The GGCT and REST positive feedback loop promotes tumor growth in Glioma.

BACKGROUND: γ-Glutamylcyclotransferase (GGCT), an enzyme crucial in glutathione metabolism, has emerged as a participant in tumorigenesis. The present study is designed to elucidate the biological role and molecular mechanisms underlying GGCT in glioma. METHODS: Gene Expression Profiling Interactive Analysis (GEPIA), Chinese Glioma Genome Atlas (CGGA), and PrognoScan online databases were utilized to examine the expressions and clinical prognosis of GGCT and REST in glioma. Cell Counting Kit-8 (CCK-8), Transwell, Wound healing, and Flow cytometric assays, and RNA-sequencing analysis were employed to uncover the molecular role of GGCT and REST. Prediction of Differentially expressed microRNA (DE-miRNAs) and miRNAs targeting GGCT 3' Untranslated Region (UTR) was performed using miRanda online datasets. Finally, Real time-quantitative Polymerase Chain Reaction (RT-qPCR), western blot and dual luciferase reporter gene activity analysis were employed to confirm a positive feedback loop involving GGCT/REST/miR-34a-5p in glioma cells. RESULTS: High expression of GGCT was correlated with poor prognosis in glioma. GGCT silencing demonstrated inhibitory effects on the proliferation, migration, and induction of apoptosis in T98G and U251 cells. Mechanistically, GGCT downregulated REST expression and modulated cancer-associated pathways in glioma cells. High expression of REST was associated with poor prognosis in glioma. In vitro and in vivo experiments showed that REST overexpression restored the repression of proliferation, invasion, migration, and xenograft tumor formation induced by GGCT knockdown. Furthermore, the study uncovered that REST inhibited miR-34a-5p mRNA expression, and miR-34a-5p suppressed GGCT expression by targeting its 3'UTR, forming a positive regulatory loop in glioma. Notably, the inhibitor of miR-34a-5p restored the role of REST silencing in decreasing GGCT expression in glioma cells. CONCLUSIONS: GGCT/REST/miR-34a-5p axis holds promising potential as a therapeutic target, offering a potential breakthrough in the treatment of glioma.

Desert Hedgehog Down-regulation Mediates Inhibition of Proliferation by γ-Glutamylcyclotransferase Knockdown in Murine Glioblastoma Stem Cells.

BACKGROUND/AIM: Glioblastoma is the most frequent type of adult-onset malignant brain tumor and has a very poor prognosis. Glioblastoma stem cells have been shown to be one of the mechanisms by which glioblastoma acquires therapy resistance. Therefore, there is a need to establish novel therapeutic strategies useful for inhibiting this cell population. γ-Glutamylcyclotransferase (GGCT) is an enzyme involved in the synthesis and metabolism of glutathione, which is highly expressed in a wide range of cancer types, including glioblastoma, and inhibition of its expression has been reported to have antitumor effects on various cancer types. The aim of this study was to clarify the function of GGCT in glioblastoma stem cells. MATERIALS AND METHODS: We searched for pathways affected by GGCT overexpression in mouse embryonic fibroblasts NIH-3T3 by comprehensive gene expression analysis. Knockdown of GGCT and overexpression of desert hedgehog (DHH), a representative ligand of the pathway, were performed in glioblastoma stem cells derived from a mouse glioblastoma model. RESULTS: GGCT overexpression activated the hedgehog pathway. Knockdown of GGCT inhibited proliferation of glioblastoma stem cells and reduced expression of DHH and the downstream target GLI family zinc finger 1 (GLI1). DHH overexpression significantly restored the growth-suppressive effect of GGCT knockdown. CONCLUSION: High GGCT expression is important for expression of DHH and activation of the hedgehog pathway, which is required to maintain glioblastoma stem cell proliferation. Therefore, inhibition of GGCT function may be useful in suppressing stemness of glioblastoma stem cells accompanied by activation of the hedgehog pathway.

Myc upregulates Ggct, γ-glutamylcyclotransferase to promote development of p53-deficient osteosarcoma.

