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1.  Massive loculated pleural effusion in a patient with pancreatic pseudocyst due to alcohol-related chronic pancreatitis 
BMJ Case Reports  2014;2014:bcr2014204032.
A 47-year-old man with a history of alcohol-related pancreatitis was admitted with dyspnoea due to a moderate right-sided pleural effusion. Diagnostic pleural tap showed an amylase of 6078 U/L. CT demonstrated a pancreatic pseudocyst with communication to the pleural cavity. Conservative medical management and chest drainage were started, but after 13 days the patient became acutely unwell with severe dyspnoea and worsening chest pain. Chest X-ray and subsequent CT demonstrated a massive pleural effusion with mediastinal shift. Ultrasound scan demonstrated loculation of the effusion requiring insertion of a large bore chest drain to relieve symptoms. He was transferred to a pancreaticobiliary centre, but subsequently made a good recovery without the need for a further procedure. This case highlights massive pleural effusion with subsequent loculation as a rare complication of chronic pancreatitis.
PMCID: PMC3987506  PMID: 24717586
2.  Cell adhesion manipulation through single cell assembly for characterization of initial cell-to-cell interaction 
Cell-to-cell interactions are complex processes that involve physical interactions, chemical binding, and biological signaling pathways. Identification of the functions of special signaling pathway in cell-to-cell interaction from the very first contact will help characterize the mechanism underlying the interaction and advance new drug discovery.
This paper reported a case study of characterizing initial interaction between leukemia cancer cells and bone marrow stromal cells, through the use of an optical tweezers-based cell manipulation tool. Optical traps were used to assemble leukemia cells at different positions of the stromal cell layer and enable their interactions by applying a small trapping force to maintain the cell contact for a few minutes. Specific drug was used to inhibit the binding of molecules during receptor-ligand-mediated adhesion.
Results and conclusions
Our results showed that the amount of adhesion molecule could affect cell adhesion during the first few minutes contact. We also found that leukemia cancer cells could migrate on the stromal cell layer, which was dependent on the adhesion state and activation triggered by specific chemokine. The reported approaches provided a new opportunity to investigate cell-to-cell interaction through single cell adhesion manipulation.
PMCID: PMC4676142  PMID: 26652601
Cell adhesion; Cell-to-cell interaction; Optical tweezers manipulation; Cell assembly; Leukemia-stromal cell interactions
3.  Reproducibility of optical coherence tomography airway imaging 
Biomedical Optics Express  2015;6(11):4365-4377.
Optical coherence tomography (OCT) is a promising imaging technique to evaluate small airway remodeling. However, the short-term insertion-reinsertion reproducibility of OCT for evaluating the same bronchial pathway has yet to be established. We evaluated 74 OCT data sets from 38 current or former smokers twice within a single imaging session. Although the overall insertion-reinsertion airway wall thickness (WT) measurement coefficient of variation (CV) was moderate at 12%, much of the variability between repeat imaging was attributed to the observer; CV for repeated measurements of the same airway (intra-observer CV) was 9%. Therefore, reproducibility may be improved by introduction of automated analysis approaches suggesting that OCT has potential to be an in-vivo method for evaluating airway remodeling in future longitudinal and intervention studies.
PMCID: PMC4646546  PMID: 26601002
(170.0170) Medical optics and biotechnology; (170.4500) Optical coherence tomography
4.  Endoscopic Doppler optical coherence tomography and autofluorescence imaging of peripheral pulmonary nodules and vasculature 
Biomedical Optics Express  2015;6(10):4191-4199.
We present the first endoscopic Doppler optical coherence tomography and co-registered autofluorescence imaging (DOCT-AFI) of peripheral pulmonary nodules and vascular networks in vivo using a small 0.9 mm diameter catheter. Using exemplary images from volumetric data sets collected from 31 patients during flexible bronchoscopy, we demonstrate how DOCT and AFI offer complementary information that may increase the ability to locate and characterize pulmonary nodules. AFI offers a sensitive visual presentation for the rapid identification of suspicious airway sites, while co-registered OCT provides detailed structural information to assess the airway morphology. We demonstrate the ability of AFI to visualize vascular networks in vivo and validate this finding using Doppler and structural OCT. Given the advantages of higher resolution, smaller probe size, and ability to visualize vasculature, DOCT-AFI has the potential to increase diagnostic accuracy and minimize bleeding to guide biopsy of pulmonary nodules compared to radial endobronchial ultrasound, the current standard of care.
PMCID: PMC4605074  PMID: 26504665
(170.0170) Medical optics and biotechnology; (170.0110) Imaging systems; (170.4500) Optical coherence tomography; (170.2520) Fluorescence microscopy
5.  Adhesion-Induced Unclasping of Cytoplasmic Tails of Integrin αIIbβ3†,‡ 
Biochemistry  2009;48(3):617-629.
Integrin αIIbβ3 plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of αIIbβ3 to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins’ cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of αIIbβ3 to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between αIIb and β3 cytoplasmic tails, we showed that its binding site in the membrane-proximal β3 715–730 segment is cryptic and becomes exposed as a result of binding of isolated αIIbβ3 to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with αIIbβ3 in resting platelets or suspended αIIbβ3-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only β3 but also the membrane-proximal 989–1000 segment of the αIIb cytoplasmic tail binds the skelemin fragment. Finally, the same residues, αIIb Val990, αIIb Arg995, and β3 His722, involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of αIIbβ3 by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between αIIb and β3 cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.
PMCID: PMC4540355  PMID: 19117493
6.  EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk 
Oncogene  2013;33(36):4464-4473.
In an effort to identify novel biallelically inactivated tumor suppressor genes (TSG) in sporadic invasive and pre-invasive non-small cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multi-‘omics approach to investigate patient matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes, and in the earliest stages of lung cancer. We find not only that decreased EYA4 expression is associated with poor survival in sporadic lung cancers, but EYA4 SNPs are associated with increased familial cancer risk, consistent with EYA4’s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we find that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross examination of EYA4 expression across multiple tumor types suggests a cell type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.
PMCID: PMC4527534  PMID: 24096489
EYA4; two-hit; hypermethylation; tumor suppressor; TSG; non-small cell lung cancer
7.  Potential application of non-small cell lung cancer-associated autoantibodies to early cancer diagnosis 
To identify a panel of tumor associated autoantibodies which can potentially be used as biomarkers for the early diagnosis of non-small cell lung cancer (NSCLC). Thirty-five unique and in-frame expressed phage proteins were isolated. Based on the gene expression profiling, four proteins were selected for further study. Both receiver operating characteristic curve analysis and leave-one-out method revealed that combined measurements of four antibodies produced have better predictive accuracies than any single marker alone. Leave-one-out validation also showed significant relevance with all stages of NSCLC patients. The panel of autoantibodies has a high potential for detecting early stage NSCLC.
PMCID: PMC4512951  PMID: 22713465
NSCLC; Tumor-associated autoantibodies; SEREX; Early diagnosis
8.  Rapid multispectral endoscopic imaging system for near real-time mapping of the mucosa blood supply in the lung 
Biomedical Optics Express  2015;6(8):2980-2990.
We have developed a fast multispectral endoscopic imaging system that is capable of acquiring images in 18 optimized spectral bands spanning 400-760 nm by combining a customized light source with six triple-band filters and a standard color CCD camera. A method is developed to calibrate the spectral response of the CCD camera. Imaging speed of 15 spectral image cubes/second is achieved. A spectral analysis algorithm based on a linear matrix inversion approach is developed and implemented in a graphics processing unit (GPU) to map the mucosa blood supply in the lung in vivo. Clinical measurements on human lung patients are demonstrated.
PMCID: PMC4541525  PMID: 26309761
(110.4234) Multispectral and hyperspectral imaging; (170.2150) Endoscopic imaging; (170.1470) Blood or tissue constituent monitoring; (170.6935) Tissue characterization; (170.3880) Medical and biological imaging; (170.1610) Clinical applications
9.  K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates 
Science (New York, N.Y.)  2014;347(6220):428-431.
The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.
PMCID: PMC4349400  PMID: 25502314
10.  Ethnicity affects EGFR and KRAS gene alterations of lung adenocarcinoma 
Oncology Letters  2015;10(3):1775-1782.
Mutations or copy number gains (CNGs) of the EGFR and KRAS genes are representative alterations in lung adenocarcinomas that are individually associated with patient characteristics such as ethnicity, smoking status and gender. However, the effects of combinations of these genetic alterations have not been statistically examined. The present study analyzed previously examined lung adenocarcinoma cases in Asian (n=166) and non-Asian (n=136) individuals in whom all four EGFR and KRAS alterations had been studied. The polynomial logistic regression models were used following adjustment for gender and smoking status, and using patients without any type of EGFR/KRAS alterations as a reference. Between the two ethnic groups, EGFR CNGs (gEGFR) occurred more frequently than EGFR mutations (mEGFR) (46 vs. 38% in Asians; 21 vs. 10% in non-Asians), whereas KRAS mutations (mKRAS) were more frequent than KRAS CNGs (gKRAS) (13 vs. 7% and 35 vs. 4%, respectively). Additionally, gEGFR and gKRAS occurred significantly more frequently in respective mutant cases, and all EGFR alterations were almost exclusive of all KRAS alterations. The polynomial logistic regression models confirmed that all types of EGFR alterations were significantly more frequent among Asian individuals than among non-Asian individuals, independent of gender and smoking status (odds ratios, 2.36–6.67). KRAS alterations occurred less frequently among Asian individuals than among non-Asian individuals, although a significant difference was not detected. The present study results indicated that the EGFR and KRAS profiles, including mutations and CNGs, differ between Asian and non-Asian individuals with lung adenocarcinoma, suggesting that ethnicity strongly affects the molecular characteristics of lung adenocarcinoma.
PMCID: PMC4533243  PMID: 26622749
lung adenocarcinoma; EGFR; KRAS; mutation; copy number gain; ethnicity
11.  Unique somatic and malignant expression patterns implicate PIWI-interacting RNAs in cancer-type specific biology 
Scientific Reports  2015;5:10423.
Human PIWI-interacting RNAs (piRNAs) are known to be expressed in germline cells, functionally silencing LINEs and SINEs. Their expression patterns in somatic tissues are largely uncharted. We analyzed 6,260 human piRNA transcriptomes derived from non-malignant and tumour tissues from 11 organs. We discovered that only 273 of the 20,831 known piRNAs are expressed in somatic non-malignant tissues. However, expression patterns of these piRNAs were able to distinguish tissue-of-origin. A total of 522 piRNAs are expressed in corresponding tumour tissues, largely distinguishing tumour from non-malignant tissues in a cancer-type specific manner. Most expressed piRNAs mapped to known transcripts, contrary to “piRNA clusters” reported in germline cells. We showed that piRNA expression can delineate clinical features, such as histological subgroups, disease stages, and survival. PiRNAs common to many cancer types might represent a core gene-set that facilitates cancer growth, while piRNAs unique to individual cancer types likely contribute to cancer-specific biology.
PMCID: PMC4444957  PMID: 26013764
12.  DNA Methylation Is Globally Disrupted and Associated with Expression Changes in Chronic Obstructive Pulmonary Disease Small Airways 
DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illumina’s Infinium HM27 and Affymetrix’s Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2–related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.
PMCID: PMC4068945  PMID: 24298892
chronic obstructive pulmonary disease; small airways; epigenetic regulation; DNA methylation; integrative omics
13.  A search for novel cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets 
Cancer research  2014;74(17):4694-4705.
Cancer/testis (CT) antigens are potential immunotherapeutic targets in cancer. However, the expression of particular antigens is limited to a subset of tumors of a given type. Thus, there is a need to identify antigens with complementary expression patterns for effective therapeutic intervention. In this study, we searched for genes that were distinctly expressed at a higher level in lung tumor tissue and the testes compared to other non-tumor tissues and identified members of the VCX/Y gene family as novel CT antigens. VCX3A, a member of the VCX/Y gene family, was expressed at the protein level in approximately 20% of lung adenocarcinomas and 35% of squamous cell carcinomas, but not expressed in normal lung tissues. Among CT antigens with concordant mRNA and protein expression levels, four CT antigens, XAGE1, VCX, IL13RA2, and SYCE1, were expressed, alone or in combination, in about 80% of lung adenocarcinoma tumors. The CT antigen VCX/Y gene family broadens the spectrum of CT antigens expressed in lung adenocarcinomas for clinical applications.
PMCID: PMC4398029  PMID: 24970476
Cancer/testis antigen; VCX/Y; lung cancer; immunotherapy
14.  Pro–Surfactant Protein B As a Biomarker for Lung Cancer Prediction 
Journal of Clinical Oncology  2013;31(36):4536-4543.
Preliminary studies have identified pro–surfactant protein B (pro-SFTPB) to be a promising blood biomarker for non–small-cell lung cancer. We conducted a study to determine the independent predictive potential of pro-SFTPB in identifying individuals who are subsequently diagnosed with lung cancer.
Patients and Methods
Pro-SFTPB levels were measured in 2,485 individuals, who enrolled onto the Pan-Canadian Early Detection of Lung Cancer Study by using plasma sample collected at the baseline visit. Multivariable logistic regression models were used to evaluate the predictive ability of pro-SFTPB in addition to known lung cancer risk factors. Calibration and discrimination were evaluated, the latter by an area under the receiver operating characteristic curve (AUC). External validation was performed with samples collected in the Carotene and Retinol Efficacy Trial (CARET) participants using a case-control study design.
Adjusted for age, sex, body mass index, personal history of cancer, family history of lung cancer, forced expiratory volume in one second percent predicted, average number of cigarettes smoked per day, and smoking duration, pro-SFTPB (log transformed) had an odds ratio of 2.220 (95% CI, 1.727 to 2.853; P < .001). The AUCs of the full model with and without pro-SFTPB were 0.741 (95% CI, 0.696 to 0.783) and 0.669 (95% CI, 0.620 to 0.717; difference in AUC P < .001). In the CARET Study, the use of pro-SFPTB yielded an AUC of 0.683 (95% CI, 0.604 to 0.761).
Pro-SFTPB in plasma is an independent predictor of lung cancer and may be a valuable addition to existing lung cancer risk prediction models.
PMCID: PMC3871515  PMID: 24248694
15.  YEATS4 is a novel oncogene amplified in non-small cell lung cancer that regulates the p53 pathway 
Cancer research  2013;73(24):7301-7312.
Genetic analyses of lung cancer have helped found new treatments in this disease. We conducted an integrative analysis of gene expression and copy number in 261 non-small cell lung cancers (NSCLC) relative to matched normal tissues to define novel candidate oncogenes, identifying 12q13-15 and more specifically the YEATS4 gene as amplified and overexpressed in ~20% of the NSCLC cases examined. Overexpression of YEATS4 abrogated senescence in human bronchial epithelial cells (HBECs). Conversely, RNAi-mediated attenuation of YEATS4 in human lung cancer cells reduced their proliferation and tumor growth, impairing colony formation and inducing cellular senescence. These effects were associated with increased levels of p21WAF1 and p53 and cleavage of PARP, implicating YEATS4 as a negative regulator of the p21-p53 pathway. We also found that YEATS4 expression affected cellular responses to cisplastin, with increased levels associated with resistance and decreased levels with sensitivity. Taken together, our findings reveal YEATS4 as a candidate oncogene amplified in NSCLC, and a novel mechanism contributing to NSCLC pathogenesis.
PMCID: PMC3959870  PMID: 24170126
YEATS4; NSCLC; oncogene; p53; integrative analysis
16.  Smoking status impacts microRNA mediated prognosis and lung adenocarcinoma biology 
BMC Cancer  2014;14:778.
Cigarette smoke is associated with the majority of lung cancers: however, 25% of lung cancer patients are non-smokers, and half of all newly diagnosed lung cancer patients are former smokers. Lung tumors exhibit distinct epidemiological, clinical, pathological, and molecular features depending on smoking status, suggesting divergent mechanisms underlie tumorigenesis in smokers and non-smokers. MicroRNAs (miRNAs) are integral contributors to tumorigenesis and mediate biological responses to smoking. Based on the hypothesis that smoking-specific miRNA differences in lung adenocarcinomas reflect distinct tumorigenic processes selected by different smoking and non-smoking environments, we investigated the contribution of miRNA disruption to lung tumor biology and patient outcome in the context of smoking status.
We applied a whole transcriptome sequencing based approach to interrogate miRNA levels in 94 patient-matched lung adenocarcinoma and non-malignant lung parenchymal tissue pairs from current, former and never smokers.
We discovered novel and distinct smoking status-specific patterns of miRNA and miRNA-mediated gene networks, and identified miRNAs that were prognostically significant in a smoking dependent manner.
We conclude that miRNAs disrupted in a smoking status-dependent manner affect distinct cellular pathways and differentially influence lung cancer patient prognosis in current, former and never smokers. Our findings may represent promising biologically relevant markers for lung cancer prognosis or therapeutic intervention.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-778) contains supplementary material, which is available to authorized users.
PMCID: PMC4216369  PMID: 25342220
Lung adenocarcinoma; miRNA; Current smoker; Former smoker; Never smoker; Reversible; Survival; Smoking specific
17.  MicroRNAs As Biomarkers For Clinical Features Of Lung Cancer 
Metabolomics : open access  2012;2(3):1000108-.
Each year about 1.4 million people die from lung cancer worldwide. Despite efforts in prevention, diagnosis and treatment, survival rate remains poor for this disease. This unfortunate situation is largely due to the fact that a high proportion of cases are diagnosed at advanced stages, highlighting the great need for identifying new biomarkers in order to improve early diagnosis and treatment. Recent studies on microRNAs have not only shed light on their involvement in tumor development and progression, but also suggested their potential utility as biomarkers for subtype diagnostics, staging and prediction of treatment response. This review article summarizes the impact of microRNAs on lung cancer biology, and highlights their role in the detection and classification of lung cancer as well as direct targets for drug development.
PMCID: PMC4159950  PMID: 25221729
18.  A novel missense mutation in CCDC88C activates the JNK pathway and causes a dominant form of spinocerebellar ataxia 
Journal of Medical Genetics  2014;51(9):590-595.
Spinocerebellar ataxias (SCAs) are a group of clinically and genetically diverse and autosomal-dominant disorders characterised by neurological deficits in the cerebellum. At present, there is no cure for SCAs. Of the different distinct subtypes of autosomal-dominant SCAs identified to date, causative genes for only a fraction of them are currently known. In this study, we investigated the cause of an autosomal-dominant SCA phenotype in a family that exhibits cerebellar ataxia and pontocerebellar atrophy along with a global reduction in brain volume.
Methods and results
Whole-exome analysis revealed a missense mutation c.G1391A (p.R464H) in the coding region of the coiled-coil domain containing 88C (CCDC88C) gene in all affected individuals. Functional studies showed that the mutant form of CCDC88C activates the c-Jun N-terminal kinase (JNK) pathway, induces caspase 3 cleavage and triggers apoptosis.
