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1.  O-6-methylguanine-deoxyribonucleic acid methyltransferase methylation enhances response to temozolomide treatment in esophageal cancer 
World-wide, esophageal cancer is a growing epidemic and patients frequently present with advanced disease that is surgically inoperable. Hence, chemotherapy is the predominate treatment. Cytotoxic platinum compounds are mostly used, but their efficacy is only moderate. Newer alkylating agents have shown promise in other tumor types, but little is known about their utility in esophageal cancer.
We utilized archived human esophageal cancer samples and esophageal cancer cell lines to evaluate O-6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT) hypermethylation status and determined sensitivity to the alkylating drug temozolomide (TMZ). Immunoblot analysis was performed to determine MGMT protein expression in cell lines. To assess and confirm the effect of TMZ treatment in a methylated esophageal cancer cell line in vivo, a mouse flank xenograft tumor model was utilized.
Nearly 71% (12/17) of adenocarcinoma and 38% (3/8) of squamous cell carcinoma (SCC) patient samples were MGMT hypermethylated. Out of four adenocarcinoma and nine SCC cell lines tested, one of each histology was hypermethylated. Immunoblot analyses confirmed that hypermethylated cell lines did not express the MGMT protein. In vitro cell viability assays showed the methylated Kyse-140 and FLO cells to be sensitive to TMZ at an IC50 of 52-420 μM, whereas unmethylated cells Kyse-410 and SKGT-4 did not respond. In an in vivo xenograft tumor model with Kyse-140 cells, which are MGMT hypermethylated, TMZ treatment abrogated tumor growth by more than 60%.
MGMT methylation may be an important biomarker in subsets of esophageal cancers and targeting by TMZ may be utilized to successfully treat these patients.
PMCID: PMC3853796  PMID: 24319345
Alkylating agents; deoxyribonucleic acid repair genes; in vivo pre-clinical; esophageal cancer; O-6-methylguanine-deoxyribonucleic acid methyltransferase hypermethylation; response to treatment; temozolomide
2.  Expression Patterns of PAX5, c-Met and Paxillin in Neuroendocrine Tumors of the Lung 
c-Met is important in the pathogenesis, invasion and spread of several forms of lung cancer and multiple c-Met inhibitors are undergoing clinical trials. PAX5 has been shown to upregulate c-Met in small cell lung carcinoma (SCLC), and co-inhibiting PAX5 and c-Met had a synergic effect in killing tumor cells. Paxillin is a downstream target of activated c-Met, and its activation leads to enhanced cell motility and tumor spread. The expression patterns of these functionally related proteins had not been systemically studied in neuroendocrine tumors of lung.
Our aim was to investigate the expression patterns of PAX5, paxillin, c-Met and phosphorylated c-Met (p-c-Met) in four categories of pulmonary neuroendocrine tumor.
Tissue microarrays of 38 typical carcinoids (TC), 6 atypical carcinoids (AC), 34 SCLC, and 11 large cell neuroendocrine carcinomas (LCNEC) were studied with immunohistochemistry.
A vast majority of four tumor types expressed c-Met, p-c-Met and paxillin. PAX5 was frequently expressed in AC, SCLC and LCNEC, but tended to be negative in TC. Coexpression of PAX5 with c-Met or p-c-Met was present in a majority of AC, SCLC and LCNEC. Significant correlation between PAX5 and paxillin was detected in SCLC and LCNEC, but not in carcinoid tumors.
The frequent coexpression of PAX5 with c-Met or p-c-Met in intermediate and high grade neuroendocrine tumors supports the therapeutic strategy of co-inhibiting these proteins. The discrepancy between high and low grade neuroendocrine tumors in terms of PAX5/paxillin expression correlation may be due to different underlying molecular genetics of these tumors.
PMCID: PMC3049158  PMID: 21043826
3.  Cardiotoxicity associated with CTLA4 and PD1 blocking immunotherapy 
Immune-checkpoint blocking antibodies have demonstrated objective antitumor responses in multiple tumor types including melanoma, non-small cell lung cancer (NSCLC), and renal cell cancer (RCC). In melanoma, an increase in overall survival has been demonstrated with anti-CTLA-4 and PD-1 inhibition. However, a plethora of immune-mediated adverse events has been reported with these agents. Immune-mediated cardiotoxicity induced by checkpoint inhibitors has been reported in single cases with variable presentation, including myocarditis and pericarditis.
Among six clinical cancer centers with substantial experience in the administration of immune-checkpoint blocking antibodies, eight cases of immune-related cardiotoxicity after ipilimumab and/or nivolumab/pembrolizumab were identified. Diagnostic findings, treatment and follow-up are reported. A large variety of cardiotoxic events with manifestations such as heart failure, cardiomyopathy, heart block, myocardial fibrosis and myocarditis was documented.
This is the largest case series to date describing cardiotoxicity of immune-checkpoint blocking antibodies. Awareness, monitoring of patients with pre-existing cardiac disorders and prompt evaluation by the treatment team is essential. Treatment including application of steroids is critical for patient safety.
PMCID: PMC4986340  PMID: 27532025
Checkpoint inhibitor; Immune-related adverse events; Melanoma; Immunotherapy; Myocarditis; Cardiomyopathy; Ipilimumab; Nivolumab; Pembrolizumab
4.  Comprehensive genetic testing identifies targetable genomic alterations in most patients with non-small cell lung cancer, specifically adenocarcinoma, single institute investigation 
Oncotarget  2016;7(14):18876-18886.
This study reviews extensive genetic analysis in advanced non-small cell lung cancer (NSCLC) patients in order to: describe how targetable mutation genes interrelate with the genes identified as variants of unknown significance; assess the percentage of patients with a potentially targetable genetic alterations; evaluate the percentage of patients who had concurrent alterations, previously considered to be mutually exclusive; and characterize the molecular subset of KRAS.
Thoracic Oncology Research Program Databases at the University of Chicago provided patient demographics, pathology, and results of genetic testing. 364 patients including 289 adenocarcinoma underwent genotype testing by various platforms such as FoundationOne, Caris Molecular Intelligence, and Response Genetics Inc. For the entire adenocarcinoma cohort, 25% of patients were African Americans; 90% of KRAS mutations were detected in smokers, including current and former smokers; 46% of EGFR and 61% of ALK alterations were detected in never smokers. 99.4% of patients, whose samples were analyzed by next-generation sequencing (NGS), had genetic alterations identified with an average of 10.8 alterations/tumor throughout different tumor subtypes. However, mutations were not mutually exclusive.
NGS in this study identified potentially targetable genetic alterations in the majority of patients tested, detected concurrent alterations and provided information on variants of unknown significance at this time but potentially targetable in the future.
PMCID: PMC4951336  PMID: 26934441
non-small cell lung cancer; genetic testing; next-generation sequencing; genomic alteration
5.  Spontaneous restoration of transplantation tolerance after acute rejection 
Nature communications  2015;6:7566.
Transplantation is a cure for end-stage organ failure but, in the absence of pharmacological immunosuppression, allogeneic organs are acutely rejected. Such rejection invariably results in allosensitization and accelerated rejection of secondary donor-matched grafts. Transplantation tolerance can be induced in animals and a subset of humans, and enables long-term acceptance of allografts without maintenance immunosuppression. However, graft rejection can occur long after a state of transplantation tolerance has been acquired. When such an allograft is rejected, it has been assumed that the same rules of allosensitization apply as to non-tolerant hosts and that immunological tolerance is permanently lost. Using a mouse model of cardiac transplantation, we show that when Listeria monocytogenes infection precipitates acute rejection, thus abrogating transplantation tolerance, the donor-specific tolerant state re-emerges, allowing spontaneous acceptance of a donor-matched second transplant. These data demonstrate a setting in which the memory of allograft tolerance dominates over the memory of transplant rejection.
PMCID: PMC4498267  PMID: 26151823
6.  Spontaneous restoration of transplantation tolerance after acute rejection 
Nature Communications  2015;6:7566.
Transplantation is a cure for end-stage organ failure but, in the absence of pharmacological immunosuppression, allogeneic organs are acutely rejected. Such rejection invariably results in allosensitization and accelerated rejection of secondary donor-matched grafts. Transplantation tolerance can be induced in animals and a subset of humans, and enables long-term acceptance of allografts without maintenance immunosuppression. However, graft rejection can occur long after a state of transplantation tolerance has been acquired. When such an allograft is rejected, it has been assumed that the same rules of allosensitization apply as to non-tolerant hosts and that immunological tolerance is permanently lost. Using a mouse model of cardiac transplantation, we show that when Listeria monocytogenes infection precipitates acute rejection, thus abrogating transplantation tolerance, the donor-specific tolerant state re-emerges, allowing spontaneous acceptance of a donor-matched second transplant. These data demonstrate a setting in which the memory of allograft tolerance dominates over the memory of transplant rejection.
An infection can break the immune tolerance of a transplanted organ, resulting in its rejection. Here the authors show that the immunological memory of transplantation tolerance dominates over the memory of allograft rejection, so that another organ transplanted later can be spontaneously accepted.
PMCID: PMC4498267  PMID: 26151823
7.  Combination MET inhibition and Topoisomerase I inhibition block cell growth of Small Cell Lung Cancer 
Molecular cancer therapeutics  2013;13(3):576-584.
Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)–induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC.
PMCID: PMC4286701  PMID: 24327519
small cell lung cancer; MET; topoisomerase-I; SU11274; SN-38
8.  A novel algorithm for simplification of complex gene classifiers in cancer 
Cancer research  2013;73(18):5625-5632.
The clinical application of complex molecular classifiers as diagnostic or prognostic tools has been limited by the time and cost needed to apply them to patients. Using an existing fifty-gene expression signature known to separate two molecular subtypes of the pediatric cancer rhabdomyosarcoma, we show that an exhaustive iterative search algorithm can distill this complex classifier down to two or three features with equal discrimination. We validated the two-gene signatures using three separate and distinct data sets, including one that uses degraded RNA extracted from formalin-fixed, paraffin-embedded material. Finally, to demonstrate the generalizability of our algorithm, we applied it to a lung cancer data set to find minimal gene signatures that can distinguish survival. Our approach can easily be generalized and coupled to existing technical platforms to facilitate the discovery of simplified signatures that are ready for routine clinical use.
PMCID: PMC4233649  PMID: 23913937
9.  MET and PI3K/mTOR as a Potential Combinatorial Therapeutic Target in Malignant Pleural Mesothelioma 
PLoS ONE  2014;9(9):e105919.
Malignant pleural mesothelioma (MPM) is an aggressive disease with a poor prognosis. Studies have shown that both MET and its key downstream intracellular signaling partners, PI3K and mTOR, are overexpressed in MPM. Here we determined the combinatorial therapeutic efficacy of a new generation small molecule inhibitor of MET, ARQ 197, and dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 in mesothelioma cell and mouse xenograft models. Cell viability results show that mesothelioma cell lines were sensitive to ARQ 197, NVP-BEZ235 and GDC-0980 inhibitors. The combined use of ARQ 197 with either NVP-BEZ235 or GDC-0980, was synergistic (CI<1). Significant delay in wound healing was observed with ARQ 197 (p<0.001) with no added advantage of combining it with either NVP-BEZ235 or GDC-0980. ARQ 197 alone mainly induced apoptosis (20±2.36%) that was preceded by suppression of MAPK activity, while all the three suppressed cell cycle progression. Both GDC-0980 and NVP-BEZ235 strongly inhibited activities of PI3K and mTOR as evidenced from the phosphorylation status of AKT and S6 kinase. The above observation was further substantiated by the finding that a majority of the MPM archival samples tested revealed highly active AKT. While the single use of ARQ 197 and GDC-0980 inhibited significantly the growth of MPM xenografts (p<0.05, p<0.001 respectively) in mice, the combination of the above two drugs was highly synergistic (p<0.001). Our results suggest that the combined use of ARQ 197/NVP-BEZ235 and ARQ 197/GDC-0980 is far more effective than the use of the drugs singly in suppressing MPM tumor growth and motility and therefore merit further translational studies.
PMCID: PMC4164360  PMID: 25221930
10.  Persistence of complex vascular lesions despite prolonged prostacyclin therapy of pulmonary arterial hypertension 
Histopathology  2012;61(4):597-609.
Continuous infusion of prostacyclin analogues improves survival in advanced pulmonary arterial hypertension. In addition to its vasodilatory effects, prostacyclin has the potential to decrease inflammation, thrombosis, and smooth muscle proliferation. The aim of this retrospective study was to determine whether pathological data support the ability of prostanoids to prevent progression of vascular disease.
Methods and results
Twenty-two autopsied patients with World Health Organization category 1 pulmonary arterial hypertension (primarily idiopathic and connective tissue disease-associated) were divided into those who received long-term prostacyclin (n = 12, PG-long, mean treatment 3.9 years) and those who received 0–1 month of prostacyclin (n = 10, PG-short). Surprisingly, PG-long patients had larger plexiform lesions (P < 0.05), with no decrease in medial and intimal thicknesses as compared with PG-short patients. Plexiform lesion size and density increased with increasing treatment time. Also, PG-long patients had fewer platelet thrombi and more frequent acute diffuse alveolar haemorrhage. Quantification of macrophages and T cells revealed no differences in inflammatory infiltrates.
Although long-term prostacyclin therapy may have an antithrombotic effect in addition to its vasodilatory actions, it was not associated with the prevention of advanced vascular lesions. The mechanism by which prostacyclin analogues improve survival in pulmonary arterial hypertension remains uncertain.
PMCID: PMC4143476  PMID: 22748137
histopathology; plexiform lesions; prostaglandins; vascular remodelling
11.  Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer 
BMC Cancer  2014;14:185.
Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC.
Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay.
Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0–3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability.
PAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.
PMCID: PMC3995599  PMID: 24628993
12.  The clinical and immunologic features of pulmonary fibrosis in sarcoidosis 
Sarcoidosis is a multisystem, granulomatous disease that most often affects the lungs. The clinical course is highly variable; many patients undergo spontaneous remission, but up to a third of patients progresses to a chronic disease course. The development of pulmonary fibrosis (PF) in a subset of patients with chronic disease has a negative impact on morbidity and mortality. While sarcoidosis-associated PF can be progressive, it is often referred to as “burnt out” disease, a designation reflecting inactive granulomatous inflammation. The immune mechanisms of sarcoidosis-associated PF are not well understood. It is not clear if fibrotic processes are active from the onset of sarcoidosis in predisposed individuals, or whether a profibrotic state develops as a response to ongoing inflammation. Transforming growth factor β (TGF-β) is an important profibrotic cytokine, and in sarcoidosis, distinct genotypes of TGF-β have been identified in those with PF. The overall cytokine profile in sarcoidosis-associated PF has not been well characterized, although a transition from a T helper 1 to a T helper 2 signature has been proposed. Macrophages have important regulatory interactions with fibroblasts, and the role of alveolar macrophages in sarcoidosis-associated PF is a compelling target for further study. Elucidating the natural history of sarcoidosis-associated PF will inform our understanding of the fundamental derangements, and will enhance prognostication and the development of therapeutic strategies.
PMCID: PMC3910531  PMID: 22683422
13.  Bronchial thermoplasty failure in severe persistent asthma: a case report 
Bronchial thermoplasty (BT) is an emerging therapy for patients with severe persistent asthma who remain poorly controlled despite standard maximal medical therapy. Thermoplasty elicits asthma control over time by applying thermal radiofrequency energy to airways to ablate underlying smooth muscle. While this therapy is suggested to eliminate such smooth muscle permanently, no human studies have examined the possibility of treatment failure.
Case report
We present a 62-year-old female with severe, refractory asthma symptoms who underwent BT without apparent complications. However, severe symptoms including multiple clinical exacerbations persisted despite BT treatment. Repeat endobronchial biopsy done six months after BT treatment demonstrated persistent smooth muscle hyperplasia in multiple airways that previously had been treated. The patient continued to have uncontrolled, refractory asthma despite multiple therapies.
This case is the first to describe a failure of BT to reduce or eliminate airway smooth muscle in a patient with severe persistent asthma. It suggests the potential for treatment failure in the management of these patients after BT and highlights the need for further study of potential BT-refractory patients.
PMCID: PMC3842589  PMID: 23651158
Bronchial thermoplasty; severe asthma
14.  The EphB4 Receptor Tyrosine Kinase Promotes Lung Cancer Growth: A Potential Novel Therapeutic Target 
PLoS ONE  2013;8(7):e67668.
Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.
PMCID: PMC3699624  PMID: 23844053
15.  Paxillin mutations affect focal adhesions and lead to altered mitochondrial dynamics 
Cancer Biology & Therapy  2013;14(7):679-691.
Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.
PMCID: PMC3742497  PMID: 23792636
mitochondria; paxillin; gene mutation; cell motility; fission; fusion; mitochondrial dynamics
16.  Genomic assessment of a multikinase inhibitor, sorafenib, in a rodent model of pulmonary hypertension 
Physiological genomics  2008;33(2):278-291.
Pulmonary hypertension (PH) and cancer pathology share growth factor- and MAPK stress-mediated signaling pathways resulting in endothelial and smooth muscle cell dysfunction and angioproliferative vasculopathy. In this study, we assessed sorafenib, an antineoplastic agent and inhibitor of multiple kinases important in angiogenesis [VEGF receptor (VEGFR)-1–3, PDGF receptor (PDGFR)-β, Raf-1 kinase] as a potential PH therapy. Two PH rat models were used: a conventional hypoxia-induced PH model and an augmented PH model combining dual VEGFR-1 and -2 inhibition (SU-5416, single 20 mg/kg injection) with hypoxia. In addition to normoxia-exposed control animals, four groups were maintained at 10% inspired O2 fraction for 3.5 wk (hypoxia/vehicle, hypoxia/SU-5416, hypoxia/sorafenib, and hypoxia/SU-5416/sorafenib). Compared with normoxic control animals, rats exposed to hypoxia/SU-5416 developed hemodynamic and histological evidence of severe PH while rats exposed to hypoxia alone displayed only mild elevations in hemodynamic values (pulmonary vascular and right ventricular pressures). Sorafenib treatment (daily gavage, 2.5 mg/kg) prevented hemodynamic changes and demonstrated dramatic attenuation of PH-associated vascular remodeling. Compared with normoxic control rats, expression profiling (Affymetrix platform) of lung RNA obtained from hypoxia [false discovery rate (FDR) 6.5%]- and hypoxia/SU-5416 (FDR 1.6%)-challenged rats yielded 1,019 and 465 differentially regulated genes (fold change >1.4), respectively. A novel molecular signature consisting of 38 differentially expressed genes between hypoxia/SU-5416 and hypoxia/SU-5416/sorafenib (FDR 6.7%) was validated by either real-time RT-PCR or immunoblotting. Finally, immunoblotting studies confirmed the upregulation of the MAPK cascade in both PH models, which was abolished by sorafenib. In summary, sorafenib represents a novel potential treatment for severe PH with the MAPK cascade a potential canonical target.
PMCID: PMC3616402  PMID: 18303084
microarrays; SU-5416; bioinformatics
17.  Functional variants of sphingosine-1-phosphate receptor 1 gene associate with asthma susceptibility 
The genetic mechanisms underlying asthma remain unclear. Increased permeability of the microvasculature is a feature of asthma and the sphingosine-1-phosphate receptor, S1PR1, is an essential participant regulating lung vascular integrity and responses to lung inflammation.
We explored the contribution of polymorphisms in the S1PR1 gene (S1PR1) to asthma susceptibility.
A combination of gene re-sequencing for SNP discovery, case-control association, functional evaluation of associated SNPs, and protein immunochemistry studies was utilized.
Immunohistochemistry studies demonstrated significantly decreased S1PR1 protein expression in pulmonary vessels in asthmatic lungs compared to non-asthmatic individuals (p<0.05). Direct DNA sequencing of 27 multiethnic samples identified 39 S1PR1 variants (18 novel SNPs). Association studies were performed based on genotyping results from cosmopolitan tagging SNPs in three case-control cohorts from Chicago and New York totaling 1061 subjects (502 cases and 559 controls). Promoter SNP rs2038366 (−1557G/T) was found to be associated with asthma (p=0.03) in European Americans. In African Americans, an association was found for both asthma and severe asthma for intronic SNP rs3753194 (c.−164+170A/G) (p=0.006 and p=0.040, respectively) and for promoter SNP rs59317557 (−532C/G) with severe asthma (p=0.028). Consistent with predicted in silico functionality, alleles of promoter SNPs rs2038366 (−1557G/T) and rs59317557 (−532C/G) influenced the activity of a luciferase S1PR1 reporter vector in transfected endothelial cells exposed to growth factors (EGF, PDGF, VEGF) known to be increased in asthmatic airways.
These data provide strong support for a role for S1PR1 gene variants in asthma susceptibility and severity.
Clinical Implications
Our results indicate S1PR1 is a novel asthma candidate gene and an attractive target for future therapeutic strategies.
Capsule summary
This study identified novel polymorphisms in S1PR1, revealed the functional implications of S1PR1 genetic variants in different populations, and their association with asthma susceptibility and severity.
PMCID: PMC3495167  PMID: 20624651
asthma; sphingosine-1-phosphate receptor 1; single nucleotide polymorphism; promoter activity
18.  A Sphingosine 1–Phosphate 1 Receptor Agonist Modulates Brain Death–Induced Neurogenic Pulmonary Injury 
Lung transplantation remains the only viable therapy for patients with end-stage lung disease. However, the full utilization of this strategy is severely compromised by a lack of donor lung availability. The vast majority of donor lungs available for transplantation are from individuals after brain death (BD). Unfortunately, the early autonomic storm that accompanies BD often results in neurogenic pulmonary edema (NPE), producing varying degrees of lung injury or leading to primary graft dysfunction after transplantation. We demonstrated that sphingosine 1–phosphate (S1P)/analogues, which are major barrier-enhancing agents, reduce vascular permeability via the S1P1 receptor, S1PR1. Because primary lung graft dysfunction is induced by lung vascular endothelial cell barrier dysfunction, we hypothesized that the S1PR1 agonist, SEW-2871, may attenuate NPE when administered to the donor shortly after BD. Significant lung injury was observed after BD, with increases of approximately 60% in bronchoalveolar lavage (BAL) total protein, cell counts, and lung tissue wet/dry (W/D) weight ratios. In contrast, rats receiving SEW-2871 (0.1 mg/kg) 15 minutes after BD and assessed after 4 hours exhibited significant lung protection (∼ 50% reduction, P = 0.01), as reflected by reduced BAL protein/albumin, cytokines, cellularity, and lung tissue wet/dry weight ratio. Microarray analysis at 4 hours revealed a global impact of both BD and SEW on lung gene expression, with a differential gene expression of enriched immune-response/inflammation pathways across all groups. Overall, SEW served to attenuate the BD-mediated up-regulation of gene expression. Two potential biomarkers, TNF and chemokine CC motif receptor-like 2, exhibited gene array dysregulation. We conclude that SEW-2871 significantly attenuates BD-induced lung injury, and may serve as a potential candidate to improve human donor availability.
