Lung Premalignancy Research Articles

Premalignant Progression in the Lung: Knowledge Gaps and Novel Opportunities for Interception of Non-Small Cell Lung Cancer. An Official American Thoracic Society Research Statement.

Rationale: Despite significant advances in precision treatments and immunotherapy, lung cancer is the most common cause of cancer death worldwide. To reduce incidence and improve survival rates, a deeper understanding of lung premalignancy and the multistep process of tumorigenesis is essential, allowing timely and effective intervention before cancer development. Objectives: To summarize existing information, identify knowledge gaps, formulate research questions, prioritize potential research topics, and propose strategies for future investigations into the premalignant progression in the lung. Methods: An international multidisciplinary team of basic, translational, and clinical scientists reviewed available data to develop and refine research questions pertaining to the transformation of premalignant lung lesions to advanced lung cancer. Results: This research statement identifies significant gaps in knowledge and proposes potential research questions aimed at expanding our understanding of the mechanisms underlying the progression of premalignant lung lesions to lung cancer in an effort to explore potential innovative modalities to intercept lung cancer at its nascent stages. Conclusions: The identified gaps in knowledge about the biological mechanisms of premalignant progression in the lung, together with ongoing challenges in screening, detection, and early intervention, highlight the critical need to prioritize research in this domain. Such focused investigations are essential to devise effective preventive strategies that may ultimately decrease lung cancer incidence and improve patient outcomes.

EP16.04-021 Chromatin Remodeling Drives Lung Pre-malignancy and is a Target for Prevention

Smoking cessation reduces lung cancer (LC) mortality; however, 50% of LC cases are diagnosed in former smokers, necessitating the need for effective preventive agents. Developing LC preventive drugs has been challenging because of the heterogeneity of this disease with respect to genetic and epigenetic alterations and the need for drugs to be non-genotoxic. Epigenetic deregulation involving methylation of cytosine to form 5-methylcytosine in conjunction with histone modifications in gene promoters to silence transcription is a key step in initiation and pre-malignancy affecting hundreds of genes involved in all aspects of cell regulation.

Chromatin remodeling by the histone methyltransferase EZH2 drives lung pre-malignancy and is a target for cancer prevention

BackgroundTrimethylation of lysine 27 and dimethylation of lysine 9 of histone-H3 catalyzed by the histone methyltransferases EZH2 and G9a impede gene transcription in cancer. Our human bronchial epithelial (HBEC) pre-malignancy model studied the role of these histone modifications in transformation. Tobacco carcinogen transformed HBEC lines were characterized for cytosine DNA methylation, transcriptome reprogramming, and the effect of inhibiting EZH2 and G9a on the transformed phenotype. The effects of targeting EZH2 and G9a on lung cancer prevention was assessed in the A/J mouse lung tumor model.ResultsCarcinogen exposure induced transformation and DNA methylation of 12–96 genes in the four HBEC transformed (T) lines that was perpetuated in malignant tumors. In contrast, 506 unmethylated genes showed reduced expression in one or more HBECTs with many becoming methylated in tumors. ChIP-on-chip for HBEC2T identified 327 and 143 genes enriched for H3K27me3 and H3K9me2. Treatment of HBEC2T and HBEC13T with DZNep, a lysine methyltransferase inhibitor depleted EZH2, reversed transformation, and induced transcriptional reprogramming. The EZH2 small molecule inhibitor EPZ6438 also affected transformation and expression in HBEC2T, while a G9a inhibitor, UNC0642 was ineffective. Genetic knock down of EZH2 dramatically reduced carcinogen-induced transformation of HBEC2. Only DZNep treatment prevented progression of hyperplasia to adenomas in the NNK mouse lung tumor model through reducing EZH2 and affecting the expression of genes regulating cell growth and invasion.ConclusionThese studies demonstrate a critical role for EZH2 catalyzed histone modifications for premalignancy and its potential as a target for chemoprevention of lung carcinogenesis.

Rationale for Lung Adenocarcinoma Prevention and Drug Development Based on Molecular Biology During Carcinogenesis.