This study expands our understanding of the cause of autosomal-dominant SCAs, a group of heterogeneous congenital neurological conditions in humans, and unveils a link between the JNK stress pathway and cerebellar atrophy.
PMCID: PMC4145425  PMID: 25062847
Neurology; Clinical Genetics
19.  A high-efficiency fiber-based imaging system for co-registered autofluorescence and optical coherence tomography 
Biomedical Optics Express  2014;5(9):2978-2987.
We present a power-efficient fiber-based imaging system capable of co-registered autofluorescence imaging and optical coherence tomography (AF/OCT). The system employs a custom fiber optic rotary joint (FORJ) with an embedded dichroic mirror to efficiently combine the OCT and AF pathways. This three-port wavelength multiplexing FORJ setup has a throughput of more than 83% for collected AF emission, significantly more efficient compared to previously reported fiber-based methods. A custom 900 µm diameter catheter ‒ consisting of a rotating lens assembly, double-clad fiber (DCF), and torque cable in a stationary plastic tube ‒ was fabricated to allow AF/OCT imaging of small airways in vivo. We demonstrate the performance of this system ex vivo in resected porcine airway specimens and in vivo in human on fingers, in the oral cavity, and in peripheral airways.
PMCID: PMC4230860  PMID: 25401011
(110.0110) Imaging systems; (110.2350) Fiber optics imaging; (110.4500) Optical coherence tomography; (170.2520) Fluorescence microscopy; (170.3890) Medical optics instrumentation
20.  EZH2 promotes E2F driven SCLC tumorigenesis through modulation of apoptosis and cell cycle regulation 
While EZH2 has been associated with both non small cell and small cell lung cancers, current observations suggest different mechanisms of EZH2 activation and overexpression in these lung cancer types. Globally, small cell lung cancer (SCLC) kills 200,000 people yearly. New clinical approaches for SCLC treatment are required to improve the poor survival rate. Given the therapeutic potential of EZH2 as a target, we sought to delineate the downstream consequences of EZH2 disruption to identify the cellular mechanisms by which EZH2 promotes tumorigenesis in SCLC.
We generated cells with stable expression of shRNA targeting EZH2 and corresponding controls (pLKO.1) and determined the consequences of EZH2 knockdown on the cell cycle and apoptosis by means of propidium iodide staining and fluorescence activated cell sorting, western blot, qRT-PCR as well as cell viability assessment using MTT assays.
We discovered that EZH2 inhibition 1) increased apoptotic activity by up-regulating the pro-apoptotic factors Puma and Bad, 2) decreased the fraction of cells in S or G2/M phases, and 3) elevated p21 protein levels, implicating EZH2 in cell death and cell cycle control in SCLC.
Our findings present evidence for the role of EZH2 in the regulation of cell cycle and apoptosis, providing a biological mechanism to explain the tumorigenicity of EZH2 in SCLC. Our work points to the great potential of EZH2 as a therapeutic target in SCLC.
PMCID: PMC3713495  PMID: 23857401
SCLC; EZH2; oncogene; RB1; E2F
21.  A Blood-Based Proteomic Classifier for the Molecular Characterization of Pulmonary Nodules 
Science translational medicine  2013;5(207):207ra142.
Each year millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. As the majority of these nodules are benign, many patients undergo unnecessary and costly invasive procedures. We present a 13-protein blood-based classifier that differentiates malignant and benign nodules with high confidence, thereby providing a diagnostic tool to avoid invasive biopsy on benign nodules. Using a systems biology strategy, 371 protein candidates were identified and a multiple reaction monitoring (MRM) assay was developed for each. The MRM assays were applied in a three-site discovery study (n = 143) on plasma samples from patients with benign and Stage IA cancer matched on nodule size, age, gender and clinical site, producing a 13-protein classifier. The classifier was validated on an independent set of plasma samples (n = 104), exhibiting a high negative predictive value (NPV) of 90%. Validation performance on samples from a non-discovery clinical site showed NPV of 94%, indicating the general effectiveness of the classifier. A pathway analysis demonstrated that the classifier proteins are likely modulated by a few transcription regulators (NF2L2, AHR, MYC, FOS) that are associated with lung cancer, lung inflammation and oxidative stress networks. The classifier score was independent of patient nodule size, smoking history and age, which are risk factors used for clinical management of pulmonary nodules. Thus this molecular test can provide a powerful complementary tool for physicians in lung cancer diagnosis.
