Exploration of potential roles of a new LOXL2 splicing variant using network knowledge in esophageal squamous cell carcinoma.

LOXL2 (lysyl oxidase-like 2), an enzyme that catalyzes oxidative deamination of lysine residue, is upregulated in esophageal squamous cell carcinoma (ESCC). A LOXL2 splice variant LOXL2-e13 and its wild type were overexpressed in ESCC cells followed by microarray analyses. In this study, we explored the potential role and molecular mechanism of LOXL2-e13 based on known protein-protein interactions (PPIs), following microarray analysis of KYSE150 ESCC cells overexpressing a LOXL2 splice variant, denoted by LOXL2-e13, or its wild-type counterpart. The differentially expressed genes (DEGs) of LOXL2-WT and LOXL2-e13 were applied to generate individual PPI subnetworks in which hundreds of DEGs interacted with thousands of other proteins. These two DEG groups were annotated by Functional Annotation Chart analysis in the DAVID bioinformatics database and compared. These results found many specific annotations indicating the potential specific role or mechanism for LOXL2-e13. The DEGs of LOXL2-e13, comparing to its wild type, were prioritized by the Random Walk with Restart algorithm. Several tumor-related genes such as ERO1L, ITGA3, and MAPK8 were found closest to LOXL2-e13. These results provide helpful information for subsequent experimental identification of the specific biological roles and molecular mechanisms of LOXL2-e13. Our study also provides a work flow to identify potential roles of splice variants with large scale data.

Quantitative proteomics reveals the downregulation of GRB2 as a prominent node of F806-targeted cell proliferation network.

High-throughput proteomics has successfully identified thousands of proteins as potential therapeutic targets during investigations into mechanisms of drug action. A novel macrolide analog, denoted F806, is a potential antitumor drug. Here, using the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS), we characterize the F806-regulating protein profiles and identify the potential target molecules or pathways of F806 in esophageal squamous cell carcinoma (ESCC) cells. From a total of 1931 quantified proteins, 181 proteins were found to be down-regulated (FDR p-value<0.1, H/L ratio<0.738), and 119 proteins were up-regulated (FDR p-value<0.1, H/L ratio>1.156). Among the down-regulated proteins, we uncovered the over- and under-represented protein clusters in biological process and molecular function respectively by Gene Ontology analysis. Furthermore, down-regulated and up-regulated proteins were significantly enriched in 37 pathways and 60 sub-pathways by bioinformatic analysis (FDR p-value<0.1), while a down-regulated molecule growth factor receptor-bound protein 2 (GRB2) was a prominent node in fourteen cell proliferation-related sub-pathways. We concluded that GRB2 downregulation would be a potential target of F806 in ESCC cells. BIOLOGICAL SIGNIFICANCE: This study used SILAC-based quantitative proteomics screen to systematically characterize molecular changes induced by a novel macrolide analog F806 in esophageal squamous cell carcinoma (ESCC) cells. Followed by bioinformatic analyses, signal pathway networks generated from the quantified proteins, would facilitate future investigation into the further mechanisms of F806 in ESCC cells. Notably, it provided information that growth factor receptor-bound protein 2 (GRB2) would be a prominent node in the F806-targeted cell proliferation network.

Desmocollin­2 affects the adhesive strength and cytoskeletal arrangement in esophageal squamous cell carcinoma cells.

Desmocollin­2 (DSC2), a transmembrane glycoprotein belonging to the desmosomal cadherin family, has been found to be differentially expressed in several types of cancer and to be involved in tumor progression. The tumor metastasis suppressing property of DSC2 in esophageal squamous cell carcinoma (ESCC) has been described, however, its contribution to cell cohesion in ESCC remains to be elucidated. In the present study, using RNA interference (RNAi), the expression of DSC2 was silenced in SHEEC and KYSE510 cells. Hanging drop and fragmentation assays were performed to investigate the role of DSC2 in cell­cell adhesion. Western blot analysis and confocal microscopy were used to analyze the expression and localization of cell adhesion molecules and cytoskeletal arrangement. The results demonstrated that DSC2 knock down by RNAi caused defects in cell­cell adhesion and a concomitant reduction in desmosomal protein expression and adherens junction molecule distribution. A decrease in the expression of DSC2 caused an increase in free γ­catenin levels, thus promoting its recruitment to the adherens junction complex. In addition, the RNAi­mediated inhibition of DSC2 led to keratin intermediate filament retraction and filamentous­actin cytoskeleton rearrangement. Taken together, these data support our previous findings and the proposal that DSC2 may be involved in the regulation of the invasive behavior of cells by a mechanism that controls cell­cell attachment and cytoskeleton rearrangement.

