Transglutaminase 2 Research Articles

Pharmacological inhibition of astrocytic transglutaminase 2 facilitates the expression of a neurosupportive astrocyte reactive phenotype in association with increased histone acetylation.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) modulates the phenotype of reactive astrocytes in a way that improves neuronal injury outcomes both in vitro and in vivo . In an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. In this study, we provide insights into the mechanisms by which TG2 deletion or inhibition result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix. To better understand how pharmacologically altering TG2 affects its ability to regulate reactive astrocyte phenotypes, we assessed how VA4 inhibition impacts TG2's interaction with Zbtb7a, a transcription factor we have previously identified as a functionally relevant TG2 nuclear interactor. The results of these studies demonstrate that VA4 significantly decreases the interaction of TG2 and Zbtb7a. TG2's interactions with Zbtb7a, as well as a wide range of other transcription factors and chromatin regulatory proteins, suggest that TG2 may act as an epigenetic regulator to modulate gene expression. To begin to understand if TG2-mediated epigenetic modification may impact astrocytic phenotypes in our models, we interrogated the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, previous RNA-sequencing and our present proteomic analysis also supported a predominant transcriptionally suppressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

DDDR-46. TARGETING TRANSGLUTAMINASE 2 (TGM2) ENHANCES SENSITIVITY OF GLIOBLASTOMA CELL LINES TO RADIOTHERAPY AND TEMOZOLOMIDE

Abstract Glioblastoma (GBM), the most aggressive and common primary brain tumor in adults, remains highly resistant to conventional therapeutics, including radiation therapy (RT), largely due to its molecular heterogeneity. Few biomarkers have been identified that successfully guide or predict treatment response. Transglutaminase 2 (TGM2), a ubiquitous multifunctional enzyme involved in numerous signal transduction pathways related to tumorigenesis, has recently been suggested to drive chemoradioresistance. Here, we investigate the effects of disrupting TGM2 by pharmacologic inhibition or CRISPR knockout (TGM2-KO) on GBM cell’s response to RT and Temozolomide (TMZ) chemotherapy. Using western blotting and clonogenic assays, we show that the allosteric TGM2 inhibitor, GK921, stabilizes p53 expression and impedes reproductive ability at IC50 and IC25 concentrations. TGM2-KO and GK921 (at IC10 and lower) sensitized GBM cell lines to RT with almost equivalent efficacy, resulting in a 2- to 3-fold suppression of clonal expansion compared to scramble controls or irradiation alone. We also demonstrate increased sensitivity of GBM cell lines to TMZ upon inhibition of TGM2 by GK921. Our findings indicate that the use of TGM2 inhibitors, such as GK921, could enhance the efficacy of RT and the potency of TMZ, positioning TGM2 as a promising therapeutic target that may improve standard treatment of GBM cells expressing TGM2.

Pharmacological inhibition of astrocytic transglutaminase 2 facilitates the expression of a neurosupportive astrocyte reactive phenotype in association with increased histone acetylation.

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) modulates the phenotype of reactive astrocytes in a way that improves neuronal injury outcomes both in vitro and in vivo. In an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. In this study, we provide insights into the mechanisms by which TG2 deletion or inhibition result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix. To better understand how pharmacologically altering TG2 affects its ability to regulate reactive astrocyte phenotypes, we assessed how VA4 inhibition impacts TG2s interaction with Zbtb7a, a transcription factor we have previously identified as a functionally relevant TG2 nuclear interactor. The results of these studies demonstrate that VA4 significantly decreases the interaction of TG2 and Zbtb7a. TG2s interactions with Zbtb7a, as well as a wide range of other transcription factors and chromatin regulatory proteins, suggest that TG2 may act as an epigenetic regulator to modulate gene expression. To begin to understand if TG2-mediated epigenetic modification may impact astrocytic phenotypes in our models, we interrogated the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, previous RNA-sequencing and our present proteomic analysis also supported a predominant transcriptionally suppressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

Transglutaminase 2 in breast cancer metastasis and drug resistance.

Transglutaminase 2 (TG2) is a widely distributed multifunctional protein with various enzymatic and non-enzymatic activities. It is becoming increasingly evident that high levels of TG2 in tumors induce the occurrence of epithelial to mesenchymal transition (EMT) and the acquisition of stem cell-like phenotypes, promoting tumor metastasis and drug resistance. By regulating intracellular and extracellular signaling pathways, TG2 promotes breast cancer metastasis to lung, brain, liver and bone, as well as resistance to various chemotherapy drugs including docetaxel, doxorubicin, platinum and neratinib. More importantly, recent studies described the involvement of TG2 in PD-1/PD-L1 inhibitors resistance. An in-depth understanding of the role that TG2 plays in the progression of metastasis and drug resistance will offer new therapeutic targets for breast cancer treatment. This review covers the extensive and rapidly growing field of the role of TG2 in breast cancer. Based on the role of TG2 in EMT, we summarize TG2-related signaling pathways in breast cancer metastasis and drug resistance and discuss TG2 as a therapeutic target.

Tumor-associated macrophages induce epithelial-mesenchymal transition and promote lung metastasis in breast cancer by activating the IL-6/STAT3/TGM2 axis

Breast cancer is one of the most common tumors in the world and metastasis is the major cause of tumor-related death. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME) and often associated with cancer metastasis. Nevertheless, the mechanism by which TAMs regulate breast cancer metastasis remain unclear. In this study, we found that transglutaminase 2 (TGM2) could serve as a crucial target in the modulation of TAMs-induced epithelial-mesenchymal transition (EMT) and invasion of breast cancer cells. Further analysis revealed that IL-6 secreted from TAMs, which was capable of inducing TGM2 expression through the activation of the JAK/STAT3 signaling pathway. Subsequent luciferase reporter assays demonstrated that STAT3 binds to the TGM2 promoter region, thereby transcriptionally enhancing TGM2 expression. In conclusion, our current research has identified the IL-6/STAT3/TGM2 axis as a pivotal regulator in breast tumorigenesis caused by TAMs, presenting a novel target for the treatment of breast cancer.

Transglutaminase 2-mediated histone monoaminylation and its role in cancer.

Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.

Transglutaminase 2 Regulates HSF1 Gene Expression in the Acute Phase of Fish Optic Nerve Regeneration.

Fish retinal ganglion cells (RGCs) can regenerate after optic nerve lesions (ONLs). We previously reported that heat shock factor 1 (HSF1) and Yamanaka factors increased in the zebrafish retina 0.5-24 h after ONLs, and they led to cell survival and the transformation of neuro-stem cells. We also showed that retinoic acid (RA) signaling and transglutaminase 2 (TG2) were activated in the fish retina, performing neurite outgrowth 5-30 days after ONLs. In this study, we found that RA signaling and TG2 increased within 0.5 h in the zebrafish retina after ONLs. We examined their interaction with the TG2-specific morpholino and inhibitor due to the significantly close initiation time of TG2 and HSF1. The inhibition of TG2 led to the complete suppression of HSF1 expression. Furthermore, the results of a ChIP assay with an anti-TG2 antibody evidenced significant anti-TG2 immunoprecipitation of HSF1 genome DNA after ONLs. The inhibition of TG2 also suppressed Yamanaka factors' gene expression. This rapid increase in TG2 expression occurred 30 min after the ONLs, and RA signaling occurred 15 min before this change. The present study demonstrates that TG2 regulates Yamanaka factors via HSF1 signals in the acute phase of fish optic nerve regeneration.

Inhibition of transglutaminase 2 inhibits ionizing radiation-induced cellular senescence in skin keratinocytes in vitro.

Senescent cells are typically characterized by a stable proliferation arrested in dividing cells accompanied with a senescence-associated secretory phenotype (SASP). Skin cellular senescence is the primary cause of skin aging, whereas the lack of identified skin senescence markers limits our understanding of the mechanisms involved in skin aging. Recent studies have revealed that intracellular calcium signaling has emerged as a key player in regulating cellular senescence and aging. However, the implication and roles of calcium signaling in skin keratinocyte senescence remain only partially understood. In this study, we developed a model for skin keratinocyte senescence using ionizing radiation (I/R) stimulation and found that the calcium-associated gene transglutaminase 2 (TGM2) was significantly induced compared with normal control. Interestingly, inhibition of TGM2 was found to delay skin keratinocyte senescence by suppressing I/R-promoted intracellular calcium signaling, accumulation of reactive oxygen species (ROS), DNA damage, as well as NF-κB-mediated SASP secretion. Taken together, our findings demonstrate that inhibition of TGM2 contributes to bypassing I/R-induced skin keratinocyte senescence and sheds light on novel strategies against skin stresses caused by I/R.

Distinct conformational states enable transglutaminase 2 to promote cancer cell survival versus cell death

Transglutaminase 2 (TG2) is a GTP-binding, protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that bind and stabilize the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotides bind and stabilize a monomeric closed conformation while calcium binds to an open state that can form higher order oligomers. SAXS analysis suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time resolved SAXS to show that LM11 increases the ability of calcium to bind and stabilize an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.

Differential miRNA and Protein Expression Reveals miR-1285, Its Targets TGM2 and CDH-1, as Well as CD166 and S100A13 as Potential New Biomarkers in Patients with Diabetes Mellitus and Pancreatic Adenocarcinoma.

Early detection of PDAC remains challenging due to the lack of early symptoms and the absence of reliable biomarkers. The aim of the present project was to identify miRNA and proteomics signatures discriminating PDAC patients with DM from nondiabetic PDAC patients. Proteomics analysis and miRNA array were used for protein and miRNA screening. We used Western blotting and Real-Time Quantitative Reverse Transcription polymerase chain reaction (qRT-PCR) for protein and miRNA validation. Comparisons between experimental groups with normal distributions were performed using one-way ANOVA followed by Tukey's post hoc test, and pairwise tests were performed using t-tests. p ≤ 0.05 was considered statistically significant. Protein clusters of differentiation 166 (CD166), glycoprotein CD63 (CD63), S100 calcium-binding protein A13 (S100A13), and tumor necrosis factor-β (TNF-β) were detected in the proteomics screening. The miRNA assay revealed a differential miRNA 1285 regulation. Previously described target proteins of miR-1285 cadherin-1 (CDH-1), cellular Jun (c-Jun), p53, mothers against decapentaplegic homolog 4 (Smad4), human transglutaminase 2 (TGM2) and yes-associated protein (YAP), were validated via Western blotting. miR-1285-3p was successfully validated as differentially regulated in PDAC + DM via qRT-PCR. Overall, our data suggest miRNA1285-3p, TGM2, CDH-1, CD166, and S100A13 as potential meaningful biomarker candidates to characterize patients with PDAC + DM. Data are available via ProteomeXchange with the identifier PXD053169.

The relationship between serum transglutaminase-2 levels and the severity of chronic spontaneous urticaria

Chronic spontaneous urticaria (CSU) is an immunological disease that is depicted by high prevalence and eminent burden for patients and society that is attributable to the arbitrary nature of symptoms and inconsistent tools for assessment of activity and severity. Transglutaminase-2 (TG2) is a posttranslational enzyme that is pervasively expressed in many cells and tissue types including mast cells. It has various biological functions, and its role in allergic disorders has been highlighted and delineated through several postulated mechanisms. This case–control study aimed at determining the relationship between serum levels TG2 and severity of CSU. To the best of our knowledge, this is the first study in Egypt to determine the relationship between serum TG2 and severity of CSU. We enrolled 60 adult patients with confirmed diagnosis of CSU. According to urticaria activity score (UAS), patients were categorized into three groups [20 with mild disease; UAS = 0, 20 with moderate disease; UAS = 1–3, 20 with severe disease; UAS = 4–6]. Another 20 healthy individuals (age and gender matched) served as a control group. All patients were subjected to detailed medical history, clinical examination, complete blood count with differential, serum total IgE, CRP, ESR, TSH, ANA, liver and renal function tests. Serum level of TG2 was done by quantitative ELISA for all enrolled patients and controls. Serum TG2 is significantly higher in patients group compared to control group (P value < 0.001). Serum TG2 levels were significantly higher in patients with severe disease compared to patients with moderate or mild disease. This is illustrated by the significant positive correlation between serum TG2 and UAS (r 0.814 and P value 0.000). Moreover, serum TG2 accurately classified CSU patients into mild, moderate and severe subgroups: as regards differentiation between mild and moderate cases (sensitivity 70%, specificity 80%, PPV 77.8, NPV 72.7) and as for the differentiation between moderate and severe cases (sensitivity 95%, specificity 90%, PPV 90.5, NPV 94.7). Serum TG2 may have a pivotal role as a marker of severity in patients with CSU.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

