Integration of pharmacoproteomic and computational approaches reveals the cellular signal transduction pathways affected by apatinib in gastric cancer cell lines.

Apatinib is known to be a highly selective vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor with anti-angiogenic and anti-tumor properties. In a phase III study, the objective response rate to apatinib was low. It remains unclear why the effectivity of apatinib varies among patients and what type of patients are candidates for the treatment. In this study, we investigated the anti-tumor efficacy of apatinib against 13 gastric cancer cell lines and found that it differed depending on the cell line. Using integrated wet and dry approaches, we showed that apatinib was a multi-kinase inhibitor of c-Kit, RAF1, VEGFR1, VEGFR2, and VEGFR3, predominantly inhibiting c-Kit. Notably, KATO-III, which was the most apatinib-sensitive among the gastric cancer cell lines investigated, was the only cell line expressing c-Kit, RAF1, VEGFR1, and VEGFR3 but not VEGFR2. Furthermore, we identified SNW1 as a molecule affected by apatinib that plays an important role in cell survival. Finally, we identified the molecular network related to SNW1 that was affected by treatment with apatinib. These results suggest that the mechanism of action of apatinib in KATO-III cells is independent of VEGFR2 and that the differential efficacy of apatinib was due to differences in expression patterns of receptor tyrosine kinases. Furthermore, our results suggest that the differential efficacy of apatinib in gastric cell lines may be attributed to SNW1 phosphorylation levels at a steady state. These findings contribute to a deeper understanding of the mechanism of action of apatinib in gastric cancer cells.

Temporal dynamics from phosphoproteomics using endoscopic biopsy specimens provides new therapeutic targets in stage IV gastric cancer.

Phosphoproteomic analysis expands our understanding of cancer biology. However, the feasibility of phosphoproteomic analysis using endoscopically collected tumor samples, especially with regards to dynamic changes upon drug treatment, remains unknown in stage IV gastric cancer. Here, we conducted a phosphoproteomic analysis using paired endoscopic biopsy specimens of pre- and post-treatment tumors (Ts) and non-tumor adjacent tissues (NATs) obtained from 4 HER2-positive gastric cancer patients who received trastuzumab-based treatment and from pre-treatment Ts and NATs of 4 HER2-negative gastric cancer patients. Our analysis identified 14,622 class 1 phosphosites with 12,749 quantified phosphosites and revealed molecular changes by HER2 positivity and treatment. An inhibitory signature of the ErbB signaling was observed in the post-treatment HER2-positive T group compared with the pre-treatment HER2-positive T group. Phosphoproteomic profiles obtained by a case-by-case review using paired pre- and post-treatment HER2-positive T could be utilized to discover predictive or resistant biomarkers. Furthermore, these data nominated therapeutic kinase targets which were exclusively activated in the patient unresponded to the treatment. The present study suggests that a phosphoproteomic analysis of endoscopic biopsy specimens provides information on dynamic molecular changes which can individually characterize biologic features upon drug treatment and identify therapeutic targets in stage IV gastric cancer.

Identification of CD14 and lipopolysaccharide-binding protein as novel biomarkers for sarcoidosis using proteomics of serum extracellular vesicles.

Sarcoidosis is a complex, polygenic, inflammatory granulomatous multi-organ disease of unknown cause. The granulomatous inflammation in sarcoidosis is driven by the interplay between T cells and macrophages. Extracellular vesicles (EVs) play important roles in intercellular communication. We subjected serum EVs, isolated by size exclusion chromatography, from seven patients with sarcoidosis and five control subjects to non-targeted proteomics analysis. Non-targeted, label-free proteomics analysis detected 2292 proteins in serum EVs; 42 proteins were up-regulated in patients with sarcoidosis relative to control subjects; and 324 proteins were down-regulated. The protein signature of EVs from patients with sarcoidosis reflected disease characteristics such as antigen presentation and immunological disease. Candidate biomarkers were further verified by targeted proteomics analysis (selected reaction monitoring) in 46 patients and 10 control subjects. Notably, CD14 and lipopolysaccharide-binding protein (LBP) were validated by targeted proteomics analysis. Up-regulation of these proteins was further confirmed by immunoblotting, and their expression was strongly increased in macrophages of lung granulomatous lesions. Consistent with these findings, CD14 levels were increased in lipopolysaccharide-stimulated macrophages during multinucleation, concomitant with increased levels of CD14 and LBP in EVs. The area under the curve values of CD14 and LBP were 0.81 and 0.84, respectively, and further increased to 0.98 in combination with angiotensin-converting enzyme and soluble interleukin-2 receptor. These findings suggest that CD14 and LBP in serum EVs, which are associated with granulomatous pathogenesis, can improve the diagnostic accuracy in patients with sarcoidosis.

Characterization of Heat Shock Protein 60 as an Interacting Partner of Superoxide Dismutase 2 in the Silkworm, Bombyx mori, and Its Response to the Molting Hormone, 20-Hydroxyecdysone.

Oxidative stress promotes pupation in some holometabolous insects. The levels of superoxide, a reactive oxygen species (ROS), are increased and superoxide dismutase 1 (BmSod1) and superoxide dismutase 2 (BmSod2) are decreased during metamorphic events in silkworm (Bombyx mori). These observations strongly suggest that pupation is initiated by oxidative stress via the down-regulation of BmSod1 and BmSod2. However, the molecular mechanisms underlying ROS production during metamorphic events in silkworm remain unknown. To investigate these molecular mechanisms, the peripheral proteins of BmSod1 and BmSod2 were identified and characterized using dry and wet approaches in this study. Based on the results, silkworm heat shock protein 60 (BmHsp60) was identified as an interacting partner of BmSod2, which belongs to the Fe/MnSOD family. Furthermore, the present study results showed that BmHsp60 mRNA expression levels were increased in response to oxidative stress caused by ultraviolet radiation and that BmHsp60 protein levels (but not mRNA levels) were decreased during metamorphic events, which are regulated by the molting hormone 20-hydroxyecdysone. These findings improve our understanding of the mechanisms by which holometabolous insects control ROS during metamorphosis.

Expression of Cancer Stem Cell Markers EpCAM and CD90 Is Correlated with Anti- and Pro-Oncogenic EphA2 Signaling in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Additionally, the efficacy of targeted molecular therapies with multiple tyrosine kinase inhibitors is limited. In this study, we focused on the cellular signaling pathways common to diverse HCC cells and used quantitative reverse phase protein array (RPPA) and statistical analyses to elucidate the molecular mechanisms determining its malignancy. We examined the heterogeneity of 17 liver cancer cell lines by performing cluster analysis of their expression of CD90 and EpCAM cancer stem cell markers. Gaussian mixture model clustering identified three dominant clusters: CD90-positive and EpCAM-negative (CD90+), EpCAM-positive and CD90-negative (EpCAM+) and EpCAM-negative and CD90-negative (Neutral). A multivariate analysis by partial least squares revealed that the former two cell populations showed distinct patterns of protein expression and phosphorylation in the EGFR and EphA2 signaling pathways. The CD90+ cells exhibited higher abundance of AKT, EphA2 and its phosphorylated form at Ser897, whereas the EpCAM+ cells exhibited higher abundance of ERK, RSK and its phosphorylated form. This demonstrates that pro-oncogenic, ligand-independent EphA2 signaling plays a dominant role in CD90+ cells with higher motility and metastatic activity than EpCAM+ cells. We also showed that an AKT inhibitor reduced the proliferation and survival of CD90+ cells but did not affect those of EpCAM+ cells. Taken together, our results suggest that AKT activation may be a key pro-oncogenic regulator in HCC.

Proteomics of serum extracellular vesicles identifies a novel COPD biomarker, fibulin-3 from elastic fibres.

There is an unmet need for novel biomarkers in the diagnosis of multifactorial COPD. We applied next-generation proteomics to serum extracellular vesicles (EVs) to discover novel COPD biomarkers. EVs from 10 patients with COPD and six healthy controls were analysed by tandem mass tag-based non-targeted proteomics, and those from elastase-treated mouse models of emphysema were also analysed by non-targeted proteomics. For validation, EVs from 23 patients with COPD and 20 healthy controls were validated by targeted proteomics. Using non-targeted proteomics, we identified 406 proteins, 34 of which were significantly upregulated in patients with COPD. Of note, the EV protein signature from patients with COPD reflected inflammation and remodelling. We also identified 63 upregulated candidates from 1956 proteins by analysing EVs isolated from mouse models. Combining human and mouse biomarker candidates, we validated 45 proteins by targeted proteomics, selected reaction monitoring. Notably, levels of fibulin-3, tripeptidyl-peptidase 2, fibulin-1, and soluble scavenger receptor cysteine-rich domain-containing protein were significantly higher in patients with COPD. Moreover, six proteins; fibulin-3, tripeptidyl-peptidase 2, UTP-glucose-1-phosphate uridylyl transferase, CD81, CD177, and oncoprotein-induced transcript 3, were correlated with emphysema. Upregulation of fibulin-3 was confirmed by immunoblotting of EVs and immunohistochemistry in lungs. Strikingly, fibulin-3 knockout mice spontaneously developed emphysema with age, as evidenced by alveolar enlargement and elastin destruction. We discovered potential pathogenic biomarkers for COPD using next-generation proteomics of EVs. This is a novel strategy for biomarker discovery and precision medicine.

Panomicon: A web-based environment for interactive, visual analysis of multi-omics data.

Multi-omics analyses, combining transcriptomics, genomics, proteomics, and so on, have led to important insights in many areas of biology and medicine. To support these analyses, software that can handle the difficulties associated with multi-omics datasets is crucial. Here, we describe Panomicon, a web-based, interactive analysis environment for multi-omics data. Building on Toxygates, a tool previously created to study single-omics data that features interactive clustering, heatmaps, and user data uploads, Panomicon introduces improvements for the storage and handling of additional omics types, as well as tools for the generation and visualization of interaction networks between different types of omics data. Panomicon is a new type of environment for the collaborative study of multi-omics data, both for users uploading data to our server and for groups wishing to host their own deployment of Panomicon. We demonstrate Panomicon's capabilities by revisiting a microRNA-mRNA interaction networks study in a non-small cell lung cancer dataset.

Metabolomic analysis of fibrotic mice combined with public RNA-Seq human lung data reveal potential diagnostic biomarker candidates for lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with poor survival that warrants early and precise diagnosis for timely therapeutic intervention. Despite accumulating genomic, transcriptomic, proteomic, and lipidomic data on IPF, evidence from water-soluble metabolomics is limited. To identify biomarkers for IPF from water-soluble metabolomic data, we measured the levels of various metabolites in bronchoalveolar lavage fluid (BALF) and serum samples from a bleomycin-induced murine pulmonary fibrotic model using gas chromatography/mass spectrometry. Thirty-two of 73 BALF metabolites and 29 of 74 serum metabolites were annotated. We observed that the levels of proline and methionine were higher in BALF but lower in serum than those in the control. Furthermore, analysis of public RNA-Seq data from the lungs of patients with IPF revealed that proline- and methionine-related genes were significantly upregulated compared to those in the lungs of healthy controls. These results suggest that proline and methionine may be potential biomarkers for IPF and may help to deepen our understanding of the pathophysiology of the disease. Based on our results, we propose a model capable of recapitulating the proline and methionine metabolism of fibrotic lungs, thereby providing better means for studying the disease and developing novel therapeutic strategies for IPF.

Superoxide dismutase down-regulation and the oxidative stress is required to initiate pupation in Bombyx mori.

