GBP2 Expression Research Articles

Weighted gene co-expression network analysis identified GBP2 connected to PPARα activity and liver cancer

Liver cancer is the fourth leading cause of cancer-related deaths with a steadily increasing rate worldwide, as a well-known hallmark of liver cancer, metabolic alterations are related to liposomal changes, a common characteristic of primary liver cancers based on recent lipidomics studies. Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor with important lipid homeostasis function, therefore we aimed to understand the molecular mechanisms and pathways that activate PPARα after using PPAR-α agonist WY-14643 and identify candidate biomarkers related to PPARα activity and evaluate their effects in liver cancer. The data from differently expressed genes (DEGs) between liver cancer tissue from obese subjects alone and liver tissue after treatment were evaluated by DESeq2 and module genes were analyzed using weighted gene co-expression network analysis (WGCNA). Final candidate genes were identified by intersecting genes among highly ranked DEGs and the brown module, which demonstrated a significant negative correlation with drug treatments. We conducted a protein–protein interaction network, and KEGG enrichment analysis, and core hub genes (CD40, CXCL9, CXCL10, TNFSF14, GBP2, GBP3, APOL3, CLDN1) were identified using the cyto-hubba plugin, among them we focused on GBP2 that plays key roles in oncogenesis and evaluate its expressional with clinical outcomes. In conclusion, the WGCNA-based co-expression network identified GBP2 as one of the hub genes with a negative relation with PPARα agonist treatments. higher expression of GBP2 was closely associated with HCC progression. Therefore, GBP2 might be a potential candidate for the study of PPARα activity in HCC.

Tryptophanyl-Transfer RNA Synthetase Is Involved in a Negative Feedback Loop Mitigating Interferon-γ-Induced Gene Expression.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes responsible for linking a transfer RNA (tRNA) with its cognate amino acid present in all the kingdoms of life. Besides their aminoacyl-tRNA synthetase activity, it was described that many of these enzymes can carry out non-canonical functions. They were shown to be involved in important biological processes such as metabolism, immunity, development, angiogenesis and tumorigenesis. In the present work, we provide evidence that tryptophanyl-tRNA synthetase might be involved in a negative feedback loop mitigating the expression of certain interferon-γ-induced genes. Mining the available TCGA and Gtex data, we found that WARS was highly expressed in cutaneous melanoma (SKCM) compared to other cancers and is of good prognosis for this particular cancer type. WARS expression correlates with genes involved in antigen processing and presentation but also transcription factors involved in IFN-γ signaling such as STAT1. In addition, WARS was found in complex with STAT1 in A375 cells treated with IFN-γ. Finally, we showed that knocking down WARS expression during IFN-γ stimulation further increases the expression of GBP2, APOL1, ISG15, HLA-A and IDO1.

Dnmt1/Tet2-mediated changes in Cmip methylation regulate the development of nonalcoholic fatty liver disease by controlling the Gbp2-Pparγ-CD36 axis

Dynamic alteration of DNA methylation leads to various human diseases, including nonalcoholic fatty liver disease (NAFLD). Although C-Maf-inducing protein (Cmip) has been reported to be associated with NAFLD, its exact underlying mechanism remains unclear. Here, we aimed to elucidate this mechanism in NAFLD in vitro and in vivo. We first identified alterations in the methylation status of the Cmip intron 1 region in mouse liver tissues with high-fat high-sucrose diet-induced NAFLD. Knockdown of DNA methyltransferase (Dnmt) 1 significantly increased Cmip expression. Chromatin immunoprecipitation assays of AML12 cells treated with oleic and palmitic acid (OPA) revealed that Dnmt1 was dissociated and that methylation of H3K27me3 was significantly decreased in the Cmip intron 1 region. Conversely, the knockdown of Tet methylcytosine dioxygenase 2 (Tet2) decreased Cmip expression. Following OPA treatment, the CCCTC-binding factor (Ctcf) was recruited, and H3K4me3 was significantly hypermethylated. Intravenous Cmip siRNA injection ameliorated NAFLD pathogenic features in ob/ob mice. Additionally, Pparγ and Cd36 expression levels were dramatically decreased in the livers of ob/ob mice administered siCmip, and RNA sequencing revealed that Gbp2 was involved. Gbp2 knockdown also induced a decrease in Pparγ and Cd36 expression, resulting in the abrogation of fatty acid uptake into cells. Our data demonstrate that Cmip and Gbp2 expression levels are enhanced in human liver tissues bearing NAFLD features. We also show that Dnmt1–Trt2/Ctcf-mediated reversible modulation of Cmip methylation regulates the Gbp2–Pparγ–Cd36 signaling pathway, indicating the potential of Cmip as a novel therapeutic target for NAFLD.

