N-nitroso-tris-chloroethylurea Research Articles

XPC Protects against Carcinogen-Induced Histologic Progression to Lung Squamous Cell Carcinoma by Reduced Basal Epithelial Cell Proliferation.

Lung squamous cell carcinoma (LUSC) is the second leading cause of lung cancer. Although characterized by high DNA mutational burdens and genomic complexity, the role of DNA repair in LUSC development is poorly understood. We sought to better understand the role of the DNA repair protein Xeroderma Pigmentosum Group C (XPC) in LUSC development. XPC knock-out (KO), heterozygous, and wild-type (WT) mice were exposed topically to N-nitroso-tris-chloroethylurea (NTCU), and lungs were evaluated for histology and pre-malignant progression in a blinded fashion at various time-points from 8-24 weeks. High-grade dysplasia and LUSC were increased in XPC KO compared with XPC WT NTCU mice (56% vs. 34%), associated with a higher mean LUSC lung involvement (p < 0.05). N-acetylcysteine pre-treatment decreased bronchoalveolar inflammation but did not prevent LUSC development. Proliferation, measured as %Ki67+ cells, increased with NTCU treatment, in high-grade dysplasia and LUSC, and in XPC deficiency (p < 0.01, ANOVA). Finally, pre-LUSC dysplasia developed earlier and progressed to higher histologic classification sooner in XPC KO compared with WT mice. Overall, this supports the protective role of XPC in squamous dysplasia progression to LUSC. Mouse models of early LUSC development are limited; this may provide a valuable model to study mechanisms of LUSC development and progression.

Aerosolized miR-138-5p and miR-200c targets PD-L1 for lung cancer prevention.

The development of chemopreventive strategies with the ability to prevent the progression of lung lesions to malignant cancers would reduce the mortality and morbidity resulting from this deadly disease. Delivery of microRNA (miRNA) by inhalation is a novel method for lung cancer prevention. In this study, we investigated the combined efficacy of aerosolized miR-138-5p and miR-200c miRNA mimics in lung cancer prevention. Combination of the two miRNAs inhibited Benzo(a)pyrene (B((a))P)-induced lung adenomas and N-nitroso-tris-chloroethylurea (NTCU)-induced lung squamous cell carcinomas with no detectable side effects. Using single-cell RNA sequencing (scRNA-seq) and imaging mass cytometry (IMC), we found that both miRNAs inhibited programmed cell death ligand 1 (PD-L1) expression. Our flow cytometry results showed that aerosolized delivery of combined miRNAs increased CD4+ and CD8+ T cells and reduced the expression of programmed cell death protein 1 (PD-1) and T-regulatory cells. Our results demonstrated that the delivery of aerosolized microRNAs targeting PD-L1 can be highly effective in preventing lung cancer development and progression in mice.

Rigid Tissue Increases Cytoplasmic pYAP Expression in Pre-Malignant Stage of Lung Squamous Cell Carcinoma (SCC) In Vivo.

Increased tissue rigidity is able to activate the Hippo signaling pathway, leading to YAP inactivation by phosphorylation and translocation into the cytoplasm. Accumulating evidence suggests that cytoplasmic pYAP serves as a tumor suppressor and could be a prognostic biomarker for several solid cancers. However, the relationship between tissue rigidity and cytoplasmic pYAP expression in the early stage of lung squamous cell carcinoma (SCC) remains elusive; this was determined in this study by using a mouse model. Female BALB/c mice were assigned into two groups (n = 6; the vehicle (VC) and the pre-malignant (PM) group, which received 70% acetone and 0.04 M N-nitroso-tris-chloroethylurea (NTCU) for 15 weeks, respectively. In this study, the formation of hyperplasia and metaplasia lesions was found in the PM group, indicating the pre-malignant stage of lung SCC. The pre-malignant tissue appeared to be more rigid as characterized by significantly higher (p < 0.05) epithelium thickness, proliferative activity, and collagen content than the VC group. The PM group also had a significantly higher (p < 0.05) cytoplasmic pYAP protein expression than the VC group. In conclusion, increased tissue rigidity may contribute to the upregulation of cytoplasmic pYAP expression, which may act as a tumor suppressor in the early stage of lung SCC.