PMCID: PMC3262681  PMID: 21617203
neurogenic pulmonary edema; lung injury; sphingosine 1–phosphate; sphingolipids; lung transplant donors
19.  Utilisation of a thoracic oncology database to capture radiological and pathological images for evaluation of response to chemotherapy in patients with malignant pleural mesothelioma 
BMJ Open  2012;2(5):e001620.
An area of need in cancer informatics is the ability to store images in a comprehensive database as part of translational cancer research. To meet this need, we have implemented a novel tandem database infrastructure that facilitates image storage and utilisation.
We had previously implemented the Thoracic Oncology Program Database Project (TOPDP) database for our translational cancer research needs. While useful for many research endeavours, it is unable to store images, hence our need to implement an imaging database which could communicate easily with the TOPDP database.
The Thoracic Oncology Research Program (TORP) imaging database was designed using the Research Electronic Data Capture (REDCap) platform, which was developed by Vanderbilt University. To demonstrate proof of principle and evaluate utility, we performed a retrospective investigation into tumour response for malignant pleural mesothelioma (MPM) patients treated at the University of Chicago Medical Center with either of two analogous chemotherapy regimens and consented to at least one of two UCMC IRB protocols, 9571 and 13473A.
A cohort of 22 MPM patients was identified using clinical data in the TOPDP database. After measurements were acquired, two representative CT images and 0–35 histological images per patient were successfully stored in the TORP database, along with clinical and demographic data.
We implemented the TORP imaging database to be used in conjunction with our comprehensive TOPDP database. While it requires an additional effort to use two databases, our database infrastructure facilitates more comprehensive translational research.
The investigation described herein demonstrates the successful implementation of this novel tandem imaging database infrastructure, as well as the potential utility of investigations enabled by it. The data model presented here can be utilised as the basis for further development of other larger, more streamlined databases in the future.
PMCID: PMC3488720  PMID: 23103606
Basic Sciences
20.  Sphingosine Kinase 1 Is Required for Mesothelioma Cell Proliferation: Role of Histone Acetylation 
PLoS ONE  2012;7(9):e45330.
Malignant pleural mesothelioma (MPM) is a devastating disease with an overall poor prognosis. Despite the recent advances in targeted molecular therapies, there is a clear and urgent need for the identification of novel mesothelioma targets for the development of highly efficacious therapeutics.
Methodology/Principal Findings
In this study, we report that the expression of Sphingosine Kinase 1 (SphK1) protein was preferentially elevated in MPM tumor tissues (49 epithelioid and 13 sarcomatoid) compared to normal tissue (n = 13). In addition, we also observed significantly elevated levels of SphK1 and SphK2 mRNA and SphK1 protein expression in MPM cell lines such as H2691, H513 and H2461 compared to the non-malignant mesothelial Met5 cells. The underlying mechanism appears to be mediated by SphK1 induced upregulation of select gene transcription programs such as that of CBP/p300 and PCAF, two histone acetyl transferases (HAT), and the down regulation of cell cycle dependent kinase inhibitor genes such as p27Kip1 and p21Cip1. In addition, using immunoprecipitates of anti-acetylated histone antibody from SphK inhibitor, SphK-I2 treated Met5A and H2691 cell lysates, we also showed activation of other cell proliferation related genes, such as Top2A (DNA replication), AKB (chromosome remodeling and mitotic spindle formation), and suppression of p21 CIP1 and p27KIP1. The CDK2, HAT1 and MYST2 were, however, unaffected in the above study. Using SphK inhibitor and specific siRNA targeting either SphK1 or SphK2, we also unequivocally established that SphK1, but not SphK2, promotes H2691 mesothelioma cell proliferation. Using a multi-walled carbon nanotubes induced peritoneal mesothelioma mouse model, we showed that the SphK1−/− null mice exhibited significantly less inflammation and granulamatous nodules compared to their wild type counterparts.
The lipid kinase SphK1 plays a positive and essential role in the growth and development of malignant mesothelioma and is therefore a likely therapeutic target.
