Steven M. Dubinett

Abstract B16: The effect of e-cigarette exposure on airway epithelial cell gene expression and transformation.

Lung cancer is the leading cause of cancer-related mortality in the United States. Despite a strong correlation between cigarette smoking and the onset of lung cancer, the prevalence of smoking still remains high. Strategies to eliminate cigarette smoking have led to the emergence of new tobacco-related products as alternatives for cigarette smoking or tools for smoking cessation. The electronic cigarette (ECIG) is a battery-powered electronic nicotine delivery system (ENDS) designed to deliver nicotine without combusting tobacco. Since nicotine is widely considered the addictive component in tobacco with limited ability to initiate cancer, ECIGs have been advertised to be a safer alternative to tobacco cigarettes (TCIGs). However, the toxicity and potential carcinogenicity of ECIGs have not previously been evaluated. In this study, we assess the impact of ECIG exposure on the carcinogenic potential of immortalized human bronchial epithelial cells on a background of silenced p53 and activated KRAS (H3mut-P53/KRAS). This model is utilized because p53 and KRAS mutations are often observed in the airway of current and former smokers at risk for lung cancer. The epithelial cells were exposed to both a low and high concentration of nicotine in the ECIG vapor- or TCIG smoke-conditioned media. The lower nicotine concentration was selected to mimic the average plasma nicotine levels in ENDS users and did not demonstrate toxic or anti-proliferative effects on the cells. The higher concentration was chosen to represent the anticipated nicotine levels to which the epithelial cells of smokers are actually exposed. In anchorage independent growth assays, the in vitro correlate of malignant transformation, we found enhanced colony growth in the H3mut-P53/KRAS cells following a 10-day treatment with the high nicotine ECIG- and TCIG-conditioned media compared to the untreated and low nicotine treatment groups. We next assessed the effect of ECIG and TCIG exposure on cell invasion using a three-dimensional air-liquid interface (ALI) model. At baseline, H3mut-P53/KRAS cells exhibit invasive behavior in the ALI model, due to the downstream effects of P53 silencing and KRAS activation. Treatment of H3mut-P53/KRAS cells with low nicotine ECIG- and TCIG-conditioned media did not further enhance the degree of invasion observed in the untreated group. We will next examine the effects of high nicotine conditioned media on cell invasion. Finally, gene expression studies show 263 differentially expressed genes following in vitro exposure to ECIG-conditioned media for 96hrs. The high nicotine ECIG-conditioned media induced a gene expression pattern similar to TCIG- conditioned media and whole cigarette smoke exposure in the H3mut-P53/KRAS cells. Preliminary analyses indicate the observed ECIG-specific gene expression changes were concordantly changed following TCIG-conditioned media exposure. We will next compare the ECIG-induced gene expression signature to carcinogenicity-related gene signatures established in previous and ongoing clinical investigations and test ECIG-altered candidate genes for their ability to drive the malignant transformation of airway epithelial cells. These studies will determine the impact of ECIG exposure on lung carcinogenicity and provide needed scientific guidance to the FDA regarding the physiologic effects of ECIGs. These studies were supported by funding from the following: NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW, SJP), NCI #U01CA152751-AS (SMD, KK), NCI #T32-CA009120-36 (SMD, SJP, PCP), NIH/NHLBI #T32HL072752 (SMD, EL), University of California Tobacco-Related Disease Research Program (TRDRP) #18FT-0060 (TCW), TRDRP #20KT-0055 (TCW), Lung Cancer SPORE P50CA70907 (JDM, JEL) Citation Format: Stacy J. Park, Tonya C. Walser, Catalina Perdomo, Teresa Wang, Paul C. Pagano, Elvira L. Liclican, Kostyantyn Krysan, Jill E. Larsen, John D. Minna, Marc E. Lenburg, Avrum Spira, Steven M. Dubinett. The effect of e-cigarette exposure on airway epithelial cell gene expression and transformation. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B16.

Abstract B07: Loss of LKB1 promotes CXCL8 production via NF-κB and WNT signaling in human bronchial epithelial cells.

