Context

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.

Objective

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.

Design

Tissue microarrays of 38 typical carcinoids (TC), 6 atypical carcinoids (AC), 34 SCLC, and 11 large cell neuroendocrine carcinomas (LCNEC) were studied with immunohistochemistry.

Results

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.

Conclusions

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.

Background

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.

Objective

We explored the contribution of polymorphisms in the S1PR1 gene (S1PR1) to asthma susceptibility.

Methods

A combination of gene re-sequencing for SNP discovery, case-control association, functional evaluation of associated SNPs, and protein immunochemistry studies was utilized.

Results

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.

Conclusion

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.

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.

Objective

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.

Conclusion

Vascular smooth muscle S100A12 accelerates atherosclerosis and augments atherosclerosis-triggered osteogenesis, reminiscent of features associated with plaque instability.

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.

Background

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.

Description

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.

Conclusion

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.

Purpose:

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.

Results:

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.

Conclusions:

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.

Background

Paxillin is a modular protein that localises to cell adhesion sites where it facilitates bidirectional communication between the intracellular actin cytoskeleton and the extracellular matrix. These complex and dynamic interactions are essential for cell adhesion, cell migration and cell survival. The authors have previously demonstrated that paxillin is overexpressed in lung cancer tissues and identified somatic paxillin mutations in 9% of lung cancers. A murine in vivo xenograft model of the most common paxillin mutation (A127T) showed increased cell proliferation and invasive tumour growth, establishing an important role for paxillin in the development of lung cancer.

Methods

The authors analysed 279 bronchoscopy-aided biopsy specimens from 92 high-risk patients. Adenocarcinoma with bronchioloalveolar features and pure bronchioloalveolar carcinoma (BAC) were analysed with fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC).

Results

Paxillin is overexpressed in premalignant areas of hyperplasia, squamous metaplasia and goblet cell metaplasia, as well as dysplastic lesions and carcinoma in high-risk patients. Concordance between increased paxillin gene copy number and paxillin overexpression was observed in cases of adenocarcinoma eusomic for chromosome 12.

Conclusions

Paxillin overexpression occurs during the earliest stages of lung cancer development. FISH and IHC analysis of lung adenocarcinoma suggests that relatively small-scale genomic rearrangements of chromosome 12 are associated with paxillin overexpression in lung adenocarcinoma.

Introduction

Chronic lymphocytic leukemia is an indolent disease that often presents with complaints of lymphadenopathy or is detected as an incidental laboratory finding. It is rarely considered in the differential diagnosis of patients presenting with tamponade or a large, bloody pericardial effusion. In patients without known cancer, a large, bloody pericardial effusion raises the possibility of tuberculosis, particularly in patients from endemic areas. However, the signs, symptoms and laboratory findings of pericarditis related to chronic lymphocytic leukemia can mimic tuberculosis.

Case Presentation

We report the case of a 58-year-old African American-Nigerian woman with a history of travel to Nigeria and a positive tuberculin skin test who presented with cardiac tamponade. She had a mild fever, lymphocytosis and a bloody pericardial effusion, but cultures and stains were negative for acid-fast bacteria. Assessment of blood by flow cytometry and pericardial biopsy by immunohistochemistry revealed CD5 (+) and CD20 (+) lymphocytes in both tissues, demonstrating this to be an unusual manifestation of early stage chronic lymphocytic leukemia.

Conclusion

Although most malignancies that involve the pericardium clinically manifest elsewhere before presenting with tamponade, this case illustrates the potential for early stage chronic lymphocytic leukemia to present as a large pericardial effusion with tamponade. Moreover, the presentation mimicked tuberculosis. This case also demonstrates that it is possible to treat chronic lymphocytic leukemia-related pericardial tamponade by removal of the fluid without chemotherapy.

MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) regulate a variety of cellular functions, many of which can be dysregulated in human cancers. Activated MET signaling can lead to cell motility and scattering, angiogenesis, proliferation, branching morphogenesis, invasion, and eventual metastasis. We performed systematic analysis of the expression of the MET receptor and its ligand HGF in tumor tissue microarrays (TMA) from human solid cancers. Standard immunohistochemistry and a computerized automated scoring system were used. DNA sequencing for MET mutations in both non-kinase and kinase domains was also performed. MET was differentially overexpressed in human solid cancers. The ligand HGF was widely expressed in both tumor, primarily intra-tumoral, and non-malignant tissues. The MET/HGF likely is functional and may be activated in autocrine fashion in vivo. MET and SCF were found to be positively stained in the bronchioalevolar junctions of lung tumors. A number of novel mutations of MET were identified, particularly in the extracellular semaphorin domain and the juxtamembrane domain. MET-HGF pathway can be assayed in TMAs and is often overexpressed in a wide variety of human solid cancers. MET can be activated through overexpression, mutation, or autocrine signaling in malignant cells. Mutations in the non-kinase regions of MET might play important role in tumorigenesis and tumor progression. MET would be an important therapeutic anti-tumor target to be inhibited, and in lung cancer, MET may represent a cancer early progenitor cell marker.