Multiomics analysis identified IL-4–induced IL1RL1high eosinophils characterized by prominent cysteinyl leukotriene metabolism

BackgroundClinical studies demonstrated that IL-4, a type 2 cytokine, plays an important role in the pathogenesis of chronic rhinosinusitis (CRS) and eosinophilic asthma (EA). However, the direct effect of IL-4 on eosinophils remains unclear. ObjectiveWe aim to elucidate the inflammatory effects of IL-4 on the functions of human eosinophils. MethodsMulti-omics analysis comprising transcriptomics, proteomics, lipidomics, quantitative RT-PCR, and flow cytometry was performed using blood eosinophils from healthy subjects stimulated with IL-4, IL-5, or their combination. ResultsTranscriptomic and proteomic analyses revealed that both IL-4 and IL-5 upregulate the expression of gamma-gultamyl transferase 5 (GGT5), a fatty acid-metabolizing enzyme that converts leukotriene C4 (LTC4) into LTD4. In addition, IL-4 specifically upregulates the expression of IL1RL1, a receptor for IL-33 and transglutaminase 2 (TGM2). Additional transcriptomic analysis of cells stimulated with IL-13 revealed altered gene expression profiles, characterized by the upregulation of GGT5, TGM2, and IL1RL1. IL-13-induced changes were not totally different from IL-4-induced one. Lipidomic analysis revealed that IL-5 and IL-4 additively increased the extracellular release of LTD4. In vitro experiments revealed that STAT6 and IL-4 receptor α control the expression of these molecules in the presence of IL-4 and IL-13. Analysis of eosinophils derived from patients with allergic disorders indicated the involvement of IL-4 and IL-13 at the inflamed sites. ConclusionsIL-4 induces the pro-allergic phenotype of IL1RL1high eosinophils with prominent cysteinyl leukotriene metabolism via STAT6. These cellular changes represent potential therapeutic targets for CRS and EA.

Transcriptomic analysis of intestine following administration of a transglutaminase 2 inhibitor to prevent gluten-induced intestinal damage in celiac disease

Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.

Bioorthogonal Labeling and Enrichment of Histone Monoaminylation Reveal Its Accumulation and Regulatory Function in Cancer Cell Chromatin.

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin.

Chemical Proteomic Profiling of Protein Dopaminylation in Colorectal Cancer Cells.

Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.

Immune cell infiltration and inflammatory landscape in primary brain tumours

BackgroundPrimary malignant brain tumours are more than one-third of all brain tumours and despite the molecular investigation to identify cancer driver mutations, the current therapeutic options available are challenging due to high intratumour heterogeneity. In addition, an immunosuppressive and inflammatory tumour microenvironment strengthens cancer progression. Therefore, we defined an immune and inflammatory profiling of meningioma and glial tumours to elucidate the role of the immune infiltration in these cancer types.MethodsUsing tissue microarrays of 158 brain tumour samples, we assessed CD3, CD4, CD8, CD20, CD138, Granzyme B (GzmB), 5-Lipoxygenase (5-LOX), Programmed Death-Ligand 1 (PD-L1), O-6-Methylguanine-DNA Methyltransferase (MGMT) and Transglutaminase 2 (TG2) expression by immunohistochemistry (IHC). IHC results were correlated using a Spearman correlation matrix. Transcript expression, correlation, and overall survival (OS) analyses were evaluated using public datasets available on GEPIA2 in Glioblastoma (GBM) and Lower Grade Glioma (LGG) cohorts.ResultsSeven out of ten markers showed a significantly different IHC expression in at least one of the evaluated cohorts whereas CD3, CD4 and 5-LOX were differentially expressed between GBMs and astrocytomas. Correlation matrix analysis revealed that 5-LOX and GzmB expression were associated in both meningiomas and GBMs, whereas 5-LOX expression was significantly and positively correlated to TG2 in both meningioma and astrocytoma cohorts. These findings were confirmed with the correlation analysis of TCGA-GBM and LGG datasets. Profiling of mRNA levels indicated a significant increase in CD3 (CD3D, CD3E), and CD138 (SDC1) expression in GBM compared to control tissues. CD4 and 5-LOX (ALOX5) mRNA levels were significantly more expressed in tumour samples than in normal tissues in both GBM and LGG. In GBM cohort, GzmB (GZMB), SDC1 and MGMT gene expression predicted a poor overall survival (OS). Moreover, in LGG cohort, an increased expression of CD3 (CD3D, CD3E, CD3G), CD8 (CD8A), GZMB, CD20 (MS4A1), SDC1, PD-L1, ALOX5, and TG2 (TGM2) genes was associated with worse OS.ConclusionsOur data have revealed that there is a positive and significant correlation between the expression of 5-LOX and GzmB, both at RNA and protein level. Further evaluation is needed to understand the interplay of 5-LOX and immune infiltration in glioma progression.