PMCID: PMC4114963  PMID: 24132637
22.  Pre-profiling factors influencing serum microRNA levels 
MicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression by preventing the translation of specific mRNA transcripts. Recent studies have shown that miRNAs are stably expressed in human serum samples, making them good candidates for the non-invasive detection of disease. However, before circulating miRNAs can be used reliably as biomarkers of disease, the pre-measurement variables that may affect serum miRNA levels must be assessed.
In this study we used quantitative RT-PCR to examine the effect of hemolysis, fasting, and smoking on the levels of 742 miRNAs in the serum of healthy individuals. We also compared serum miRNA profiles of samples taken from healthy individuals over different time periods to assess normal serum miRNA fluctuations.
We have found that mechanical hemolysis of blood samples can significantly alter serum miRNA quantification and have identified 162 miRNAs that are significantly up-regulated in hemolysed serum samples. Conversely, fasting and smoking were demonstrated to not have a significant effect on the overall serum miRNA profiles of healthy individuals. The serum miRNA profiles of matched samples taken from individuals over varying time periods showed a high correlation and no miRNAs were significantly differentially expressed in these samples further suggesting the utility of serum miRNAs as biomarkers of disease. Taking the above results into consideration, we have identified miR-99a-5p and miR-139-5p as novel endogenous controls for serum miRNA studies due to their consistency across all sample sets.
These results identify important pre-profiling factors that should be taken into consideration when identifying endogenous controls and candidate biomarkers for circulating miRNA studies.
PMCID: PMC4107491  PMID: 25093010
MicroRNA; Cancer; Biomarker; Serum; miR-99a-5p; miR-139-5p
23.  Validation of Airway Wall Measurements by Optical Coherence Tomography in Porcine Airways 
PLoS ONE  2014;9(6):e100145.
Examining and quantifying changes in airway morphology is critical for studying longitudinal pathogenesis and interventions in diseases such as chronic obstructive pulmonary disease and asthma. Here we present fiber-optic optical coherence tomography (OCT) as a nondestructive technique to precisely and accurately measure the 2-dimensional cross-sectional areas of airway wall substructure divided into the mucosa (WAmuc), submucosa (WAsub), cartilage (WAcart), and the airway total wall area (WAt). Porcine lung airway specimens were dissected from freshly resected lung lobes (N = 10). Three-dimensional OCT imaging using a fiber-optic rotary-pullback probe was performed immediately on airways greater than 0.9 mm in diameter on the fresh airway specimens and subsequently on the same specimens post-formalin-fixation. The fixed specimens were serially sectioned and stained with H&E. OCT images carefully matched to selected sections stained with Movat’s pentachrome demonstrated that OCT effectively identifies airway epithelium, lamina propria, and cartilage. Selected H&E sections were digitally scanned and airway total wall areas were measured. Traced measurements of WAmuc, WAsub, WAcart, and WAt from OCT images of fresh specimens by two independent observers found there were no significant differences (p>0.05) between the observer’s measurements. The same wall area measurements from OCT images of formalin-fixed specimens found no significant differences for WAsub, WAcart and WAt, and a small but significant difference for WAmuc. Bland-Altman analysis indicated there were negligible biases between the observers for OCT wall area measurements in both fresh and formalin-fixed specimens. Bland-Altman analysis also indicated there was negligible bias between histology and OCT wall area measurements for both fresh and formalin-fixed specimens. We believe this study sets the groundwork for quantitatively monitoring pathogenesis and interventions in the airways using OCT.