Osteosarcoma (OS) in humans is characterized by alterations in the TP53 gene. In mice, loss of p53 triggers OS development, for which c-Myc (Myc) oncogenicity is indispensable. However, little is known about which genes are targeted by Myc to promote tumorigenesis. Here, we examined the role of γ-glutamylcyclotransferase (Ggct) which is a component enzyme of the γ-glutamyl cycle essential for glutathione homeostasis, in human and mouse OS development. We found that GGCT is a poor prognostic factor for human OS, and that deletion of Ggct suppresses p53-deficient osteosarcomagenesis in mice. Myc upregulates Ggct directly by binding to the Ggct promoter, and deletion of a Myc binding site therein by genome editing attenuated the tumorigenic potential of p53-deficient OS cells. Taken together, these results show a rationale that GGCT is widely upregulated in cancer cells and solidify its suitability as a target for anticancer drugs.

Identified γ-glutamyl cyclotransferase (GGCT) as a novel regulator in the progression and immunotherapy of pancreatic ductal adenocarcinoma through multi-omics analysis and experiments.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is renowned for its formidable and lethal nature, earning it a notorious reputation among malignant tumors. Due to its challenging early diagnosis, high malignancy, and resistance to chemotherapy drugs, the treatment of pancreatic cancer has long been exceedingly difficult in the realm of oncology. γ-Glutamyl cyclotransferase (GGCT), a vital enzyme in glutathione metabolism, has been implicated in the proliferation and progression of several tumor types, while the biological function of GGCT in pancreatic ductal adenocarcinoma remains unknown. METHODS: The expression profile of GGCT was validated through western blotting, immunohistochemistry, and RT-qPCR in both pancreatic cancer tissue samples and cell lines. Functional enrichment analyses including GSVA, ssGSEA, GO, and KEGG were conducted to explore the biological role of GGCT. Additionally, CCK8, Edu, colony formation, migration, and invasion assays were employed to evaluate the impact of GGCT on the proliferation and migration abilities of pancreatic cancer cells. Furthermore, the LASSO machine learning algorithm was utilized to develop a prognostic model associated with GGCT. RESULTS: Our study revealed heightened expression of GGCT in pancreatic cancer tissues and cells, suggesting an association with poorer patient prognosis. Additionally, we explored the immunomodulatory effects of GGCT in both pan-cancer and pancreatic cancer contexts, found that GGCT may be associated with immunosuppressive regulation in various types of tumors. Specifically, in patients with high expression of GGCT in pancreatic cancer, there is a reduction in the infiltration of various immune cells, leading to poorer responsiveness to immunotherapy and worse survival rates. In vivo and in vitro assays indicate that downregulation of GGCT markedly suppresses the proliferation and metastasis of pancreatic cancer cells. Moreover, this inhibitory effect appears to be linked to the regulation of GGCT on c-Myc. A prognostic model was constructed based on genes derived from GGCT, demonstrating robust predictive ability for favorable survival prognosis and response to immunotherapy.

Genome-wide identification and analysis of the GGCT gene family in wheat.

BACKGROUND: γ-glutamylcyclotransferase (GGCT), an enzyme to maintain glutathione homeostasis, plays a vital role in the response to plant growth and development as well as the adaptation to various stresses. Although the GGCT gene family analysis has been conducted in Arabidopsis and rice, the family genes have not yet been well identified and analyzed at the genome-wide level in wheat (Triticum aestivum L.). RESULTS: In the present study, 20 TaGGCT genes were identified in the wheat genome and widely distributed on chromosomes 2A, 2B, 2D, 3A, 4A, 5A, 5B, 5D, 6A, 6B, 6D, 7A, 7B, and 7D. Phylogenetic and structural analyses showed that these TaGGCT genes could be classified into three subfamilies: ChaC, GGGACT, and GGCT-PS. They exhibited similar motif compositions and distribution patterns in the same subgroup. Gene duplication analysis suggested that the expansion of TaGGCT family genes was facilitated by segmental duplications and tandem repeats in the wheat evolutionary events. Identification of diverse cis-acting response elements in TaGGCT promoters indicated their potential fundamental roles in response to plant development and abiotic stresses. The analysis of transcriptome data combined with RT-qPCR results revealed that the TaGGCTs genes exhibited ubiquitous expression across plant organs, with highly expressed in roots, stems, and developing grains. Most TaGGCT genes were up-regulated after 6 h under 20% PEG6000 and ABA treatments. Association analysis revealed that two haplotypes of TaGGCT20 gene displayed significantly different Thousand-kernel weight (TKW), Kernel length (KL), and Kernel width (KW) in wheat. The geographical and annual distribution of the two haplotypes of TaGGCT20 gene further revealed that the frequency of the favorable haplotype TaGGCT20-Hap-I was positively selected in the historical breeding process of wheat. CONCLUSION: This study investigated the genome-wide identification, structure, evolution, and expression analysis of TaGGCT genes in wheat. The motifs of TaGGCTs were highly conserved throughout the evolutionary history of wheat. Most TaGGCT genes were highly expressed in roots, stems, and developing grains, and involved in the response to drought stresses. Two haplotypes were developed in the TaGGCT20 gene, where TaGGCT20-Hap-I, as a favorable haplotype, was significantly associated with higher TKW, KL, and KW in wheat, suggesting that the haplotype is used as a function marker for the selection in grain yield in wheat breeding.