Protein-protein interaction network analyses for elucidating the roles of LOXL2-delta72 in esophageal squamous cell carcinoma.

Lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase (LOX) family, is a copper-dependent enzyme that catalyzes oxidative deamination of lysine residues on protein substrates. LOXL2 was found to be overexpressed in esophageal squamous cell carcinoma (ESCC) in our previous research. We later identified a LOXL2 splicing variant LOXL2-delta72 and we overexpressed LOXL2-delta72 and its wild type counterpart in ESCC cells following microarray analyses. First, the differentially expressed genes (DEGs) of LOXL2 and LOXL2-delta72 compared to empty plasmid were applied to generate protein-protein interaction (PPI) sub-networks. Comparison of these two sub-networks showed hundreds of different proteins. To reveal the potential specific roles of LOXL2- delta72 compared to its wild type, the DEGs of LOXL2-delta72 vs LOXL2 were also applied to construct a PPI sub-network which was annotated by Gene Ontology. The functional annotation map indicated the third PPI sub-network involved hundreds of GO terms, such as "cell cycle arrest", "G1/S transition of mitotic cell cycle", "interphase", "cell-matrix adhesion" and "cell-substrate adhesion", as well as significant "immunity" related terms, such as "innate immune response", "regulation of defense response" and "Toll signaling pathway". These results provide important clues for experimental identification of the specific biological roles and molecular mechanisms of LOXL2-delta72. This study also provided a work flow to test the different roles of a splicing variant with high-throughput data.

Functional analysis of the mRNA profile of neutrophil gelatinase­associated lipocalin overexpression in esophageal squamous cell carcinoma using multiple bioinformatic tools.

Neutrophil gelatinase-associated lipocalin (NGAL) is a member of the lipocalin superfamily; dysregulated expression of NGAL has been observed in several benign and malignant diseases. In the present study, differentially expressed genes, in comparison with those of control cells, in the mRNA expression profile of EC109 esophageal squamous cell carcinoma (ESCC) cells following NGAL overexpression were analyzed by multiple bioinformatic tools for a comprehensive understanding. A total of 29 gene ontology (GO) terms associated with immune function, chromatin structure and gene transcription were identified among the differentially expressed genes (DEGs) in NGAL overexpressing cells. In addition to the detected GO categories, the results from the functional annotation chart revealed that the differentially expressed genes were also associated with 101 functional annotation category terms. A total of 59 subpathways associated locally with the differentially expressed genes were identified by subpathway analysis, a markedly greater total that detected by traditional pathway enrichment analysis only. Promoter analysis indicated that the potential transcription factors Snail, deltaEF1, Mycn, Arnt, MNB1A, PBF, E74A, Ubx, SPI1 and GATA2 were unique to the downregulated DEG promoters, while bZIP910, ZNF42 and SOX9 were unique for the upregulated DEG promoters. In conclusion, the understanding of the role of NGAL overexpression in ESCC has been improved through the present bioinformatic analysis.

Comprehensive bioinformation analysis of the mRNA profile of fascin knockdown in esophageal squamous cell carcinoma.