Background:

Treatment of non-small cell lung cancer (NSCLC) remains a difficult task in oncology. Targeted inhibition of oncogenic proteins is promising. In this study, we evaluate the expression of MET and PKCß and in vitro effects of their inhibition using SU11274 and enzastaurin (LY317615.HCl) respectively.

Materials and Methods:

Patient samples were analyzed by immunohistochemistry for expression of PKCß and MET, utilizing tissue microarrays under an IRB-approved protocol. Expression of PKCß and MET was evaluated in cell lines by immunoblotting. Treatment with SU1174 against MET and enzastaurin against PKCß was performed in H1993 and H358 cell lines, and cell proliferation and downstream signaling (phosphorylation of MET, AKT, FAK, and GSK3ß) were evaluated by immunoblotting. Statistical analysis was performed using SPSS 16.0.

Results:

Expression of MET positively correlated with lymph node metastases (p=.0004), whereas PKCß showed no correlation (p=0.204). MET and PKCß expression were also strongly correlated (p<0.001). Expression of MET was observed in 5/8 cell lines (H358, H1703, A549, H1993, H2170; absent from H522, H661, or SW1573), whereas PKCß expression was observed in 8/8 cell lines. Cell proliferation was significantly impaired by treatment with SU11274 and enzastaurin, and their effects were synergistic in combination (CI=0.32 and 0.09). Phosphorylation of MET, FAK, AKT, and GSK3ß were strongly inhibited with both agents in combination.

Conclusions:

Concomitant inhibition of MET and PKCß significantly increased cytotoxicity in vitro against NSCLC, disrupting important downstream signaling pathways. Further evaluation in animal models is warranted.

Rationale: We previously demonstrated pre–B-cell colony enhancing factor (PBEF) as a biomarker in sepsis and sepsis-induced acute lung injury (ALI) with genetic variants conferring ALI susceptibility.

Objectives: To explore mechanistic participation of PBEF in ALI and ventilator-induced lung injury (VILI).

Methods: Two models of VILI were utilized to explore the role of PBEF using either recombinant PBEF or PBEF+/− mice.

Measurements and Main Results: Initial in vitro studies demonstrated recombinant human PBEF (rhPBEF) as a direct rat neutrophil chemotactic factor with in vivo studies demonstrating marked increases in bronchoalveolar lavage (BAL) leukocytes (PMNs) after intratracheal injection in C57BL/6J mice. These changes were accompanied by increased BAL levels of PMN chemoattractants (KC and MIP-2) and modest increases in lung vascular and alveolar permeability. We next explored the potential synergism between rhPBEF challenge (intratracheal) and a model of limited VILI (4 h, 30 ml/kg tidal volume) and observed dramatic increases in BAL PMNs, BAL protein, and cytokine levels (IL-6, TNF-α, KC) compared with either challenge alone. Gene expression profiling identified induction of ALI- and VILI-associated gene modules (nuclear factor-κB, leukocyte extravasation, apoptosis, Toll receptor pathways). Heterozygous PBEF+/− mice were significantly protected (reduced BAL protein, BAL IL-6 levels, peak inspiratory pressures) when exposed to a model of severe VILI (4 h, 40 ml/kg tidal volume) and exhibited significantly reduced expression of VILI-associated gene expression modules. Finally, strategies to reduce PBEF availability (neutralizing antibody) resulted in significant protection from VILI.

Conclusions: These studies implicate PBEF as a key inflammatory mediator intimately involved in both the development and severity of ventilator-induced ALI.

PAX5 is a nuclear transcription factor required for B cell development and its expression was evaluated in upper aerodigestive malignancies and pancreatic cancer by immunoblotting. PAX5 protein expression was relatively strong in small cell lung cancer (SCLC, 11/12), however its expression was not detected in non-SCLC (NSCLC, n=13), mesothelioma (n=7), pancreatic (n=6), esophageal (n=6) and head and neck cancer cell lines (n=12). In comparison, PAX8 and PAX3 expression was absent or non-detectable in SCLC cell lines, however PAX8 was expressed in most of the NSCLC cell lines (13/13) tested and also frequently in all the other cell lines . We also detected frequent expression of PAX2 and PAX9 protein in the various cell lines. Utilizing neuroendocrine tumor samples, we found that the frequency as well as the average intensity of expression of PAX5 increased from pulmonary carcinoid (9%, moderate and strong PAX5 expression, n=44), to large cell neuroendocrine carcinoma (LCNC, 27% n=11) to SCLC (33%, n=76). FISH analysis revealed no translocations of PAX5 gene, but polyploidy in some SCLC tumor tissues (6 /37). We determined that PAX5 could regulate the transcription of c-Met using luciferase coupled reporter and ChIP analysis. In addition the phospho-c-Met (active form) and PAX5 were both localized to the same intra-nuclear compartment in HGF treated SCLC cells and interacted with each other. Finally, we determined the therapeutic translational potential of PAX5 using PAX5 knockdown SCLC cells in conjunction with Topoisomerase 1 (SN38) and c-Met (SU11274) inhibitors. Loss of endogenous PAX5 significantly decreased the viability of SCLC cells, especially when combined with SN38 or SU11274 and maximum effect was seen when both inhibitors were used. We therefore propose that PAX5 could be an important regulator of cMet transcription and a potential target for therapy in SCLC.

Purpose

Malignant pleural mesothelioma (MPM) is a disease with few therapeutic options. Protein kinase C beta (PKCß) is involved in important cellular functions. Enzastaurin (LY317615.HCl) is a novel inhibitor of PKC in clinical development.

Experimental Design

MPM cell lines (7) and patient tumor tissues (24) were evaluated for expression of PKCß by immunoblotting and immunohistochemistry, respectively. In-vitro cell-growth assays were performed with enzastaurin with or without cisplatin. Cell migration was evaluated with the wound healing assay. Downstream signaling (survival and focal adhesion pathways) was studied by immunobloting for related molecules in the presence of phorbol-ester with or without enzastaurin.

Results

Expression for PKCß1 was seen in all cases, with a mean integrated optical density (IOD) of 152.5 (standard deviation = 95.47, n=24), whereas PKCß2 expression was less intense, with a mean IOD of 11.45 (standard deviation = 16.27, n=21). There was a trend toward lower overall survival among patients expressing above-median PKCß1 (p=.064), but not PKCß2. Robust expression of PKCß1 and low expression of PKCß2 was observed in MPM cell lines. Treatment of MPM cell lines with enzastaurin revealed IC50 of 5 μM, and strong synergism was observed when combined with cisplatin. Wound healing assay revealed that treatment of H2461 cells with enzastaurin reduced migration by 59.2 %. Enzastaurin treatment led to disruption of F-actin architecture. Downstream signaling showed reduced phosphorylation of: AKT, FAK, p130Cas, S6 ribosomal protein and paxillin.

Conclusions

PKCß1 was expressed in the majority of MPM samples. Enzastaurin has pre-clinical activity against MPM, and exhibited synergism with cisplatin. PKCß inhibition in MPM might be able to reduce the invasiveness of MPM by affecting cytoskeletal function.

Objective

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.

Background

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.

Methods

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.

Results

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.

Discussion

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.

Conclusions

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.

Background

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.

Conclusions/Significance

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.

Background

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.

Conclusions

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.

Background

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.

Description

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.

Conclusion

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.

Patients with systemic sclerosis (SSc) can develop pulmonary hypertension (PH; mean pulmonary artery pressure ≥ 25 mm Hg) caused by pulmonary arterial hypertension (PAH), left ventricular disease, or pulmonary fibrosis. PAH is a pulmonary vascular disease, the diagnosis of which requires pulmonary capillary wedge pressure less than 15 mm Hg, pulmonary vascular resistance greater than 3 Wood Units, and exclusion of thromboembolism and parenchymal lung disease. Molecular mechanisms underlying PAH-SSc include activation of inflammatory and fibrogenic pathways in the vasculature and right ventricle. Circulating autoantibodies trigger endothelial damage and fibroblast activation. PAH most commonly occurs as a late complication in patients with limited cutaneous disease and anticentromere antibodies. Although echocardiography is a useful screening tool, heart catheterization is required to diagnose PAH before initiating therapy. Prognosis and therapeutic response are worse in PAH-SSc than in other PAH categories (median survival, 1–3 y). Approved therapies include prostacyclins, endothelin antagonists, and phosphodiesterase type 5 inhibitors. Research is needed to define disease mechanisms and develop effective therapies.

Lung cancer is characterized by abnormal cell growth and invasion, and the actin cytoskeleton plays a major role in these processes. The focal adhesion protein paxillin is a target of a number of oncogenes involved in key signal transduction and important in cell motility and migration. In lung cancer tissues, we have found that paxillin was highly expressed (compared with normal lung), amplified (12.1%, 8 of 66) and correlated with increased MET and epidermal growth factor receptor (EGFR) gene copy numbers, or mutated (somatic mutation rate of 9.4%, 18 of 191). Paxillin mutations (19 of 21) were clustered between LD motifs 1 and 2 and the LIM domains. The most frequent point mutation (A127T) enhanced lung cancer cell growth, colony formation, focal adhesion formation, and colocalized with Bcl-2 in vitro. Gene silencing from RNA interference of mutant paxillin led to reduction of cell viability. A murine in vivo xenograft model of A127T paxillin showed an increase in tumor growth, cell proliferation, and invasion. These results establish an important role for paxillin in lung cancer.