• Analysis of TWEAK Receptor (Fn14) Expression and Function in Non-Small Cell Lung Cancer Cells

      Cheng, Emily; Winkles, Jeffrey Allan (2014)
      The cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a TNF superfamily member that is expressed by multiple cell types and is involved in many functions including proliferation, migration, survival, differentiation, de-differentiation, or cell death. It is the only ligand for the TNF receptor (TNFR) superfamily member Fibroblast Growth Factor-Inducible 14 (Fn14). The TWEAK:Fn14 signaling axis mediates multiple cellular processes including inflammation, angiogenesis, cell growth and death, and progenitor differentiation to aid in wound repair. Fn14 is overexpressed in over a dozen solid tumor types and constitutive signaling of the receptor is thought to be involved in tumor growth and metastasis. We previously showed that Fn14 levels are elevated in non-small cell lung cancer (NSCLC) tumors and NSCLC cell lines expressing constitutively activated Epidermal Growth Factor Receptor (EGFR) mutants. Also, we found that treatment of EGFR-mutant cells with erlotinib, (an EGFR tyrosine kinase inhibitor that is FDA-approved for use in the treatment of advanced NSCLC) decreases Fn14 levels and that Fn14 levels regulate NSCLC cell migration in vitro. In the present study, we extended these findings by showing that Fn14 levels also regulate NSCLC cell invasion. We also provide evidence that EGFR-mutant NSCLC cells that express high levels of Fn14 exhibit constitutive activation of the cytoplasmic tyrosine kinase Src. We found that inhibition of Src activity in NSCLC cells by dasatinib decreases Fn14 gene expression at both the mRNA and protein levels. Src depletion in NSCLC cells by siRNA also downregulates Fn14 protein expression. Finally, we show that Fn14 expression is significantly higher in an NIH 3T3 cell line engineered to express the constitutively active v-Src oncoprotein in comparison to parental NIH 3T3 cells, and that the NIH 3T3/v-Src cells require Fn14 expression for full invasive capacity. Taken together, these data demonstrate a functional role for Fn14 in NSCLC cell invasion and identify the Src tyrosine kinase as a new regulator of Fn14 gene expression.
    • Association between History of Chronic Lung Disease and Non-Small Cell Lung Carcinoma in Maryland

      Gardner, Lisa Dawn Marie; Amr, Sania; 0000-0003-3340-2818 (2016)
      Although cigarette smoking is the primary risk factor for non-small cell lung cancer (NSCLC), 25% of cases are not due to smoking or other established risk factors. Chronic lung diseases (chronic bronchitis, emphysema, and asthma) are major sources of inflammation in lung tissue, and a history of these diseases may increase one's risk of NSCLC, especially among never smokers. We used data from the Maryland Lung Cancer Study to investigate whether a history of chronic lung disease is associated with NSCLC risk, and if use of aspirin and/or other non-steroidal anti-inflammatory drugs (NSAIDs) decreases such risk, independently of smoking status. In the present case-control study, 1,660 NSCLC cases and 1,959 population controls were interviewed using a standardized questionnaire. Logistic regression estimated adjusted odds ratios (OR) for having NSCLC by: 1) history and 2) mean duration of chronic lung disease; and 3) regular use, 4) mean frequency, and 5) mean duration of aspirin and/or other NSAIDs. A history of chronic lung disease was statistically significantly associated with having NSCLC (OR = 1.87, 95% confidence interval (CI) 1.54-2.28). When stratified by smoking status, a history of chronic lung disease significantly increased the odds of having NSCLC in never (OR = 1.99, 95% CI 1.19-3.34), former (OR = 1.68, 95% CI 1.29-2.20), and current smokers (OR = 2.40, 95% CI 1.62-3.57), compared to those without chronic lung disease. Regular aspirin use was significantly associated with a 36% decreased risk of NSCLC, compared to non-regular use (OR = 0.64, 95% CI 0.52-0.79), but this association remained significant only in former (OR = 0.62, 95% CI 0.47-0.82) and current smokers (OR = 0.55, 95% CI 0.37-0.81). Regular use of other NSAIDs was associated with a statistically significant increase in the risk of having NSCLC compared to non-regular use (OR = 1.55, 95% CI 1.08-2.22); this association remained significant in former smokers (OR = 1.89, 95% CI 1.12-3.21). This study provides support for: 1) chronic inflammation as a potential contributing factor to NSCLC risk, regardless of smoking status, sex, and race; and 2) regular use of aspirin as a protective factor in former and current smokers.
    • The Elucidation of Mutation-Specific Glycoprotein and Exosomal Non-small Cell Lung Cancer Biomarkers