Lung adenocarcinoma (LUAD) is the most common and aggressive subtype of lung cancer with the greatest heterogeneity and aggression. Inspite of recent years’ achievements in understanding the pathogenesis of this disease, as well as the development of new therapeutic approaches, our knowledge on crucial early molecular events during its development is still rudimentary. Recent classification and grading of LUAD has postulated that LUAD does not arise spontaneously, but through a stepwise process from lung adenomatous premalignancy atypical adenomatous hyperplasia to adenocarcinoma in situ, minimally invasive adenocarcinoma, and eventually frankly invasive predominant adenocarcinoma. In this review, we discuss the molecular processes that drive the evolutionary process that results in the formation of LUAD. We also describe how to handle lung premalignancy in clinical settings based on the most recent advances in genomic biology and our own understanding of lung cancer prevention.

High-Throughput Drug Screening Identifies a Potent Wnt Inhibitor that Promotes Airway Basal Stem Cell Homeostasis

SUMMARYMechanisms underpinning airway epithelial homeostatic maintenance and ways to prevent its dysregulation remain elusive. Herein, we identify that β-catenin phosphorylated at Y489 (p-β-cateninY489) emerges during human squamous lung cancer progression. This led us to develop a model of airway basal stem cell (ABSC) hyperproliferation by driving Wnt/β-catenin signaling, resulting in a morphology that resembles premalignant lesions and loss of ciliated cell differentiation. To identify small molecules that could reverse this process, we performed a high-throughput drug screen for inhibitors of Wnt/β-catenin signaling. Our studies unveil Wnt inhibitor compound 1 (WIC1), which suppresses T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) activity, reduces ABSC proliferation, induces ciliated cell differentiation, and decreases nuclear p-β-cateninY489. Collectively, our work elucidates a dysregulated Wnt/p-β-cateninY489 axis in lung premalignancy that can be modeled in vitro and identifies a Wnt/β-catenin inhibitor that promotes airway homeostasis. WIC1 may therefore serve as a tool compound in regenerative medicine studies with implications for restoring normal airway homeostasis after injury.

S02.05 The Importance of Elucidating Genomic Events is Lung Premalignancy

S02.05 The Importance of Elucidating Genomic Events is Lung Premalignancy

Abstract 4224: SGLT2 is a diagnostic target for early stage lung adenocarcinoma

Abstract Lung cancer is the leading cause of cancer-related death, and lung adenocarcinoma (LUAD) is the most frequent histology. Early diagnosis and treatment are essential; however, there are no targeted diagnostic and therapeutic approaches for early LUAD. An important hallmark of cancer is the increased glucose uptake via GLUT transporters. GLUT activity is imaged in cancer by positron emission tomography (PET) with 2-[18F] fluorodeoxyglucose (FDG). FDG PET is a standard tool for staging advanced lung cancer, but has low sensitivity for early-stage LUAD. This is considered a consequence of slow growth and low requirement of glucose. However, we discovered a novel system of metabolic supply not detected by FDG PET: the sodium glucose transporter 2 (SGLT2)1. SGLT2 activity can be measured in vivo with the PET tracer methyl-4-[18F] fluorodeoxyglucose (Me4FDG). We identified high expression of SGLT2 in human lung premalignancy and early-stage LUAD. Me4FDG detects early-stage, FDG-negative LUAD in mouse models. Targeting SGLT2 with FDA-approved inhibitors significantly reduces tumor growth and prolongs survival in genetic and patient-derived murine models, confirming an important role of SGLT2 in early-stage LUAD2. The reliance of early stage LUAD on SGLT2 opens exciting diagnostic and therapeutic opportunities. The National Lung Screening Trial showed a 20% reduction in lung cancer mortality in high risk individuals using low-dose helical computed tomography (CT). CT is highly sensitive for detecting lung nodules, but is limited by low specificity, especially for LUAD. On CT, LUAD may appear as solid or subsolid nodules. Most subsolid nodules are not cancer, and many will remain stable or resolve; however, subsolid lesions can represent premalignant lesions or adenocarcinoma in situ. These lesions in the early spectrum of LUAD may persist for months to years before transforming into invasive disease. As a result, current standard of care is to follow these patients with CT imaging to monitor these indeterminate lesions for radiologic signs of malignant progression. The identification of novel biomarkers to predict the malignant potential of these nodules at their initial identification is of paramount importance. 1Scafoglio et al. Proc Natl Acad Sci U S A 2015;112(30):E4111-4119 2Scafoglio et al. Sci. Transl. Med. 10, eaat5933 (2018) Citation Format: Brendon Villegas, Eva Koziolek, Jie Liu, W. Dean Wallace, David Elashoff, Denise R. Aberle, David B. Shackelford, Jorge R. Barrio, Jane Yanagawa, Steven M. Dubinett, Claudio Scafoglio. SGLT2 is a diagnostic target for early stage lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4224.