Loss of LKB1/STK11 , a tumor suppressor gene, characterizes 5-39% of primary non-small cell lung cancers (NSCLC). In a murine model of lung carcinogenesis with inducible KRAS activation, concurrent mutation of LKB1 ( KrasG12D, Lkb1-/- ) yields a higher frequency of NSCLC tumors and metastasis compared to KRAS mutation only. In vitro studies have shown that following LKB1 loss, tumor cells acquire increased migration and invasion through activation of the SRC kinase family. Although these previous findings offer a possible explanation for LKB1 -dependent tumor progression, few mechanisms have been proposed to delineate how LKB1 deficiency contributes to pulmonary carcinogenesis. In this study, we utilize human bronchial epithelial cells (HBECs) immortalized in the absence of viral onco-proteins that represent a useful model to study the early events of lung carcinogenesis. Knockdown of LKB1 in HBECs (LKB1-KD HBECs) leads to numerous cancer-associated phenotypes, including increased proliferation (when combined with KRAS mutation), preference for glycolytic metabolism and induction of epithelial-to-mesenchymal transition. More importantly, LKB1-KD HBECs secrete higher levels of multiple inflammatory factors, most prominent of which is the pro-angiogenic chemokine CXCL8. By binding to its membrane receptors CXCR1/2 which are universally expressed by neutrophils, macrophages, endothelial cells and pulmonary epithelial cells, CXCL8 is associated with chemotaxis, angiogenesis, tumorigenicity and metastasis. Our data indicate that knockdown of LKB1 in HBECs leads to transcriptional upregulation and elevated secretion of CXCL8. Moreover, the NF-κB and WNT pathways are activated and mediate the increased release of CXCL8 following LKB1 loss in HBECs. Conversely, re-introduction of wildtype LKB1 in LKB1-null NSCLC tumor cells decreases CXCL8 production and reduces NF-κB and WNT signaling activity. These findings suggest that LKB1 deficiency drives augmentation of CXCL8 in the developing tumor microenvironment via LKB1-dependent NF-κB and WNT signaling in HBECs. In future functional studies, we will determine if the LKB1-CXCL8 axis yields LKB1-dependent angiogenesis and tumorigenesis. Delineating the contribution of LKB1 to these malignant phenotypes may yield a more thorough understanding of the pathogenesis of NSCLC, which is needed to create a pathway for the application of novel early detection and chemoprevention strategies. NIH/NCI #U01CA152751 (SMD, TCW), NCI #U01CA152751-S1 (SMD, TCW), NCI #U01CA152751-AS (SMD, KK), NCI SPORE #P50CA70907 (JDM, JEL) Citation Format: RUI LI, Tonya C. Walser, Kostyantyn Krysan, David Shackelford, Jill E. Larsen, John D. Minna, Steven M. Dubinett. Loss of LKB1 promotes CXCL8 production via NF-κB and WNT signaling in human bronchial epithelial cells. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B07.

Lung Tumor Microenvironment and Myelomonocytic Cells

The lung tumor microenvironment consists of tumor cells, stroma, blood vessels, immune infiltrates and the extracellular matrix. Genetic alterations in oncogenes and tumor suppressor genes or epigenetic changes in the tumor that modulate tumor growth and invasion into the surrounding tissue orchestrate the persistence of inflammatory infiltrates. These cellular infiltrates modulate tumor development and progression. The infiltrates vary by size and composition in diverse tumor types and at different stages of tumor development. The lung tumor programs the cellular infiltrates and dysregulates inflammation to sustain tumor growth, progression and hypo responsiveness of the tumor. Characterization of the complex interactions among the infiltrates and lung cancer will aid in defining their role in tumor progression. This understanding will be important for the development of novel anticancer therapies. Although this is not a trivial undertaking, the information garnered will take us a step closer to personalized medicine. If we know an individual’s lung tumor inflammatory infiltrates, we will be able to predict the risk of tumor progression and then give specific treatment to reprogram the tumor microenvironment to control the disease.

Abstract A15: Snail upregulation of SPARC leads to increased invasion in models of both early-and late-stage NSCLC

The zinc-finger protein Snail is upregulated in human non-small cell lung cancer (NSCLC) tissues and is associated with poor patient prognosis. We have shown that Snail upregulation in lung cancer cell lines leads to morphologic changes indicative of epithelial-mesenchymal transition (EMT) in vitro and tumor progression in vivo. Snail overexpression is associated with differential gene expression related to diverse aspects of lung cancer progression. One gene upregulated by Snail in both NSCLC and histologically normal human bronchial epithelial cell lines is secreted protein, acidic and rich in cysteine (SPARC). Expression of SPARC modulates reversible interactions between cells and their extracellular matrix and is known to play an important role in the wound healing process of normal epithelial cells. Its upregulation in several cancer types is associated with increased migration, invasion, and poor patient prognosis. Here, we show that overexpression of Snail leads to increased invasion in a modified Boyden chamber assay in models of both early and late stage NSCLC. Knockdown of SPARC by shRNA leads to a reversal of invasion, indicating that SPARC is at least partially responsible for the effect of Snail on invasion. Computational analysis indicates that Snail does not directly enhance the transcription of SPARC; therefore, we have investigated the indirect mechanism for this relationship. Both ERK1/2 and the chaperone protein HSP27 have emerged as potential components of the pathway. Snail overexpression also leads to activation of ERK1/2, and chemical inhibition of this pathway leads to a decrease in SPARC protein. Snail-overexpressing cell lines show increased transcription of HSP27 mRNA. As Snail has been shown to be overexpressed in the inflammatory microenvironment of premalignancy and in established tumors, SPARC-driven invasion supports both the parallel and linear models of tumor progression. Delineating pathways involved in Snail-dependent and SPARC-mediated tumor progression may yield new targets for lung cancer prevention and treatment.