Faculty Bibliography

The development of lung cancer in humans can be divided into three steps: initiation, promotion and progression. This process is driven by alterations in related signal transduction pathways. These pathways signal the aberrant activation of NF-kappaB, a transcription factor that regulates the expression of genes important for lung tumorigenesis. Our current knowledge about the role of the NF-kappaB signaling pathway in the development of lung cancer has been bolstered by animal models demonstrating the connection between K-ras and tobacco induced lung transformation with NF-kappaB. Activation of downstream genes leads to cell proliferation, inhibition of apoptosis, angiogenesis, inflammation, invasion, and metastasis.

Lung cancer is the leading cause of cancer deaths, with an overall survival of 15% at five years. Biomarkers that can sensitively and specifically detect lung cancer at early stage are crucial for improving this poor survival rate. Sputum has been the target for the discovery of non-invasive biomarkers for lung cancer because it contains airway epithelial cells, and molecular alterations identified in sputum are most likely to reflect tumor-associated changes or field cancerization caused by smoking in the lung. Sputum-based molecular biomarkers include morphology, allelic imbalance, promoter hypermethylation, gene mutations and, recently, differential miRNA expression. To improve the sensitivity and reproducibility of sputum-based biomarkers, we recommend standardization of processing protocols, bronchial epithelial cell enrichment, and identification of field cancerization biomarkers.

Rationale: Lung cancer is the leading cause of death from all types of cancer worldwide. The high mortality rate is partly attributed to the lack of methods for early detection and identification of patients who are at risk for developing disease. Since adenocarcinoma in the lung periphery is the most common type of lung cancer and the earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the peripheral airways, we hypothesize that identifying gene expression signatures in the peripheral airways will provide the best approach for better understanding field carcinogenesis of lung adenocarcinomas. To this end, we performed gene and miRNA profiling of cells obtained from brushings of peripheral airways of smokers with and without lung adenocarcinomas. These studies will provide not only mechanistic insights into field carcinogenesis of adenocarcinoma, but more importantly, potential tools for the early detection of lung adenocarcinomas. To this end, we performed gene expression profiling of the peripheral small airway epithelium of patients with lung cancer and compared it to that of non-cancerous smokers to identify gene expression signatures of field cancerization in lung cancer. Methods: Fiberoptic bronchoscopies with brushings were used to collect peripheral airway epithelial cells from the 10^th-12^th-order bronchi in non-cancerous smokers or from the unaffected lobe of suspected lung cancer patients. Clinical samples obtained from the suspected lung cancer patients confirmed the histological diagnosis of primary lung cancer. Using Affymetrix HG-U133A Plus 2.0 microarrays, we performed gene expression profiling of over 47,000 genes using total RNA isolated from small airway epithelial cells obtained from brushings of both lung cancer patients and non-cancerous smokers. Results: Microarray analysis of the gene expression profile in the peripheral airway epithelium demonstrated several up-regulated and down-regulated genes which could represent significant alterations in lung cancer. Significantly up-regulated genes included pro-platelet basic protein, gremlin 1, aminolevulinate synthase 2, sparc/osteonectin, and wnt inhibitory factor 1 while down-regulated genes include cytochrome P450 1B1 and 2A6, mucin 2, aldedyhe dehydrognease 3A1, and glutathione peroxidase 2 (p value < 0.05). Conclusion: We had identified several up-regulated and down-regulated genes in peripheral airway epithelial cells of cancer patients which were significantly different when compared to non-cancerous smokers. Identification of the gene expression signatures in field carcinogenesis will not only provide mechanistic insights into lung cancer, but also allow us to identify novel biomarkers for the early detection of lung cancer