Interaction of MRPL9 and GGCT Promotes Cell Proliferation and Migration by Activating the MAPK/ERK Pathway in Papillary Thyroid Cancer.

Thyroid cancer remains the most common endocrine malignancy worldwide, and its incidence has steadily increased over the past four years. Papillary Thyroid Cancer (PTC) is the most common differentiated thyroid cancer, accounting for 80-85% of all thyroid cancers. Mitochondrial proteins (MRPs) are an important part of the structural and functional integrity of the mitochondrial ribosomal complex. It has been reported that MRPL9 is highly expressed in liver cancer and promotes cell proliferation and migration, but it has not been reported in PTC. In the present study we found that MRPL9 was highly expressed in PTC tissues and cell lines, and lentivirus-mediated overexpression of MRPL9 promoted the proliferation and migration ability of PTC cells, whereas knockdown of MRPL9 had the opposite effect. The interaction between MRPL9 and GGCT (γ-glutamylcyclotransferase) was found by immunofluorescence and co-immunoprecipitation experiments (Co-IP). In addition, GGCT is highly expressed in PTC tissues and cell lines, and knockdown of GGCT/MRPL9 in vivo inhibited the growth of subcutaneous xenografts in nude mice and inhibited the formation of lung metastases. Mechanistically, we found that knockdown of GGCT/MRPL9 inhibited the MAPK/ERK signaling pathway. In conclusion, our study found that the interaction of GGCT and MRPL9 modulates the MAPK/ERK pathway, affecting the proliferation and migration of PTC cells. Therefore, GGCT/MRPL9 may serve as a potential biomarker for PTC monitoring and PTC treatment.

MiR-205-5p/GGCT Attenuates Growth and Metastasis of Papillary Thyroid Cancer by Regulating CD44.

Papillary thyroid cancer (PTC) remains the most common endocrine malignancy, despite marked achieves in recent decades, and the mechanisms underlying the pathogenesis and progression for PTC are incompletely elucidated. Accumulating evidence show that γ-glutamylcyclotransferase (GGCT), an enzyme participating in glutathione homeostasis and is elevated in multiple types of tumors, represents an attractive therapeutic target. Using bioinformatics, immunohistochemistry, qRT-PCR, and Western blot assays, we found that GGCT expression was upregulated in PTC and correlated with more aggressive clinicopathological characteristics and worse prognosis. GGCT knockdown inhibited the growth and metastasis ability of PTC cells both in vitro and in vivo and reduced the expression of mesenchymal markers (N-cadherin, CD44, MMP2, and MMP9) while increasing epithelial marker (E-cadherin) in PTC cells. We confirmed binding of microRNA-205-5p (miR-205-5p) on the 3'-UTR regions of GGCT by dual-luciferase reporter assay and RNA-RNA pull-down assay. Delivery of miR-205-5p reversed the pro-malignant capacity of GGCT both in vitro and in vivo. Lastly, we found that GGCT interacted with and stabilized CD44 in PTC cells by co-immunoprecipitation and immunohistochemistry assays. Our findings illustrate a novel signaling pathway, miR-205-5p/GGCT/CD44, that involves in the carcinogenesis and progression of PTC. Development of miR-205-mimics or GGCT inhibitors as potential therapeutics for PTC may have remarkable applications.

Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy.

γ-Glutamylcyclotransferase (GGCT), which is one of the major enzymes involved in glutathione metabolism, is upregulated in a wide range of cancers-glioma, breast, lung, esophageal, gastric, colorectal, urinary bladder, prostate, cervical, ovarian cancers and osteosarcoma-and promotes cancer progression; its depletion leads to the suppression of proliferation, invasion, and migration of cancer cells. It has been demonstrated that the suppression or inhibition of GGCT has an antitumor effect in cancer-bearing xenograft mice. Based on these observations, GGCT is now recognized as a promising therapeutic target in various cancers. This review summarizes recent advances on the mechanisms of the antitumor activity of GGCT inhibition.