Abstract 660: The molecular characterization of a murine lung premalignancy model for lung cancer interception

Abstract There is a critical need to identify strategies for lung cancer prevention, as it remains the leading cause of cancer-related death in the US. A critical barrier to developing new approaches to prevent lung cancer is the lack of understanding of key molecular and cellular changes that lead to cancer initiation and progression. Our group has initiated a Pre-Cancer Genome Atlas (PCGA) to define the early molecular alterations that drive premalignant progression of lung cancer. This work has identified transcriptomic alterations in immune pathways associated with progression of bronchial dysplasia (BD) that werefurther validated by immunofluorescent staining showing shifts in immune populations in adjacent bronchial biopsy sections. A challenge to further explore interactions between epithelial and immune cells driving lesion progression is the lack of in vivo models of premalignant transitions ofBD that progress into lung squamous cell carcinoma (LUSC). However, in the NTCU mouse model of LUSC, mice treated with carcinogen develop lesions histologically comparable to human BD. The NTCU-induced murine model serves to act as a robust model for LUSC premalignancy, however, the molecular and cellular alterations driving disease in this model have not been well characterized. This study seeks to investigate the molecular pathogenesis of NTCU-induced BD progression to carcinoma to that of human BD progression to determine the pre-clinical utility of the model for intervention studies. RNA was isolated from normal, dysplastic and tumor tissue from NTCU-treated mice and sequenced (n=22). Gene expression alterations associated with histological grade were identified using regression models. Concordance between the murine and human data was investigated using GSEA (Gene Set Enrichment Analysis). We identified 503 genes differentially expressed with increasing histological severity in the mouse dysplasia (FDR<0.05) and involved in cancer signaling and immune regulation. We also identified significant concordance of gene expression differences associated with histology between mouse and human dysplastic lesions (FDR<0.05). Furthermore, fluorescent cytometry analysis of lung leucocytes detected immune cell infiltration associated with increasing dose anddisease progression (n=30), and identified a dose-dependent suppression of several lymphoid populations and increase in myeloid populations frequencies associated with disease progression. These observations were concordant with transcriptional and histologic findings in both mouse and human samples. In summary, our data suggest molecular and cellular concordance between human and murine lesions that precede the development of LUSC, and suggest NTCU-induced BD progression to LUSC be a relevant pre-clinical model for testing targeted immunoprevention strategies. Citation Format: Sarah A. Mazzilli, Kahkeshan Hijazi, Riley Pihl, Anna Belkina, Xiaohui Zhang, Gang Liu, Marc Lenburg, Christopher Stevenson, Avrum Spira, Jennifer Beane. The molecular characterization of a murine lung premalignancy model for lung cancer interception [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 660.

DNA-PKc deficiency drives pre-malignant transformation by reducing DNA repair capacity in concert with reprogramming the epigenome in human bronchial epithelial cells

The expression of DNA-dependent protein kinase catalytic subunit (DNA-PKc) is highly variable in smokers and reduced enzyme activity has been associated with risk for lung cancer. An in vitro model of lung pre-malignancy was used to evaluate the role of double-strand break DNA repair capacity in transformation of hTERT/CDK4 immortalized human bronchial epithelial cells (HBECs) and reprograming of the epigenome. Here we show that knockdown of DNA-PKc to levels simulating haploinsufficiency dramatically reduced DNA repair capacity following challenge with bleomycin and significantly increased transformation efficiency of HBEC lines exposed weekly for 12 weeks to this radiomimetic. Transformed HBEC lines with wild type or knockdown of DNA-PKc showed altered expression of more than 1,000 genes linked to major cell regulatory pathways involved in lung cancer. While lung cancer driver mutations were not detected in transformed clones, more than 300 genes that showed reduced expression associated with promoter methylation in transformed clones or predictive for methylation in malignant tumors were identified. These studies support reduced DNA repair capacity as a key factor in the initiation and clonal expansion of pre-neoplastic cells and double-strand break DNA damage as causal for epigenetic mediated silencing of many lung cancer-associated genes. The fact that DNA damage, repair, and epigenetic silencing of genes are causal for many other cancers that include colon and prostate extends the generalizability and impact of these findings.

