iTRAQ-Based Quantitative Proteomics Approach Identifies Novel Diagnostic Biomarkers That Were Essential for Glutamine Metabolism and Redox Homeostasis for Gastric Cancer.

PURPOSE: To screen the novel biomarkers for gastric cancer and to determine the values of glutaminase 1 (GLS1) and gamma-glutamylcyclotransferase (GGCT) for detecting gastric cancer. EXPERIMENTAL DESIGN: A discovery group of four paired gastric cancer tissue samples are labeled with Isobaric tag for relative and absolute quantitation agents and identified with LC-ESI-MS/MS. A validation group of 168 gastric cancer samples and 30 healthy controls are used to validate the expression of GLS1 and GGCT. RESULTS: Four hundred and thirty-one proteins are found differentially expressed in gastric cancer tissues. Of these proteins, GLS1 and GGCT are found overexpressed in gastric cancer patients, with sensitivity of 75.6% (95% CI: 69-82.2%) and specificity of 81% (95% CI: 75-87%) for GLS1, and with sensitivity of 63.1% (95% CI: 55.7-71.5%) and specificity of 60.7% (95% CI: 53.3-68.2%) for GGCT. The co-expression of GLS1 and GGCT in gastric cancer tissues has sensitivity of 78.1% (95% CI: 70.1-86.1%) and specificity of 86.5% (95% CI: 79.5-93.4%). Moreover, both GLS1 and GGCT present higher expression of 82.6% (95% CI: 68.5-99.4%) and 73.9% (95% CI: 54.5-93.3%) in lymph node metastasis specimen than those in non-lymph node metastasis specimen. The areas under ROC curves are up to 0.734 for the co-expression of GLS1 and GGCT in gastric cancer. The co-expression of GLS1 and GGCT is strongly associated with histological grade, lymph node metastasis, and TNM stage Ⅲ/Ⅳ. CONCLUSIONS AND CLINICAL RELEVANCE: The present study provides the quantitative proteomic analysis of gastric cancer tissues to identify prognostic biomarkers of gastric cancer. The co-expression level of GLS1 and GGCT is of great clinical value to serve as diagnostic and therapeutic biomarkers for early gastric cancer.

γ-Glutamyl cyclotransferase contributes to tumor progression in high grade serous ovarian cancer by regulating epithelial-mesenchymal transition via activating PI3K/AKT/mTOR pathway.

OBJECTIVE: High grade serous ovarian cancer (HGSC) remains one of the most lethal malignancies in females. We previously reported that γ-glutamyl cyclotransferase (GGCT) was significantly upregulated in serous ovarian cancer. The current study was aimed to explore the function and underlying mechanism of GGCT in HGSC. METHODS: GGCT expression was assessed by immunohistochemistry in 128 HGSC patients. Stable cell lines with GGCT gene overexpression or knockdown were established to investigate the function of GGCT in HGSC in vitro and in vivo. RESULTS: GGCT is highly upregulated in HGSC tissues and associated with FIGO stage, lymph node metastasis and ascitic fluid volume. High expression of GGCT is associated with poor survival in HGSC patients. The Harrell's c-indexes of the prognostic models for overall survival and progression-free survival prediction were 0.758 and 0.726, respectively. GGCT knockdown suppresses proliferation, clone formation, migration, and invasion of tumor cells in vitro while forced GGCT overexpression presents opposite results. Furthermore, GGCT silencing inhibits tumor growth and spread in vivo. Epithelial-mesenchymal transition (EMT) and PI3K/AKT/mTOR signaling pathway are suppressed in GGCT silenced cells and enhanced in GGCT overexpressed cells. Inactivation of PI3K/AKT/mTOR signaling pathway in GGCT overexpressed cells induces EMT inhibition. CONCLUSIONS: Our data reveals an important role of GGCT in regulating EMT and progression of HGSC, providing a valuable prognostic marker and potential target for treatment of HGSC patients.

Gamma-Glutamylcyclotransferase: A Novel Target Molecule for Cancer Diagnosis and Treatment.

Gamma-glutamylcyclotransferase (GGCT) is one of the major enzymes involved in glutathione metabolism. However, its gene locus was unknown for many years. Recently, the gene for GGCT was found to be identical to C7orf24, which is registered as a hypothetical protein. Orthologs have been found in bacteria, plants, and nematodes as well as higher organisms, and the GGCT gene is highly preserved among a wide range of species. GGCT (C7orf24) was also reported as an upregulated protein in various cancers. Although the function of GGCT in cancer cells has not been determined, the following important activities have been reported: (1) high expression in various cancer tissues and cancer cell lines, (2) low expression in normal tissues, (3) inhibition of cancer cell proliferation via anti-GGCT RNAi, (4) inhibition of cancer cell invasion and migration via anti-GGCT RNAi, (5) an epigenetic transcriptional regulation in cancer cells, and (6) an antitumor effect in cancer-bearing xenograft mice. Therefore, GGCT is promising as a diagnostic marker and a therapeutic target for various cancers. This review summarizes these interesting findings.

Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element.

Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the -267 ATF/cAMP response element (CRE) site and a novel -248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein ß binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease.

Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis.

Chemoresistance is a poor prognostic factor in breast cancer and is a major obstacle to the successful treatment of patients receiving chemotherapy. However, the precise mechanism of resistance remains unclear. In this study, a pair of breast cancer cell lines, MCF-7 and its adriamycin-resistant counterpart MCF-7/ADR was used to examine resistance-dependent cellular responses and to identify potential therapeutic targets. We applied nanoflow liquid chromatography (nLC) and tandem mass tags (TmT) quantitative mass spectrometry to distinguish the differentially expressed proteins (DEPs) between the two cell lines. Bioinformatics analyses were used to identify functionally active proteins and networks. 80 DEPs were identified with either up- or down-regulation. Basing on the human protein-protein interactions (PPI), we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions. Six different signaling pathways and most of the DEPs strongly linked to chemoresistance, invasion, metastasis development, proliferation, and apoptosis. The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot. The glucose-6-phosphate dehydrogenase (G6PD), gamma-glutamyl cyclotransferase (GGCT), isocitrate dehydrogenase 1 (NADP+,soluble)(IDH1), isocitrate dehydrogenase 2 (NADP+,mitochondrial) (IDH2) and glutathione S-transferase pi 1(GSTP1), five of the critical components of GSH pathway, contribute to chemoresistance.