A five-protein prognostic signature with GBP2 functioning in immune cell infiltration of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is of poor clinical outcomes, and currently lacks reliable prognostic biomarkers. By analyzing the datasets of the Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), we established a five-protein prognostic signature containing GBP2, HLA-DRA, ISG15, ISG20 and ITGAX. Our data indicate that this signature was closely correlated with advanced stage, higher pathological grade, and unfavorable survivals in patients with ccRCC. We further functionally characterized GBP2. Overexpression of GBP2 enhanced the phosphorylation of STAT2 and STAT3 to trigger JAK-STAT signaling and promote cell migration and invasion in ccRCC. Treatment of Ruxolitinib, a specific inhibitor of JAK/STAT, attenuated the GBP2-mediated phenotypes. Patients with high GBP2 expression were accompanied with more infiltration of immune cells positively stained with CD3, CD8, CD68, and immune checkpoint markers PD-1 and CTLA4, which was validated by Opal multiplex immunohistochemistry in ccRCC tissues. More CD8 + T cells and CD68 + macrophages were observed in patients expressing high GBP2. Taken together, a five-protein prognostic signature was constructed in our study. GBP2 has an oncogenic role via modulating JAK-STAT signaling and tumor immune infiltration, and thus may serve as a potential therapeutic target in ccRCC.

Single-cell RNA-seq reveals interferon-induced guanylate-binding proteins are linked with sarcopenia.

Sarcopenia is defined as an age-related progressive loss of muscle mass and/or strength. Although different factors can contribute to this disease, the underlying mechanisms remain unclear. We assessed transcriptional heterogeneity in skeletal muscles from sarcopenic and control mice at single-cell resolution. A mouse model was established to study sarcopenic skeletal muscles. Single-cell RNA-seq was performed on tibialis anterior (TA) muscle cells collected from sarcopenic and control mice. A series of bioinformatic analyses were carried out to identify and compare different cell types under different conditions. Immunofluorescence staining and western blotting were used to validate the findings from single-cell experiments. Tube formation assays were conducted to further evaluate the effects of Gbp2 on endothelial cells during angiogenesis. A murine sarcopenia model was successfully established using a senescence-accelerated mouse strain (SAMP6, n=5). Sarcopenia phenotype was induced by administration of dexamethasone (20mg/kg) and reduced physical activity. Senescence-resistant mice strain (SAMR1) and SAMP6 strain with similar activity but injected with PBS were recruited as two control groups. As signs of sarcopenia, body weight, muscle cell counts and cross-sectional fibre area were all significantly decreased in sarcopenic mice (P value=0.004, 0.03 and 0.035, respectively). After quality control, 13 612 TA muscle single-cell transcriptomes were retained for analysis. Fourteen cell clusters were identified from the profiled cells. Among them, two distinct endothelial subtypes were found to be dominant in the sarcopenia group (42.2% cells) and in the two control groups (59.1% and 47.9% cells), respectively. 191 differentially expressed genes were detected between the two endothelial subtypes. Sarcopenia-specific endothelial cell subtype exhibited a dramatic increase in the interferon family genes and the interferon-inducible guanylate-binding protein (GBP) family gene expressions. For example, Igtp and Gbp2 in sarcopenic endothelial cells were 5.4 and 13.3 times higher than those in the control groups, respectively. We further validated our findings in muscle specimens of sarcopenia patients and observed that GBP2 levels were increased in endothelial cells of a subset of patients (11 of 40 patients, 27.5%), and we identified significantly higher CD31 and GBP2 co-localization (P value=0.001128). Finally, we overexpressed Gbp2 in human umbilical vein endothelial cells in vitro. The endothelial cells with elevated Gbp2 expression displayed compromised tube formation. Our single-cell-based results suggested that endothelial cells may play critical roles in sarcopenia development through interferon-GBP signalling pathways, highlighting new therapeutic directions to slow down or even reverse age-related sarcopenia.

The Large GTPase, GBP-2, Regulates Rho Family GTPases to Inhibit Migration and Invadosome Formation in Breast Cancer Cells.