Tissue Rigidity Increased during Carcinogenesis of NTCU-Induced Lung Squamous Cell Carcinoma In Vivo.

Increased tissue rigidity is an emerging hallmark of cancer as it plays a critical role in promoting cancer growth. However, the field lacks a defined characterization of tissue rigidity in dual-stage carcinogenesis of lung squamous cell carcinoma (SCC) in vivo. Pre-malignant and malignant lung SCC was developed in BALB/c mice using N-nitroso-tris-chloroethylurea (NTCU). Picro sirius red staining and atomic force microscopy were performed to measure collagen content and collagen (diameter and rigidity), respectively. Then, the expression of tenascin C (TNC) protein was determined using immunohistochemistry staining. Briefly, all tissue rigidity parameters were found to be increased in the Cancer group as compared with the Vehicle group. Importantly, collagen content (33.63 ± 2.39%) and TNC expression (7.97 ± 2.04%) were found to be significantly higher (p < 0.05) in the Malignant Cancer group, as compared with the collagen content (18.08 ± 1.75%) and TNC expression (0.45 ± 0.53%) in the Pre-malignant Cancer group, indicating increased tissue rigidity during carcinogenesis of lung SCC. Overall, tissue rigidity of lung SCC was suggested to be increased during carcinogenesis as indicated by the overexpression of collagen and TNC protein, which may warrant further research as novel therapeutic targets to treat lung SCC effectively.

PPARgamma agonism inhibits progression of premalignant lesions in a murine lung squamous cell carcinoma model.

The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. H&amp;E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

Two cell line models to study multiorganic metastasis and immunotherapy in lung squamous cell carcinoma.

ABSTRACTThere is a paucity of adequate mouse models and cell lines available to study lung squamous cell carcinoma (LUSC). We have generated and characterized two models of phenotypically different transplantable LUSC cell lines, i.e. UN-SCC679 and UN-SCC680, derived from A/J mice that had been chemically induced with N-nitroso-tris-chloroethylurea (NTCU). Furthermore, we genetically characterized and compared both LUSC cell lines by performing whole-exome and RNA sequencing. These experiments revealed similar genetic and transcriptomic patterns that may correspond to the classic LUSC human subtype. In addition, we compared the immune landscape generated by both tumor cells lines in vivo and assessed their response to immune checkpoint inhibition. The differences between the two cell lines are a good model for the remarkable heterogeneity of human squamous cell carcinoma. Study of the metastatic potential of these models revealed that both cell lines represent the organotropism of LUSC in humans, i.e. affinity to the brain, bones, liver and adrenal glands. In summary, we have generated valuable cell line tools for LUSC research, which recapitulates the complexity of the human disease.

NTCU induced pre-malignant and malignant stages of lung squamous cell carcinoma in mice model

Mice have served as an excellent model to understand the etiology of lung cancer for years. However, data regarding dual-stage carcinogenesis of lung squamous cell carcinoma (SCC) remain elusive. Therefore, we aim to develop pre-malignant (PM) and malignant (M) lung SCC in vivo using N-nitroso-tris-chloroethylurea (NTCU). BALB/C mice were allotted into two main groups; PM and M groups which received treatment for 15 and 30 weeks, respectively. Then, the mice in each main group were allotted into three groups; control, vehicle, and cancer (n = 6), which received normal saline, 70% acetone, and 0.04 M NTCU by skin painting, respectively. Histopathologically, we discovered a mix of hyperplasia, metaplasia, and dysplasia lesions in the PM group and intracellular bridge; an SCC feature in the M group. The M group was positive for cytokeratin 5/6 protein which confirmed the lung SCC subtype. We also found significantly higher (P < 0.05) epithelium thickness in the cancer groups as compared to the vehicle and control groups at both the PM and M. Overall, this study discovered that NTCU is capable of developing PM and M lung SCC in mice model at appropriate weeks and the vehicle group was suggested to be adequate as control group for future research.