Introduction: Lung cancer is the leading causes of cancer death with an overall survival of 15% at 5 years. The poor survival is primarily because most of the patients present at advanced stage when a surgical resection is no longer feasible. Tobacco smoking is the single most important cause of lung cancer. We hypothesized that sputum would be a specific and sensitive biomarker that is easily accessible and non-invasive to detect lung tumorigenesis at early stage among high-risk smokers. Rationale: Sputum contains upper and lower airway epithelial cells, suggesting the molecular alterations identified in sputum likely reflects tumor-associated changes in the lung. Three genes were chosen to be examined using DNA derived from sputum based on their implication in lung tumorigenesis. STK11 is a serine/threonine kinase that regulates cell polarity and metabolism and functions as a tumor suppressor. Germline mutation of STK11 results in the Peutz-Jeghers syndrome, a familial cancer syndrome characterized an increased risk for epithelial cancers, including non-small cell lung cancer (NSCLC) where STK11 is found to be mutated at 30-40% frequency. CDKN2A (also known as p16) is another tumor suppressor protein that plays an important role in regulating the cell cycle, and mutations in p16 increase the risk of developing a variety of cancers including lung cancer and importantly, its loss of function has been implicated in the early stage of tumorigenesis. p53, a tumor suppressor that is the most frequently mutated gene in lung adenocarcinoma, responds to diverse cellular stresses to regulate target genes that induce cell cycle arrest, apoptosis, senescence, or DNA repair. This DNA-binding protein is postulated to bind to a p53-binding site and activate expression of downstream genes that inhibit growth and/or invasion, which is often impaired by mutations identified in various human cancers. Methods: Spontaneous sputum was collected from normal smokers and lung cancer patients and processed to extract genomic DNA. Primers were designed to span exons 1-2 in CDKN2A, exons 2-9 in TP53 and exons 1-9 in STK11. Amplicons were analyzed by direct sequencing. Results: We identified numerous sequence variations in all three genes examined, including verified polymorphisms and mutations in the introns and exons. Most frequently, we detected G to A substitution in the 5' UTR region in CDKN2A and C to G substitution, which results in P72R in exon 4 in TP53. Conclusion: This panel of genes may be useful biomarkers for early detection of lung cancer among smokers

Occupational exposure to nickel compounds has been associated with lung and nasal cancers. We have previously shown that exposure of the human lung adenocarcinoma A549 cells to NiCl(2) for 24 hr significantly increased global levels of trimethylated H3K4 (H3K4me3), a transcriptional activating mark that maps to the promoters of transcribed genes. To further understand the potential epigenetic mechanism(s) underlying nickel carcinogenesis, we performed genome-wide mapping of H3K4me3 by chromatin immunoprecipitation and direct genome sequencing (ChIP-seq) and correlated with transcriptome genome-wide mapping of RNA transcripts by massive parallel sequencing of cDNA (RNA-seq). The effect of NiCl(2) treatment on H3K4me3 peaks within 5,000 bp of transcription start sites (TSSs) on a set of genes highly induced by nickel in both A549 cells and human peripheral blood mononuclear cells were analyzed. Nickel exposure increased the level of H3K4 trimethylation in both the promoters and coding regions of several genes including CA9 and NDRG1 that were increased in expression in A549 cells. We have also compared the extent of the H3K4 trimethylation in the absence and presence of formaldehyde crosslinking and observed that crosslinking of chromatin was required to observe H3K4 trimethylation in the coding regions immediately downstream of TSSs of some nickel-induced genes including ADM and IGFBP3. This is the first genome-wide mapping of trimethylated H3K4 in the promoter and coding regions of genes induced after exposure to NiCl(2). This study may provide insights into the epigenetic mechanism(s) underlying the carcinogenicity of nickel compounds