Sodium-glucose transporter 2 is a diagnostic and therapeutic target for early-stage lung adenocarcinoma.

The diagnostic definition of indeterminate lung nodules as malignant or benign poses a major challenge for clinicians. We discovered a potential marker, the sodium-dependent glucose transporter 2 (SGLT2), whose activity identified metabolically active lung premalignancy and early-stage lung adenocarcinoma (LADC). We found that SGLT2 is expressed early in lung tumorigenesis and is found specifically in premalignant lesions and well-differentiated adenocarcinomas. SGLT2 activity could be detected in vivo by positron emission tomography (PET) with the tracer methyl 4-deoxy-4-[18F] fluoro-alpha-d-glucopyranoside (Me4FDG), which specifically detects SGLT activity. Using a combination of immunohistochemistry and Me4FDG PET, we identified high expression and functional activity of SGLT2 in lung premalignancy and early-stage/low-grade LADC. Furthermore, selective targeting of SGLT2 with FDA-approved small-molecule inhibitors, the gliflozins, greatly reduced tumor growth and prolonged survival in autochthonous mouse models and patient-derived xenografts of LADC. Targeting SGLT2 in lung tumors may intercept lung cancer progression at early stages of development by pairing Me4FDG PET imaging with therapy using SGLT2 inhibitors.

Abstract A16: Sodium-dependent glucose transporter 2 is a novel diagnostic and therapeutic target for early-stage lung adenocarcinoma

Abstract Lung cancer claims approximately 160,000 lives in the United States every year, and lung adenocarcinoma (LADC) is the most frequent type. Early diagnosis is crucial; the National Lung Screening Trial showed a 20% reduction in mortality in high-risk individuals screened by computed tomography (CT). However, CT has low specificity in early-stage LADC. In vivo detection of glucose uptake by FDG PET is a standard tool for lung cancer staging, but has low sensitivity in early-stage LADC. Here, we demonstrate that GLUT transporters, responsible for FDG uptake, are expressed in advanced, poorly differentiated LADC, whereas early LADC utilizes a different glucose transport system, the sodium-dependent glucose transporter 2 (SGLT2), that is not detected by FDG. This can be imaged with the novel tracer Me4FDG. We investigated the expression of two different glucose transporters, GLUT1 and SGLT2, in a cohort of 56 human LADC specimens, in patient-derived xenografts, and in a KrasG12D-driven, p53-null genetically engineered mouse model of LADC. We observed a switch in the modality of glucose transport during lung carcinogenesis: SGLT2 was highly expressed in premalignant lesions and well-differentiated LADC, whereas GLUT1 was upregulated in advanced, poorly differentiated lesions. This pattern was observed both in human samples and in murine models. This observation led us to hypothesize that early-stage LADCs are often negative on FDG PET because this imaging modality does not detect the activity of SGLT2, which is expressed in early lesions. Therefore, we performed PET imaging with the tracer Me4FDG, which measures SGLT2 activity, in our mouse model, and observed that Me4FDG accumulated in small nodules that were negative with FDG. We confirmed the functionality of SGLT2 in human LADC by Me4FDG PET in patient-derived xenografts. Finally, we tested the hypothesis that sodium-dependent glucose transport is a critical metabolic supply strategy in the early stages of lung adenocarcinoma development. We performed therapeutic trials in genetically engineered mouse models with the FDA-approved SGLT2 inhibitor canagliflozin, which slowed down tumor progression and prolonged mouse survival, by specifically reducing the proliferation rate of premalignant lesions. In conclusion, Me4FDG PET is a novel diagnostic test to image lung premalignancy and early-stage LADC. We propose that Me4FDG-PET will identify the patients who are likely to respond to SGLT2 inhibition, as a cancer interception strategy to prevent the progression of premalignancy to invasive disease, by blocking the supply of glucose required for proliferation of premalignant lesions. Citation Format: Claudio Scafoglio, Sean T. Bailey, Gihad Abdelhady, Jie Liu, Jane Yanagawa, Aditya Shirali, Dean W. Wallace, Jorge R. Barrio, Ernest M. Wright, Tonya Walser, Steven M. Dubinett, David B. Shackelford. Sodium-dependent glucose transporter 2 is a novel diagnostic and therapeutic target for early-stage lung adenocarcinoma [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A16.