FP16.01 Development and Characterization of Two Novel Squamous Cell Lung Cancer Mouse Cell Lines

Lung cancer is the leading cause of cancer-related death worldwide. The incidence and mortality associated with lung cancer is a major Public Health challenge in advanced societies. About 20-30% of all lung cancers are classified as lung squamous cell carcinoma (LUSC), also known as squamous cell carcinoma (SSC or SqCC) or epidermoid carcinoma. The absence of preclinical models makes LUSC disease analysis and research more challenging. The development of well-characterized cellular and molecular tools is essential for the advance in lung cancer research. In the present work, we have performed an exhaustive molecular and functional characterization of a new LUSC research model, recently developed in our laboratory. This model consists of two novel A/J mouse-derived syngeneic cell lines from NTCU-induced LUSC.In our opinion, these cell lines may become a robust tool for the study of squamous cell lung cancer in a reliable and reproducible manner. Carcinogenesis and cell line generation: LUSC tumors were induced by N-nitroso-tris-chloroethylurea (NTCU) (Toronto Research Chemicals) treatment applying 0.04 M NTCU by skin painting twice a week for 20 weeks to 8-week-old A/J mice. Immunohistochemical staining: Dissected tumors were fixed in 4% paraformaldehyde and embedded in paraffin. Three-micrometer-thick sections were used for tumors immunohistochemical phenotypic characterization. Exome sequencing: Whole exome sequencing was performed on extracted DNA from the tumor-derived cell lines. RNA sequencing analysis: Samples were prepared with the Illumina TruSeq Stranded mRNA kit and sequenced as reverse paired-end (50 bp) runs on the Nextseq sequencer. Tumor immunotherapy experiments: 2x106 cells were subcutaneously inoculated in one flank of 8-week-old female A/J mice. When the tumors reached a volume of 75 mm3, mice were randomized and treated intraperitoneally with 3 doses of 100 μg of anti-PD1 or anti-CTLA4. Tumor immune landscape analysis: 2x106 cells were subcutaneously inoculated in one flank of 6 8-week-old female A/J mice . Thirteen days after cell inoculation, tumors were collected and processed for flow cytometry analysis. Metastasis in vivo model: Cells were transduced with a triple modality construct containing a triple fusion protein GFP, luciferase and thymidine kinase. Eight-week old female mice were inoculated in the left cardiac ventricle with 1 × 105 cells. Cells were followed by bioluminescence. We have generated two transplantable LUSC cell lines (UN-SCC679 and UN-SCC680) derived from an N-nitroso-tris-chloroethylurea (NTCU) chemically-induced mouse model in A/J mice. Both cells lines expressed cytokeratins and p40, and lacked thyroid transcription factor 1 (TTF1) expression. Furthermore, we genetically characterized the LUSC cell lines by performing whole exome and RNA sequencing. In addition, we characterized the immune landscape of both tumors in vivo and assessed their response to immunotherapy studies combining the gold-standard checkpoint inhibitors in clinic. Finally, we studied the metastatic potential of each of these two models and confirmed that they reflect the human LUSC organotropism to the brain, bones, liver and adrenal glands. We have generated a very valuable tool for LUSC research that recapitulates the complexity of the human disease.

Lrig1 expression identifies airway basal cells with high proliferative capacity and restricts lung squamous cell carcinoma growth

BackgroundLung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 (leucine-rich repeats and immunoglobulin-like domains 1) is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Methods and resultsUsing an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression.ConclusionTogether, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

Chemopreventive effects of pterostilbene through p53 and cell cycle in mouse lung of squamous cell carcinoma model

