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Sugarbaker, David

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Sugarbaker

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David

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Sugarbaker, David
Author's Publications: search.filters.isAuthorOfPublication.62758d0f-3c23-4491-90f2-fa89d951b067

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    Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression
    (Public Library of Science, 2012) Barbone, Dario; Cheung, Priscilla; Battula, Sailaja; Busacca, Sara; Gray, Steven G.; Longley, Daniel B.; Bueno, Raphael; Sugarbaker, David; Bueno, Raphael; Broaddus, V. Courtney
    When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies.
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    Asbestos Burden Predicts Survival in Pleural Mesothelioma
    (National Institute of Environmental Health Sciences, 2008) Christensen, Brock C.; Godleski, John; Roelofs, Cora R.; Longacker, Jennifer L.; Bueno, Raphael; Sugarbaker, David; Marsit, Carmen J.; Nelson, Heather H.; Kelsey, Karl T.
    Background: Malignant pleural mesothelioma (MPM) is a rapidly fatal asbestos-associated malignancy with a median survival time of < 1 year following diagnosis. Treatment strategy is determined in part using known prognostic factors. Objective: The aim of this study was to examine the relationship between asbestos exposure and survival outcome in MPM in an effort to advance the understanding of the contribution of asbestos exposure to MPM prognosis. Methods: We studied incident cases of MPM patients enrolled through the International Mesothelioma Program at Brigham and Women’s Hospital in Boston, Massachusetts, using survival follow-up, self-reported asbestos exposure (n = 128), and a subset of cases (n = 80) with quantitative asbestos fiber burden measures. Results: Consistent with the established literature, we found independent, significant associations between male sex and reduced survival (p < 0.04), as well as between nonepithelioid tumor histology and reduced survival (p < 0.02). Although self-reported exposure to asbestos was not predictive of survival among our cases, stratifying quantitative asbestos fiber burden [number of asbestos bodies per gram of lung (wet weight)] into groups of low (0–99 asbestos bodies), moderate (100–1,099), and high fiber burden (> 1,099), suggested a survival duration association among these groups (p = 0.06). After adjusting for covariates in a Cox model, we found that patients with a low asbestos burden had a 3-fold elevated risk of death compared to patients with a moderate fiber burden [95% confidence interval (CI), 0.95–9.5; p = 0.06], and patients with a high asbestos burden had a 4.8-fold elevated risk of death (95% CI, 1.5–15.0; p < 0.01) versus those with moderate exposure. Conclusion: Our data suggest that patient survival is associated with asbestos fiber burden in MPM and is perhaps modified by susceptibility.
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    Differentially Expressed Alternatively Spliced Genes in Malignant Pleural Mesothelioma Identified Using Massively Parallel Transcriptome Sequencing
    (BioMed Central, 2009) Dong, Lingsheng; Jensen, Roderick V; De Rienzo, Assunta; Gordon, Gavin J.; Xu, Yanlong; Sugarbaker, David; Bueno, Raphael
    Background: Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples. Methods: We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens. Results: We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively).Conclusion Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.
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    Aging and Environmental Exposures Alter Tissue-Specific DNA Methylation Dependent upon CpG Island Context
    (Public Library of Science, 2009) Christensen, Brock C.; Marsit, Carmen J.; Zheng, Shichun; Wrensch, Margaret R.; Wiemels, Joseph L.; Nelson, Heather H.; Karagas, Margaret R.; Padbury, James F.; Yeh, Ru-Fang; Wiencke, John K.; Kelsey, Karl T.; Houseman, Eugene Andres; Bueno, Raphael; Sugarbaker, David
    Epigenetic control of gene transcription is critical for normal human development and cellular differentiation. While alterations of epigenetic marks such as DNA methylation have been linked to cancers and many other human diseases, interindividual epigenetic variations in normal tissues due to aging, environmental factors, or innate susceptibility are poorly characterized. The plasticity, tissue-specific nature, and variability of gene expression are related to epigenomic states that vary across individuals. Thus, population-based investigations are needed to further our understanding of the fundamental dynamics of normal individual epigenomes. We analyzed 217 non-pathologic human tissues from 10 anatomic sites at 1,413 autosomal CpG loci associated with 773 genes to investigate tissue-specific differences in DNA methylation and to discern how aging and exposures contribute to normal variation in methylation. Methylation profile classes derived from unsupervised modeling were significantly