Whole Genome Sequencing of Mesothelioma Cell Lines
ABSTRACT: Malignant pleural mesothelioma (MPM) is characterized by dismal prognosis. Consequently, dissection of the molecular mechanisms driving malignancy is of key importance. Here we investigate whether activating mutations in the telomerase reverse transcriptase (TERT) gene promoter are present in MPM and associated with disease progression, cell immortalization, and genomic alteration patterns.
Project description:Malignant pleural mesothelioma (MPM) is a highly lethal, poorly understood neoplasm that is typically associated with asbestos exposure. We performed transcriptional profiling using high-density oligonucleotide microarrays containing approximately 22,000 genes to elucidate potential molecular and pathobiological pathways in MPM using discarded human MPM tumor specimens (n=40), normal lung specimens (n=4), normal pleura specimens (n=5), and MPM and SV40-immortalized mesothelial cell lines (n=5). In global expression analysis using unsupervised clustering techniques, we found two potential subclasses of mesothelioma which correlate loosely with tumor histology. We also identified sets of genes with expression levels that distinguish between multiple tumor subclasses, normal and tumor tissues, and tumors with different morphologies. Microarray gene expression data were confirmed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and protein analysis for three novel candidate oncogenes (NME2, CRI1, and PDGFC) and one candidate tumor suppressor (GSN). Finally, we used bioinformatics tools (i.e. software) to create and explore complex physiological pathways that may be relevant in mesothelioma tumorigenesis, pathobiology, or both. Tissues and cell lines profiled using microarrays. Discarded MPM surgical specimens (n=40), normal pleura specimens (n=5), and normal lung specimens (n=4) were freshly collected (and snap frozen) from patients who underwent surgery at Boston’s Brigham and Women’s Hospital (BWH) between October 1998 and August 2000. All of these patients underwent extrapleural pneumonectomy with heated intra-pleural cisplatin chemotherapy delivered after the specimens were removed. All normal specimens were obtained from patients who were never diagnosed with MPM. Two human MPM cell lines (MS589 and MS428) were kindly provided by Jonathan A. Fletcher, M.D., Department of Pathology, BWH. The JMN1B MPM cell line19,20 has been described previously. The SV40-immortalized, non-tumorigenic mesothelial cell line (Met-5A)21 and the MPM cell line MSTO-211H22 were purchased from the American Type Culture Collection. Normal tissues were obtained from additional consented patients undergoing treatment for diseases other than MPM. All MPM samples used in these studies contained relatively pure tumor (greater than 50% tumor cells per high power field examined in a section adjacent to the tissue used). The microscopic slides from the patients’ resection specimens were reviewed by one of the authors (J.G.), and the diagnosis and histologic subclassification of MPM confirmed in all cases. Linked clinical and pathological data were obtained for all patients who contributed tumor specimens. Specimens and data were rendered anonymous to protect patient confidentiality. Studies utilizing human tissues were approved by and conducted in accordance with the policies of the Institutional Review Board at BWH. SUBMITTER_CITATION: Gordon, G. J., Rockwell, G. N., Jensen, R. V., Rheinwald, J. G., Glickman, J.N., Aronson, J. P., Pottorf, B. J., Nitz, M. D., Richards, W. G., Sugarbaker, D. J., and Bueno, R. Identification of novel candidate oncogenes and tumor suppressors in malignant pleural mesothelioma using large-scale transcriptional profiling. American Journal of Pathology, 166: 1827-1840, 2005.
Project description:In this study we performed proteogenomic analysis for 9 cell lines of malignant melanoma. The main objectives of the study were identifying the variants originating from point mutations and analyzing the effect of exome data filtering on the outcome of variant identification.
Project description:Malignant Pleural Mesothelioma (MPM) is an aggressive cancer that is often diagnosed at an advanced stage and is characterized by a long latency period (20-40 years between initial exposure and diagnosis) and prior exposure to asbestos. Currently accurate diagnosis of MPM is difficult due to the lack of sensitive biomarkers, and despite minor improvements in treatment, median survival rates do not exceed 12 months. Accumulating evidence suggests that aberrant expression of long non-coding RNAs (lncRNAs) play an important functional role in cancer biology. LncRNAs are a class of recently discovered non-protein coding RNAs >200 nucleotides in length with a role in regulating transcription. Here we used NCode long noncoding microarrays to identify differentially expressed lncRNAs potentially involved in MPM pathogenesis. High priority candidate lncRNAs were selected on the basis of statistical (P<0.05) and biological significance (>3-fold difference). Expression levels of 9 candidate lncRNAs were technically validated using RT-qPCR, and biologically validated in three independent test sets: (1) 57 archived MPM tissues obtained from extrapleural pneumonectomy patients, (2) 15 cryopreserved MPM and 3 benign pleura, and (3) an extended panel of 10 MPM cell lines. RT-qPCR analysis demonstrated consistent up-regulation of these lncRNAs in independent datasets. ROC curve analysis showed that two candidates were able to separate benign pleura and MPM with high sensitivity and specificity, and were associated with nodal metastases and survival following induction chemotherapy. These results suggest that lncRNAs have potential to serve as biomarkers in MPM. To identify mRNA and lncRNA biomarkers associated with malignant pleural mesothelioma (MPM), we performed gene expression array analysis on 4 MPM cell lines (H28, MM05, MSTO-211H, H226) compared to the immortalised mesothelial line (MeT-5A). All cell lines were profiled in duplicate. Synthesis of the labelled first strand cDNA was conducted using the Superscript Plus Direct cDNA labeling system with starting material of 10ug total RNA. The labeled dNTP mix was added to the reaction to generate labeled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA was purified using low elution volume spin cartridges included in the purification module. Samples were labelled using Alexa Fluor 555 dyes. Samples were then hybridised to NCode Long Noncoding RNA Microarrays. Slides were scanned using an Agilent Scanner. Genes differentially expressed between Met-5A and the MPM cell lines were identified on the basis of P-value and fold change.