      Clark, David Joseph; Mao, Li, M.D.; 0000-0003-0527-8469 (2015)
      Lung cancer is the leading cause of cancer-related death in the world. This high rate of mortality is often a result of late-stage diagnosis, which occurs in the majority of lung cancer cases. The high false positive rate associated with the current screening technique leads to unnecessary and invasive follow-up procedures, thus newer diagnostics must be developed. Here, we evaluated the potential role of exosomes as "biomarker vesicles" in non-small cell lung cancer (NSCLC). We employed quantitative proteomics to evaluate both N-linked glycoprotein expression patterns, and exosomal protein cargo abundance differences between an immortalized bronchial epithelial cell line and two NSCLC cell lines each harboring distinct, non-overlapping mutations in cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR). The inherent benefits of this strategy included: (i) the identification of several glycoproteins that were overexpressed in both NSCLC cells, as well as glycoproteins uniquely overexpressed in each profiled NSCLC cell line, and (ii) characterization of NSCLC exosomes to determine proteins enriched in NSCLC exosomes. These results allowed us to generate a preliminary panel of overexpressed glycoproteins that could be further validated as enriched NSCLC exosomal protein cargo, and identify several potential biomarkers of NSCLC to be examined in future clinical studies. We also developed a novel strategy to unbiasedly characterize the exosome proteome by pairing quantitative proteomics and multivariate cluster analysis. Using the breast cancer cell line SKBR3B as a proof-of-concept, we were able to calculate the probability of an identified protein being exosomal in origin. With this method, we were able to determine proteins enriched in the exosome fraction in an unbiased manner, and identify potential residual contaminant proteins that were identified in the final analyte. In summary, our study provides an in-depth characterization of NSCLC exosomes and the identification of several exosomal proteins that could serve as candidate markers of NSCLC In addition, we described a methodology to classify a protein as a "true" constituent of the exosome proteome. Taken together, these studies provide a foundation for the development of a platform that utilizes exosomes in a diagnostic setting.
    • Lung cancer risk associated with pulmonary tuberculosis and inflammatory lesions in the lungs

      Yu, Ying-Ying; Baumgarten, Mona (2007)
      Background. The inflammatory response induced by tuberculosis, scarring, and granulomas may increase the risk for lung cancer. Methods. (I) SEER-Medicare linked database was used to examine the association of lung cancer and history of tuberculosis among persons aged 65 and older. We included lung cancer cases from SEER registries and frequency-matched population-based controls. Medicare claims data were used to ascertain of tuberculosis. We analyzed the association between case/control status and tuberculosis using logistic regression models adjusting for sex, age, race, SEER registry, and year of selection. To explore the possibility of confounding by smoking, we also assessed the association with larynx and bladder cancers, because these two types of cancer are known to be associated with smoking but not tuberculosis. (II) Data from the PLCO cancer screening trial were examined to test the association between lung cancer and baseline chest radiographic (CXR) diagnosis of granulomas and scarring. The screening cohort received a baseline CXR and completed a study questionnaire. We used multivariable Cox proportional hazards models to estimate the HRs adjusted for age at randomization, sex, race and cumulative tobacco smoking. Results. (I) We identified 107,389 lung cancer cases and the same number of controls. Odds of having tuberculosis within the previous 17 years were 4.2 times higher among cases than controls (95% CI 3.5-5.1) adjusted for confounders. The association was stronger when history of tuberculosis was defined in terms of inpatient claims (OR 4.7) compared to outpatient claims (OR 3.8). The association was strongest in the year preceding lung cancer diagnosis (OR 10.4), but was still significant in the 1-10 year period prior (OR 2.5). We also found increased odds of having tuberculosis among larynx cancer cases (OR 3.6), but not among bladder cancer cases (OR 1.2), compared to controls. (II) 809 lung cancers were identified following baseline CXRs. Increased lung cancer risk was associated with the presence of scars (HR 1.5, 95% CI 1.2-1.8) but not granulomas (HR 1.1, 95% CI 0.9-1.4). The findings were consistent across time (1-10 years). Conclusion. The possible etiologic associations between lung cancer and pulmonary damage induced by tuberculosis or other scarring conditions may be mediated through chronic inflammation.
    • Novel effects of piRNAs and pfeRNAs in lung somatic cells