Early Events in the Molecular Pathogenesis of Lung Cancer.

The majority of cancer-related deaths in the United States and worldwide are attributed to lung cancer. There are more than 90 million smokers in the United States who represent a significant population at elevated risk for lung malignancy. In other epithelial tumors, it has been shown that if neoplastic lesions can be detected and treated at their intraepithelial stage, patient prognosis is significantly improved. Thus, new strategies to detect and treat lung preinvasive lesions are urgently needed in order to decrease the overwhelming public health burden of lung cancer. Limiting these advances is a poor knowledge of the earliest events that underlie lung cancer development and that would constitute markers and targets for early detection and prevention. This review summarizes the state of knowledge of human lung cancer pathogenesis and the molecular pathology of premalignant lung lesions, with a focus on the molecular premalignant field that associates with lung cancer development. Lastly, we highlight new approaches and models to study genome-wide alterations in human lung premalignancy in order to facilitate the discovery of new markers for early detection and prevention of this fatal disease. Cancer Prev Res; 9(7); 518-27. ©2016 AACR.

Abstract A2-63: Airway gene-coexpression network rewiring in the presence of bronchial dysplasia and lung cancer

Abstract Lung cancer is a deadly disease, killing 160,000 a year in the US. The 5-year survival rate of lung cancer patients is invariably low, partially due to a lack of early diagnoses and limited access to CT screening programs during the early, most-curable stages of the disease. The urgent need for alternative early-stage diagnostics has been addressed in previous work, which focused on smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells. These lung cancer associated alterations have been shown to serve as a clinically relevant biomarker for the early detection of lung cancer. In this study, we have expanded upon this work by leveraging gene expression from airway epithelial cells collected in the presence of bronchial premalignant lesions. Specifically, we conducted network-based analyses to further explore alterations in bronchial gene coexpression (GCE) patterns in the uninvolved large airway of patients with tobacco-induced premalignancy and those with lung cancer. By studying airway GCE patterns present in patients with lung cancer and premalignancy, but absent in disease-free smokers, we hope to discover the earliest changes in process of lung carcinogenesis that may serve as novel targets for lung cancer treatment and prevention. Coexpression networks use expression-profile correlations to cluster genes to provide system-wide context for single genes implicated in a disease and elucidate the influence of interconnectedness of cellular components on phenotype. Here, we employed Weighted Gene Coexpression Network Analysis (WGCNA) to build GCE networks derived from cytologically normal airway epithelial cells obtained from current and former smokers with lung cancer (n=409), dysplastic airway lesions (n=121), and neither condition (n=412). Samples were collected from subjects undergoing white-light bronchoscopy as part of the AEGIS trial by Allegro Dx, or from subjects undergoing autofluorescence bronchoscopy in the British Columbia Lung Health Study. RNA was extracted from cells and profiled on microarrays or by mRNA-Seq. In order to detect and quantify reconfiguration of networks under different disease states, the Modular Differential Connectivity (MDC) measure was calculated to determine the degree of topological similarity between any pair of GCE modules. In addition, Fisher's Exact Test (FET) was used to calculate significance of gene-member overlap between pairs of GCE modules originating from disease networks to distinguish modules conserved in premalignancy and cancer. To identify modules conserved between disease states, but dissimilar from the healthy smoker modules, we focused on those with (a) significant overlap by FET, (b) non-differentiating premalignancy vs. cancer MDC and (c) differentiating normal vs. disease MDC. Hub genes within each network were identified based on intra-modular connectivity, a measure of module essentiality. We prioritized genes with increased connectivity upon entering the premalignant state that remained strong in the lung cancer network. We identified 16 module pairs conserved between disease states, i.e. cancer modules similarly connected (MDC FDR<0.05) and significantly overlapping (pFET<0.01) with premalignancy modules, one of which was weakly connected in the disease-free networks (MDC FDR<0.05). Functional annotation of these modules revealed associations with pathways and transcription factors implicated in carcinogenesis and cell proliferation. Furthermore, we identified two hub genes linked to pathways regulating cigarette smoke-induced signaling and inhibition of lung cancer cell growth. Integration of differential connectivity and GCE network analyses represents a novel approach to discovering pathways associated with early lung carcinogenesis. Our results suggest the occurrence of transcriptomic alterations that arise in patients with premalignancy and remain present in patients with lung cancer. Future work will include experimental validation of these findings. The computational approach outlined above may aid in the identification of candidate master regulators of early lung carcinogenesis that have potential utility both as indicators of increased lung cancer risk and as early-stage drug targets for chemoprevention or therapy. Citation Format: Anna Tassinari, Bin Zhang, Katrina Steiling, Duncan Whitney, Kate Porta, Stephen Lam, Marc Lenburg, Avrum Spira, Jennifer Beane. Airway gene-coexpression network rewiring in the presence of bronchial dysplasia and lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-63.