Project description:Leptospirosis is a re-emerging tropical infectious disease caused by the pathogenic Leptospira spp. The different host innate immune responses were partially related to the different severity of leptospirosis. In this study, we employed transcriptomics and cytokine array to comparatively calculate the responses of murine peritoneal macrophages (MPM) and human peripheral blood monocytes (HBM) to leptospiral infection, and uncover a series of different expression regulations of these two immune cells. The regulation percentages in several biological processes of MPM, such as the antigen process and presentation, the regulation of membrane potential, and the innate immune response, etc., were much greater than those of HBM (>2 folds). In HBM and MPM, the CASP8 and FADD-like apoptosis regulator genes were significantly up-regulated, which supported previous results that the caspase-8/3 pathway plays an important role in macrophage apoptosis during leptospiral infection. The key component of complement pathway, C3, was only up-regulated in MPM. Several cytokines (e.g. IL-10, TNF-alpha) were differently expressed at both mRNA and protein level in MPM and HBM. The differences in the transcriptomics and the cytokine expression revealed in this study were partially consistent with the different outcomes of the chronic and acute leptospirosis; thus, these findings facilitated further molecular study on the innate immune response to leptospiral infection. Murine peritoneal macrophages (MPM) and human peripheral blood monocytes (HBM) were infected by pathogenic Leptospira. The host cells were collected at 1h, 2h, and 4h after infection. The uninfected macrophage sample was designed as a negative control. Three biological replicates were designed for each sample type. There were totally 24 hybridizations (12 for MPM, and 12 for HBM) included in this submission.
Project description:Purpose: Celiac disease (CD) is a risk factor for developing Small Bowel Carcinoma (SBC) with a 14 folds higher risk than that of the general population. As SBCs associated with CD (CD-SBCs) are extremely rare, very few molecular data are currently available about their pathogenesis and information about CD-SBC transcriptomic profiling is completely lacking. Patients and Methods: We generated RNA-seq data on 13 CD-SBCs selected from the largest and well-characterized series of CD-SBCs published so far that was collected by the Small Bowel Cancer Italian Consortium. In all cases we compared the CD-SBC transcriptional signatures with the four consensus molecular subtypes (CMS) of colorectal carcinoma (CRC) applying the ‘CMS classifier’. CpG Island Methylator Phenotype (CIMP+) was evaluated in all cases using methylation-sensitive multiple ligation-dependent probe amplification. Results: RNA-based signatures of CD-SBCs exhibited strong similarities with CRCs. Twelve of 13 CD-SBCs (92%) fell within the two main subtypes exhibiting high immune and inflammatory signatures, i.e. CMS1 and CMS4. CMS1 CD-SBCs (62% of cases) were commonly MSI/CIMP+ tumors and showed increased expression of genes associated with a diffuse TH1 and cytotoxic T immune infiltrate, up-regulation of apoptosis, cell cycle progression and proteasome pathways. CMS4 CD-SBCs (31% of cases) showed prominent TGF-β activation and were characterized by complement-associated inflammation, matrix remodeling, stromal invasion and angiogenesis Conclusions: RNA-based signatures of CD-SBCs strongly recapitulate CMS classification of CRC, give a promising tool to improve our knowledge of this rare entity and provide clinically useful information using the wealth of data available for CRC.
Project description:We used microarrays to detail the global gene expression of mithramycin treated cells, xenografts and sp1 depleted cells with p53 overexpression. MPM cells and xenografts were treated with mithramycin or SP1 depleted/p53 overexpressed MPM cells were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to see the invitro and invivo effect of mithramycin (pharmacological model) and depletion of SP1 and overexpression of p53 (genetic model).
Project description:Here we directly compare for the first time how the longstanding static model of mouse Dentate Gyrus (DG) development compares with a comprehensive high-resolution live-cell multiphoton (live-MPM) imaging approach. We took advantage of multiple fluorescent protein-based cell-type specific reporters to identify Neural Stem Cells (NSC), Intermediate Neurogenic Progenitors (INPs), and Granule Neurons (GNs) to generate live 4D cellular datasets across embryonic, postnatal and adult ages. Live-MPM revealed that INPs and NSCs migrated long distances along multiple routes to seed the SGZ from multiple directions, and from mosaic progenitor zones along the septo-temporal axis of the hippocampus. We found that dynamic INPs processes and interactions contributed to the architecture of both transient and permanent NSC niches during embryonic development, and that INP cellular plasticity is maintained in the adult SGZ NSC niche. We also used a Molecular Systems (MS) approach to determine the basis for maintained INP cellular plasticity that revealed an overlapping signaling network infrastructure based largely on Rho-family mediated regulation of cytoskeletal dynamics. Our combined strategies revealed that dynamic INPs are a major molecular signaling transition state in the adult SGZ, and that Tbr2 expression defines the initial stage of GN commitment. Our novel findings reveal fundamental new insight into one of the most well studied brain regions key for normal cognitive function, and the importance of analyzing the development of live stem cell niches in vivo. In concert with live-cell imaging, we used microarray analysis to identify genes that may be involved in the development of the Dentate Gyrus NSC niche.
Project description:The data in this submission relate to whole exome sequencing from murine ovarian cancer cell line ID8. All sequencing was performed by Beckman Coulter Genomics, Grenoble, France in February 2013.