Transglutaminase 2 serves as a pathogenic hub gene of KRAS mutant colon cancer based on integrated analysis.

Colon cancer is acknowledged as one of the most common malignancies worldwide, ranking third in United States regarding incidence and mortality. Notably, approximately 40% of colon cancer cases harbor oncogenic KRAS mutations, resulting in the continuous activation of epidermal growth factor receptor signaling. To investigate the key pathogenic genes in KRAS mutant colon cancer holds considerable importance. Weighted gene co-expression network analysis, in combination with additional bioinformatics analysis, were conducted to screen the key factors driving the progression of KRAS mutant colon cancer. Meanwhile, various in vitro experiments were also conducted to explore the biological function of transglutaminase 2 (TGM2). Integrated analysis demonstrated that TGM2 acted as an independent prognostic factor for progression-free survival. Immunohistochemical analysis on tissue microarrays revealed that TGM2 was associated with an elevated probability of perineural invasion in patients with KRAS mutant colon cancer. Additionally, biological roles of the key gene TGM2 was also assessed, suggesting that the downregulation of TGM2 attenuated the proliferation, invasion, and migration of the KRAS mutant colon cancer cell line. This study underscores the potential significance of TGM2 in the progression of KRAS mutant colon cancer. This insight not only offers a theoretical foundation for therapeutic approaches but also highlights the need for additional clinical trials and fundamental research to support our preliminary findings.

Transcriptomics Studies Reveal Functions of Transglutaminase 2 in Breast Cancer Cells Using Membrane Permeable and Impermeable Inhibitors

Transglutaminase 2 (TG2) performs many functions both under physiological and pathological conditions. In cancer, its expression is associated with aggressiveness, propensity to epithelial-mesenchymal transition, and metastasis. Since TG2 performs key functions both outside and inside the cell, using inhibitors with different membrane permeability we analyzed the changes in the transcriptome induced in two triple-negative cell lines (MDA-MB-436 and MDA-MB-231) with aggressive features. By characterizing pathways and gene networks, we were able to define the effects of TG2 inhibitors (AA9, membrane-permeable, and NCEG2, impermeable) in relation to the roles of the enzyme in the intra- and extracellular space within the context of breast cancer.The deregulated genes revealed p53 and integrin signaling to be the common pathways with some genes showing opposite changes in expression. In MDA-MB-436, AA9 induced apoptosis, modulated cadherin, Wnt, gastrin and cholecystokinin receptors (CCKR) mediated signaling, with RHOB and GNG2 playing significant roles, and affected the Warburg effect by decreasing glycolytic enzymes. In MDA-MB-231 cells, AA9 strongly impacted HIF-mediated hypoxia, including AKT and mTOR pathway. These effects suggest an anti-tumor activity by blocking intracellular TG2 functions. Conversely, the use of NCEG2 stimulated the expression of ATP synthase and proteins involved in DNA replication, indicating a potential promotion of cell proliferation through inhibition of extracellular TG2. To effectively utilize these molecules as an anti-tumor strategy, an appropriate delivery system should be evaluated to target specific functions and avoid adverse effects. Additionally, considering combinations with other pathway modulators is crucial.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.