A Bioinformatics Resource for TWEAK-Fn14 Signaling Pathway

TNF-related weak inducer of apoptosis (TWEAK) is a new member of the TNF superfamily. It signals through TNFRSF12A, commonly known as Fn14. The TWEAK-Fn14 interaction regulates cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, tissue remodeling and inflammation. Although TWEAK has been reported to be associated with autoimmune diseases, cancers, stroke, and kidney-related disorders, the downstream molecular events of TWEAK-Fn14 signaling are yet not available in any signaling pathway repository. In this paper, we manually compiled from the literature, in particular those reported in human systems, the downstream reactions stimulated by TWEAK-Fn14 interactions. Our manual amassment of the TWEAK-Fn14 pathway has resulted in cataloging of 46 proteins involved in various biochemical reactions and TWEAK-Fn14 induced expression of 28 genes. We have enabled the availability of data in various standard exchange formats from NetPath, a repository for signaling pathways. We believe that this composite molecular interaction pathway will enable identification of new signaling components in TWEAK signaling pathway. This in turn may lead to the identification of potential therapeutic targets in TWEAK-associated disorders

Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes

© 2017 Wong et al.; Published by Cold Spring Harbor Laboratory Press. Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions, 192 novel protein start sites, 19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted noncoding RNAs to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes

miRNA and proteomic dysregulation in non-small cell lung cancer in response to cigarette smoke

Dysregulation of miRNAs is well associated with the development of non-small cell lung cancer (NSCLC). It is imperative that dysregulation of miRNAs by cigarette smoke will affect the expression of their targets, either leading to the activation of oncoproteins or suppression of tumor suppressor proteins. In this study, we have carried out miRNA sequencing and SILAC-based proteomics analysis of H358 cells chronically exposed to cigarette smoke condensate. miRNA sequencing resulted in the identification of 208 miRNAs, of which 6 miRNAs were found to be significantly dysregulated (fold change ≥ 4, p-value ≤ 0.05) in H358-smoke exposed cells. Proteomic analysis of the smoke exposed cells compared to the parental cells resulted in the quantification of 2,396 proteins, of which 681 proteins were found to be differentially expressed (fold change ≥ 2). Gene ontology based analysis of target proteins revealed enrichment of proteins involved in biological processes driving metabolism and a decrease in expression of proteins associated with immune response in the cells exposed to cigarette smoke. Pathway analysis using Ingenuity Pathway Analysis (IPA) revealed activation of ERK/MAPK and integrin signaling and repression of RhoGDI signaling in H358 smoke exposed cells. We also identified 5 novel miRNA in H358 smoke exposed cells using unassigned reads of small RNA-Seq dataset. In summary, this study indicates that chronic exposure to cigarette smoke leads to widespread dysregulation of miRNAs and their targets, resulting in signaling aberrations in NSCLC. The miRNAs and their targets identified in the study need to be further investigated to explore their role as potential targets and/or molecular markers in NSCLC especially in smokers

Identification of potential biomarkers of head and neck squamous cell carcinoma using iTRAQ based quantitative proteomic approach

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in India. Despite improvements in treatment strategy, the survival rates of HNSCC patients remain poor. Thus, it is necessary to identify biomarkers that can be used for early detection of disease. In this study, we employed iTRAQ-based quantitative mass spectrometry analysis to identify dysregulated proteins from a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. We identified 2468 proteins, of which 496 proteins were found to be dysregulated in at least two out of three HNSCC cell lines compared to immortalized normal oral keratinocytes. We detected increased expression of replication protein A1 (RPA1) and heat shock protein family H (Hsp110) member 1 (HSPH1), in HNSCC cell lines compared to control. The differentially expressed proteins were further validated using parallel reaction monitoring (PRM) and western blot analysis in HNSCC cell lines. Immunohistochemistry-based validation using HNSCC tissue microarrays revealed overexpression of RPA1 and HSPH1 in 15.7% and 32.2% of the tested cases, respectively. Our study illustrates quantitative proteomics as a robust approach for identification of potential HNSCC biomarkers. The proteomic data has been submitted to ProteomeXchange Consortium (http://www.proteomecentral.proteomexchange.org) via the PRIDE public data repository accessible using the data identifier - PXD009241. Keywords: HNSCC, iTRAQ, Parallel reaction monitoring, Mass spectrometry, OKF6/TERT

Integrated multi-omics analysis reveals potential mechanisms of acquired resistance to erlotinib in head and neck cancer cells