Genetic Control of Rod Bipolar Cell Number in the Mouse Retina.

Genetic variants modulate the numbers of various retinal cell types in mice. For instance, there is minimal variation in the number of rod bipolar cells (RBCs) in two inbred strains of mice (A/J and C57BL/6J), yet their F1 offspring contain significantly more RBCs than either parental strain. To investigate the genetic source of this variation, we mapped the variation in the number of RBCs across 24 genetically distinct recombinant inbred (RI) strains (the AXB/BXA strain-set), seeking to identify quantitative trait loci (QTL). We then sought to identify candidate genes and potential casual variants at those genomic loci. Variation in RBC number mapped to three genomic loci, each modulating cell number in excess of one-third of the range observed across the RI strains. At each of these loci, we identified candidate genes containing variants that might alter gene function or expression. The latter genes were also analyzed using a transcriptome database, revealing a subset for which expression correlated with variation in RBC number. Using an electroporation strategy, we demonstrate that early postnatal expression of one of them, Ggct (gamma-glutamyl cyclotransferase), modulates bipolar cell number. We identify candidate regulatory variants for this gene, finding a large structural variant (SV) in the putative promoter that reduces expression using a luciferase assay. This SV reducing Ggct expression in vitro is likely the causal variant within the gene associated with the variation in Ggct expression in vivo, implicating it as a quantitative trait variant (QTV) participating in the control of RBC number.

Knockdown of GGCT inhibits cell proliferation and induces late apoptosis in human gastric cancer.

BACKGROUND: Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown. METHODS: The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown. RESULTS: We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What's more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer. CONCLUSION: This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.

Gamma-glutamylcyclotransferase promotes the growth of human glioma cells by activating Notch-Akt signaling.

Glioma as an aggressive type tumor is rapidly growing and has become one of the leading cause of cancer-related death worldwide. γ-Glutamylcyclotransferase (GGCT) has been shown as a diagnostic marker in various cancers. To reveal whether there is a correlation between GGCT and human glioma, GGCT expression in human glioma tissues and cell lines was first determined. We found that GGCT expression was up-regulated in human glioma tissues and cell lines. Further, we demonstrate that GGCT knockdown inhibits glioma cell T98G and U251 proliferation and colony formation, whereas GGCT overexpression leads to oppose effects. GGCT overexpression promotes the expression of Notch receptors and activates Akt signaling in glioma cells, and Notch-Akt signaling is activated in glioma tissues with high expression of GGCT. Finally, we show that inhibition of Notch-Akt signaling with Notch inhibitor MK-0752 blocks the effects of GGCT on glioma proliferation and colony formation. In conclusion, GGCT plays a critical role in glioma cell proliferation and may be a potential cancer therapeutic target.

γ-Glutamylcyclotransferase as a novel immunohistochemical biomarker for the malignancy of esophageal squamous tumors.

Recently, overexpression of γ-glutamylcyclotransferase (GGCT) has been reported in various cancer tissues suggesting that it has significant potential as a diagnostic marker. The aim of this study was to examine the suitability of GGCT for the detection of high-risk lesions at an early stage of esophageal squamous cell carcinoma (ESCC). A total of 200 lesions, including 120 invasive ESCC, 80 esophageal squamous intraepithelial neoplasia consisting of 40 low-grade intraepithelial neoplasia (LGIEN) and 40 high-grade intraepithelial neoplasia (HGIEN), as well as 20 confounding lesions, were examined by immunohistochemical (IHC) staining for GGCT. IHC staining for Ki-67 and p53 was also performed in esophageal squamous intraepithelial neoplasia to compare the diagnostic power of GGCT. Increased expression of GGCT was common in invasive ESCC and HGIEN (87.5% and 85.0%, respectively), but was much less frequently observed in LGIEN (17.5%). GGCT expression significantly correlated with the presence of lymph node metastasis and the degree of differentiation. In the differential diagnosis of LGIEN and HGIEN, GGCT possessed both high sensitivity and high specificity, while Ki-67 and p53 only possessed either high sensitivity or high specificity. Additionally, GGCT expression was higher in 7 out of 8 ESCC cell lines (KYSE series) than in a normal esophageal squamous cell line (Het-1A). The expression levels strongly correlated with enzymatic activity (r=.92; P<.001). These results indicate that overexpressed GGCT retains its enzymatic activity and can become a valuable biomarker for the diagnosis of HGIEN that is likely to progress to subepithelial invasion.