Simple SummaryToo many women still die of breast cancer each year. Those breast cancers that kill are those with cells that have migrated away from the primary tumor in the breast and established new tumors at other sites in the body. These tumors are not reached when the original tumor in the breast is removed. This study was designed to determine why some breast cancers move away from their primary tumor and others do not. We have identified a protein that inhibits this movement. Understanding this finding may provide us with ways to inhibit tumor cell movement in patients.Breast cancer is the most common cancer in women. Despite advances in early detection and treatment, it is predicted that over 43,000 women will die of breast cancer in 2021. To lower this number, more information about the molecular players in breast cancer are needed. Guanylate-Binding Protein-2 has been correlated with better prognosis in breast cancer. In this study, we asked if the expression of GBP-2 in breast cancer merely provided a biomarker for improved prognosis or whether it actually contributed to improving outcome. To answer this, the 4T1 model of murine breast cancer was used. 4T1 cells themselves are highly aggressive and highly metastatic, while 67NR cells, isolated from the same tumor, do not leave the primary site. The expression of GBP-2 was examined in the two cell lines and found to be inversely correlated with aggressiveness/metastasis. Proliferation, migration, and invadosome formation were analyzed after altering the expression levels of GBP-2. Our experiments show that GBP-2 does not alter the proliferation of these cells but inhibits migration and invadosome formation downstream of regulation of Rho GTPases. Together these data demonstrate that GBP-2 is responsible for cell autonomous activities that make breast cancer cells less aggressive.

Guanylate-binding proteins induce apoptosis of leukemia cells by regulating MCL-1 and BAK

Interferon-inducible guanylate-binding proteins (GBPs) are well-known for mediating host-defense mechanisms against cellular pathogens. Emerging evidence suggests that GBPs are also implicated in tumorigenesis; however, their underlying molecular mechanism is still unknown. In this study, we identified that GBP1 and GBP2 interact with MCL-1, the key prosurvival member of the BCL-2 family, via its BH3 domain. GBPs induce caspase-dependent apoptosis in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells, where the proapoptotic BCL-2 member, BAK, is an indispensable mediator. In particular, GBP2 completely inhibited the MCL-1-mediated promotion of the survival of CML cells through competitive inhibition, resulting in BAK liberation from MCL-1. Concurrently, GBP2 dramatically upregulates BAK expression via its inhibition of the PI3K/AKT pathway. Moreover, paclitaxel upregulates GBP2 expression, and paclitaxel-induced apoptotic activity was distinctively compromised by knockout of GBP2 in CML cells. Bioinformatics analyses of leukemia databases revealed that transcripts of GBPs were generally downregulated in leukemia patients and that GBPs were favorable prognosis markers. Thus, these findings provide molecular evidence of GBPs as apoptosis-inducing proteins of leukemia cells and suggest that GBPs are attractive targets for the development of chemotherapeutics.

GBP2 as a potential prognostic biomarker in pancreatic adenocarcinoma

BackgroundPancreatic adenocarcinoma (PAAD) is a disease with atypical symptoms, an unfavorable response to therapy, and a poor outcome. Abnormal guanylate-binding proteins (GBPs) play an important role in the host’s defense against viral infection and may be related to carcinogenesis. In this study, we sought to determine the relationship between GBP2 expression and phenotype in patients with PAAD and explored the possible underlying biological mechanism.MethodWe analyzed the expression of GBP2 in PAAD tissues using a multiple gene expression database and a cohort of 42 PAAD patients. We evaluated GBP2’s prognostic value using Kaplan–Meier analysis and the Cox regression model. GO and KEGG enrichment analysis, co-expression analysis, and GSEA were performed to illustrate the possible underlying biological mechanism. CIBERSORT and the relative expression of immune checkpoints were used to estimate the relationship between GBP2 expression and tumor immunology.ResultGBP2 was remarkably overexpressed in PAAD tissue. The overexpression of GBP2 was correlated with an advanced T stage and poor overall survival (OS) and GBP2 expression was an independent risk factor for OS in PAAD patients. Functional analysis demonstrated that positively co-expressed genes of GBP2 were closely associated with pathways in cancer and the NOD-like receptor signaling pathway. Most of the characteristic immune checkpoints, including PDCD1, PDCDL1, CTLA4, CD80, TIGIT, LAG3, IDO2, and VISTA, were significantly expressed in the high-GBP2 expression group compared with the low-GBP2 expression group.ConclusionGBP2 acted as a potential prognostic biomarker and was associated with immune infiltration and the expression of immune checkpoints in PAAD.