      Gable, Tyler; Mao, Li, M.D.; Schneider, Abraham (2017)
      Aberrant expression and function of PIWI-interacting RNAs (piRNAs) and piRNA-Likes (pilRNAs or piR-Ls) have been reported in various cancers. The majority of current reports have identified or assumed roles of these pilRNA that require association with PIWI family proteins to affect either transposable element silencing or mRNA transcript silencing through base pair matching. However, new reports are describing pilRNA which are capable of regulating physiological and pathological conditions through interactions with non-PIWI and non-PIWI-related proteins and whose interaction is necessary for the function of the binding partner protein. Specific cases examined in lung tissue have also found evidence that such interactions may be occurring at critical phosphorylated residues on the target proteins and suggests an early mechanism through which such interactions may occur. Therefore, we hypothesized that somatic expression of piRNA and piRNA-Like non-coding RNAs play active and dynamic roles in the progression of lung squamous cell and adenocarcinoma via phosphorylation-site interactions of PIWI-independent mechanisms. We began our investigations with RNA sequencing profiles of differentially expressed piRNA and pilRNA in lung somatic cells. Guided by expression data, we explored the phenotype and mechanism of action of potentially significant pilRNA species. We began with our study investigating a piR-L species which induces chemoresistance to cisplatin-based therapy by inhibiting apoptosis in lung squamous cell carcinoma. Next, we examined mitochondrial piRNA57125 which associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis. Finally, we performed a phosphorylation-wide sncRNA screen which reveals Protein Functional Effector sncRNAs (pfeRNAs) in human lung somatic cells. Collectively, these studies have supported the hypothesis that pilRNA and pfeRNA do play critical roles in the progression of lung squamous cell and adenocarcinomas and may, in fact, do so through phosphorylation-site interactions. As a result of these studies, we have re-named these pilRNA as pfeRNA, a more encompassing and descriptive terminology which is described below.
    • Role of Cell Cycle Regulators Enhancer of Zeste Homolog 2 and Cell Division Cycle 25A in Tumorigenesis of the Aerodigestive Tract

      Younis, Rania Hassan M.; Mao, Li, M.D. (2011)
      Oral Squamous Cell Carcinoma (OSCC) and Non Small Cell Lung Cancer (NSCLC) represent the most common aerodigestive tract malignancies and are a major public health burden worldwide. The prognosis of NSCLC patients is dismal with a 5-year survival rate of 16%. Although the 5-year survival rate of patients with OSCC is more than 50%, many of the survivors suffer significant treatment side effects and disfigurement. Therefore, further understanding of the biologic basis of tumorigenic processes of these cancers is critical for development of novel diagnostic and therapeutic strategies. One of the common features of these cancers is the deregulation of cell cycle process during carcinogenesis. The transcriptional repressor Enhancer of Zeste Homolog 2 (EZH2) has been implicated in cell cycle regulation and tumorigenesis. While the cell cycle promoter Cell Division Cycle 25A (CDC25A) has been proposed as a critical cell cycle promoter linked to several malignancies. Here we investigated the role of the epigenetic EZH2 on cell cycle progression and malignant phenotypes in oral premalignancy and NSCLC. We also studied CDC25A expression and modulation of the cell cycle in NSCLC. Our results provide evidence that EZH2 promotes malignancy in Leuk-1 cell line and is a prognostic marker to OSCC onset in patients with oral premalignancy. EZH2 expression directly correlated with expression of cell cycle promoters in NSCLC NCI-H1299 and Leuk-1 cells. We also identified CDC25A-Q110del a stable isoform in NSCLC that confers CDC25A protein stability and promotes its activity. CDC25A-Q110del confers more cellular survival upon DNA damage and correlates with poor overall survival in NSCLC patients. These data unravel a novel role of EZH2 in cell cycle regulation of oral premalignancy and describe a new mode of regulation and expression of CDC25A in NSCLC. Given the potential role of the two molecules in the tumorigenic process, they may also serve as targets for blocking tumor development or progression. The link between EZH2 and CDC25A in cell cycle regulation may warrant additional investigation regarding the cross talk between the two molecules.
    • Smoking, estrogen, and lung cancer in women

