172H mutant knock-in mice. Loss of AKT1 significantly reduced the incidence of tumor development and extended the life span from around 3 months in AKT1-proficient and p53R172H mutant mice (AKT1+/+; p53R172H/R172H) to about 12 months in AKT1-deficient and p53R172H mutant mice (AKT1−/−; p53R172H/R172H). To understand how the activated AKT conferred mutant p53 gain-of-function, we demonstrated that p53R172H mutant activated AKT-mTOR signaling pathway that led to an inhibition of autophagy both in vitro and in vivo. Furthermore, induction of autophagy in p53R172H mutant lung cancer cells through suppressing AKT-mTOR pathway pharmacologically by the dual kinase inhibitor NVP-BEZ235 treatment or genetically by RNAi-mediated knockdown of mTOR dramatically inhibited p53R172H mutant-induced colony formation in soft agar and tumor growth in SCID mice. Our results support that AKT plays a crucial role in mediating mutant p53 gain-of-function phenotypes in vivo through the inhibition of autophagy by the activated AKT-mTOR signaling pathway. These findings provide the proof-of-principle that targeting the AKT-mTOR-autophagy pathway might have promising therapeutic benefits for cancer patients carrying such p53 mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1173. doi:1538-7445.AM2012-1173

Abstract 505: Jagged 2 knockdown inhibits invasion in colorectal cancer cell lines

Abstract Background: The notch pathway is involved in control of cell differentiation in various tissues including pancreas, muscle and bone marrow. Jagged 2 (JAG2) is a ligand for the notch pathway and was found to be overexpressed in malignant plasma cells from patients with multiple myeloma, causing the secretion and release of IL-6, VEGF, and IGF-1 in the microenvironment supporting myeloma cell growth. In breast cancers, it was found to be overexpressed at the hypoxic invasive front and was significantly correlated with metastasis free survival. Furthermore, up-regulation of JAG2 expression in mouse lung adenocarcinoma cells promoted its metastasis in mice. However, the expression pattern and function of JAG2 in colorectal cancer (CRC) is not clear. Therefore in this study, we examined the expression of JAG2 in CRC cell lines and determined the effect(s) of JAG2 knockdown by RNA interference in CRC cell lines. Materials and Methods: The expression of JAG2 protein was examined in four CRC cell lines (SW480, SW620, HCT116, DLD-1) and also in 1 normal colon epithelial cell line (CCD18co) using the western blot. To investigate the effects JAG2 knockdown in CRC cell lines, a pool of 4 siRNAs against JAG2 was transfected into the 4 CRC cell lines listed above. The effects on cell growth was then determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (the MTS assay) and the Matrigel invasion assay was performed to assess its effects on invasive capability. Results: Western blot results showed that JAG2 protein was expressed in all 4 colorectal cancer cell lines including SW480, SW620, HCT116 and DLD-1 but not in the CCD18co normal colon epithelial cell line. The MTS assay showed that cell growth was not affected by JAG2 knockdown in the CRC cell lines. Interestingly, the invasive capability of HCT116 and DLD-1cells was reduced after JAG2 knockdown. Conclusions: Results from this study suggest that JAG2 may play a role in the invasiveness of CRC cell lines but not in cell proliferation. JAG2 may be a promising therapeutic target for inhibition of metastasis in colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 505. doi:1538-7445.AM2012-505