Perhaps, oxidative stress progresses pupation in some Lepidopteran insects; however, the reasons for this remain obscure. In our previous study, we clarified Bombyx mori SOD1 (BmSOD1) and B. mori SOD2 (BmSOD2) proteins respond in common to ultraviolet irradiation (UV) oxidative stress and metamorphosis. This result strongly suggested pupation initiates by oxidative stress and might mediate by down-regulation of expression of BmSOD1 and BmSOD2 proteins. Thus, we examined about these relationships in B. mori in this study. In the microarray data reanalysis, we found the Notch signaling pathways as the common pathways in pupation and UV oxidative stress in B. mori. Also, we showed a molting hormone, 20-hydroxyecdysone, leads not only generation of superoxide but also downregulation of the expression of BmSOD proteins during pupation in B. mori. Our findings can contribute to a deeper understanding of how biological defense systems work against environmental oxidative stress.

The role of glycogen in development and adult fitness in Drosophila.

The polysaccharide glycogen is an evolutionarily conserved storage form of glucose. However, the physiological significance of glycogen metabolism on homeostatic control throughout the animal life cycle remains incomplete. Here, we describe Drosophila mutants that have defective glycogen metabolism. Null mutants of glycogen synthase (GlyS) and glycogen phosphorylase (GlyP) displayed growth defects and larval lethality, indicating that glycogen plays a crucial role in larval development. Unexpectedly, however, a certain population of larvae developed into adults with normal morphology. Semi-lethality in glycogen mutants during the larval period can be attributed to the presence of circulating sugar trehalose. Homozygous glycogen mutants produced offspring, indicating that glycogen stored in oocytes is dispensable for embryogenesis. GlyS and GlyP mutants showed distinct metabolic defects in the levels of circulating sugars and triglycerides in a life stage-specific manner. In adults, glycogen as an energy reserve is not crucial for physical fitness and lifespan under nourished conditions, but glycogen becomes important under energy stress conditions. This study provides a fundamental understanding of the stage-specific requirements for glycogen metabolism in the fruit fly.

Comparative analysis of seven types of superoxide dismutases for their ability to respond to oxidative stress in Bombyx mori.

Insects are well adapted to changing environmental conditions. They have unique systems for eliminating reactive oxygen species (ROS). Superoxide dismutase (SOD) is a key enzyme that plays a primary role in removing ROS. Bombyx mori is a lepidopteran insect, whose body size is larger than the model insect Drosophila melanogaster, which enabled us to more easily examine gene expression at the tissue level. We searched B. mori SOD (BmSOD) genes using genome database, and we analyzed their function under different type of oxidative stress. Consequently, we identified four new types of BmSODs in addition to the three types already known. Two of the seven types had a unique domain architecture that has not been discovered previously in the SOD family, and they were expressed in different tissues and developmental stages. Furthermore, these BmSODs responded differently to several kinds of stressors. Our results showed that the seven types of BmSODs are likely to play different roles in B. mori; therefore, B. mori could be used to distinguish the functions of each SOD for resistance to oxidative stress that changes with the environmental conditions.

Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.

BACKGROUND: Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. RESULTS: Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. CONCLUSIONS: We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.

Superoxide dismutases, SOD1 and SOD2, play a distinct role in the fat body during pupation in silkworm Bombyx mori.

One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation.

Production of wild-type and mutant-type human DAP12 proteins by Bombyx mori nucleopolyhedrovirus vector.

A Japanese patient with Nasu-Hakola disease was found to have a serine-to-asparagine (S39N) substitution in human DNAX-activation protein 12 (DAP12). To elucidate the functional abnormalities of mutant-type DAP12, we expressed wild-type and mutant-type recombinant DAP12 protein with Bombyx mori nucleopolyhedrovirus (BmNPV) vector, and successfully purified the respective proteins from the hemolymph of recombinant BmNPV infected B. mori larvae.