PMCID: PMC4064993  PMID: 24949633
24.  Novel FOXF1 mutations in sporadic and familial cases of Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins imply a role for its DNA binding domain 
Sen, Partha | Yang, Yaping | Navarro, Colby | Silva, Iris | Szafranski, Przemyslaw | Kolodziejska, Katarzyna E. | Dharmadhikari, Avinash V. | Mostafa, Hasnaa | Kozakewich, Harry | Kearney, Debra | Cahill, John B. | Whitt, Merrissa | Bilic, Masha | Margraf, Linda | Charles, Adrian | Goldblatt, Jack | Gibson, Kathleen | Lantz, Patrick | Garvin, Julian | Petty, John | Kiblawi, Zeina | Zuppan, Craig | McConkie-Rosell, Allyn | McDonald, Marie T. | Peterson-Carmichael, Stacey L. | Gaede, Jane T. | Shivanna, Binoy | Schady, Deborah | Friedlich, Philippe S. | Hays, Stephen R. | Palafoll, Irene Valenzuela | Siebers-Renelt, Ulrike | Bohring, Axel | Finn, Laura S. | Siebert, Joseph R. | Galambos, Csaba | Nguyen, Lananh | Riley, Melissa | Chassaing, Nicolas | Vigouroux, Adeline | Rocha, Gustavo | Fernandes, Susana | Brumbaugh, Jane | Roberts, Kari | Ho-ming, Luk | Lo, Ivan | Lam, Stephen | Gerychova, Romana | Jezova, Marta | Valaskova, Iveta | Fellmann, Florence | Afshar, Katayoun | Giannoni, Eric | Muhlethaler, Vincent | Liang, Jinlong | Beckmann, Jacques S. | Lioy, Janet | Deshmukh, Hitesh | Srinivasan, Lakshmi | Swarr, Daniel T. | Sloman, Melissa | Shaw-Smith, Charles | van Loon, Rosa Laura | Hagman, Cecilia | Sznajer, Yves | Barrea, Catherine | Galant, Christine | Detaille, Thierry | Wambach, Jennifer A. | Cole, F. Sessions | Hamvas, Aaron | Prince, Lawrence S. | Diderich, Karin E.M. | Brooks, Alice S. | Verdijk, Rob M. | Ravindranathan, Hari | Sugo, Ella | Mowat, David | Baker, Michael L. | Langston, Claire | Welty, Stephen | Stankiewicz, Pawel
Human mutation  2013;34(6):801-811.
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare and lethal developmental disorder of the lung defined by a constellation of characteristic histopathological features. Non-pulmonary anomalies involving organs of gastrointestinal, cardiovascular, and genitourinary systems have been identified in approximately 80% of patients with ACD/MPV. We have collected DNA and pathological samples from more than 90 infants with ACD/MPV and their family members. Since the publication of our initial report of four point mutations and ten deletions, we have identified an additional thirty eight novel nonsynonymous mutations of FOXF1 (nine nonsense, seven frameshift, one inframe deletion, twenty missense, and one no stop). This report represents an up to date list of all known FOXF1 mutations to the best of our knowledge. Majority of the cases are sporadic whereas four familial cases with three showing maternal inheritance, consistent with paternal imprinting of the gene. Twenty five mutations (60%) are located within the putative DNA binding domain, indicating its plausible role in gene regulation. Five mutations map to the second exon. We identified two additional genic and eight genomic deletions upstream to FOXF1. These results corroborate and extend our previous observations and further establish involvement of FOXF1 in ACD/MPV and lung organogenesis.
PMCID: PMC3663886  PMID: 23505205
Lung; Development; Angiogenesis; ACD/MPV; FOXF1; Imprinting

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