associated with age (P<0.0001) and were significant predictors of tissue origin (P<0.0001). In solid tissues (n = 119) we found striking, highly significant CpG island–dependent correlations between age and methylation; loci in CpG islands gained methylation with age, loci not in CpG islands lost methylation with age (P<0.001), and this pattern was consistent across tissues and in an analysis of blood-derived DNA. Our data clearly demonstrate age- and exposure-related differences in tissue-specific methylation and significant age-associated methylation patterns which are CpG island context-dependent. This work provides novel insight into the role of aging and the environment in susceptibility to diseases such as cancer and critically informs the field of epigenomics by providing evidence of epigenetic dysregulation by age-related methylation alterations. Collectively we reveal key issues to consider both in the construction of reference and disease-related epigenomes and in the interpretation of potentially pathologically important alterations.
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    Asbestos exposure predicts cell cycle control gene promoter methylation in pleural mesothelioma
    (Oxford University Press, 2008) Christensen, Brock C.; Godleski, John; Marsit, Carmen J.; Houseman, Eugene Andres; Lopez-Fagundo, Cristina Y.; Longacker, Jennifer L.; Bueno, Raphael; Sugarbaker, David; Nelson, Heather H.; Kelsey, Karl T.
    Malignant pleural mesothelioma (MPM) is a rapidly fatal tumor with increasing incidence worldwide responsible for many thousands of deaths annually. Although there is a clear link between exposure to asbestos and mesothelioma, and asbestos is known to be both clastogenic and cytotoxic to mesothelial cells, the mechanisms of causation of MPM remain largely unknown. However, there is a rapidly emerging literature that describes inactivation of a diverse array of tumor suppressor genes (TSGs) via promoter DNA CpG methylation in MPM, although the etiology of these alterations remains unclear. We studied the relationships among promoter methylation silencing, asbestos exposure, patient demographics and tumor histology using a directed approach; examining six cell cycle control pathway TSGs in an incident case series of 70 MPMs. Promoter hypermethylation of APC, CCND2, CDKN2A, CDKN2B, HPPBP1 and RASSF1 were assessed. We observed significantly higher lung asbestos body burden if any of these cell cycle genes were methylated (P < 0.02), and there was a significant trend of increasing asbestos body counts as the number of methylated cell cycle pathway genes increased from 0 to 1 to >1 (P < 0.005). This trend of increasing asbestos body count and increasing number of methylated cell cycle pathway genes remained significant (P < 0.05) after controlling for age, gender and tumor histology. These data suggest a novel tumorigenic mechanism of action of asbestos and may contribute to the understanding of precisely how asbestos exposure influences the etiology and clinical course of malignant mesothelioma.
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    Genes Associated with Prognosis after Surgery for Malignant Pleural Mesothelioma Promote Tumor Cell Survival In Vitro
    (BioMed Central, 2011) Gordon, Gavin J.; Bueno, Raphael; Sugarbaker, David
    Background: Mesothelioma is an aggressive neoplasm with few effective treatments, one being cytoreductive surgery. We previously described a test, based on differential expression levels of four genes, to predict clinical outcome in prospectively consented mesothelioma patients after surgery. In this study, we determined whether any of these four genes could be linked to a cancer relevant phenotype. Methods: We conducted a high-throughput RNA inhibition screen to knockdown gene expression levels of the four genes comprising the test (ARHGDIA, COBLL1, PKM2, TM4SF1) in both a human lung-derived normal and a tumor cell line using three different small inhibitory RNA molecules per gene. Successful knockdown was confirmed using quantitative RT-PCR. Detection of statistically significant changes in apoptosis and mitosis was performed using immunological assays and quantified using video-assisted microscopy at a single time-point. Changes in nuclear shape, size, and numbers were used to provide additional support of initial findings. Each experiment was conducted in triplicate. Specificity was assured by requiring that at least 2 different siRNAs produced the observed change in each cell line/time-point/gene/assay combination. Results: Knockdown of ARHGDIA, COBLL1, and TM4SF1 resulted in 2- to 4-fold increased levels of apoptosis in normal cells (ARHGDIA only) and tumor cells (all three genes). No statistically significant changes were observed in apoptosis after knockdown of PKM2 or for mitosis after knockdown of any gene. Conclusions: We provide evidence that ARHGDIA, COBLL1, and TM4SF1 are negative regulators of apoptosis in cultured tumor cells. These genes, and their related intracellular signaling pathways, may represent potential therapeutic targets in mesothelioma.