BACKGROUND: Because associations have been reported between inhaled ambient ultrafine particles and increased risk of cardiopulmonary disease, it has been suggested that inhaled engineered nanoparticles (NPs) may also induce adverse effects on the cardiovascular system. OBJECTIVE: We examined the long-term cardiovascular effects of inhaled nickel hydroxide NPs (nano-NH) using a sensitive mouse model. METHODS: Hyperlipidemic, apoprotein E-deficient (ApoE-/-) mice were exposed to nano-NH at either 0 or 79 mug Ni/m3, via a whole-body inhalation system, for 5 hr/day, 5 days/week, for either 1 week or 5 months. We measured various indicators of oxidative stress and inflammation in the lung and cardiovascular tissue, and we determined plaque formation on the ascending aorta. RESULTS: Inhaled nano-NH induced significant oxidative stress and inflammation in the pulmonary and extrapulmonary organs, indicated by up-regulated mRNA levels of certain antioxidant enzyme and inflammatory cytokine genes; increased mitochondrial DNA damage in the aorta; significant signs of inflammation in bronchoalveolar lavage fluid; changes in lung histopathology; and induction of acute-phase response. In addition, after 5-month exposures, nano-NH exacerbated the progression of atherosclerosis in ApoE-/- mice. CONCLUSIONS: This is the first study to report long-term cardiovascular toxicity of an inhaled nanomaterial. Our results clearly demonstrate that long-term exposure to inhaled nano-NH can induce oxidative stress and inflammation, not only in the lung but also in the cardiovascular system, and that this stress and inflammation can ultimately contribute to progression of atherosclerosis in ApoE-/- mice

The authors provide an update on a previously reported patient with in-transit metastatic melanoma of the scalp treated with topical diphencyprone (DPCP). Molecular studies implicate the thymus-derived TH17 lymphocyte subset in a remarkable immunotherapeutic regression. The authors performed RT-PCR of total RNA from paraffin-embedded tissue before and after treatment with DPCP. Before treatment with DPCP, the authors found elevated expression of IL 17C/D/E/F; after treatment there was no detectable expression. Conversely, increased expression of PLZF/CD27 and CTLA4 was seen after treatment with no expression before treatment. No expression of IL17A/B, CD7, RORgTand FoxP3 were before or after treatment. Conclusions are limited to only the time samples were obtained. Remarkable regression of an in-transit metastatic melanoma treated with the immunomodulatory agent DPCP showed gain and loss of gene expression of the TH17 pathway. Further study of this pathway from NK to NK-T to TH7 and TH1 cells both with and without accessory or dendritic cells will improve understanding of contact sensitizers as topical immunomodulators

AIM: To evaluate the ability of anti-ricin A-chain antibodies, delivered intracellularly, to protect against ricin-induced cytotoxicity in RAW264.7 cells. METHODS: Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide. RAW264.7 cells were incubated with these antibodies either before or after ricin exposure. The changes in cytotoxicity were estimated by MTT assay. Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. RESULTS: Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies. Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. CONCLUSION: Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for post-exposure treatment of ricin intoxication

Short and long-term pulmonary response to inhaled nickel hydroxide nanoparticles (nano-Ni(OH)(2), CMD = 40 nm) in C57BL/6 mice was assessed using a whole body exposure system. For short-term studies mice were exposed for 4 h to nominal concentrations of 100, 500, and 1000 mg/m(3). For long-term studies mice were exposed for 5 h/d, 5 d/w, for up to 5 months (m) to a nominal concentration of 100 mg/m(3). Particle morphology, size distribution, chemical composition, solubility, and intrinsic oxidative capacity were determined. Markers of lung injury and inflammation in bronchoalveolar lavage fluid (BALF); histopathology; and lung tissue elemental nickel content and mRNA changes in macrophage inflammatory protein-2 (Mip-2), chemokine ligand 2 (Ccl2), interleukin 1-alpha (Il-1alpha), and tumor necrosis factor-alpha (Tnf-alpha) were assessed. Dose-related changes in BALF analyses were observed 24 h after short-term studies while significant changes were noted after 3 m and/or 5 m of exposure (24 h). Nickel content was detected in lung tissue, Ccl2 was most pronouncedly expressed, and histological changes were noted after 5 m of exposure. Collectively, data illustrates nano-Ni(OH)(2) can induce inflammatory responses in C57BL/6 mice

AIM: To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated. METHODS: To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium]. RESULTS: Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells. CONCLUSION: We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication