TG2 Expression Research Articles

Transglutaminases Derived from Astrocytes Accelerate Amyloid β Aggregation.

Activation of astrocytes has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Transglutaminase (TG) is a crosslinking enzyme and contributes to cell adhesion, cytoskeleton construct, extracellular matrix formation, and so on. One of the isozymes, tissue-type TG (TG2) is reported to be activated in AD. Moreover, amyloid β1-42 (Aβ), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be a substrate of TG2. However, contribution and derivation of TGs in brain for Aβ aggregation remain to be clarified. In the present study, we examined the effects of cultured astrocytes prepared from rat embryonic brain cortex on Aβ aggregation. When freshly prepared Aβ was added to cultured astrocytes for 7days, Aβ monomer decreased and Aβ oligomer unchanged. On the other hand, when Aβ monomer was diluted with astrocytes conditioned medium, Aβ oligomer increased time-dependently, and an inhibitor of TGs, cystamine, blocked it. Furthermore, when cultured astrocytes were stimulated with aggregated Aβ, TG2 expression significantly increased. These results suggest that astrocytes could uptake Aβ monomer to eliminate from brain; however, TGs derived from astrocytes might accelerate Aβ aggregation and the aggregated Aβ might enhance TG2 in astrocytes as a vicious cycle in pathological conditions. Adequate control of TGs expression and function in astrocytes would be an important factor in AD pathology.

Amphotericin B Increases Transglutaminase 2 Expression Associated with Upregulation of Endocytotic Activity in Mouse Microglial Cell Line BV-2.

Amphotericin B (AmB), a polyene antibiotic, is reported to cause the microglial activation to induce nitric oxide (NO) production and proinflammatory cytokines expression, and change neurotrophic factors expression in cultured microglia (Motoyoshi et al. in Neurochem Int 52:1290-1296, 2008). On the other hand, tissue-type transglutaminase (TG2) is involved in connection to phagocytes with apoptotic cells. Engulfment of neurons by activated microglia is thought to cause neurodegenerative diseases but detail is unclear, and involvement of TG2 in phagocytosis has been reported in our previous study using lipopolysaccharide-stimulated BV-2 cells (Kawabe et al. in Neuroimmunomodulation 22(4):243-249, 2015). In the present study, we examined the changes of TG2 expression, phagocytosis and pinocytosis in BV-2 cells stimulated by AmB. AmB stimulation increased TG2 expression and TG activity. Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were also up-regulated by AmB stimulation in BV-2 cells. Blockade of TG activity by cystamine, an inhibitor of TGs, suppressed AmB-enhanced TG2 expression, TG activity, NO production, phagocytosis and pinocytosis. Excessive NO production from microglia and/or facilitation of phagocytosis might be involved in neuronal death. To control TG activity might make possible to protect neurons and care for CNS diseases.

Tissue transglutaminase induces Epithelial-Mesenchymal-Transition and the acquisition of stem cell like characteristics in colorectal cancer cells.

Human colon cancer cell lines (CRCs) RKO, SW480 and SW620 were investigated for TG2 involvement in tumour advancement and aggression. TG2 expression correlated with tumour advancement and expression of markers of epithelial-mesenchymal transition (EMT). The metastatic cell line SW620 showed high TG2 expression compared to the primary tumour cell lines SW480 and RKO and could form tumour spheroids under non- adherent conditions. TG2 manipulation in the CRCs by shRNA or TG2 transduction confirmed the relationship between TG2 and EMT. TGFβ1 expression in CRC cells, and its level in the cell medium and extracellular matrix was increased in primary tumour CRCs overexpressing TG2 and could regulate TG2 expression and EMT by both canonical (RKO) and non-canonical (RKO and SW480) signalling. TGFβ1 regulation was not observed in the metastatic SW620 cell line, but TG2 knockdown or inhibition in SW620 reversed EMT. In SW620, TG2 expression and EMT was associated with increased presence of nuclear β-catenin which could be mediated by association of TG2 with the Wnt signalling co-receptor LRP5. TG2 inhibition/knockdown increased interaction between β-catenin and ubiquitin shown by co-immunoprecipitation, suggesting that TG2 could be important in β-catenin regulation. β-Catenin and TG2 was also upregulated in SW620 spheroid cells enriched with cancer stem cell marker CD44 and TG2 inhibition/knockdown reduced the spheroid forming potential of SW620 cells. Our data suggests that TG2 could hold both prognostic and therapeutic significance in colon cancer.

Transglutaminase Interaction with α6/β4-Integrin Stimulates YAP1-Dependent ΔNp63α Stabilization and Leads to Enhanced Cancer Stem Cell Survival and Tumor Formation.

Transglutaminase 2 (TG2) expression is required for epidermal squamous cell carcinoma cancer stem cell survival. However, the molecular signaling mechanisms triggered by TG2 that mediate this survival action are not well understood. Here we show that TG2 is constitutively expressed in ECS cells, where it interacts with α6/β4 integrin to stimulate FAK and Src signaling, leading to PI3K activation of phosphoinositide-dependent kinase 1 (PDK1). PDK1 inhibits Hippo signaling, leading to enhanced nuclear accumulation of YAP1, which interacted with and stabilized ΔNp63α to enhance epidermal squamous cell carcinoma spheroid formation, invasion, and migration. Overall, these findings suggest that constitutive TG2 expression results in stabilization of ΔNp63α, leading to maintenance of cancer stem cell properties and enhanced tumor formation. Cancer Res; 76(24); 7265-76. ©2016 AACR.

Silencing TG2 in Acute Myeloid Leukemia. Possible Role of TG2 in Microenvironment-Mediated Chemoresistance

Background: Tissue transglutaminase (TG2) is a multi-domain, multi-functional enzyme with diverse biological functions including calcium-dependent posttranslational modification of proteins, extracellular matrix formation, integrin-mediated signaling, and signal transduction. In many cancer types, increased TG2 expression has been associated with malignancy and resistance to chemotherapy. Previous studies using reverse phase proteomic arrays (RPPA) on 511 AML samples indicated in many cases that TG2 expression was elevated in samples from patients with relapsed AML when compared to samples collected at AML diagnosis (PMID: 23576428). In addition, elevated TG2 expression correlated with increased expression of proteins involved in cell-adhesion and the regulation of apoptosis. These findings suggest that the expression of TG2, in combination with other makers, may be suitable to predict survival outcome in AML patients and that targeting TG2 might sensitize AML cells to standard chemotherapy.Results: Our preliminary data demonstrated that the expression of TG2 mRNA, as determined by cDNA array analysis and validated by qRT-PCR, is induced (>30 fold increase) in OCI-AML3 leukemia cells when co-cultured with normal donor-derived Bone Marrow-derived Mesenchymal Stromal Cells (BM-MSC) in hypoxic conditions (1% pO2) for 48hs. Increase in mRNA correlated with an increase in protein level as determined by Western Blot. In order to test the hypothesis that the expression of TG2 in leukemia cells is related to resistance to chemotherapy and poor prognosis we down-regulated or over-expressed TG2 by lentiviral transduction with TG2-shRNA or TG2-over-expressing vector, respectively, in OCI-AML3 cells. Our in vitro studies showed that although the over-expression of TG2 in OCI-AML3 cells did not have a significant impact on resistance to chemotherapy, down-regulation of TG2 had a moderate effect on sensitizing cells to AraC (13% increase in apoptosis; P = 0.0006). To further investigate these findings we performed in vivo experiments in NGS mice engrafted with OCI-AML3 stably-transduced with either scramble-shRNA or TG2-shRNA. After confirmation of engraftment by bioluminescence imaging, leukemia-bearing mice were treated with a combination of AraC and doxorubicin. Consistent with the in vitro data, the down-regulation of TG2 rendered OCI-AML3 cells more susceptible to chemotherapy and prolonged the survival of leukemia-bearing mice suggesting a role for TG2 in resistance to chemotherapy (median survival of mice transplanted with scramble-shRNA cells, 44 days; compared to mice transplanted with TG2-shRNA cells, 51 days; P = 0.0027).Conclusions: Our data indicate that TG2 is up-regulated in response to microenvironmental signals like hypoxia and cell-cell contact with stromal cells. Silencing of TG2 in AML cells sensitized cells to chemotherapy in vitro and in vivo and extended the survival of the leukemia-bearing mice suggesting that targeting TG2 in combination with standard chemotherapy in AML could be developed into a novel therapeutic approach. DisclosuresKonopleva:Cellectis: Research Funding; Calithera: Research Funding.

Tissue Transglutaminase Activates Cancer-Associated Fibroblasts and Contributes to Gemcitabine Resistance in Pancreatic Cancer

Resistance to chemotherapy is a hallmark of pancreatic ductal adenocarcinoma (PDA) and has been partly attributed to the dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme, is secreted by PDA cells and cross-links proteins in the tumor microenvironment (TME) through acyl-transfer between glutamine and lysine residues, promoting PDA growth. The objective of the current study was to determine whether secreted TG2 by PDA cells alters the response of pancreatic tumors to gemcitabine. Orthotopic pancreatic xenografts and co-culture of PDA and stromal cells were employed to determine the mechanisms by which TG2 alters tumor-stroma interactions and response to gemcitabine. Analysis of the pancreatic The Cancer Genome Atlas (TCGA) database demonstrated that increased TG2 expression levels correlate with worse overall survival (hazard ratio=1.37). Stable TG2 knockdown in PDA cells led to decreased size of pancreatic xenografts and increased sensitivity to gemcitabine in vivo. However, TG2 downregulation did not increase cytotoxicity of gemcitabine in vitro. Additionally, multivessel density and gemcitabine uptake in pancreatic tumor tissue, as measured by mass spectrometry (MS-HPLC), were not significantly different in tumors expressing TG2 versus tumors in which TG2 was knocked down. Fibroblasts, stimulated by TG2 secreted by PDA cells, secrete laminin A1, which protects cancer cells from gemcitabine-induced cytotoxicity. In all, our results demonstrate that TG2 secreted in the pancreatic TME orchestrates the cross talk between cancer cells and stroma, impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition to increase the antitumor effects of gemcitabine in PDA.

Effect of Some Growth Factors on Tissue Transglutaminase Overexpression Induced by β-Amyloid in Olfactory Ensheathing Cells.

Herein, we assessed in a particular glial cell type, called olfactory ensheathing cells (OECs), the effect of some growth factors (GFs) on tissue transglutaminase (TG2) overexpression induced by amyloid-beta (Aβ) with native full-length peptide 1-42 or by fragments, 25-35 or 35-25, as control. Previously, we demonstrated that TG2 overexpression induced by some stressors was down-regulated by GFs exposure in OECs. To monitor cell viability, an MTT test was used, while TG2 expression was examined using immunocytochemical and Western blot analysis. We also considered the involvement of the TG2-mediated apoptotic pathway. Vimentin expression was evaluated as well. Reactive oxygen species and reduced glutathione levels were utilized to test the oxidative intracellular status. Lactate dehydrogenase released into the medium, as a marker of necrotic cell death, was evaluated. We found that in OECs exposed to Aβ(1-42) or Aβ(25-35) for 24h, TG2 expression increased, and we observed that the protein appeared prevalently localized in the cytosol. The pre-treatment with GFs, basic fibroblast growth factor (bFGF) or glial-derived neurotrophic factor (GDNF), down-regulated the TG2 level, which was prevalently limited to the nuclear compartment. Vimentin expression and caspase cleavage showed a significant enhancement in Aβ(1-42) and Aβ(25-35) exposed cells. The pre-treatment with bFGF or GDNF was able to restore the levels of the proteins to control values, and the intracellular oxidative status modified by the exposure to Aβ(1-42) or Aβ(25-35). Our data suggest that both bFGF or GDNF could be an innovative mechanism to contrast TG2 expression, which plays a key role in Alzheimer's disease.

Expression of Transglutaminase in Foreskin of Children with Balanitis Xerotica Obliterans.

Balanitis xerotica obliterans (BXO) is a chronic inflammatory skin disorder of unclear etiology. The etiology and the exact molecular mechanisms underlying the disease are still unknown. The human transglutaminase (TG) family consists of several proteins with catalytic activity essential for biological processes. In the present research we investigated the transcript levels of three TGs in patients operated on for congenital phimosis without or with histologically confirmed BXO; Thirty children with acquired phimosis were enrolled. The removed foreskins were sent both for histological diagnosis and for quantitative real-time PCR to evaluate the transcript levels of keratinocyte (TG1), tissue (TG2), and epidermal (TG3) transglutaminase; We observed a decrease in TG1 and TG3 transcripts by about 70% (p < 0.001) in foreskins from patients with BXO (n = 15) in comparison with patients without BXO (n = 15) and an increase in TG2 mRNA levels by 2.9 folds (p < 0.001); Reduced expression of both TG1 and TG3 was associated with the altered structure of the foreskin in BXO and can be a consequence of damage to keratinocytes. Increased expression of TG2 can be the result of chronic inflammation. TG2 overexpression can play a pivotal role in triggering and maintaining the inflammatory response in BXO patients.

Celiac anti-type 2 transglutaminase antibodies induce differential effects in fibroblasts from celiac disease patients and from healthy subjects.

Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

Role of tissue transglutaminase in age-associated ventricular stiffness.

Aging is associated with increased cardiomyocyte loss, left-ventricular hypertrophy, and the accumulation of extracellular matrix, which results in declining cardiac function. The role of the matrix crosslinking enzyme, tissue transglutaminase (TG2), in age-related myocardial stiffness, and contractile function remains incompletely understood. In this study, we examined the role of TG2 in cardiac function, and determined whether TG2 inhibition can prevent age-associated changes in cardiac function. Male Fisher rats (18-month-old) were administered the transglutaminase inhibitor cystamine (study group) or saline (age-matched controls) for 12weeks via osmotic mini-pumps. Cardiac function was determined by echocardiography and invasive pressure-volume loops. Rat hearts were dissected out, and TG2 expression, activity, and S-nitrosation were determined. Young (6-month-old) males were used as controls. TG2 activity significantly increased in the saline-treated but not in the cystamine-treated aging rat hearts. TG2 expression also increased with age and was unaltered by cystamine treatment. Aged rats showed increased left ventricular (LV) end-systolic dimension and a decrease in fractional shortening compared with young, which was not affected by cystamine. However, cystamine treatment preserved the preload-independent index of LV filling pressure and restored end-diastolic pressure, end-diastolic pressure-volume relationships, and arterial elastance toward young. An increase in TG2 activity contributes to age-associated increase in diastolic stiffness, thereby contributing to age-associated diastolic dysfunction. TG2 may thus represent a novel target for age-associated diastolic heart failure.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System.

Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system.

Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System.

Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system.

Tissue Transglutaminase Regulates Steroid Resistance in Acute Lymphoblastic Leukemia Via Nuclear Factor-Kappa B Related Signaling Pathways

Steroid resistance is lethal to the patients with acute lymphoblastic leukemia (ALL). To improve survival for steroid-resistant ALL patients, it is necessary to develop innovative therapeutic strategies to overcome steroid resistance. Tissue transglutaminase (TG2) is an intracellular calcium-dependent protein cross-linking enzyme reported to be over-expressed in various metastatic or chemotherapy-resistant cancer cells including lymphoma cells. Chronic expression of TG2 constitutively activates nuclear factor kappa B (NF-kappaB) and it is directed linked to NF-kappaB-related signaling networks including hypoxia-inducible factor-1 (HIF-1), Zeb, Snail, or Twist that influence drug resistance or metastasis in inflammatory or cancer cells. In the present study, we used biochemical and molecular methodologies to demonstrate that steroid resistance was associated with TG2-regulated signaling pathways including NF-kappaB and its downstream transcriptional factors in steroid-resistant ALL. We generated steroid-adapted subclones of ALL cell lines that were extremely more resistant to steroid than the parent cells. We found that steroid-resistant ALL cells expressed elevated levels of TG2 and the modification of TG2 activities altered NF-kappaB expression in this cells. TG2-related NF-kappaB activation modulated the expression of HIF-1 and NF-kappaB-targeted genes such as Zeb, Snail, and Twist in steroid-resistant ALL cells. Also, TG2 inhibition improved the cytotoxicity of steroid in steroid-resistant ALL cells. This is the first study to show the link between TG2, NF-kappaB, its downstream signaling networks, and steroid resistance in ALL. TG2 inhibition could be used as an alternative target to overcome steroid resistance in ALL. Furthermore, TG2 and HIF-1 expression could serve as new biomarkers of steroid resistance evolution or cancer recurrence in ALL. DisclosuresNo relevant conflicts of interest to declare.

Study of the mechanism underlying the inhibitory effects of transglutaminase II on apoptosis in the osteosarcoma MG-63 cell line under hypoxic conditions.

The aim of the present study was to investigate the association between the apoptosis phenomenon in the MG-63 osteosarcoma cell line, and transglutaminase II (TG2) expression. The relationship between the anti-apoptotic mechanism of TG2 and the expression of cytochrome c as well as caspase-3 under hypoxic conditions was also verified. A hypoxic culture of MG-63 cells was prepared. The hypoxia and TG2 siRNA hypoxia groups were established, and the cultures were incubated for 12 h under hypoxic conditions. TG2 activity, TG2 protein expression and its mRNA level were investigated. Cytochrome c and caspase-3 protein levels in the TG2 nucleus and cytoplasm were measured. The apoptotic rate was also monitored. The results showed that TG2 activity, TG2 protein expression and its mRNA level in the hypoxia group were significantly higher than those of the siRNA hypoxia group. The results showed statistically insignificant differences (P<0.05). By contrast, a comparison of the two groups in the cytoplasm yielded no statistically significant differences (P>0.05). Cytochrome c and caspase-3 protein levels in the hypoxia group were significantly higher than those of the TG2 siRNA hypoxia group. The results showed statistically significant differences (P<0.05). By contrast, the protein levels in the cytoplasm were significantly lower than those of the TG2 siRNA hypoxia group, with differences being statistically significant (P<0.05). The differences in apoptotic rates between the hypoxia and TG2 siRNA hypoxia groups were also statistically significant (P<0.05). Under hypoxic conditions, a high TG2 expression inhibited the apoptosis of the MG-63 osteosarcoma cell line. This effect was probably associated with its suppressive activity on the transportation of cytochrome c and caspase-3 from nucleus to cytoplasm.

Silencing of TGM2 reverses epithelial to mesenchymal transition and modulates the chemosensitivity of breast cancer to docetaxel

Epithelial to mesenchymal transition (EMT) plays a critical role in drug resistance. The aim of the present study was to further elucidate its role by examining the effect of tissue transglutaminase (TG2) on EMT and drug resistance in breast cancer. An antisense lentiviral (LV) short hairpin (sh)RNA construct specific to the TG2 gene (TGM2) was designed, synthesized and stably transfected into MDA-MB-231 cells to silence TGM2 by RNA interference (RNAi). The transfected cells expressed low levels of TG2 and constituted the RNAi (TGM2-shRNA) group. A control (NC) group was also established by transfecting MDA-MB-231 cells with scrambled shRNA. The expression levels of TG2, E-cadherin, vimentin and B-cell lymphoma (Bcl)-2 in the cells were examined via western blotting. The transfected MDA-MB-231 cells were divided into four groups, two of which were treated with doxetaxel (TXT): NC, RNAi, TXT and RNAi + TXT groups,. Cell proliferation was analyzed by MTT assay and cell apoptosis was detected by flow cytometry. An in vivo assay was also conducted, in which MDA-MB-231 cells transfected with scrambled shRNA or TGM2-shRNA were subcutaneously implanted into nude mice. After 2 weeks, TXT or vehicle was intraperitoneally administered at a dose of 10 mg/kg on day 1 of every week and tumor growth was monitored. Following the silencing of TGM2 in the MDA-MB-231 cells, the cells showed changes in morphology, indicating that an increased expression of TG2 was associated with a mesenchymal morphology. Transfection of the cells with TGM2-shRNA affected the expression of TG2, E-cadherin, vimentin and Bcl-2. In the MTT assay, the proliferation of MDA-MB-231 cells was significantly inhibited in the RNAi group compared with the control group (P<0.05), and the inhibitory effect increased in a time-dependent manner. Following treatment with TXT for 48 h, apoptosis was significantly promoted in the RNAi + TXT group compared with that in the other groups (P<0.05). Measurement of the tumors in the nude mice indicated that the combination of RNAi and TXT brought about a stronger antitumor effect than either treatment alone. These results suggest that the downregulation of TG2 reversed EMT and modulated the chemosensitivity of breast cancer to TXT. TG2 may be an important predictive and prognostic factor for the treatment efficacy of chemotherapy in patients with breast cancer.

Transglutaminase is a tumor cell and cancer stem cell survival factor.

Recent studies indicate that cancer cells express elevated levels of type II transglutaminase (TG2), and that expression is further highly enriched in cancer stem cells derived from these cancers. Moreover, elevated TG2 expression is associated with enhanced cancer stem cell marker expression, survival signaling, proliferation, migration, invasion, integrin-mediated adhesion, epithelial-mesenchymal transition, and drug resistance. TG2 expression is also associated with formation of aggressive and metastatic tumors that are resistant to conventional therapeutic intervention. This review summarizes the role of TG2 as a cancer cell survival factor in a range of tumor types, and as a target for preventive and therapeutic intervention. The literature supports the idea that TG2, in the closed/GTP-binding/signaling conformation, drives cancer cell and cancer stem cell survival, and that TG2, in the open/crosslinking conformation, is associated with cell death.

MiR-19-Mediated Inhibition of Transglutaminase-2 Leads to Enhanced Invasion and Metastasis in Colorectal Cancer.

Transglutaminase-2 (TG2) is a critical cross-linking enzyme in the extracellular matrix (ECM) and tumor microenvironment (TME). Although its expression has been linked to colorectal cancer, its functional role in the processes that drive disease appears to be context dependent. There is now considerable evidence of a role for microRNAs (miRNA) in the development and progression of cancer, including metastasis. A cell model of metastatic colon adenocarcinoma was used to investigate the contribution of miRNAs to the differential expression of TG2, and functional effects on inflammatory and invasive behavior. The impact of TG2 in colorectal cancer was analyzed in human colorectal tumor specimens and by manipulations in SW480 and SW620 cells. Effects on invasive behavior were measured using Transwell invasion assays, and cytokine production was assessed by ELISA. TG2 was identified as a target for miR-19 by in silico analysis, which was confirmed experimentally. Functional effects were evaluated by overexpression of pre-miR-19a in SW480 cells. Expression of TG2 correlated inversely with invasive behavior, with knockdown in SW480 cells leading to enhanced invasion, and overexpression in SW620 cells the opposite. TG2 expression was observed in colorectal cancer primary tumors but lost in liver metastases. Finally, miR-19 overexpression and subsequent decreased TG2 expression was linked to chromosome-13 amplification events, leading to altered invasive behavior in colorectal cancer cells. Chromosome-13 amplification in advanced colorectal cancer contributes to invasion and metastasis by upregulating miR-19, which targets TG2.

Tissue transglutaminase-interleukin-6 axis facilitates peritoneal tumor spreading and metastasis of human ovarian cancer cells.

Inflammation has recently been implicated in cancer formation and progression. As tissue transglutaminase (TG2) has been associated with both inflammatory signaling and tumor cell behavior, we propose that TG2 may be an important link inducing interleukin-6 (IL-6)-mediated cancer cell aggressiveness, including cancer stem cell-like characteristics and distant hematogenous metastasis. We evaluated the effect of differential TG2 and IL-6 expression on in vivo distant metastasis of human ovarian cancer cells. IL-6 production in human ovarian cancer cells was dependent on their TG2 expression levels. The size and efficiency of tumor sphere formation were correlated with TG2 expression levels and were dependent on TG2-mediated IL-6 secretion in human ovarian cancer cells. Primary tumor growth and propagation in the peritoneum and distant hematogenous metastasis into the liver and lung were also dependent on TG2 and downstream IL-6 expression levels in human ovarian cancer cells. In this report, we provide evidence that TG2 is an important link in IL-6-mediated tumor cell aggressiveness, and that TG2 and downstream IL-6 could be important mediators of distant hematogenous metastasis of human ovarian cancer cells. Intervention specific to TG2 and/or downstream IL-6 in ovarian cancer cells could provide a promising means to control tumor metastasis.

Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2–syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.