Epidermal growth factor receptor (EGFR) is overexpressed in 90% of head and neck squamous cell carcinomas (HNSCC). However, most clinical trials with tyrosine kinase inhibitors (TKIs) have shown modest response rates due to development of acquired resistance. We performed whole exome sequencing (WES) of an isogenic pair of erlotinib-sensitive (SCC-S) and resistant (SCC-R) HNSCC cell lines to elucidate the molecular mechanisms that govern acquired resistance to erlotinib. Exome sequencing resulted in identification of 148 non-synonymous single nucleotide variants (SNVs) in 139 genes and copy number alterations (CNA) (≥2 fold) affecting 339 genes in SCC-R cells compared to SCC-S cells. Comparison of SNVs from SCC-R against post-translational modification databases resulted in identification of loss of ubiquitinylation site at p.K57E in dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) which was validated using in-house high-throughput proteomic data. Substitution mutation K57N in MAP2K1 is shown to result in its constitutive activation and subsequent gefitinib resistance in lung adenocarcinoma. We also identified a well-known driver mutation p.G13R in Harvey rat sarcoma viral oncogene homolog (HRAS) (AllF: 20.41%). In addition, we also observed CNA in other genes of this pathway including RAC-beta serine/threonine-protein kinase (AKT2), glycogen synthase kinase-3 alpha (GSK3A), Rho guanine nucleotide exchange factor 1 (ARHGEF1) amongst others. Corresponding protein expression changes of these genes were also observed in proteomics data. Quantitative phosphoproteomics revealed hyperphosphorylation of other proteins involved in MAPK pathway such as serine/threonine-protein kinase B-raf (BRAF), MAP2K2, mitogen-activated protein kinases such as MAPK1 and MAPK3. Integrative multi-omic analysis revealed constitutive activation of key intermediates of MAPK pathway in SCC-R cells compared to SCC-S cells which may be essential in the development of acquired resistance to erlotinib in these cells. We hypothesize that combinatorial treatment regime involving inhibition of putative targets such as MAP2K1 with erlotinib therapy may aid in tackling acquired erlotinib resistance in HNSCC patients

Dysregulation of splicing proteins in head and neck squamous cell carcinoma

Signaling plays an important role in regulating all cellular pathways. Altered signaling is one of the hallmarks of cancers. Phosphoproteomics enables interrogation of kinase mediated signaling pathways in biological systems. In cancers, this approach can be utilized to identify aberrantly activated pathways that potentially drive proliferation and tumorigenesis. To identify signaling alterations in head and neck squamous cell carcinoma (HNSCC), we carried out proteomic and phosphoproteomic analysis of HNSCC cell lines using a combination of tandem mass tag (TMT) labeling approach and titanium dioxide-based enrichment. We identified 4,920 phosphosites corresponding to 2,212 proteins in six HNSCC cell lines compared to a normal oral cell line. Our data indicated significant enrichment of proteins associated with splicing. We observed hyperphosphorylation of SRSF protein kinase 2 (SRPK2) and its downstream substrates in HNSCC cell lines. SRPK2 is a splicing kinase, known to phosphorylate serine/arginine (SR) rich domain proteins and regulate splicing process in eukaryotes. Although genome-wide studies have reported the contribution of alternative splicing events of several genes in the progression of cancer, the involvement of splicing kinases in HNSCC is not known. In this study, we studied the role of SRPK2 in HNSCC. Inhibition of SRPK2 resulted in significant decrease in colony forming and invasive ability in a panel of HNSCC cell lines. Our results indicate that phosphorylation of SRPK2 plays a crucial role in the regulation of splicing process in HNSCC and that splicing kinases can be developed as a new class of therapeutic target in HNSCC

Histological analysis for effect of polysaccharide on tissue morphology.

<p>Microscopic examination of histological paraffin sections (H & E staining) showed standard and consistent morphology of reconstructed human epidermis (RHE). Thin and broad arrows indicate intact stratum corneum and Epidermis respectively.</p

Effect of polysaccharide fraction treatment on median chronological lifespan and AUC in yeast.

<p>Effect of polysaccharide fraction treatment on median chronological lifespan and AUC in yeast.</p

Expression of CD44 and HA in human reconstructed epidermis after treatment with polysaccharide.

<p>Proteins were quantified using ELISA in two separate EpiSkin^TM D6 tissue samples and at three different concentrations of polysaccharide (1, 10 and 100 μg/mL). Control was the tissue sample treated with solvent DMSO.</p