Abstract B1-17: Airway gene-coexpression network rewiring in the presence of bronchial dysplasia and lung cancer

Abstract Lung cancer is a deadly disease, killing 160,000 a year in the US. The 5-year survival rate of lung cancer patients is invariably low, partially due to a lack of early diagnoses and limited access to CT screening programs during the early, most-curable stages of the disease. The urgent need for alternative early-stage diagnostics has been addressed in previous work, which focused on smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells. These lung cancer associated alterations have been shown to serve as a clinically relevant biomarker for the early detection of lung cancer. In this study, we have expanded upon this work by leveraging gene expression from airway epithelial cells collected in the presence of bronchial premalignant lesions. Specifically, we conducted network-based analyses to further explore alterations in bronchial gene coexpression (GCE) patterns in the uninvolved large airway of patients with tobacco-induced premalignancy and those with lung cancer. By studying airway GCE patterns present in patients with lung cancer and premalignancy, but absent in disease-free smokers, we hope to discover the earliest changes in process of lung carcinogenesis that may serve as novel targets for lung cancer treatment and prevention. Coexpression networks use expression-profile correlations to cluster genes to provide system-wide context for single genes implicated in a disease and elucidate the influence of interconnectedness of cellular components on phenotype. Here, we employed Weighted Gene Coexpression Network Analysis (WGCNA) to build GCE networks derived from cytologically normal airway epithelial cells obtained from current and former smokers with lung cancer (n=409), dysplastic airway lesions (n=121), and neither condition (n=412). Samples were collected from subjects undergoing white-light bronchoscopy as part of the AEGIS trial by Allegro Dx, or from subjects undergoing autofluorescence bronchoscopy in the British Columbia Lung Health Study. RNA was extracted from cells and profiled on microarrays or by mRNA-Seq. In order to detect and quantify reconfiguration of networks under different disease states, the Modular Differential Connectivity (MDC) measure was calculated to determine the degree of topological similarity between pairs of GCE modules. In addition, Fisher's Exact Test (FET) was used to calculate significance of gene-member overlap between pairs of GCE modules originating from disease networks to distinguish modules conserved in premalignancy and cancer. To identify modules conserved between disease states, but dissimilar from the healthy smoker modules, we focused on those with (a) significant overlap by FET, (b) non-differentiating premalignancy vs. cancer MDC and (c) differentiating normal vs. disease MDC. Hub genes within each network were identified based on intra-modular connectivity, a measure of module essentiality. We prioritized genes with increased connectivity upon entering the premalignant state that remained strong in the lung cancer network. We identified 16 module pairs conserved between disease states, i.e. cancer modules similarly connected (MDC FDR<0.05) and significantly overlapping (pFET<0.01) with premalignancy modules, one of which was weakly connected in the disease-free networks (MDC FDR<0.05). Functional annotation of these modules revealed associations with pathways and transcription factors implicated in carcinogenesis and cell proliferation. Furthermore, we identified two hub genes linked to pathways regulating cigarette smoke-induced signaling and inhibition of lung cancer cell growth. Integration of differential connectivity and GCE network analyses represents a novel approach to discovering pathways associated with early lung carcinogenesis. Our results suggest the occurrence of transcriptomic alterations that arise in patients with premalignancy and remain present in patients with lung cancer. Future work will include experimental validation of these findings. The computational approach outlined above may aid in the identification of candidate master regulators of early lung carcinogenesis that have potential utility both as indicators of increased lung cancer risk and as early-stage drug targets for chemoprevention or therapy. Citation Format: Anna Tassinari, Bin Zhang, Katrina Steiling, Duncan Whitney, Kate Porta, Stephen Lam, Marc Lenburg, Avrum Spira, Jennifer Beane. Airway gene-coexpression network rewiring in the presence of bronchial dysplasia and lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-17.