BACKGROUND: Fascin, an actin-bundling protein forming actin bundles including filopodia and stress fibers, is overexpressed in multiple human epithelial cancers including esophageal squamous cell carcinoma (ESCC). Previously we conducted a microarray experiment to analyze fascin knockdown by RNAi in ESCC. METHOD: In this study, the differentially expressed genes from mRNA expression profilomg of fascin knockdown were analyzed by multiple bioinformatics methods for a comprehensive understanding of the role of fascin. RESULTS: Gene Ontology enrichment found terms associated with cytoskeleton organization, including cell adhesion, actin filament binding and actin cytoskeleton, which might be related to fascin function. Except GO categories, the differentially expressed genes were annotated by 45 functional categories from the Functional Annotation Chart of DAVID. Subpathway analysis showed thirty-nine pathways were disturbed by the differentially expressed genes, providing more detailed information than traditional pathway enrichment analysis. Two subpathways derivated from regulation of the actin cytoskeleton were shown. Promoter analysis results indicated distinguishing sequence patterns and transcription factors in response to the co-expression of downregulated or upregulated differentially expressed genes. MNB1A, c-ETS, GATA2 and Prrx2 potentially regulate the transcription of the downregulated gene set, while Arnt-Ahr, ZNF42, Ubx and TCF11-MafG might co-regulate the upregulated genes. CONCLUSIONS: This multiple bioinformatic analysis helps provide a comprehensive understanding of the roles of fascin after its knockdown in ESCC.

Regulation of neutrophil gelatinase-associated lipocalin expression by C/EBPß in lung carcinoma cells.

Neutrophil gelatinase-associated lipocalin (NGAL), a member of the lipocalin family, has been found to be overexpressed in a variety of tumors, including lung adenocarcinomas. However, the mechanism by which NGAL expression is regulated in lung carcinoma needs further evaluation. In this study, immunohistochemistry was employed to analyze the expression of NGAL in lung carcinoma tissue samples, including lung squamous carcinomas, adenocarcinomas, adenosquamous carcinomas and bronchial alveolar cell carcinomas. The results showed that NGAL was expressed in 82.61% (19/23) of the samples. RT-PCR and immunofluorescent staining showed that NGAL was localized to the cytoplasm in lung carcinoma cell lines. To explore the transcriptional regulation mechanism of NGAL basal expression in lung carcinoma, a 1515-bp fragment (-1431 to +84) of the NGAL promoter region was cloned and a series of deletion and mutation constructs were generated. These constructs were analyzed using the luciferase reporter assay. The results indicated that the cis-acting elements important for the basal activity of NGAL transcription were likely located between -152 and -141. Further analysis using site-directed mutagenesis and the luciferase reporter assay suggested that the C/EBP binding sites were responsible for the activity of the NGAL promoter. Finally, the binding ability and specificity of the transcription factors were determined by electrophoretic mobility-shift assay (EMSA). The results showed that C/EBPß was able to bind to the -152 and -141 segments. Taken together, these findings suggest that NGAL is expressed in lung carcinomas and that NGAL expression is mediated by the binding of C/EBPß to the -152 and -141 segment of the NGAL promoter.

Expression of NGAL and NGALR in human embryonic, fetal and normal adult tissues.

This study investigated the distribution of neutrophil gelatinase-associated lipocalin (NGAL) and neutrophil gelatinase-associated lipocalin receptor (NGALR) in human embryonic, fetal and normal adult tissues. Tissue microarray technology was used to perform immunohistochemical examination on human embryos, fetuses at 4-22 weeks of gestation and adult tissue specimens. Results demonstrated that during the development of the nervous system, NGALR was prevalent in the neural tube and cerebrum, and NGAL was only detected in the stellate cells of the cerebrum and the Purkinje cells of the cerebellum. NGAL and NGALR were expressed in the lung alveolar epithelium and in the gastrointestinal tract in embryos, but were almost undetectable in later developmental stages. In the embryonic adrenal glands, the two proteins demonstrated moderate positivity in the cortex and the medulla. In adults, NGAL was particularly present in the cells of the inner and outer layers of the cortex and was absent in the medulla, while NGALR exhibited strong positivity in the cortex and the medulla. Evident expression of NGAL and NGALR was observed throughout development in the neutrophil-rich sites, the renal tubule epithelium and certain gland epithelia of the gastrointestinal tract, but was undetectable in the heart, liver and thyroid gland. Taken together, these results demonstrated that the expression of NGAL and NGALR was time-specific and highly tissue­specific. Correlations between their expression in embryogenesis and carcinogenesis should be examined.