Cell proliferation and cell death abnormalities are strongly linked to the development of cancer, including lung cancer. The purpose of this study was to investigate the effect of pterostilbene on cell proliferation and cell death via cell cycle arrest during the transition from G1 to S phase and the p53 pathway. A total of 24 female Balb/C mice were randomly categorized into four groups (n = 6): N-nitroso-tris-chloroethyl urea (NTCU) induced SCC of the lungs, vehicle control, low dose of 10 mg/kg PS + NTCU (PS10), and high dose of 50 mg/kg PS + NTCU (PS50). At week 26, all lungs were harvested for immunohistochemistry and Western blotting analysis. Ki-67 expression is significantly lower, while caspase-3 expression is significantly higher in PS10 and PS50 as compared to the NTCU (p < 0.05). There was a significant decrease in cyclin D1 and cyclin E2 protein expression in PS10 and PS50 when compared to the NTCU (p < 0.05). PS50 significantly increased p53, p21, and p27 protein expression when compared to NTCU (p < 0.05). Pterostilbene is a potential chemoprevention agent for lung SCC as it has the ability to upregulate the p53/p21 pathway, causing cell cycle arrest.

An Improved Murine Premalignant Squamous Cell Model: Tobacco Smoke Exposure Augments NTCU-Induced Murine Airway Dysplasia.

Tobacco smoke-induced squamous cell lung cancer (SCC) develops from endobronchial dysplastic lesions that progress to invasive disease. A reproducible murine model recapitulating histologic progression observed in current and former smokers will advance testing of new preventive and therapeutic strategies. Previous studies show that prolonged topical application of N-nitroso-tris-chloroethylurea (NTCU) generates a range of airway lesions in sensitive mice similar to those induced by chronic tobacco smoke exposure in humans. To improve the current NTCU model and better align it with human disease, NTCU was applied to mice twice weekly for 4-5 weeks followed by a recovery period before cigarette smoke (CS) or ambient air (control) exposure for an additional 3-6 weeks. Despite the short time course, the addition of CS led to significantly more premalignant lesions (PML; 2.6 vs. 0.5; P < 0.02) and resulted in fewer alveolar macrophages (52,000 macrophages/mL BALF vs. 68,000; P < 0.05) compared with control mice. This improved NTCU + CS model is the first murine SCC model to incorporate tobacco smoke and is more amenable to preclinical studies because of the increased number of PML, decreased number of mice required, and reduced time needed for PML development.

Airway brushing as a new experimental methodology to detect airway gene expression signatures in mouse lung squamous cell carcinoma

As a consequence of exposure to environmental toxicants, a “field cancerization” effect occurs in the lung, resulting in the development of a field of initiated, but morphologically normal appearing cells within a damaged epithelium containing mutations in oncogene or tumor suppressor genes. Unlike humans, whose airway field of injury associated with lung cancer has long been investigated with airway brushings obtained via bronchoscopy, no methods are available for similar studies in the mouse due to the small size of the murine airways. In this protocol, we describe a detailed method for performing airway brushing from a live mouse, which enables repeated sampling from the same mouse and thus, mimicking the bronchoscopy protocol used in humans. Using this approach in the N-nitroso-tris-chloroethylurea (NTCU)-induced mouse lung squamous cell carcinoma (SCC) model, we isolated airway epithelial cells with intact cell membrane structure and then performed transcriptome sequencing (RNA-Seq). We found activation of the PI3K signaling network to be the most significant in cytologically normal bronchial airway epithelial cells of mice with preneoplastic lung SCC lesions. Prolonged exposure to NTCU also induced activation of NF-kappaB (NFƙB), the downstream pathway of PI3K; this NTCU-induced lung SCC progression can be reversed by blocking the NFƙB pathway.

Abstract 1252: A pre-clinical model for lung cancer interception: gene expression similarities between human and mouse bronchial premalignant lesions

Abstract The molecular events that drive the development of the premalignant lesions (PMLs) that precede lung squamous cell carcinoma (SCC) are poorly understood. A major limitation to understanding PMLs and developing interventions is the lack of preclinical models to test candidates derived from human studies. Previous work by our group and others suggests that the N-nitroso-tris-chloroethylurea (NTCU) mouse model of lung SCC may be a candidate for modeling human PMLs based on histologic similarities, but its molecular relationship to human disease is limited. In this study, we investigate the transcriptomic similarities between PMLs from the NTCU treated mice and humans. RNA from 40 whole lung sections (curls) and laser capture microdissected (LCM) of PMLs of varying histology from SWR/J and A/J mice treated with NTCU were profiled by RNA-Seq. RNA from 131 human endobronchial biopsies representing analogous histological grades were also profiled by RNA-Seq as part of the Pre-Cancer Genome Atlas (PCGA). Linear modeling was used to identify gene expression differences associated with increasing histological severity, mouse strain and sample type. Concordance of gene expression associated with histology between species was investigated using GSEA and GSVA. Biological pathways were inferred using EnrichR and GSEA. A gene expression signature associated with increasing histological severity across all mouse samples was identified (1195 genes, FDR&amp;lt;0.05), which was concordant with gene expression differences associated with increasing histological severity of human PMLs (FDR&amp;lt;0.05). We identified a 51-gene (p&amp;lt;0.001) and a 178-gene (p&amp;lt;0.001) signatures associated with increasing histology in the all SWR/J and A/J samples respectively. These gene expression signatures are enriched for biological pathways involved in immune modulation, occurring with progressing lesions. Conversely, genes whose expression changed with increasing histology in a model that included only LCM samples (153 genes at p&amp;lt;0.001) revealed epithelial-specific processes including the up-regulation of KRT5 and p63 and loss of TTF1. Additionally, in the LCM samples, we identified up-regulation of genes involved in proliferation including a number of genes involved in cell structure, metabolism, transcription and translation, anti-apoptosis as well as several immunosuppressive genes. The major finding of these studies is that there are molecular similarities between PMLs from human and NTCU treated mice, which suggest that this mouse model may be useful to investigate targeted early intervention to halt or reverse PML progression towards lung cancer. Citation Format: Sarah A. Mazzilli, Anna Tassinari, Xiaohui Xiao, Gang Liu, Samjot Dhillon,, Candace Johnson, Mary Reid, Marc Lenburg, Avrum Spira, Jennifer Beane. A pre-clinical model for lung cancer interception: gene expression similarities between human and mouse bronchial premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1252. doi:10.1158/1538-7445.AM2017-1252

Abstract 188: Preventive effect of aerosolized bexarotene in three major subtypes of lung cancer: adenocarcinoma, squamous cell carcinoma and small cell lung cancer in mice

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States. The 5 year survival rate for lung cancer patients has remained a dismal 15% for 3 decades. Thus, the development of chemopreventive agents that could prevent lung cancer could potentially reduce the incidence and mortality of pulmonary neoplasms. Bexarotene has exhibited inhibitory effects in preclinical in vivo models of mammary and lung tumorigenesis, has been approved for clinical use in the treatment of cutaneous T-cell lymphoma, and has shown efficacy in phase I/II trials of non-small cell lung cancer (NSCLC). Preclinical studies have demonstrated that it is highly effective in the prevention of all three major subtypes of lung cancer in mouse models: adenocarcinoma (AD), squamous cell carcinoma (SCC), and small cell lung cancer (SCLC). The major side effects of bexarotene when administered orally to rodents or human patients have been hypertriglyceridemia and hypercholesterolemia. Previous studies in a mouse model of lung AD have demonstrated that aerosol delivery of bexarotene through nasal inhalation exhibited potent chemopreventive activity similar to that observed following oral administration (59 to74% reductions in lung tumor multiplicity). The significant decreases in tumor multiplicity and tumor load were achieved without hypertriglycerides that accompany oral bexarotene administration. In this study, aerosolized delivery of 10-30 mg/ml bexarotene showed a significant chemopreventive effect in all three major subtypes. In the N-nitroso-tris-chloroethylurea (NTCU) induced mouse SCC model, 1 week after the first dose of NTCU a dose dependent decrease in tumor formation was observed, with the highest dose causing 75% (p&amp;lt;0.001) and 42% (p&amp;lt;0.01) decreases in SCC tumor burden and the percentage of SCC tumors, respectively. Tumor load was decreased by 73% in A/Jp53 mouse AD model and by 41% in A/Jp53Rb mouse SCLC model. Aerosol delivery of bexarotene had no effect on animal body weight and other signs of toxicity, and no effect on triglyceride and cholesterol level. Aerosolized bexarotene formulation was effective against all 3 major lung cancer cell types and would be a major advance achieving significant reductions in preventing lung cancer incidence in persons at high risk of lung cancer e.g. former or present smokers. Citation Format: Qi Zhang, Jing Pan, Marker S. Miller, Ronald A. Lubet, Yian Wang, Ming You. Preventive effect of aerosolized bexarotene in three major subtypes of lung cancer: adenocarcinoma, squamous cell carcinoma and small cell lung cancer in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 188. doi:10.1158/1538-7445.AM2017-188

Role of deltaNp63(pos)CD44v(pos) cells in the development of N-nitroso-tris-chloroethylurea-induced peripheral-type mouse lung squamous cell carcinomas.

The role of cells expressing stem cell markers deltaNp63 and CD44v has not yet been elucidated in peripheral‐type lung squamous cell carcinoma (pLSCC) carcinogenesis. Female A/J mice were painted topically with N‐nitroso‐tris‐chloroethylurea (NTCU) for induction of pLSCC, and the histopathological and molecular characteristics of NTCU‐induced lung lesions were examined. Histopathologically, we found atypical bronchiolar hyperplasia, squamous metaplasia, squamous dysplasia, and pLSCCs in the treated mice. Furthermore, we identified deltaNp63pos CD44vpos CK5/6pos CC10pos clara cells as key constituents of early precancerous atypical bronchiolar hyperplasia. In addition, deltaNp63pos CD44vpos cells existed throughout the atypical bronchiolar hyperplasias, squamous metaplasias, squamous dysplasias, and pLSCCs. Overall, our findings suggest that NTCU induces pLSCC through an atypical bronchiolar hyperplasia–metaplasia–dysplasia–SCC sequence in mouse lung bronchioles. Notably, Ki67‐positive deltaNp63pos CD44vpos cancer cells, cancer cells overexpressing phosphorylated epidermal growth factor receptor and signal transducer and activator of transcription 3, and tumor‐associated macrophages were all present in far greater numbers in the peripheral area of the pLSCCs compared with the central area. These findings suggest that deltaNp63pos CD44vpos clara cells in mouse lung bronchioles might be the origin of the NTCU‐induced pLSCCs. Our findings also suggest that tumor‐associated macrophages may contribute to creating a tumor microenvironment in the peripheral area of pLSCCs that allows deltaNp63pos CD44vpos cancer cell expansion through activation of epidermal growth factor receptor signaling, and that exerts an immunosuppressive effect through activation of signal transducer and activator of transcription 3 signaling.

Vitamin D Repletion Reduces the Progression of Premalignant Squamous Lesions in the NTCU Lung Squamous Cell Carcinoma Mouse Model.

The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2,000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C; 80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the percentages of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D-deficient + NTCU (DN), 22.7% [P < 0.05 compared with vitamin D-sufficient +NTCU (SN)]; DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared with SN mice, DN mice exhibited a three-fold increase (P < 0.005) in circulating white blood cells (WBC), a 20% (P < 0.05) increase in IL6 levels, and a four-fold (P < 0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted.

Abstract A11: Airway gene expression indicates an increase in oxidative phosphorylation in the field of injury of individuals with premalignant lesions

Abstract Lung squamous cell carcinoma (SCC) claims the lives of approximately 50,000 individuals in the United States annually due to late stage diagnosis and lack of effective treatment options. Lung SCC arises in the epithelial layer of the bronchial airways and is preceded by the development of premalignant lesions (PMLs). The molecular events involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs that develop go on to form carcinoma. Our group is now using high-throughput genomic techniques to characterize the process of premalignant progression by examining PMLs and non-lesion areas in the airway of individuals with lesions (i.e. the “field of injury”,) to identify events that lead to the development of SCC. mRNA-Seq was conducted on cytologically-normal proximal airway epithelium collected by brushing the mainstem bronchus from current and former smokers with (n=43) and without (n=32) PMLs (metaplatic lesions excluded). Linear modeling strategies were used to identify genes altered between subjects with and without PMLs (n=801 out of 13,916 genes at FDR&amp;lt;0.01). Genes were significantly concordantly enriched (FDR&amp;lt;0.01) in an overlapping sample set profiled by microarray (n=57 out of the 75 samples, 33/57 with PMLs) and enriched among up-regulated genes in the independent sample set (n=158, 38/158 with PMLs). Pathway analysis by GSEA revealed enrichment (FDR&amp;lt;0.05) of oxidative phosphorylation (OXPHOS) /respiratory electron transport among genes up-regulated in the airways of subjects with PMLs. OXPHOS is the most efficient metabolic pathway that generates energy in the form of ATP by utilizing the structures and enzymes of the mitochondria and OXPHOS is often elevated during repair epithelial tissue. However, OXPHOS metabolism is often superseded in the development of cancer by glycolysis. To validate these computational findings related to the OXPHOS pathway, we examined oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) in lung cell lines. These cell lines have either high OXPHOS and low glycolytic gene expression (H1299) or low OXPHOS and high glycolytic gene expression (HCC4006). Consistent with the gene expression pattern in these cell lines, The OCR for H1299s and HBECs were 10% higher than HCC4006 cells reflecting higher OXPHOS activity (p = 0.06). Whereas the ECAR was 39% higher in HCC4006 than H1299 cells and 46 % higher than HBECs (p&amp;lt;0.001and p&amp;lt;0.001 respectively), reflecting higher glycolytic metabolism corresponding to the gene expression. Next, we measured protein expression of genes that were elevated in the field of injury of PMLs, which included: mitochondrial membrane protein, translocase of the outer membrane (TOMM 22) and a subunit of complex IV of the electron transport chain, cytochrome C oxidase (COX-IV), in FFPE sections of lung tissue from the N-nitroso-tris-chloroethylurea (NTCU) mouse model of lung SCC. The NTCU model develops PMLs in the bronchial epithelium that progress to lung SCC in a histologically similar pattern to that observed in humans; it is important to note the carcinogen is administered cutaneously and not inhaled thus the field of injury may differ from inhaled carcinogens such as tobacco smoke. Protein levels of TOMM 22, and COX-IV were found to be elevated in dysplastic lesions compared to untreated controls. Together these data suggest that metabolism-associated gene expression is correlated with cellular metabolism and furthermore there is an increase in OXPHOS associated with the development of PMLs. While we are currently in the process of validating these findings in human normal and PMLs, our results suggest that there is potential that therapeutically increasing or maintaining OXPHOS in premalignant lesions or the field of injury may be a mechanism of prevention for lung cancer. Citation Format: Sarah A. Mazzilli, Gang Liu, Xiaohui Zhang, Huiqing Si, Stephen Lam, Marc Lenburg, Avrum Spira, Jennifer E. Beane. Airway gene expression indicates an increase in oxidative phosphorylation in the field of injury of individuals with premalignant lesions. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A11.

Tracheal Dysplasia Precedes Bronchial Dysplasia in Mouse Model of N-Nitroso Trischloroethylurea Induced Squamous Cell Lung Cancer

Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US and has a 5-year survival rate of only 16%. Histological changes in the bronchial epithelium termed dysplasia are precursors to invasive SCC. However, the cellular mechanisms that cause dysplasia are unknown. To fill this knowledge gap, we used topical application of N-nitroso-tris chloroethylurea (NTCU) for 32 weeks to induce squamous dysplasia and SCC in mice. At 32 weeks the predominant cell type in the dysplastic airways was Keratin (K) 5 and K14 expressing basal cells. Notably, basal cells are extremely rare in the normal mouse bronchial epithelium but are abundant in the trachea. We therefore evaluated time-dependent changes in tracheal and bronchial histopathology after NTCU exposure (4, 8, 12, 16, 25 and 32 weeks). We show that tracheal dysplasia occurs significantly earlier than that of the bronchial epithelium (12 weeks vs. 25 weeks). This was associated with increased numbers of K5+/K14+ tracheal basal cells and a complete loss of secretory (Club cell secretory protein expressing CCSP+) and ciliated cells. TUNEL staining of NTCU treated tissues confirmed that the loss of CCSP+ and ciliated cells was not due to apoptosis. However, mitotic index (measured by bromodeoxyuridine incorporation) showed that NTCU treatment increased proliferation of K5+ basal cells in the trachea, and altered bronchial mitotic population from CCSP+ to K5+ basal cells. Thus, we demonstrate that NTCU-induced lung epithelial dysplasia starts in the tracheal epithelium, and is followed by basal cell metaplasia of the bronchial epithelium. This analysis extends our knowledge of the NTCU-SCC model by defining the early changes in epithelial cell phenotypes in distinct airway locations, and this may assist in identifying new targets for future chemoprevention studies.

Dietary Diindolylmethane Suppresses Inflammation-Driven Lung Squamous Cell Carcinoma in Mice

Inflammatory conditions of the lung such as chronic obstructive pulmonary disease (COPD) are known to increase lung cancer risk, particularly lung squamous cell carcinoma (LSCC). In the present study, we developed a mouse model of inflammation-driven LSCC that was induced by N-nitroso-trischloroethylurea (NTCU) and enhanced by lipopolysaccharide (LPS), a potent proinflammatory agent contained in tobacco and tobacco smoke, and determined the chemopreventive effects of BioResponse diindolylmethane (DIM) in the same model. Compared with mice treated with NTCU alone, mice treated with the combination of NTCU and LPS had a 9-fold increase in the number of bronchioles with LSCC. Also, compared with mice treated with LPS alone, mice treated with NTCU plus LPS showed significantly increased expression of the inflammatory cytokines IL1α, IL6, and TNFα (all three increased about 7-fold). Parallel to the increased cytokine gene expression, the NTCU plus LPS-treated group exhibited significantly enhanced activation of NF-κB, STAT3, ERK, p-38, and Akt, expression of p53, COX-2, and Mcl-1, and NF-κB- and STAT3-DNA binding in the lung. Dietary administration of DIM (10 μmol/g diet or 2,460 ppm) to mice treated with NTCU plus LPS reduced the incidence of LSCC by 2-fold, suppressed activation/expression of proinflammatory and procarcinogenic proteins and NF-κB- and STAT3-DNA binding, but not the expression of cytokines and p53. This study highlights the potential significance of our mouse model to identify promising drugs or dietary agents for the chemoprevention of human LSCC and that DIM is a very good candidate for clinical lung cancer chemoprevention trials.

Honokiol inhibits lung tumorigenesis through inhibition of mitochondrial function.

Honokiol is an important bioactive compound found in the bark of Magnolia tree. It is a nonadipogenic PPARγ agonist and capable of inhibiting the growth of a variety of tumor types both in vitro and in xenograft models. However, to fully appreciate the potential chemopreventive activity of honokiol, a less artificial model system is required. To that end, this study examined the chemopreventive efficacy of honokiol in an initiation model of lung squamous cell carcinoma (SCC). This model system uses the carcinogen N-nitroso-trischloroethylurea (NTCU), which is applied topically, reliably triggering the development of SCC within 24 to 26 weeks. Administration of honokiol significantly reduced the percentage of bronchial that exhibit abnormal lung SCC histology from 24.4% bronchial in control to 11.0% bronchial in honokiol-treated group (P = 0.01) while protecting normal bronchial histology (present in 20.5% of bronchial in control group and 38.5% of bronchial in honokiol-treated group. P = 0.004). P63 staining at the SCC site confirmed the lung SCCs phenotype. In vitro studies revealed that honokiol inhibited lung SCC cells proliferation, arrested cells at the G1-S cell-cycle checkpoint, while also leading to increased apoptosis. Our study showed that interfering with mitochondrial respiration is a novel mechanism by which honokiol changed redox status in the mitochondria, triggered apoptosis, and finally leads to the inhibition of lung SCC. This novel mechanism of targeting mitochondrial suggests honokiol as a potential lung SCC chemopreventive agent.