PMCID: PMC3444486  PMID: 23028939
21.  S100A12 in Vascular Smooth Muscle Accelerates Vascular Calcification in Apolipoprotein E–Null Mice by Activating an Osteogenic Gene Regulatory Program 
The proinflammatory cytokine S100A12 is associated with coronary atherosclerotic plaque rupture. We previously generated transgenic mice with vascular smooth muscle–targeted expression of human S100A12 and found that these mice developed aortic aneurysmal dilation of the thoracic aorta. In the current study, we tested the hypothesis that S100A12 expressed in vascular smooth muscle in atherosclerosis-prone apolipoprotein E (ApoE)–null mice would accelerate atherosclerosis.
Methods and Results
ApoE-null mice with or without the S100A12 transgene were analyzed. We found a 1.4-fold increase in atherosclerotic plaque size and more specifically a large increase in calcified plaque area (45% versus 7% of innominate artery plaques and 18% versus 10% of aortic root plaques) in S100A12/ApoE-null mice compared with wild-type/ApoE-null littermates. Expression of bone morphogenic protein and other osteoblastic genes was increased in aorta and cultured vascular smooth muscle, and importantly, these changes in gene expression preceded the development of vascular calcification in S100A12/ApoE-null mice. Accelerated atherosclerosis and vascular calcification were mediated, at least in part, by oxidative stress because inhibition of NADPH oxidase attenuated S100A12-mediated osteogenesis in cultured vascular smooth muscle cells. S100A12 transgenic mice in the wild-type background (ApoE+/+) showed minimal vascular calcification, suggesting that S100A12 requires a proinflammatory/proatherosclerotic environment to induce osteoblastic differentiation and vascular calcification.
Vascular smooth muscle S100A12 accelerates atherosclerosis and augments atherosclerosis-triggered osteogenesis, reminiscent of features associated with plaque instability.
PMCID: PMC3364048  PMID: 20966394
calcification; coronary artery disease; genetically altered mice; vascular biology
22.  Simvastatin Attenuates Radiation-Induced Murine Lung Injury and Dysregulated Lung Gene Expression 
Novel therapies are desperately needed for radiation-induced lung injury (RILI), which, despite aggressive corticosteroid therapy, remains a potentially fatal and dose-limiting complication of thoracic radiotherapy. We assessed the utility of simvastatin, an anti-inflammatory and lung barrier–protective agent, in a dose- and time-dependent murine model of RILI (18–(25 Gy). Simvastatin reduced multiple RILI indices, including vascular leak, leukocyte infiltration, and histological evidence of oxidative stress, while reversing RILI-associated dysregulated gene expression, including p53, nuclear factor–erythroid-2–related factor, and sphingolipid metabolic pathway genes. To identify key regulators of simvastatin-mediated RILI protection, we integrated whole-lung gene expression data obtained from radiated and simvastatin-treated mice with protein–protein interaction network analysis (single-network analysis of proteins). Topological analysis of the gene product interaction network identified eight top-prioritized genes (Ccna2a, Cdc2, fcer1 g, Syk, Vav3, Mmp9, Itgam, Cd44) as regulatory nodes within an activated RILI network. These studies identify the involvement of specific genes and gene networks in RILI pathobiology, and confirm that statins represent a novel strategy to limit RILI.
PMCID: PMC3095940  PMID: 20508068
radiation pneumonitis; lung vascular permeability; simvastatin; gene dysregulation; protein–protein interaction
23.  Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 (SOD2) in Pulmonary Arterial Hypertension 
Circulation  2010;121(24):2661-2671.
Excessive proliferation and impaired apoptosis of pulmonary artery smooth muscle cells (PASMC) contributes to vascular obstruction in patients and fawn-hooded rats (FHR) with pulmonary arterial hypertension (PAH). Expression and activity of mitochondrial superoxide dismutase-2 (SOD2), the major generator of H2O2, is known to be reduced in PAH; however, the mechanism and therapeutic relevance of this is unknown.
Methods and Results
SOD2 expression in PASMC is decreased in PAH patients and FHR with PAH. FHR PASMC have higher proliferation and lower apoptosis rates than Sprague-Dawley PASMC. Moreover, FHR PASMC have hyperpolarized mitochondria, low H2O2 production and a reduced cytoplasmic and mitochondrial redox state. Administration of SOD2 siRNA to normal PASMC recapitulates the FHR-PAH phenotype, hyperpolarizing mitochondria, decreasing H2O2 and inhibiting caspase activity. Conversely, SOD2 over-expression in FHR PASMC, or therapy with the SOD-mimetic MnTBAP, reverses the hyperproliferative PAH phenotype. Importantly, SOD-mimetic therapy regresses PAH in vivo. Investigation of the SOD2 gene revealed no mutation, suggesting a possible epigenetic dysregulation. Genomic bisulfite sequencing demonstrates selective hypermethylation of a CpG island in an enhancer region of intron 2 and another in the promoter. Differential methylation occurs selectively in PA versus aortic SMC and is reversed by the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, restoring both SOD2 expression and the proliferation/apoptosis ratio. The expression of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs.