Guanylate-binding protein 2 orchestrates innate immune responses against murine norovirus and is antagonized by the viral protein NS7

Noroviruses are the main causative agents of acute viral gastroenteritis, but the host factors that restrict their replication remain poorly identified. Guanylate-binding proteins (GBPs) are interferon (IFN)-inducible GTPases that exert broad antiviral activity and are important mediators of host defenses against viral infections. Here, we show that both IFN-γ stimulation and murine norovirus (MNV) infection induce GBP2 expression in murine macrophages. Results from loss- and gain-of-function assays indicated that GBP2 is important for IFN-γ-dependent anti-MNV activity in murine macrophages. Ectopic expression of MNV receptor (CD300lf) in human HEK293T epithelial cells conferred susceptibility to MNV infection. Importantly, GBP2 potently inhibited MNV in these human epithelial cells. Results from mechanistic dissection experiments revealed that the N-terminal G domain of GBP2 mediates these anti-MNV effects. R48A and K51A substitutions in GBP2, associated with loss of GBP2 GTPase activity, attenuated the anti-MNV effects of GBP2. Finally, we found that nonstructural protein 7 (NS7) of MNV co-localizes with GBP2 and antagonizes the anti-MNV activity of GBP2. These findings reveal that GBP2 is an important mediator of host defenses against murine norovirus.

Guanylate‐binding protein‐2 inhibits colorectal cancer cell growth and increases the sensitivity to paclitaxel of paclitaxel‐resistant colorectal cancer cells by interfering Wnt signaling

Among the GTPase family members, guanylate-binding protein-1 (GBP-1) is the most thoroughly studied member in a plethora of human cancers. GBP-2, on the other hand, remains limitedly studied. We wonder how GBP-2 participates in colorectal carcinoma (CRC) as well as the paclitaxel (PTX)-resistance of CRC. In this study, the authors are determined to dig into the role that GBP-2 plays in the sensitivity of CRC to PTX, therefore, possibly indicating a promising gene therapy target for CRC. Forced expression of GBP-2 gene was done by plasmid transfection. Reverse transcriptase-polymerase chain reaction and immunoblot were conducted to detect the expression of GBP-2 messenger RNA (mRNA) and protein, respectively. Colony foci formation assay, transwell invasion assay, and flow cytofluorometry were done to determine the proliferation, invasion, and apoptosis of PTX-resistant and PTX-sensitive CRC cell lines, respectively. The level of GBP-2 mRNA and protein in PTX-resistant CRC cell lines was significantly lower than in nonresistant cell lines. Forced exogenous expression of GBP-2 in PTX-resistant CRC cell lines resulted in more sensitivity to PTX because of the demonstration of less cell proliferation, invasion, and more apoptosis. Wnt signaling was suppressed when GBP-2 was upregulated by transfection of GBP-2 overexpression plasmids, and Wnt signaling did not affect GBP-2 expression. GBP-2 upregulation could enhance the killing effect of PTX in both PTX-sensitive CRC cells and PTX-resistant CRC cells by suppressing Wnt signaling.

C57BL/6 and 129 inbred mouse strains differ in Gbp2 and Gbp2b expression in response to inflammatory stimuli in vivo

Background: Infections cause the production of inflammatory cytokines such as Interferon gamma (IFNγ). IFNγ in turn prompts the upregulation of a range of host defence proteins including members of the family of guanylate binding proteins (Gbps). In humans and mice alike, GBPs restrict the intracellular replication of invasive microbes and promote inflammation. To study the physiological functions of Gbp family members, the most commonly chosen in vivo models are mice harbouring loss-of-function mutations in either individual Gbp genes or the entire Gbp gene cluster on mouse chromosome 3. Individual Gbp deletion strains differ in their design, as some strains exist on a pure C57BL/6 genetic background, while other strains contain a 129-derived genetic interval encompassing the Gbp gene cluster on an otherwise C57BL/6 genetic background. Methods: To determine whether the presence of 129 alleles of paralogous Gbps could influence the phenotypes of 129-congenic Gbp-deficient strains, we studied the expression of Gbps in both C57BL/6J and 129/Sv mice following in vivo stimulation with adjuvants and after infection with either Toxoplasma gondii or Shigella flexneri. Results: We show that C57BL/6J relative to 129/Sv mice display moderately elevated expression of Gbp2, but more prominently, are also defective for Gbp2b (formerly Gbp1) mRNA induction upon immune priming. Notably, Toxoplasma infections induce robust Gbp2b protein expression in both strains of mice, suggestive of a Toxoplasma-activated mechanism driving Gbp2b protein translation. We further find that the higher expression of Gbp2b mRNA in 129/Sv mice correlates with a gene duplication event at the Gbp2b locus resulting in two copies of the Gbp2b gene on the haploid genome of the 129/Sv strain. Conclusions: Our findings demonstrate functional differences between 129 and C57BL/6 Gbp alleles which need to be considered in the design and interpretation of studies utilizing mouse models, particularly for phenotypes influenced by Gbp2 or Gbp2b expression.