      Blackman, Janine Annette; Bush, Trudy (1997)
      Mortality rates from lung cancer in women have been rising since the 1940s, and surpassed breast cancer rates in 1987. Smoking is clearly a major risk factor for lung cancer, although other factors also may influence risk among smokers. Recently, estrogen receptors have been detected in lung tumors. One study suggested that adenocarcinoma of the lung was increased among users of estrogen replacement therapy (ERT). Another report suggested reduced risk of death from lung cancer in ERT users. Given the large numbers of women taking ERT, the association between ERT and lung cancer needs to be further addressed. A case-control study design, including women from several cities in the U.S., was used to investigate the association between lung cancer (adenocarcinoma, squamous, small, and other cell types) and exposure to ERT. Over 500 cases of lung cancer and 2500 hospital-based controls with diagnoses unrelated to hormone use or smoking were utilized. Adjusted risk estimates among current and past smokers showed no association between lung cancer and long-term (>=12 months) use of ERT (OR = 0.9, 95%CI = 0.6-1.3). For small cell lung cancer, however, a significant protective effect was observed (OR = 0.4, 95%CI = 0.2-1.0). On the other hand, for adenocarcinoma, there was a suggestion of increased risk with long-term use (OR = 1.5. 95%CI = 0.9-2.4), and the difference in the risk estimates between adenocarcinoma and small cell lung cancer was significant (two-tailed p<.02). No significant associations were observed between long-term use of ERT and squamous cell lung cancer (OR = 0.8, 95%CI = 0.1-1.9), or with other types of lung cancer (OR = 0.6, 95%CI = 0.3-1.4). Based on these findings, there is no suggestion of an increased risk of lung cancer (all cell types combined) due to ERT. Further, ERT may be protective against small cell lung cancer, while perhaps leading to an increased risk of adenocarcinoma. Although numerous potential confounders were evaluated in the analyses, confounding due to diet or smoking of filtered (versus non-filtered) cigarettes could not be ruled out, as these data were unavailable. Future studies are needed on the association between ERT and lung cancer which evaluate these variables as potential confounders. Also, the significant difference observed in the risk estimates for adenocarcinoma and small cell lung cancer demonstrates the need to study each cell type of lung cancer separately.
    • A study of ligand-induced erbB receptor interactions and downstream signaling in an erbB-2 overexpression model for lung cancer

      Fernandes, Audrey Maria; Hamburger, Anne, Ph.D. (1998)
      Overexpression of erbB-2 is associated with a poor prognosis in lung cancer (Kern, 1990). We have found that overexpression of erbB-2 alone in a human bronchial epithelial cell model system is not sufficient for tumor production (Hamburger, 1998). Characterization of our cell model system revealed the presence of constitutive production of the erbB family of ligands: TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF and heregulin. Of these endogenously-expressed ligands, only TGF-alpha expressed and secreted at high levels in the presence of overexpressed erbB-2 was found to correlate with tumorigenicity. Of the four constitutively expressed erbB receptors, erbB-1 and erbB-2 were the major species, while erbB-3 and erbB-4 represented a minor fraction of the population. Endogenously produced TGF-alpha was found to induce formation of erbB-1/erbB-2, erbB-1/erbB-3, erbB-1/erbB-4, erbB-2/erbB-4, erbB-2/erbB-3 and erbB-3/erbB-4 heterodimer complexes. However, only the TGF-alpha-induced erbB-1/erbB-2 heterodimer was formed in tumorigenic E6TM cells but not in non-tumorigenic E6TMA cells (Hamburger, 1998). Activation of the PI-3-kinase signaling pathway was comparable in both cell lines, however, activation of the ras-MAPK signaling pathway, however, was found to correlate with formation of the TGF-alpha induced erbB-1/erbB-2 heterodimer complex and tumorigenicity. We demonstrated that both Stats 1 and 3 co-immunoprecipitate with the constitutively produced erbB-1/erbB-2 heterodimer complex in tumorigenic E6TM cells. Activation of Stats 1 and 3 was shown to be dependent on high levels of TGF-alpha. We demonstrated that the erbS-1/erbB-2 heterodimer and erbB-2 kinase activity was required for Stat activation, while the erbB-1 homodimer was not sufficient for the TGF-alpha-induced activation of Stats 1 and 3. Our results support the hypothesis that the erbB-1/erbB-2 heterodimer, formed and activated in response to TGF-alpha, is critical to the tumorigenic conversion of these lung epithelial cells through the activation of signaling cascades including the MAPK and Stat pathways, but not PI-3-kinase.