Tissue-specific, epigenetic SOD2 deficiency initiates and sustains a heritable form of PAH by impairing redox signaling and creating a proliferative, apoptosis-resistant PASMC. SOD augmentation regresses experimental PAH. The discovery of an epigenetic component to PAH may offer new therapeutic targets.
PMCID: PMC2914302  PMID: 20529999
Pulmonary arterial hypertension; Voltage-gated potassium channels (Kv1.5); Hypoxia-inducible factor-1α (HIF-1α); Epigenetic gene methylation; DNA methyltransferase
24.  Proteomic characterization of non-small cell lung cancer in a comprehensive translational thoracic oncology database 
In recent years, there has been tremendous growth and interest in translational research, particularly in cancer biology. This area of study clearly establishes the connection between laboratory experimentation and practical human application. Though it is common for laboratory and clinical data regarding patient specimens to be maintained separately, the storage of such heterogeneous data in one database offers many benefits as it may facilitate more rapid accession of data and provide researchers access to greater numbers of tissue samples.
The Thoracic Oncology Program Database Project was developed to serve as a repository for well-annotated cancer specimen, clinical, genomic, and proteomic data obtained from tumor tissue studies. The TOPDP is not merely a library—it is a dynamic tool that may be used for data mining and exploratory analysis. Using the example of non-small cell lung cancer cases within the database, this study will demonstrate how clinical data may be combined with proteomic analyses of patient tissue samples in determining the functional relevance of protein over and under expression in this disease.
Clinical data for 1323 patients with non-small cell lung cancer has been captured to date. Proteomic studies have been performed on tissue samples from 105 of these patients. These tissues have been analyzed for the expression of 33 different protein biomarkers using tissue microarrays. The expression of 15 potential biomarkers was found to be significantly higher in tumor versus matched normal tissue. Proteins belonging to the receptor tyrosine kinase family were particularly likely to be over expressed in tumor tissues. There was no difference in protein expression across various histologies or stages of non-small cell lung cancer. Though not differentially expressed between tumor and non-tumor tissues, the over expression of the glucocorticoid receptor (GR) was associated improved overall survival. However, this finding is preliminary and warrants further investigation.
Though the database project is still under development, the application of such a database has the potential to enhance our understanding of cancer biology and will help researchers to identify targets to modify the course of thoracic malignancies.
PMCID: PMC3097094  PMID: 21884620
25.  Role of protein kinase C β and vascular endothelial growth factor receptor in malignant pleural mesothelioma: Therapeutic implications and the usefulness of Caenorhabditis elegans model organism 
To examine the role of both protein kinase C (PKC)-β and vascular endothelial growth factor receptor (VEGFR)-2 in malignant pleural mesothelioma (MPM) using respective inhibitors, enzastaurin and KRN633.
Materials and Methods:
MPM cell lines, control cells, and a variety of archived MPM tumor samples were used to determine the protein expression levels of PKC-β, VEGFR-2, VEGF, and p-AKT. Effects of enzastaurin and KRN633 on phosphorylation status of key signaling molecules and viability of the mesothelioma cells were determined. The common soil nematode, Caenorhabditis elegans, was treated with enzastaurin to determine its suitability to screen for highly potent kinase inhibitors.
PKC-β1, PKC-β2 and VEGFR-2/KDR were overexpressed in MPM cell lines and MPM tumor tissues. Enzastaurin treatment resulted in significant loss in viability of VEGF induced cell proliferation; however, the effect of KRN633 was much less. Enzastaurin also dramatically decreased the phosphorylation of PKC-β, its downstream target p-AKT, and surprisingly, the upstream VEGFR-2. The combination of the two drugs at best was additive and similar results were obtained with respect to cell viability. Treatment of C. elegans with enzastaurin resulted in clear phenotypic changes and the worms were hypermotile with abnormal pattern and shape of eggs, suggesting altered fecundity.
PKC-β1 and VEGFR-2 are both excellent therapeutic targets in MPM. Enzastaurin was better at killing MPM cells than KRN633 and the combination lacked synergy. In addition, we show here that C. elegans can be used to screen for the next generation inhibitors as treatment with enzastaurin resulted in clear phenotypic changes that could be assayed.
PMCID: PMC3049271  PMID: 21383961
Enzastaurin; KRN633; malignant pleural mesothelioma; PKC-β; VEGFR-2

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