C57BL/6 and 129 inbred mouse strains differ in Gbp2 and Gbp2b expression in response to inflammatory stimuli in vivo.

Background: Infections cause the production of inflammatory cytokines such as Interferon gamma (IFNγ). IFNγ in turn prompts the upregulation of a range of host defence proteins including members of the family of guanylate binding proteins (Gbps). In humans and mice alike, GBPs restrict the intracellular replication of invasive microbes and promote inflammation. To study the physiological functions of Gbp family members, the most commonly chosen in vivo models are mice harbouring loss-of-function mutations in either individual Gbp genes or the entire Gbp gene cluster on mouse chromosome 3. Individual Gbp deletion strains differ in their design, as some strains exist on a pure C57BL/6 genetic background, while other strains contain a 129-derived genetic interval encompassing the Gbp gene cluster on an otherwise C57BL/6 genetic background. Methods: To determine whether the presence of 129 alleles of paralogous Gbps could influence the phenotypes of 129-congenic Gbp-deficient strains, we studied the expression of Gbps in both C57BL/6J and 129/Sv mice following in vivo stimulation with adjuvants and after infection with either Toxoplasma gondii or Shigella flexneri. Results: We show that C57BL/6J relative to 129/Sv mice display moderately elevated expression of Gbp2, but more prominently, are also defective for Gbp2b (formerly Gbp1) mRNA induction upon immune priming. Notably, Toxoplasma infections induce robust Gbp2b protein expression in both strains of mice, suggestive of a Toxoplasma-activated mechanism driving Gbp2b protein translation. We further find that the higher expression of Gbp2b mRNA in 129/Sv mice correlates with a gene duplication event at the Gbp2b locus resulting in two copies of the Gbp2b gene on the haploid genome of the 129/Sv strain. Conclusions: Our findings demonstrate functional differences between 129 and C57BL/6 Gbp alleles which need to be considered in the design and interpretation of studies utilizing mouse models, particularly for phenotypes influenced by Gbp2 or Gbp2b expression.

Inflammasome Activation by Bacterial Outer Membrane Vesicles Requires Guanylate Binding Proteins.

ABSTRACTThe Gram-negative bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as “free” LPS aggregates or actively secreted by live bacteria as a component of outer membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli. In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1β (IL-1β) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types.

Interferon-inducible guanylate binding protein (GBP2) is associated with better prognosis in breast cancer and indicates an efficient T cell response

Recently, interferon-inducible guanylate binding protein (GBP2) has been discussed as a possible control factor in tumor development, which is controlled by p53, and inhibits NF-Kappa B and Rac protein as well as expression of matrix metalloproteinase 9. However, the potential role that GBP2 plays in tumor development and prognosis has not yet been studied. We analyzed whether GBP2 mRNA levels are associated with metastasis-free interval in 766 patients with node negative breast carcinomas who did not receive systemic chemotherapy. Furthermore, response to anthracycline-based chemotherapy was studied in 768 breast cancer patients. High expression of GBP2 in breast carcinomas was associated with better prognosis in the univariate (P < 0.001, hazard ratio 0.763, 95 % CI 0.650-0.896) as well as in the multivariate Cox analysis (P = 0.008, hazard ratio 0.731, 95 % CI 0.580-0.920) adjusted to the established clinical factors age, pT stage, grading, hormone and ERBB2 receptor status. The association was particularly strong in subgroups with high proliferation and positive estrogen receptor status but did not reach significance in carcinomas with low expression of proliferation associated genes. Besides its prognostic capacity, GBP2 also predicted pathologically complete response to anthracycline-based chemotherapy (P = 0.0037, odds ratio 1.39, 95 % CI 1.11-1.74). Interestingly, GBP2 correlated with a recently established T cell signature, indicating tumor infiltration with T cells (R = 0.607, P < 0.001). GBP2 is associated with better prognosis in fast proliferating tumors and probably represents a marker of an efficient T cell response.