Abstract ED02-03: DNA methylation as a target in cancer prevention

Abstract Epigenetic silencing of genes and microRNAs (miRs) via chromatin remodeling and cytosine methylation has emerged as a major mechanism in cancer etiology. The reversal of gene and miR silencing by affecting cytosine-DNA methyltransferases (DNMTs), histone methyltransferases (HMTs), or histone deacetylases (HDACs) offers new strategies for cancer prevention and intervention for lung cancer. This presentation will provide an overview of studies using natural chemopreventive agents (e.g., lycopenes, isoflavones, omega-3 fatty acids) that reprogram the epigenome for affecting lung, colon, breast and prostate cancer either through generalized effects or targeting the expression of specific microRNAs to modulate genes involved in cancer. Focused studies from our laboratory using an in vitro model of hTERT/cdk4 immortalized human bronchial epithelial cell lines (HBECs) will describe the sequence of events driving gene and microRNA silencing for lung pre-malignancy and novel preventive strategies to reverse transformation. New concepts that incorporate tissue surrogates and biomarkers to assess in humans the efficacy of chemopreventives for lung cancer will be presented. Citation Format: Steven A. Belinsky. DNA methylation as a target in cancer prevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr ED02-03.

Abstract 5592: Lovastatin and low dose celecoxib decrease proliferation and inflammation in a cellular model of lung pre-malignancy

Abstract Statin drugs, such as lovastatin and pravastatin, block the cellular synthesis of cholesterol by inhibiting the function of HMG-CoA reductase. In a prospective study, patients undergoing statin treatment for prevention of atherosclerosis were at a lower risk of developing certain cancers, including lung and breast, suggesting a possible use for lovastatin in the chemoprevention of lung cancer. Here, we show that lovastatin dose-dependently decreases proliferation in both histologically normal and Kras-mutated human bronchial epithelial cells (HBECs) through the inhibition of cholesterol production. However, increased expression of PGE2, which we and others have shown to contribute to lung cancer progression, was noted with lovastatin treatment alone. Lovastatin up-regulates the production of PGE2 by up-regulating COX-2 expression and simultaneously down-regulating metabolism of PGE2 via down-regulation of 15-PGDH expression. Treatment with the cholesterol precursor, mevalonic acid, inhibited up-regulation of PGE2 and blocked dose-dependent anti-proliferative activity of lovastatin, indicating sterol-dependence. Treatment with both lovastatin and low dose (0.1uM) celecoxib, a COX-2 inhibitor, decreased PGE2 protein production in vector control and Kras-mutated HBECs. These findings suggest that the combination of lovastatin and low dose celecoxib overcomes up-regulation of PGE2 by lovastatin alone and that the combination represents a potentially effective lung cancer chemopreventative regimen worthy of further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5592.

Abstract 3472: Inhaled carcinogen induced lung cancer in mice

Abstract Inhaled carcinogen-induced lung cancer originates at the bronchial epithelium. Traditional animal models with tumor grafting or intraperitoneal/intravenous injection of tobacco carcinogens either lack the natural stages of tumor initiation and progression, or lack the advantage of modeling the disease through direct exposure of the airway epithelium to the causative agents. To overcome these problems, we used intratracheal inoculation of the major tobacco carcinogens benzo[a]pyrene (BP) and nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to induce lung premalignancy and lung cancer in two different strains of mice. This mouse model of inhaled carcinogen-induced lung cancer consistently mimics the carcinogenesis process in tobacco-induced human lung cancer that progresses through a series of identifiable histopathological stages. In our model the lung carcinogenesis clearly progresses through three different stages: 1. Early transitional stage with no pathological lesions (0-15 weeks after first dose of the carcinogens); 2. Premalignant stage with dysplasia in the lungs in 100% of both A/J and ICR mice at 16 weeks after the first dose of the carcinogens; 3. Malignant stage with tumor nodules developed in the lungs in 100% of A/J and > 60% of ICR mice at 20-24 weeks. We have been using this model in trying to identify key molecular players in each stage that could possibly serve as therapeutic targets for lung cancer treatment. Currently it is one of best available models for studying environmental carcinogen-induced lung cancer and warrants further characterization. Future studies using this model are expected to enhance our understanding of the disease and facilitate our efforts of developing early therapeutic interventions. Supported by NIH grants CA96515 and CA104297. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3472.

A selective small molecule inhibitor of c-Met, PHA-665752, reverses lung premalignancy induced by mutant K-ras.

The c-Met receptor tyrosine kinase has been implicated in cellular transformation induced by mutant Ras, a commonly activated proto-oncogene in non-small cell lung cancer (NSCLC). However, the role of c-Met has not been defined in K-ras-mutant NSCLC, a disease for which no effective targeted therapeutic options currently exist. To acquire a greater understanding of its role, we used genetic and pharmacologic approaches to inhibit c-Met in mice and cultured cells. In Kras(LA1) mice, which develop premalignant lung lesions that progress to multifocal lung adenocarcinomas owing to somatic mutations in K-ras, c-Met was expressed in multiple cell types within premalignant lung lesions, and high concentrations of HGF were detected in bronchoalveolar lavage samples. Short-term treatment with PHA-665752, a c-Met inhibitor, decreased the numbers of premalignant lung lesions and induced apoptosis in tumor cells and vascular endothelial cells within lesions. In cell culture, PHA-665752 induced apoptosis of a lung adenocarcinoma cell line derived from Kras(LA1) mice (LKR-13) and a murine lung endothelial cell line (MEC). c-Met depletion by siRNA transfection induced apoptosis of MECs but not LKR-13 cells. Collectively, these findings suggest that apoptosis was an on-target effect of PHA-665752 in MECs but not in LKR-13 cells. We conclude that PHA-665752 inhibited lung tumorigenesis in Kras(LA1) mice and may provide a novel therapeutic approach to the prevention of K-ras-mutant NSCLC.

Lung premalignancy induced by mutant B-Raf, what is thy fate? To senesce or not to senesce, that is the question: Figure 1.

Lung premalignancy induced by mutant B-Raf, what is thy fate? To senesce or not to senesce, that is the question: Figure 1.

Lung cancer chemoprevention.

Lung cancer is the leading cause of cancer death in the United States. The persisting grim lung cancer incidence and mortality figures argue powerfully for new approaches such as chemoprevention for controlling this disease. Retinoids are among the most intensively studied cancer chemoprevention agents, including in the lung. Several randomized clinical or translational chemoprevention trials (e.g., of retinoids, beta-carotene, or combined folic acid and vitamin B(12)) have been conducted in lung pre-malignancy. Retinoid studies have produced important data on molecular/cellular markers of lung carcinogenesis, e.g., loss of heterozygosity (LOH) at 3p and 9p and retinoic acid receptor-beta (RAR-beta). Two large randomized trials with a lung cancer endpoint, the Alpha-Tocopherol, Beta-Carotene (ATBC) Prevention Study and the Beta-Carotene and Retinol Efficacy Trial (CARET), found that beta-carotene (+/- retinol) was harmful (in smokers). Recently completed lung-second-primary-tumor-prevention trials include the retinoids retinyl palmitate and 13-cis-retinoic acid (13cRA) and N-acetylcysteine (NAC). Vitamin E and selenium show promise for lung cancer prevention, based on positive secondary/subset analyses of three large-scale, randomized National Cancer Institute (NCI) cancer prevention trials. Future directions of lung cancer chemoprevention include the study of molecular markers of risk and drug activity, molecular targeting study, improved imaging techniques (e.g., molecular imaging) and new drug delivery systems.