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O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy
Abstract
O-GlcNAc is a monosaccharide attached to serine or threonine hydroxyl moieties on numerous nuclear and cytoplasmic proteins; O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Although recent studies have shown that O-GlcNAcylation plays essential roles in breast cancer progression, it is also necessary to know whether O-GlcNAcylation is involved in other types of human cancer. In this study, O-GlcNAcylation levels and the expressions of OGT and OGA in human lung and colon cancer tissues were examined by immunohistochemistry analysis. We found that O-GlcNAcylation as well as OGT expression was significantly elevated in the cancer tissues compared with that in the corresponding adjacent tissues. Additionally, the roles of O-GlcNAcylation in the malignancy of lung and colon cancer were investigated in vitro. The results showed that O-GlcNAcylation markedly enhanced the anchorage-independent growth of lung and colon cancer cells; O-GlcNAcylation could also enhance lung and colon cancer invasion in a context-dependent manner. All together, this study suggests that O-GlcNAcylation might play important roles in lung and colon cancer formation and progression, and may be a valuable target for diagnosis and therapy of cancer.
... Oxygen levels have an effect on the cellular metabolism and mitochondrial activity [6,125]. By using 3D culture models, we have aimed to achieve this naturally via the spheroid's hypoxic core [113,[126][127][128]. ...
... Phosphorylation of histones has been found to be a significant marker of tumor grade and mitotic index in breast cancer [123] and proliferative marker in bladder cancer [124]. Increased O-GlcNAc glycosylation is consistently found in cancer as well (breast [125,126], prostate [127], lung [128], colorectal [128,129], liver [130], and nonsolid cancers such as chronic lymphocytic leukemia [131]) and has been correlated with increased metastatic potential. Unique modifications of histones for degradation also reveal the importance of histone turnover and homeostasis (and proteostasis) in epigenetic regulation [132]. ...
... Phosphorylation of histones has been found to be a significant marker of tumor grade and mitotic index in breast cancer [123] and proliferative marker in bladder cancer [124]. Increased O-GlcNAc glycosylation is consistently found in cancer as well (breast [125,126], prostate [127], lung [128], colorectal [128,129], liver [130], and nonsolid cancers such as chronic lymphocytic leukemia [131]) and has been correlated with increased metastatic potential. Unique modifications of histones for degradation also reveal the importance of histone turnover and homeostasis (and proteostasis) in epigenetic regulation [132]. ...
Current drug screening protocols use in vitro cancer cell panels grown in 2D to evaluate drug response and select the most promising candidates for further in vivo testing. Most drug candidates fail at this stage, not showing the same efficacy in vivo as seen in vitro. An improved first screening that is more translatable to the in vivo tumor situation could aid in reducing both time and cost of cancer drug development. 3D cell cultures are an emerging standard for in vitro cancer cell models, being more representative of in vivo tumour conditions. To overcome the translational challenges with 2D cell cultures, 3D systems better model the more complex cell-to-cell contact and nutrient levels present in a tumour, improving our understanding of cancer complexity. Furthermore, cancer cells exhibit altered metabolism, a phenomenon described a century ago by Otto Warburg, and possibly related to changes in nutrient access. However, there are few reports on how 3D cultures differ metabolically from 2D cultures, especially when grown in physiological glucose conditions. Along with this, metabolic drug targeting is considered an underutilized and poorly understood area of cancer therapy. Therefore, the aim of this work was to investigate the effect of culture conditions on response to metabolic drugs and study the metabolism of 3D spheroid cultures in detail. To achieve this, multiple cancer cell lines were studied in high and low glucose concentrations and in 2D and 3D cultures. We found that glucose concentration is important at a basic level for growth properties of cell lines with different metabolic phenotypes and it affects sensitivity to metformin. Furthermore, metformin is able to shift metabolic phenotype away from OXPHOS dependency. There are significant differences in glucose metabolism of 3D cultures compared to 2D cultures, both related to glycolysis and oxidative phosphorylation. Spheroids have higher ATP-linked respiration in standard nutrient conditions and higher non-aerobic ATP production in the absence of supplemented glucose. Multi-round treatment of spheroids is able to show more robust response than standard 2D drug screening, including resistance to therapy. Results from 2D cultures both over and underestimate drug response at different concentrations of 5-fluorouracil (5-FU). A higher maximum effect of 5-FU is seen in models with lower OCR/ECAR ratios, an indication of a more glycolytic metabolic phenotype. In conclusion, both culture method and nutrient conditions are important consideration for in vitro cancer models. There is good reason to not maintain in vitro cultures in artificially high glucose conditions. It can have downstream affects on drug response and likely other important metrics. If possible, assays should also be implemented in 3D. If not in everyday assays, at least as a required increase in complexity to validate 2D results. Finally, metabolism even in the small scope presented here, is complex in terms of phenotypic variation. This shows the importance of metabolic screening in vitro to better understand the effects of these small changes and to model how a specific tumor may behave based on its complex metabolism.
... Therefore, as a downstream pathway of HBP, O-GlcNAcylation in cancer cells is largely affected by this particular mode of metabolism. Hyper-O-GlcNAcylation has been observed in almost all types of cancer (Ma and Vosseller 2014), including ovarian cancer (Queiroz et al. 2016), cervical cancer (Zeng et al. 2016), breast cancer (Caldwell et al. 2010), endometrial cancer , liver cancer (Xu et al. 2017), colorectal cancer (Mi et al. 2011), cholangiocarcinoma (Phoomak et al. 2012), pancreatic cancer , gastric cancer (Jiang et al. 2016), esophageal squamous cell carcinoma (Qiao et al. 2012), bladder cancer, (Jin et al. 2020a) prostate cancer (Lynch et al. 2012), renal cell carcinoma (Wang et al. 2019), lung cancer (Lin et al. 2018), laryngeal cancer (Starska et al. 2015), thyroid papilloma , chronic and acute lymphoblastic leukemia (Shi et al. 2010;Zhang et al. 2017a) and glioblastoma (Ciraku et al. 2022). In addition to participating in metabolic reprogramming of cancer cells, O-GlcNAcylation also linked to various hallmarks of cancer, including cancer cell survival, The TP53 S149Ffs*32 mutation is likely oncogenic Prostate Adenocarcinoma S149 S149Pfs*21 The TP53 S149Pfs*21 mutation is likely oncogenic Pancreatic Adenocarcinoma S149 S149Ffs*32 ...
... Hyper-O-GlcNAcylation and elevated OGT levels were observed in colorectal cancer (CRC) tissues compared with those in adjacent normal tissues, while OGA expression showed no significant difference between tumor and normal tissues. (Mi et al. 2011;Phueaouan et al. 2013;Steenackers et al. 2016) Hyper-O-GlcNAcylation participates in the progression of CRC through multiple pathways. Recently, Zhu et al. (Zhu et al. 2019) showed that O-GlcNAcylation of transcription factor YIN-YANG-1 (YY1) at Thr236 could enhance the expression of solute carrier family 22 member 15 (SLC22A15) and aralkylamine N-acetyltransferase (AANAT) and increased its own protein stability, thereby exerting an oncogenic effect in CRC cells. ...
... Many studies have reported the role of O-GlcNAcylation in malignant tumors of the respiratory system. Mi et al. (Mi et al. 2011) not only observed elevated OGT and O-GlcNAcylation in lung cancer, but also indicated that hyper-O-GlcNAcylation would increase the growth and invasion of lung cancer cells. Lin et al. (Lin et al. 2018) showed a clinical relationship between OGT and lung cancer: Hyper-O-GlcNAcylation independently predicted a worse prognosis in patients with stage I lung adenocarcinoma. ...
O -linked β- d - N -acetylglucosamine ( O -GlcNAc) is an important post-translational modification of serine or threonine residues on thousands of proteins in the nucleus and cytoplasm of all animals and plants. In eukaryotes, only two conserved enzymes are involved in this process. O -GlcNAc transferase is responsible for adding O -GlcNAc to proteins, while O -GlcNAcase is responsible for removing it. Aberrant O -GlcNAcylation is associated with a variety of human diseases, such as diabetes, cancer, neurodegenerative diseases, and cardiovascular diseases. Numerous studies have confirmed that O -GlcNAcylation is involved in the occurrence and progression of cancers in multiple systems throughout the body. It is also involved in regulating multiple cancer hallmarks, such as metabolic reprogramming, proliferation, invasion, metastasis, and angiogenesis. In this review, we first describe the process of O -GlcNAcylation and the structure and function of O -GlcNAc cycling enzymes. In addition, we detail the occurrence of O -GlcNAc in various cancers and the role it plays. Finally, we discuss the potential of O -GlcNAc as a promising biomarker and novel therapeutic target for cancer diagnosis, treatment, and prognosis.
... Néanmoins, en fonction des conditions environnementales, les cellules tumorales peuvent présenter une flexibilité métabolique et conserver leur activité mitochondriale (Obre and Rossignol, 2015). (Caldwell et al., 2010;Gu et al., 2010;Champattanachai et al., 2013), LLC (Leucémiques Lymphoïdes Chroniques) (Shi et al., 2010), pulmonaires (Mi et al., 2011), hépatiques , prostatiques (Lynch et al., 2012), endométriaux (Krześlak et al., 2012), pancréatiques , colorectaux (Mi et al., 2011;Steenackers et al., 2016;Yu et al., 2019, p.5), laryngés (Starska et al., 2015) et vésicaux . (Champattanachai et al., 2013;Trinca et al., 2018). ...
... Néanmoins, en fonction des conditions environnementales, les cellules tumorales peuvent présenter une flexibilité métabolique et conserver leur activité mitochondriale (Obre and Rossignol, 2015). (Caldwell et al., 2010;Gu et al., 2010;Champattanachai et al., 2013), LLC (Leucémiques Lymphoïdes Chroniques) (Shi et al., 2010), pulmonaires (Mi et al., 2011), hépatiques , prostatiques (Lynch et al., 2012), endométriaux (Krześlak et al., 2012), pancréatiques , colorectaux (Mi et al., 2011;Steenackers et al., 2016;Yu et al., 2019, p.5), laryngés (Starska et al., 2015) et vésicaux . (Champattanachai et al., 2013;Trinca et al., 2018). ...
... Ainsi, l'augmentation du métabolisme glucidique associée à une surexpression de l'OGT et à une hyper-O-GlcNAcylation des protéines, notamment des oncoprotéines, a été décrite dans les cancers (cf. 8.4.3 page 202) dont le CCR(Mi et al., 2011;Steenackers et al., 2016;Yu et al., 2019). Plus particulièrement, notre équipe a montré que la O-GlcNAcylation participerait à la tumorigenèse du CCR par glycosylation et stabilisation de l'oncoprotéine β-caténine sur la Thr 41. ...
La O-GlcNAcylation (O-N-acétylglucosaminylation) est une MPT (modification post-traductionnelle) dynamique et réversible catalysée par un unique couple d’enzymes antagonistes : l’OGT (O-GlcNAc transférase) et l’OGA (O GlcNAcase). Elle est considérée comme un véritable senseur nutritionnel et régule un grand nombre de mécanismes cellulaires fondamentaux. En ciblant des oncoprotéines et des suppresseurs de tumeur, sa dérégulation est associée à la cancérogenèse et la progression tumorale. En revanche, son rôle dans la réponse aux thérapies anti-cancéreuses est très peu étudié. Il a été néanmoins montré récemment que l’hyper-O-GlcNAcylation impacte la réponse de certains cancers à des drogues telles que le tamoxifène, le cisplatine, le bortézomib et le 5-FU (5-fluorouracile). Le 5-FU est la chimiothérapie de référence du CCR (cancer colorectal) et la TS (Thymidylate Synthase) sa cible principale. La surexpression de la TS est un biomarqueur de résistance au 5-FU utilisé en clinique. La TS a été montrée comme étant O-GlcNAcylée mais le rôle de cette MPT n’a pas été élucidé. Il nous est donc paru intéressant d’analyser le « cross-talk » entre O-GlcNAcylation et réponse au 5-FU dans le CCR dans l’hypothèse que la O-GlcNAcylation pourrait impacter la sensibilité au 5-FU en régulant sa cible TS. Un modèle murin in vivo de CCR humains et des cellules coliques non cancéreuses et cancéreuses ont été utilisés pour analyser l’effet du 5-FU sur la O-GlcNAcylation globale des protéines et réciproquement l’impact de la O-GlcNAcylation sur le niveau et l’activité de la TS, et la réponse au 5-FU. Nos données in vitro corroborent nos résultats in vivo et soutiennent que le 5-FU diminue la O-GlcNAcylation globale et que, réciproquement, la O-GlcNAcylation augmente le niveau de TS et sensibilise le CCR au 5-FU. Nous avons déchiffré le mécanisme moléculaire sous-jacent mettant en lumière le rôle de la O-GlcNAcylation dans la stabilisation de la TS et sa protection contre la dégradation protéasomale. Deux sites de O-GlcNAcylation de la TS ont été identifiés : la Thr251 à l’interface de dimérisation de l’enzyme et la Thr306 dans la séquence dégron carboxy-terminale connue pour contrôler sa dégradation. Ensemble nos résultats proposent une nouvelle stratégie thérapeutique combinant le 5-FU à un inhibiteur de l’OGA afin d’améliorer la réponse du CCR à la chimiothérapie à base de 5-FU.
... It has recently been shown that many oncogene and tumor suppressor genes can be acetylated with OGlcNAc, demonstrating the importance of O-GlcNAc glycosylation as a cancer regulator [43]. O-GlcNAc glycosylation has been shown to increase the in vitro invasion of breast cancer cells and in vivo lung metastasis [44]. It has also been recorded that with increased OGT expression, O-GlcNAc glycosylation increases significantly in human lung and colon cancer tissues compared to the relevant [44]. ...
... O-GlcNAc glycosylation has been shown to increase the in vitro invasion of breast cancer cells and in vivo lung metastasis [44]. It has also been recorded that with increased OGT expression, O-GlcNAc glycosylation increases significantly in human lung and colon cancer tissues compared to the relevant [44]. Toivonen et al. reported that the increase in OGT gene expression was significantly higher in the patient group than in the control group in postmenopausal women receiving hormone therapy. ...
... In another study, it was recorded that O-GlcNAc glycosylation and in parallel to this, OGT expression levels, increased in premalignant and malignant skin tumors [45]. However, previous research has reported that augmentation of O-GlcNAcylation and OGT levels are associated with the malignant phenotypes of most cancers including breast [17,46], colon [44,47,48], liver [49][50][51] and prostate [52,53]. ...
Objectives
The aim of this study was to investigate BAP1 , OGT and YY1 genes and protein levels in 12 samples (8 males, 4 females) of eyelid tumor tissue with basal cell carcinoma (BCC) and 12 normal control subjects (8 males, 4 females).
Methods
The expression levels of these genes were determined with RT-PCR and the protein levels and expression using ELISA and IHC methods, respectively.
Results
In RT-PCR analysis, statistically significant upregulated expression was determined of 1.84-fold of BAP1 , 2.85-fold of OGT and 3.06-fold of YY1 genes (p < 0.05). In the patient group, compared to the control group, there was a similar statistically significant strong correlation between the proteins (BAP1 and YY1; r = 0.850, BAP1 and OGT; r = 0.811, OGT and YY1; r = 0.755) (p < 0.05). In the ELISA and IHC analysis methods, a significant increase in BAP1 and YY1 protein expression levels was observed compared to the control group (p < 0.05).
Conclusions
The study results demonstrated that BAP1 and YY1 genes and protein levels were upregulated in eyelid tumor tissue with BCC.
... Several studies have shown that cancers, including prostate and colorectal cancers, harboring hyper-O-GlcNAcylation are associated with worse prognosis [77,78]. In squamous cell lung cancer, O-GlcNAcylation and increased OGT levels were observed in lung cancer cells compared with the adjacent lung tissue [78]. ...
... Several studies have shown that cancers, including prostate and colorectal cancers, harboring hyper-O-GlcNAcylation are associated with worse prognosis [77,78]. In squamous cell lung cancer, O-GlcNAcylation and increased OGT levels were observed in lung cancer cells compared with the adjacent lung tissue [78]. The expression of OGT in patients with stages II, III, and IV lung adenocarcinomas was higher than that in patients with stage I lung adenocarcinoma [79]. ...
... Cell lines of breast, colorectal, prostate, and hepatocellular carcinoma treated with investigational OGT inhibitors have shown a significant decrease in tumor growth [105][106][107][108][109]. Recently, tamoxifen-resistant breast cancer cell lines treated with OGT small molecule inhibitor OSMI-1 showed anti-tumor activity via epigenetic activation of the tumor-suppressor ERRFI1 [62]. Similarly, reductions in OGT levels have shown to inhibit growth of lung cancer cells; however, the role of OGT inhibitor in cancers have not been elucidated using an investigational agent [78]. ...
Cancer cells are able to reprogram their glucose metabolism and retain energy via glycolysis even under aerobic conditions. They activate the hexosamine biosynthetic pathway (HBP), and the complex interplay of O-linked N-acetylglucosaminylation (O-GlcNAcylation) via deprivation of nutrients or increase in cellular stress results in the proliferation, progression, and metastasis of cancer cells. Notably, cancer is one of the emerging diseases associated with O-GlcNAcylation. In this review, we summarize studies that delineate the role of O-GlcNAcylation in cancer, including its modulation in metastasis, function with receptor tyrosine kinases, and resistance to chemotherapeutic agents, such as cisplatin. In addition, we discuss the function of O-GlcNAcylation in eliciting immune responses associated with immune surveillance in the tumor microenvironment. O-GlcNAcylation is increasingly accepted as one of the key players involved in the activation and differentiation of T cells and macrophages. Finally, we discuss the prognostic role of O-GlcNAcylation and potential therapeutic agents such as O-linked β-N-acetylglucosamine-transferase inhibitors, which may help overcome the resistance mechanism associated with the reprogramming of glucose metabolism.
... En el carcinoma escamoso de pulmón, así como en el adenocarcinoma de colon, existe un aumento de la O-GlcNAcilación y de la OGT en el tejido tumoral, en comparación con el tejido no tumoral circundante. En ensayos in vitro, de las líneas celulares de ambos tipos de cáncer, la O-GlcNAcilación favorece el crecimiento independiente del anclaje celular, sugiriendo que la O-GlcNAcilación tiene un papel importante en la progresión tumoral en estos tipos de cáncer, por lo que se ha considerado como un blanco de referencia para el diagnóstico y la terapia en cáncer (Mi et al., 2011) (Tabla III). ...
... Por otra parte, es pertinente destacar que el aumento de la O-GlcNAc tiene diversas consecuencias en las células tumorales, un efecto común y frecuente, es la disminución en los niveles de E-cadherina, que ocasiona una pérdida de la adhesión celular, lo que facilita el proceso de metástasis de las células de cáncer de mama, de ovario, de colon y de próstata (Mi et al., 2011;Jin, Yu, Zhao, Wu & Yang, 2013;Gu et al., 2014, Jiang et al., 2019. ...
La O-GlcNAcilación, es una modificación postraduccional sensible al estado nutricional de la célula, en la que un residuo de N-acetil-D-glucosamina es añadido al grupo hidroxilo de Serina o Treonina en proteínas, a partir del sustrato UDP-GlcNAc por la O-GlcNAc transferasa y eliminado por la O-GlcNAcasa. La importancia de esta modificación radica en su capacidad para regular diferentes eventos celulares como son: el metabolismo, la proliferación, la resistencia a la muerte, el crecimiento y las vías de señalización, que impactan de forma positiva en la transformación y la progresión tumoral. Se ha observado cómo la O-GlcNAcilación tiene la capacidad de modificar la expresión de las moléculas de adhesión, y favorecer el proceso de migración y metástasis. El objetivo de esta revisión es describir cómo la O-GlcNAcilación impacta en las moléculas de adhesión en diversos carcinomas, y una reflexión sobre diversas direcciones de investigación en este tema.
... Dysregulation of O-GlcNAcylation is involved in the development of various diseases, such as type 2 diabetes, neurodegenerative diseases, and cancer [7,8]. O-GlcNAcylation level is relatively high in a majority of cancer cells, such as in breast cancer [9], ovarian cancer, cervical cancer [10], lung cancer and colon cancer [11], and this is attributable to an increase in OGT and/or GFAT activity [12]. The elevated steady-state levels of O-GlcNAcylated targets observed in many cancers provide these cells with a selective advantage for their growth, metastasis, and immune evasion [13]. ...
... Since the elevation of cellular O-GlcNAcylation levels in certain cancer cells (including HeLa and HCT-116) contributes to their migration [10,11,14], we hypothesized that treatment with ZQN-17 might suppress their migration. To validate this hypothesis, we performed a transwell migration assay using HeLa and HCT-116 cells. ...
Suppression of cellular O-GlcNAcylation can repress proliferation and migration of various cancer cells, which opens a new avenue for cancer therapy. Based on the regulation of insulin gene transcription, we designed a cell-based fluorescent reporter capable of sensing cellular O-GlcNAcylation in HEK293T cells. The fluorescent reporter mainly consists of a reporter (GFP), an internal reference (RFP), and an operator (NeuroD1), which serves as a “sweet switch” to control GFP expression in response to cellular O-GlcNAcylation changes. The fluorescent reporter can efficiently sense reduced levels of cellular O-GlcNAcylation in several cell lines. Using the fluorescent reporter, we screened 120 natural products and obtained one compound, sesamin, which could markedly inhibit protein O-GlcNAcylation in HeLa and HCT-116 cells and repress their migration in vitro. Altogether, the present study demonstrated the development of a novel strategy for anti-tumor drug screening, as well as for conducting gene transcription studies.
... It has been reported that the HBP influx is increased in nearly all cancers [23], and aberrant protein glycosylation is frequently associated with the onset and progression of CRC [27][28][29]. Studies show that OGT and O-GlcNAcylation are significantly elevated in human colon cancer tissues [42] and enhance the proliferation and migration of CRC cells [43]. GFPT1 overexpression also results in increased O-GlcNAcylation and regulates stem-like properties in colon and lung cancer cells [44]. ...
Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine–fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.
... We have previously reported that OGT/O-GlcNAc regulates pulmonary arterial smooth muscle cell proliferation and vascular angiogenesis in idiopathic pulmonary arterial hypertension (IPAH) (72). In addition, elevated O-GlcNAc has also been widely reported in lung cancer and in EMT (76)(77)(78)(79). These findings have been linked to increased glucose metabolism and metabolic shifts in glycolysis (Warburg effect), which both have been documented during the metabolic reprogramming of fibroblasts to myofibroblasts in IPF (21,22,80,81). ...
Background
Idiopathic pulmonary fibrosis (IPF) is a chronic pulmonary disease that is characterized by an excessive accumulation of extracellular matrix (ECM) proteins (e.g. collagens) in the parenchyma, which ultimately leads to respiratory failure and death. While current therapies exist to slow the progression, no therapies are available to resolve fibrosis.
Methods
We characterized the O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT)/O-GlcNAc axis in IPF using single-cell RNA-sequencing (scRNA-seq) data and human lung sections and isolated fibroblasts from IPF and non-IPF donors. The underlying mechanism(s) of IPF were further investigated using multiple experimental models to modulate collagen expression and accumulation by genetically and pharmacologically targeting OGT. Furthermore, we hone in on the transforming growth factor-beta (TGF-β) effector molecule, Smad3, by co-expressing it with OGT to determine if it is modified and its subsequent effect on Smad3 activation.
Results
We found that OGT and O-GlcNAc levels are upregulated in patients with IPF compared to non-IPF. We report that the OGT regulates collagen deposition and fibrosis resolution, which is an evolutionarily conserved process demonstrated across multiple species. Co-expression of OGT and Smad3 showed that Smad3 is O-GlcNAc modified. Blocking OGT activity resulted in decreased phosphorylation at Ser-423/425 of Smad3 attenuating the effects of TGF-β1 induced collagen expression/deposition.
Conclusion
OGT inhibition or knockdown successfully blocked and reversed collagen expression and accumulation, respectively. Smad3 is discovered to be a substrate of OGT and its O-GlcNAc modification(s) directly affects its phosphorylation state. These data identify OGT as a potential target in pulmonary fibrosis resolution, as well as other diseases that might have aberrant ECM/collagen accumulation.
... Specifically, the majority of upregulated immune genes in WT mice code for negative regulation of cell migration and positive regulation of leukocyte activation. For instance, we observed fivefold induced expression of O-linked N-acetylglucosamine transferase, and this enzyme is upregulated in lung cancer (Mi et al. 2011) and was reported to promote an inflammatory phenotype of macrophages (Chang et al. 2020b). Conversely, with cRaf transgenic animals, orosomucoid 1 (Orm1) is the only immune response gene that is upregulated (threefold, Fig. 2e) and a recent study demonstrated Orm1 to enhance the immunosuppressive function of tumor-associated macrophages (TAMs) (Matsusaka et al. 2021). ...
Tobacco smoke (TS) is the leading cause for lung cancer (LC), and female smokers are at a greater risk for LC. Yet, the underlying causes are unknown. We performed whole genome scans in TS exposed wild type and histologically characterized tumor lesions of cRaf transgenic mice. We constructed miRNA-gene and transcription factor-miRNA/gene regulatory networks and determined sex-specific gene regulations by evaluating hormone receptor activities. We validated the findings from TS exposed cRaf mice in a large cohort of smoking and never-smoking LC patients. When compared to males, TS prompted a sevenfold increase in tumor multiplicity in cRaf females. Genome-wide scans of tumor lesions identified 161 and 53 genes and miRNAs, which code for EGFR/MAPK signaling, cell proliferation, oncomirs and oncogenes, and 50% of DEGs code for immune response and tumor evasion. Outstandingly, in transgenic males, TS elicited upregulation of 20 tumor suppressors, some of which are the targets of the androgen and estrogen receptor. Conversely, in females, 18 tumor suppressors were downregulated, and five were specifically repressed by the estrogen receptor. We found TS to perturb the circadian clock in a sex-specific manner and identified a female-specific regulatory loop that consisted of the estrogen receptor, miR-22-3p and circadian genes to support LC growth. Finally, we confirmed sex-dependent tumor promoting effects of TS in a large cohort of LC patients. Our study highlights the sex-dependent genomic responses to TS and the interplay of circadian clock genes and hormone receptors in the regulation of oncogenes and oncomirs in LC growth.
... N-acetylglucosamine is an intracellular metabolite that is indirectly involved in cancer cell biology. This metabolite has been shown to be upregulated during cancer progression, as seen in breast [44], prostate [45], colon [46], lung [47], liver [48], bladder [49], pancreatic cancer [50], and leukemia [51]. Elevated N-acetylglucosamine levels were also shown to play a clinical importance in primary and metastatic tumors [45]. ...
The aim of this systematic review and meta-analysis is to assess the diagnostic potential of salivary metabolomics in the detection of oral potentially malignant disorders (OPMDs) and oral cancer (OC).
A systematic review was performed in accordance with the 3rd edition of the Centre for Reviews and Dissemination (CRD) and Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. Electronic searches for articles were carried out in the PubMed, Web of Science, and Scopus databases. The quality assessment of the included studies was evaluated using the Newcastle-Ottawa Quality Assessment Scale (NOS) and the new version of the QUADOMICS tool. Meta-analysis was conducted whenever possible. The effect size was presented using the Forest plot, whereas the presence of publication bias was examined through Begg’s funnel plot.
A total of nine studies were included in the systematic review. The metabolite profiling was heterogeneous across all the studies. The expression of several salivary metabolites was found to be significantly altered in OPMDs and OCs as compared to healthy controls. Meta-analysis was able to be conducted only for N-acetylglucosamine. There was no significant difference (SMD = 0.15; 95% CI − 0.25–0.56) in the level of N-acetylglucosamine between OPMDs, OC, and the control group.
Evidence for N-acetylglucosamine as a salivary biomarker for oral cancer is lacking. Although several salivary metabolites show changes between healthy, OPMDs, and OC, their diagnostic potential cannot be assessed in this review due to a lack of data. Therefore, further high-quality studies with detailed analysis and reporting are required to establish the diagnostic potential of the salivary metabolites in OPMDs and OC.
While some salivary metabolites exhibit significant changes in oral potentially malignant disorders (OPMDs) and oral cancer (OC) compared to healthy controls, the current evidence, especially for N-acetylglucosamine, is inadequate to confirm their reliability as diagnostic biomarkers. Additional high-quality studies are needed for a more conclusive assessment of salivary metabolites in oral disease diagnosis.
... Abnormal O-GlcNAcylation is a common phenomenon in CRC [86][87][88], which also predicts a poor prognosis [89,90]. When colorectal cancer tissues were compared to paracancerous tissues, the researchers discovered that O-GlcNAcylation levels in the former were substantially higher than in the latter [91]. As can be seen, O-GlcNAcylation, a new post-translational alteration, is strongly linked to CRC [92]. ...
O-GlcNAcylation, a recently discovered post-translational modification of proteins, plays a crucial role in regulating protein structure and function, and is closely associated with multiple diseases. Research has shown that O-GlcNAcylation is abnormally upregulated in most cancers, promoting disease progression. To elucidate the roles of O-GlcNAcylation in cancer, this review summarizes various cancer-associated biological events regulated by O-GlcNAcylation and the corresponding signaling pathways. This work may provide insights for future studies on the function or underlying mechanisms of O-GlcNAcylation in cancer.
... In lung cancer, O-GlcNAcylation and expression of OGT are increased, potentially promoting tumorigenesis and cancer progression (166). O-GlcNAcylation can accelerate the Kras G12Dinduced lung tumorigenesis (167). ...
Protein glycosylation is a widespread posttranslational modification that can impact the function of proteins. Dysregulated protein glycosylation has been linked to several diseases, including chronic respiratory diseases (CRDs). CRDs pose a significant public health threat globally, affecting the airways and other lung structures. Emerging researches suggest that glycosylation plays a significant role in regulating inflammation associated with CRDs. This review offers an overview of the abnormal glycoenzyme activity and corresponding glycosylation changes involved in various CRDs, including chronic obstructive pulmonary disease, asthma, cystic fibrosis, idiopathic pulmonary fibrosis, pulmonary arterial hypertension, non-cystic fibrosis bronchiectasis, and lung cancer. Additionally, this review summarizes recent advances in glycomics and glycoproteomics-based protein glycosylation analysis of CRDs. The potential of glycoenzymes and glycoproteins for clinical use in the diagnosis and treatment of CRDs is also discussed.
... Por lo que el incremento en la ingesta de glucosa debido al efecto Warburg, así como el incremento de la ingesta de glutamina, cooperan para provocar un incremento en el flujo de HBP, contribuyendo finalmente a un incremento en las concentraciones de UDP-GlcNAc (55), sustrato donador de la OGT. Confirmando lo anterior, se han observado niveles de O-GlcNAcilación aberrante con una tendencia al incremento, fenómeno conocido como hiper-O-GlcNAcilación en diferentes tipos de cáncer: en cáncer de mama (56,57), de próstata (58), de pulmón (59), colo-rectal (59,60), de hígado (61), y en leucemia linfoide crónica (62). Existe evidencia creciente que ha mostrado una relación entre los niveles de O-GlcNAc y diversas características distintivas del cáncer, como angiogénesis y metástasis; además, muchos supresores tumorales y oncogenes conocidos por ser iniciadores clave del fenotipo maligno canceroso, están regulados por O-GlcNac (p53, NFκB, c-Myc, HIF-1 y AKT) (63). ...
... Our work is in line with the observation that elevated O-GlcNAcylation correlates with different cancer types, such as breast cancer, prostate cancer, and bladder cancer (43). In colon cancer, both O-GlcNAc and OGT abundance increased in clinical patient samples (44). Here, we found that YTHDF1 O-GlcNAcylation boost the expression of c-Myc, at least in SW620 cells. ...
O-linked N-acetylglucosamine (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein we show that O-GlcNAc modifies the m6A mRNA reader YTHDF1 and fine-tunes its nuclear translocation by the exportin protein Crm1. First we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.
... The synthesis of mucin-type O-glycans is catalyzed by GALNT enzymes 51 . Different expression of GALNT and GCNT genes coding for GalNAc and GlcNAc transferase enzymes has already been described in several types of cancer, including lung cancer 52,53 . It is hypothesized that-similarly to these enzymes-glycosyltransferases involved in CS and HS biosynthesis may also be upregulated. ...
Lung cancer is one of the most common types of cancer with limited therapeutic options, therefore a detailed understanding of the underlying molecular changes is of utmost importance. In this pilot study, we investigated the proteomic and glycosaminoglycan (GAG) profile of ALK rearranged lung tumor tissue regions based on the morphological classification, mucin and stromal content. Principal component analysis and hierarchical clustering revealed that both the proteomic and GAG-omic profiles are highly dependent on mucin content and to a lesser extent on morphology. We found that differentially expressed proteins between morphologically different tumor types are primarily involved in the regulation of protein synthesis, whereas those between adjacent normal and different tumor regions take part in several other biological processes (e.g. extracellular matrix organization, oxidation–reduction processes, protein folding) as well. The total amount and the sulfation profile of heparan sulfate and chondroitin sulfate showed small differences based on morphology and larger differences based on mucin content of the tumor, while an increase was observed in both the total amount and the average rate of sulfation in tumors compared to adjacent normal regions.
... Further findings from this study showed that inhibition of OGT enzyme in NSCLC cell lines decreases O-GlcNAcylation. This decrease significantly affects two hallmarks in nsclc such as invasion and anchorage independent growth, these findings collectively imply that O-GlcNAcylation may contribute to the growth of lung cancer [32]. Certain glycolytic enzymes may be O-GlcNAc glycosylated, which could help control glycolysis and which in lung cancer cause distinct metabolic states. ...
Non-Small Cell Lung Cancer (NSCLC) is the most prevalent kind of lung cancer with around 85% of total lung cancer cases. Despite vast therapies being available, the survival rate is low (5 year survival rate is 15%) making it essential to comprehend the mechanism for NSCLC cell survival and progression. The plethora of evidences suggests that the Post Translational Modification (PTM) such as phosphorylation, methylation, acetylation, glycosylation, ubiquitination and SUMOylation are involved in various types of cancer progression and metastasis including NSCLC. Indeed, an in-depth understanding of PTM associated with NSCLC biology will provide novel therapeutic targets and insight into the current sophisticated therapeutic paradigm. Herein, we reviewed the key PTMs, epigenetic modulation, PTMs crosstalk along with proteogenomics to analyze PTMs in NSCLC and also, highlighted how epi‑miRNA, miRNA and PTM inhibitors are key modulators and serve as promising therapeutics.
... Changes in the glycosylation of proteins are a common phenomenon in cancer [11]. The increased proportion of branched N-glycans and the increased amount of O-glycans have already been reported in relation to lung cancer [8,12,13]. Proteoglycans (PGs), a specific class of glycoproteins, are macromolecules whose structure consists of a core protein and one or more glycosaminoglycan (GAG) chains covalently attached to it via a tetrasaccharide linker. PGs are predominantly located in the extracellular matrix and the plasma membrane. ...
Lung cancer is one of the most commonly diagnosed cancer types. Studying the molecular changes that occur in lung cancer is important to understand tumor formation and identify new therapeutic targets and early markers of the disease to decrease mortality. Glycosaminoglycan chains play important roles in various signaling events in the tumor microenvironment. Therefore, we have determined the quantity and sulfation characteristics of chondroitin sulfate and heparan sulfate in formalin-fixed paraffin-embedded human lung tissue samples belonging to different lung cancer types as well as tumor adjacent normal areas. Glycosaminoglycan disaccharide analysis was performed using HPLC-MS following on-surface lyase digestion. Significant changes were identified predominantly in the case of chondroitin sulfate; for example, the total amount was higher in tumor tissue compared to the adjacent normal tissue. We also observed differences in the degree of sulfation and relative proportions of individual chondroitin sulfate disaccharides between lung cancer types and adjacent normal tissue. Furthermore, the differences in the 6-O-/4-O-sulfation ratio of chondroitin sulfate were different between the lung cancer types. Our pilot study revealed that further investigation of the role of chondroitin sulfate chains and enzymes involved in their biosynthesis is an important aspect of lung cancer research.
... 57,58 O-GlcNAcylation is generally increased in human cancers, 59-61 directly impacting several nucleocytoplasmic signaling pathways. 16,32,[62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78] An example of a key cancer-related process impacted by O-GlcNAc is epithelial to mesenchymal transition (EMT). TGFβ activates Smad transcription factors leading to the transcription of EMT-related genes. ...
Glycosylation has a clear role in cancer initiation and progression, with numerous studies identifying distinct glycan features or specific glycoproteoforms associated with cancer. Common findings include that aggressive cancers tend to have higher expression levels of enzymes that regulate glycosylation as well as glycoproteins with greater levels of complexity, increased branching, and enhanced chain length1. Research in cancer glycoproteomics over the last 50-plus years has mainly focused on technology development used to observe global changes in glycosylation. Efforts have also been made to connect glycans to their protein carriers as well as to delineate the role of these modifications in intracellular signaling and subsequent cell function. This review discusses currently available techniques utilizing mass spectrometry-based technologies used to study glycosylation and highlights areas for future advancement.
... Dysregulated metabolism is one of the hallmarks of cancer, and many of the proteins that play important roles in the proteasome/ mTOR/mitochondrial axis (SI Appendix, Fig. S16) are aberrantly expressed in cancer. Increased levels of OGT expression and O-GlcNAc modification are often observed in cancer cells (50)(51)(52). Hyperactivation of mTOR signaling is observed in many types of cancers; mTOR signaling is the second most frequently altered signaling pathway in cancer, and multiple mTOR inhibitors have been used in cancer clinical trials (53,54). Increased expression of proteasome subunits and high proteasome activity are also characteristic of many cancer types (55)(56)(57), and proteasome inhibitors have been used in the clinic for the treatment of hematological malignancies (58). ...
O-GlcNAc transferase (OGT) modifies serine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We performed a genome-wide CRISPR-Cas9 screen in mouse embryonic stem cells (mESCs) to identify candidates whose depletion rescued the block in cell proliferation induced by OGT deficiency. We show that the block in cell proliferation in OGT-deficient cells stems from mitochondrial dysfunction secondary to mTOR (mechanistic target of rapamycin) hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phospho-proteomic screen. Our study highlights a unique series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness, and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.
... Upregulated in prostate cancer cell lines [67]. Upregulated in lung and colon cancer tissues [68]. ...
It is thought that accurate risk assessment and early diagnosis of breast cancer (BC) can help reduce cancer-related mortality. Proteomics analysis of breast milk may provide biomarkers of risk and occult disease. Our group works on the analysis of human milk samples from women with BC and controls to investigate alterations in protein patterns of milk that could be related to BC. In the current study, we used mass spectrometry (MS)-based proteomics analysis of 12 milk samples from donors with BC and matched controls. Specifically, we used one-dimensional (1D)-polyacrylamide gel electrophoresis (PAGE) coupled with nanoliquid chromatography tandem MS (nanoLC-MS/MS), followed by bioinformatics analysis. We confirmed the dysregulation of several proteins identified previously in a different set of milk samples. We also identified additional dysregulations in milk proteins shown to play a role in cancer development, such as Lactadherin isoform A, O-linked N-acetylglucosamine (GlcNAc) transferase, galactosyltransferase, recoverin, perilipin-3 isoform 1, histone-lysine methyltransferase, or clathrin heavy chain. Our results expand our current understanding of using milk as a biological fluid for identification of BC-related dysregulated proteins. Overall, our results also indicate that milk has the potential to be used for BC biomarker discovery, early detection and risk assessment in young, reproductively active women.
... O-GlcNAc primarily has a biological function in carcinogenesis through O-GlcNAcylating proteins such as p53 and β-catenin [87,88]. Recently, it has been observed that O-GlcNAcylation plays a role in a variety of human malignant tumors, including prostate cancer, colon cancer, lung cancer, breast cancer, etc. [89,90]. Wang L et al. discovered that O-GlcNAcylation levels and OGT expression in renal cancer cell lines and tissue samples were considerably higher than in normal controls. ...
Abstract
Renal cell carcinoma (RCC) is one of the most prevalent malignant tumors of the urinary system, accounting for around 2% of all cancer diagnoses and deaths worldwide. Clear cell RCC (ccRCC) is the most prevalent and aggressive histology with an unfavorable prognosis and inadequate treatment. Patients’ progression-free survival is considerably improved by surgery; however, 30% of patients develop metastases following surgery. Identifying novel targets and molecular markers for RCC prognostic detection is crucial for more accurate clinical diagnosis and therapy. Glycosylation is a critical post-translational modification (PMT) for cancer cell growth, migration, and invasion, involving the transfer of glycosyl moieties to specific amino acid residues in proteins to form glycosidic bonds through the activity of glycosyltransferases. Most cancers, including RCC, undergo glycosylation changes such as branching, sialylation, and fucosylation. In this review, we discuss the latest findings on the significance of aberrant glycans in the initiation, development, and progression of RCC. The potential biomarkers of altered glycans for the diagnosis and their implications in RCC have been further highlighted.
... A fine "Yin-Yang" occupancy competition mechanism between O-GlcNAcylation and phosphorylation on the same or adjacent Ser/Thr residues regulates protein's interaction, stability, subcellular localization, and enzymatic activity of their common target proteins (8). Elevated nutrients uptake and metabolism are correlated with increased HBP flow, UDP-GlcNAc level, and global protein O-GlcNAcylation in a wide variety of cancers including chronic lymphocytic leukemia (CLL), breast, lung, liver, prostate, endometrium, pancreas, colon, larynx and bladder (9)(10)(11)(12)(13)(14)(15)(16)(17). This aberrant hyper-O-GlcNAcylation is also the result of an alteration in the expression and activity of HBP enzymes and OGT (18)(19)(20)(21). ...
In cancer cells, metabolic reprogramming is associated with an alteration of the O-GlcNAcylation homeostasis. This post-translational modification (PTM) that attaches O-GlcNAc moiety to intracellular proteins is dynamically and finely regulated by the O-GlcNAc Transferase (OGT) and the O-GlcNAcase (OGA). It is now established that O-GlcNAcylation participates in many features of cancer cells including a high rate of cell growth, invasion, and metastasis but little is known about its impact on the response to therapies. The purpose of this review is to highlight the role of O-GlcNAc protein modification in cancer resistance to therapies. We summarize the current knowledge about the crosstalk between O-GlcNAcylation and molecular mechanisms underlying tumor sensitivity/resistance to targeted therapies, chemotherapies, immunotherapy, and radiotherapy. We also discuss potential benefits and strategies of targeting O-GlcNAcylation to overcome cancer resistance.
... We previously found that OGT expression is an independent prognostic factor in patients with lung adenocarcinoma [21]. It has been reported that knockdown of OGT expression in A549 lung cancer cells decreases their invasiveness in vitro [22]. TGF-βinduced epithelial-mesenchymal transition of A549 cells is accompanied by significantly increased OGT expression and cellular O-GlcNAcylation [23]. ...
Simple Summary
Lung cancer claims the most lives annually among cancers; to date, invasion and metastasis still pose challenges to effective treatment. O-GlcNAcylation, an enzymatic modification of proteins after biosynthesis, modulates the functions of many proteins. Aberrant O-GlcNAcylation is linked to pathogenic mechanisms of cancer, including invasion and metastasis. However, little is known about the profile of O-GlcNAcylated proteins involved in cancer aggressiveness. Here, by comparing profiles of O-GlcNAcylated proteins from two lung cancer cell lines different in their invasive potential, we identified candidates for O-GlcNAcylated proteins that may be involved in cancer aggressiveness. One of these candidates, SAM68, was further characterized. Results confirmed O-GlcNAcylation of SAM68; functional analyses on SAM68 with mutations at O-GlcNAcylation sites suggested a role of O-GlcNAcylated SAM68 in modulating lung cancer cell migration/invasion. Future elucidation of the functional significance of differential O-GlcNAcylation of proteins identified in this study may provide new insights into mechanisms of lung cancer progression.
Abstract
O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.
... 277 In the study of lung and colon cancer, not only O-GlcNAcylation and global O-GlcNAc transferase expression but also the OGT mRNA levels were evaluated in tumor tissues. 278 Similarly, a high level of O-GlcNAcylation in target proteins was detected and was associated with the pathogenesis of characterizes chronic lymphocytic leukemia. 279 O-GlcNAcylation targets are involved in various biological processes and signal cascades. ...
More and more in-depth studies have revealed that the occurrence and development of tumors depend on gene mutation and tumor heterogeneity. The most important manifestation of tumor heterogeneity is the dynamic change of tumor microenvironment (TME) heterogeneity. This depends not only on the tumor cells themselves in the microenvironment where the infiltrating immune cells and matrix together forming an antitumor and/or pro-tumor network. TME has resulted in novel therapeutic interventions as a place beyond tumor beds. The malignant cancer cells, tumor infiltrate immune cells, angiogenic vascular cells, lymphatic endothelial cells, cancer-associated fibroblastic cells, and the released factors including intracellular metabolites, hormonal signals and inflammatory mediators all contribute actively to cancer progression. Protein post-translational modification (PTM) is often regarded as a degradative mechanism in protein destruction or turnover to maintain physiological homeostasis. Advances in quantitative transcriptomics, proteomics, and nuclease-based gene editing are now paving the global ways for exploring PTMs. In this review, we focus on recent developments in the PTM area and speculate on their importance as a critical functional readout for the regulation of TME. A wealth of information has been emerging to prove useful in the search for conventional therapies and the development of global therapeutic strategies.
... To test this conjecture, we first investigated TS expression and O-GlcNAcylation levels in CRC tissues. Consistent with previously published data, O-GlcNAcylation levels are increased in colorectal tumors compared to normal tissues [32][33][34]. AOM/DSS treatment induces tumorigenesis that activates glucose and glutamine consumption to support cell proliferation. Increased nutrient availability, subsequent flux through Hexosamine Biosynthetic Pathway (HBP) and/or OGT activity lead to hyper-O-GlcNAcylation in Fig. 6 O-GlcNAcylation at T251 and T306 increases TS dimer stabilization by generating intra-and inter-monomer interactions. ...
Alteration of O-GlcNAcylation, a dynamic posttranslational modification, is associated with tumorigenesis and tumor progression. Its role in chemotherapy response is poorly investigated. Standard treatment for colorectal cancer (CRC), 5-fluorouracil (5-FU), mainly targets Thymidylate Synthase (TS). TS O-GlcNAcylation was reported but not investigated yet. We hypothesize that O-GlcNAcylation interferes with 5-FU CRC sensitivity by regulating TS. In vivo, we observed that combined 5-FU with Thiamet-G (O-GlcNAcase (OGA) inhibitor) treatment had a synergistic inhibitory effect on grade and tumor progression. 5-FU decreased O-GlcNAcylation and, reciprocally, elevation of O-GlcNAcylation was associated with TS increase. In vitro in non-cancerous and cancerous colon cells, we showed that 5-FU impacts O-GlcNAcylation by decreasing O-GlcNAc Transferase (OGT) expression both at mRNA and protein levels. Reciprocally, OGT knockdown decreased 5-FU-induced cancer cell apoptosis by reducing TS protein level and activity. Mass spectrometry, mutagenesis and structural studies mapped O-GlcNAcylated sites on T251 and T306 residues and deciphered their role in TS proteasomal degradation. We reveal a crosstalk between O-GlcNAcylation and 5-FU metabolism in vitro and in vivo that converges to 5-FU CRC sensitization by stabilizing TS. Overall, our data propose that combining 5-FU-based chemotherapy with Thiamet-G could be a new way to enhance CRC response to 5-FU.
... Instead, C-MYC increases OGT through HSP90A, an important molecular Ovary O-GlcNAcylation, cell migration and changes in E-Catherin level are correlated [28] HCC O-GlcNAcylation is linked with tumor recurrence [29] Bladder Content of OGT/OGA mRNAs helps predicting bladder cancer [30] Cholangiocarcinoma OGT overexpression and aggressiveness are correlated [31] Prostate Overexpression of OGT contributes to invasion, angiogenesis, and metastasis, associates with poor prognosis of patients [32][33][34] Endometrium Clinicpathologic conditions are correlated with OGT and OGA mRNA expression [35] Lung and colon OGT is overexpressed in cancer compared to adjacent tissues. Treatment with TMG increased cell anchorage-independent growth and invasion in cell lines [36,37] Chronic lymphocytic leukemia Indolent and aggressive clinical behavior of CLL cells were found to correlate with higher and lower O-GlcNAc levels, respectively [38] Thyroid cancer OGA enzyme activity is increased, cancer has less O-GlcNAcylated proteins [39] Breast Increased OGT level, increased o-GlcNAcylation level enhances the migration/invasion. Higher level of OGT and lower OGA was seen in higher grade of breast cancer tissues [40,41] Esophageal cancer OGT is overexpressed and promotes O-linked protein glycosylation EGF GFPT1 Increase expression [131] hypoxia Gfpt2 ...
Prevalent dysregulation of epigenetic modifications plays a pivotal role in cancer. Targeting epigenetic abnormality is a new strategy for cancer therapy. Understanding how conventional oncogenic factors cause epigenetic abnormality is of great basic and translational value. O-GlcNAcylation is a protein modification which affects physiology and pathophysiology. In mammals, O-GlcNAcylation is catalyzed by one single enzyme OGT and removed by one single enzyme OGA. O-GlcNAcylation is affected by the availability of the donor, UDP-GlcNAc, generated by the serial enzymatic reactions in the hexoamine biogenesis pathway (HBP). O-GlcNAcylation regulates a wide spectrum of substrates including many proteins involved in epigenetic modification. Like epigenetic modifications, abnormality of O-GlcNAcylation is also common in cancer. Studies have revealed substantial impact on HBP enzymes and OGT/OGA by oncogenic signals. In this review, we will first summarize how oncogenic signals regulate HBP enzymes, OGT and OGA in cancer. We will then integrate this knowledge with the up to date understanding how O-GlcNAcylation regulates epigenetic machinery. With this, we propose a signal axis from oncogenic signals through O-GlcNAcylation dysregulation to epigenetic abnormality in cancer. Further elucidation of this axis will not only advance our understanding of cancer biology but also provide new revenues towards cancer therapy.
... In cell physiology, UDP-GlcNAc is a substrate required for O-GlcNAcylation and N-/O-glycosylation to form glycoproteins and glycoconjugates [25]. The enhancement of O-GlcNAcylation was reported for lung tumor tissues [29]. Moreover, numerous enzymes related to glycosylation as well as the level of UDP-GlcNAc were increased in lung ADC compared to control tissues [8]. ...
Lung cancer represents one of the leading worldwide causes of cancer death, but the pathobiochemistry of this disease is still not fully understood. Here we characterize the lipidomic and metabolomic profiles of the tumor and surrounding normal tissues for 23 patients with non-small cell lung cancer. In total, 500 molecular species were identified and quantified by a combination of the lipidomic shotgun tandem mass spectrometry (MS/MS) analysis and the targeted metabolomic approach using liquid chromatography (LC) – MS/MS. The statistical evaluation includes multivariate and univariate methods with the emphasis on paired statistical approaches. Our research revealed significant changes in several biochemical pathways related to the central carbon metabolism, acylcarnitines, dipeptides as well as the disruption in the lipid metabolism observed mainly for glycerophospholipids, sphingolipids, and cholesteryl esters.
... Elevated global protein O-GlcNAcylation is a common finding in several solid cancers including breast (Caldwell et al., 2010;Krześlak et al., 2012), prostate (Lynch et al., 2012), colorectal (Mi et al., 2011), and liver (Zhu et al., 2012). These cancers are also associated with dysregulated NF-κB function, which contributes to tumorigenesis (Xia et al., 2014). ...
Nuclear factor-kappaB (NF-κB) is a pleiotropic, evolutionarily conserved transcription factor family that plays a central role in regulating immune responses, inflammation, cell survival, and apoptosis. Great strides have been made in the past three decades to understand the role of NF-κB in physiological and pathological conditions. Carcinogenesis is associated with constitutive activation of NF-κB that promotes tumor cell proliferation, angiogenesis, and apoptosis evasion. NF-κB is ubiquitously expressed, however, its activity is under tight regulation by inhibitors of the pathway and through multiple posttranslational modifications. O-GlcNAcylation is a dynamic posttranslational modification that controls NF-κB-dependent transactivation. O-GlcNAcylation acts as a nutrient-dependent rheostat of cellular signaling. Increased uptake of glucose and glutamine by cancer cells enhances NF-κB O-GlcNAcylation. Growing evidence indicates that O-GlcNAcylation of NF-κB is a key molecular mechanism that regulates cancer cell proliferation, survival and metastasis and acts as link between inflammation and cancer. In this review, we are attempting to summarize the current understanding of the cohesive role of NF-κB O-GlcNAcylation in inflammation and cancer.
... Aberrant metabolism is one of the hallmarks of cancer, and many of the proteins that play important roles in the proteasome/ mTOR/ mitochondrial axis (Supplementary Fig. 8) are aberrantly expressed in cancer. Increased levels of OGT expression and O-GlcNAc modification are often observed in cancer cells [30][31][32] . Hyperactivation of mTOR signaling is observed in many types of cancers; mTOR signaling is the second most frequently altered signaling pathway in cancer; and multiple mTOR inhibitors have been used in cancer clinical trials 33,34 . ...
O-GlcNAc transferase (OGT) catalyzes the modification of serine and threonine residues on nuclear and cytosolic proteins with O-linked N-acetylglucosamine (GlcNAc). OGT is essential for mammalian cell viability, but the underlying mechanisms are still enigmatic. We employed a genome-wide CRISPR-Cas9 viability screen in mouse embryonic stem cells (mESCs) with inducible Ogt gene deletion and showed that the block in cell viability induced by OGT deficiency stems from mitochondrial dysfunction secondary to mTOR hyperactivation. In normal cells, OGT maintains low mTOR activity and mitochondrial fitness through suppression of proteasome activity; in the absence of OGT, increased proteasome activity results in increased steady-state amino acid levels, which in turn promote mTOR lysosomal translocation and activation, and increased oxidative phosphorylation. mTOR activation in OGT-deficient mESCs was confirmed by an independent phosphoproteomic screen. Our study highlights a novel series of events whereby OGT regulates the proteasome/ mTOR/ mitochondrial axis in a manner that maintains homeostasis of intracellular amino acid levels, mitochondrial fitness and cell viability. A similar mechanism operates in CD8+ T cells, indicating its generality across mammalian cell types. Manipulating OGT activity may have therapeutic potential in diseases in which this signaling pathway is impaired.
... Glycosyltransferase OGT has been shown to enhance cell proliferation by stabilizing the c-Myc protein by combining it with β-Nacetylglucosamine [101]. Increased OGT expression was found in many tumors, including prostate [102], breast [103], lung, and colon cancers [104]. Lowering the level of OGT mRNA leads to a decrease in c-Myc protein in prostate cancer cells [102]. ...
Alterations in the expression level of the MYC gene are often found in the cells of various malignant tumors. Overexpressed MYC has been shown to stimulate the main processes of oncogenesis: uncontrolled growth, unlimited cell divisions, avoidance of apoptosis and immune response, changes in cellular metabolism, genomic instability, metastasis, and angiogenesis. Thus, controlling the expression of MYC is considered as an approach for targeted cancer treatment. Since c-Myc is also a crucial regulator of many cellular processes in healthy cells, it is necessary to find ways for selective regulation of MYC expression in tumor cells. Many recent studies have demonstrated that non-coding RNAs play an important role in the regulation of the transcription and translation of this gene and some RNAs directly interact with the c-Myc protein, affecting its stability. In this review, we summarize current data on the regulation of MYC by various non-coding RNAs that can potentially be targeted in specific tumor types.
... Therefore, in view of its importance in cellular processes, the disruption of O-GlcNAcylation is related to the pathological processes of many kinds of tumors. Several studies showed decreased O-GlcNAc levels in some tumor samples compared with matched adjacent tissues (Slawson et al., 2001), whereas other studies found accumulated O-GlcNAc, enhanced O-GlcNAcylation and increased OGT and OGA expression in breast, lung, and colon tumor tissues compared with the respective corresponding control tissues (Gu et al., 2010;Mi et al., 2011). A similar phenomenon was evident in patients with chronic lymphocytic leukemia (Shi et al., 2010). ...
Metabolic rewiring and epigenetic remodeling, which are closely linked and reciprocally regulate each other, are among the well-known cancer hallmarks. Recent evidence suggests that many metabolites serve as substrates or cofactors of chromatin-modifying enzymes as a consequence of the translocation or spatial regionalization of enzymes or metabolites. Various metabolic alterations and epigenetic modifications also reportedly drive immune escape or impede immunosurveillance within certain contexts, playing important roles in tumor progression. In this review, we focus on how metabolic reprogramming of tumor cells and immune cells reshapes epigenetic alterations, in particular the acetylation and methylation of histone proteins and DNA. We also discuss other eminent metabolic modifications such as, succinylation, hydroxybutyrylation, and lactylation, and update the current advances in metabolism- and epigenetic modification-based therapeutic prospects in cancer.
... Inactivation of VHL accounts for up to 92% of ccRCC [47], but rare mutation of VHL has been found in lung cancer [4], gastric cancer [6] and breast cancer [7], suggesting that non-mutational mechanisms might contribute to the loss of tumor suppressor function of pVHL in these types of cancers. Accumulating evidence has confirmed that elevated O-GlcNAcylation has been described in various types of cancers [48], including lung cancer [49]. O-GlcNAcylation indeed plays an essential role in regulating a number of proteins stability and functions [50]. ...
Yes-associated protein (YAP) is a vital transcriptional co-activator that activates cell proliferation and evasion of apoptosis for the promotion of tumorigenesis. The von Hippel-Lindau tumor suppressor protein (pVHL), as a critical component of E3 ubiquitin ligase, targets various substrates to regulate tumor progression. However, the precise molecular mechanisms of pVHL during tumorigenesis remain largely unclear. Herein, we found that there was a significant negative correlation between pVHL and YAP at protein level in the TCGA-LUAD dataset and our cohort. Over-expression of pVHL decreased YAP protein expression and reduced its transcriptional activity. Further study indicated that pVHL did not affect YAP mRNA level but decreased YAP protein stability in a lysosome-dependent manner. In addition, the pVHL-mediated degradation of YAP inhibited cellular proliferation, migration, and enhanced chemosensitivity to cisplatin in lung adenocarcinoma cells. Interestingly, the pVHL-mediated YAP degradation was blocked by elevated O-GlcNAcylation. Collectively, our findings demonstrate that pVHL modulates the lysosomal degradation of YAP, and may provide more clues to better understanding the tumor suppressive effects of pVHL.
Tripartite Motif 14 (TRIM14) is an oncoprotein that belongs to the E3 ligase TRIM family, which is involved in the progression of various tumors except for non-small cell lung carcinoma (NSCLC). However, little is currently known regarding the function and related mechanisms of TRIM14 in NSCLC. Here, we found that the TRIM14 protein was downregulated in lung adenocarcinoma tissues compared with the adjacent tissues, which can suppress tumor cell proliferation and migration both in vitro and in vivo. Moreover, TRIM14 can directly bind to glutamine fructose-6-phosphate amidotransferase 1 (GFAT1), which in turn results in the degradation of GFAT1 and reduced O-glycosylation levels. GFAT1 is a key enzyme in the rate-limiting step of the hexosamine biosynthetic pathway (HBP). Replenishment of N-acetyl-d-glucosamine can successfully reverse the inhibitory effect of TRIM14 on the NSCLC cell growth and migration as expected. Collectively, our data revealed that TRIM14 suppressed NSCLC cell proliferation and migration through ubiquitination and degradation of GFAT1, providing a new regulatory role for TRIM14 on HBP.
Cell cycle errors can lead to mutations, chromosomal instability, or death; thus, the precise control of cell cycle progression is essential for viability. The nutrient-sensing posttranslational modification, O-GlcNAc, regulates the cell cycle allowing one central control point directing progression of the cell cycle. O-GlcNAc is a single N-acetylglucosamine sugar modification to intracellular proteins that is dynamically added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. These enzymes act as a rheostat to fine-tune protein function in response to a plethora of stimuli from nutrients to hormones. O-GlcNAc modulates mitogenic growth signaling, senses nutrient flux through the hexosamine biosynthetic pathway, and coordinates with other nutrient-sensing enzymes to progress cells through Gap phase 1 (G1). At the G1/S transition, O-GlcNAc modulates checkpoint control, while in S Phase, O-GlcNAcylation coordinates the replication fork. DNA replication errors activate O-GlcNAcylation to control the function of the tumor-suppressor p53 at Gap Phase 2 (G2). Finally, in mitosis (M phase), O-GlcNAc controls M phase progression and the organization of the mitotic spindle and midbody. Critical for M phase control is the interplay between OGT and OGA with mitotic kinases. Importantly, disruptions in OGT and OGA activity induce M phase defects and aneuploidy. These data point to an essential role for the O-GlcNAc rheostat in regulating cell division. In this review, we highlight O-GlcNAc nutrient sensing regulating G1, O-GlcNAc control of DNA replication and repair, and finally, O-GlcNAc organization of mitotic progression and spindle dynamics.
The enzymatic modification of protein serine or threonine residues by N-acetylglucosamine, namely O-GlcNAcylation, is a ubiquitous post-translational modification that frequently occurs in the nucleus and cytoplasm. O-GlcNAcylation is dynamically regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase, and regulates nearly all cellular processes in epigenetics, transcription, translation, cell division, metabolism, signal transduction and stress. Aberrant O-GlcNAcylation has been shown in a variety of diseases, including diabetes, neurodegenerative diseases and cancers. Deciphering O-GlcNAcylation remains a challenge due to its low abundance, low stoichiometry and extreme lability in most tandem mass spectrometry. Separation or enrichment of O-GlcNAc proteins or peptides from complex mixtures has been of great interest because quantitative analysis of protein O-GlcNAcylation can elucidate their functions and regulatory mechanisms in disease. However, valid and specific analytical methods are still lacking, and efforts are needed to further advance this direction. Here, we provide an overview of recent advances in various analytical methods, focusing on chemical oxidation, affinity of antibodies and lectins, hydrophilic interaction, and enzymatic addition of monosaccharides in conjugation with these methods. O-GlcNAcylation quantification has been described in detail using mass-spectrometric or non-mass-spectrometric techniques. We briefly summarized dysregulated changes in O-GlcNAcylation in disease.
Despite the remarkable progress in cancer treatment up to now, we are still far from conquering the disease. The most substantial change after the malignant transformation of normal cells into cancer cells is the alteration in their metabolism. Cancer cells reprogram their metabolism to support the elevated energy demand as well as the acquisition and maintenance of their malignancy, even in nutrient-poor environments. The metabolic alterations, even under aerobic conditions, such as the upregulation of the glucose uptake and glycolysis (the Warburg effect), increase the ROS (reactive oxygen species) and glutamine dependence, which are the prominent features of cancer metabolism. Among these metabolic alterations, high glutamine dependency has attracted serious attention in the cancer research community. In addition, the oncogenic signaling pathways of the well-known important genetic mutations play important regulatory roles, either directly or indirectly, in the central carbon metabolism. The identification of the convergent metabolic phenotypes is crucial to the targeting of cancer cells. In this review, we investigate the relationship between cancer metabolism and the signal transduction pathways, and we highlight the recent developments in anti-cancer therapy that target metabolism.
The expression of O-GlcNAc transferase (OGT) and its catalytic product, O-GlcNAcylation (O-GlcNAc), are elevated in many types of cancers, including prostate cancer (PC). Inhibition of OGT serves as a potential strategy for PC treatment alone or combinational therapy. PC is the second common cancer type in male worldwide, for which chemotherapy is still the first-line treatment. However, the function of inhibition of OGT on chemotherapeutic response in PC cells is still unknown. In this study, we show that inhibition of OGT by genetic knockdown using shRNA or by chemical inhibition using OGT inhibitors sensitize PC cells to docetaxel, which is the most common chemotherapeutic agent in PC chemotherapy. Furthermore, we identified that microRNA-140 (miR-140) directly binds to OGT mRNA 3′ untranslated region and inhibits OGT expression. Moreover, docetaxel treatment stimulates miR-140 expression, whereas represses OGT expression in PC cells. Overexpression of miR-140 enhanced the drug sensitivity of PC cells to docetaxel, which could be reversed by overexpression of OGT. Overall, this study demonstrates miR-140/OGT axis as therapeutic target in PC treatment and provides a promising adjuvant therapeutic strategy for PC therapy.
Untargeted Nuclear Magnetic Resonance (NMR) metabolomics of polar extracts from the pancreata of a caerulin-induced mouse model of pancreatitis (Pt) and of a transgenic mouse model of pancreatic cancer (PCa) were used to find metabolic markers of Pt and to characterize the metabolic changes accompanying PCa progression. Using multivariate analysis a 10-metabolite metabolic signature specific to Pt tissue was found to distinguish the benign condition from both normal tissue and precancerous tissue (low grade pancreatic intraepithelial neoplasia, PanIN, lesions). The mice pancreata showed significant changes in the progression from normal tissue, through low-grade and high-grade PanIN lesions to pancreatic ductal adenocarcinoma (PDA). These included increased lactate production, amino acid changes consistent with enhanced anaplerosis, decreased concentrations of intermediates in membrane biosynthesis (phosphocholine and phosphoethanolamine) and decreased glycosylated uridine phosphates, reflecting activation of the hexosamine biosynthesis pathway and protein glycosylation.
Le carcinome hépatocellulaire (CHC) est la tumeur primitive la plus fréquente dans le foie se développant essentiellement sur un foie cirrhotique. Le CHC est au 6ème rang en termes d’incidence et au 2nd en termes de mortalité. La compréhension imparfaite des mécanismes impliqués dans le CHC empêche le développement de traitements efficaces contre ce cancer. Notre étude a porté sur les effets d’une lectine humaine de type C, REG3A, sur la carcinogenèse hépatique. Il s’agit d’une protéine secrétée de 16 kDa liant certains poly-oligosaccharides agissant par voie autocrine et paracrine sur différents organes inflammatoires et tumoraux. REG3A déploie des activités biologiques variées dans la prolifération et la survie des cellules, le contrôle de l’inflammation et du stress oxydatif. Son rôle dans la carcinogenèse est controversé. Nous montrons que la surexpression de REG3A retarde l’apparition du CHC dans deux modèles murins de carcinogenèse chimique et génétique. Nous montrons que cet effet de REG3A est associé à une réduction significative des protéines O-GlcNAcylées. Nous décrivons comment la O-GlcNAcylation via REG3A déséquilibre la balance stabilité / dégradation de l’oncogène c-MYC, favorise la dégradation de c-MYC et freine le développement tumoral dans les modèles étudiés. Au total, notre étude révèle un lien entre lectine, O-GlcNAcylation et contrôle tumoral. Les mécanismes par lesquels REG3A régule la O-GlcNAcylation des protéines restent cependant à élucider.
Several studies have demonstrated a role of O-GlcNAcylation (O-GlcNAc) in tumorigenesis of various carcinomas by modification of tumor-associated proteins. However, its implication in the pathogenesis of osteosarcoma remains unclear. This study aimed to investigate the levels of O-GlcNAc and the expressions of O-linked N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA) in human osteosarcoma tissues, by using immunohistochemistry; and to find correlations between the levels or expressions and several clinicopathologic parameters. There were 109 first diagnosed osteosarcoma patients, including Enneking stage IIB (n=70) and III (n=39). Correlations between the immunoreactive score (IRS) and clinicopathologic parameters, overall survival, and metastasis-free survival were evaluated. A positive correlation was found between the IRS of OGA and the percentage of postchemotherapeutic tumor necrosis (r=0.308; P=0.017). Univariate analysis revealed significantly lower OGA IRS in metastatic patients (P=0.020) and poor chemotherapeutic-responder patients (P=0.001). By multivariate analysis, presence of tumor metastasis (P=0.002) and lower OGA IRS (P=0.004) was significantly associated with shorter overall survival. Subgroup analysis in stage IIB osteosarcoma (n=70) demonstrated that male sex (P=0.019), presence of tumor recurrence (P=0.026), poor chemotherapeutic responder (P=0.022), and lower OGA IRS (P=0.019) were significantly correlated with short metastasis-free survival. But, lower OGA IRS was the only independent predictor for short metastasis-free survival (P=0.006). Our findings suggested that O-GlcNAc pathway, especially OGA, may involve in pathogenesis and aggressiveness of osteosarcoma. Low level of OGA expression may be used as a poor prognostic indicator.
Background
O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) is a key enzyme that regulates O-GlcNAc modification, which is significantly up-regulated and participates in the regulation of tumorigenesis. Although previous research indicated that OGT promotes epithelial-mesenchymal transition (EMT) of lung cancer, the underlying molecular mechanisms, especially within the tumor inflammatory microenvironment, require further elucidation.
Methods
The role of the inflammatory signaling Interleukin 6/Signal Transducer and activator of transcription 3 (IL-6/STAT3) in Non-small cell lung cancer (NSCLC) cells A549 were confirmed by Transwell assay, Scratch wound healing assay, Western blot, Immunofluorescence staining, and Nuclear and cytoplasmic extraction experiment. Western blot detected OGT expression and whole protein O-GlycNacylation after IL-6 stimulation in NSCLCs cells. The biological effects and related mechanism of OGT in NSCLC cells were investigated by Western blot, Transwell assay, Immunofluorescence staining and Immunoprecipitation. The up-stream mechanism of OGT expression was explored by employing the specific chemical inhibitors, and the expression and distribution of OGT and phosphorylated STAT3 in NSCLC samples were confirmed by immunohistochemical analysis.
Results
IL-6/STAT3 promoted the migration and invasion of NSCLC cells. IL-6 stimulation elevated OGT expression and the total protein O-GlycNacylation in A549 cells. Silencing OGT by shRNA significantly inhibited the IL-6 induced EMT marker (N-cadherin and Slug) expression, migration and invasion in A549 cells. OGT interacted with and mediated O-GlycNacylation of STAT3, which promoted STAT3 Y705 phosphorylation in IL-6 treated NSCLC cells. OGT expression was positively regulated by NF-κB p65 signaling pathway after IL-6 stimulation, instead of STAT3 signaling. OGT and phosphorylated STAT3 had an obviously higher expression in human NSCLC tissues, and phosphorylated STAT3 was mainly expressed in the nucleus.
Conclusion
The above results showed that OGT regulated O-GlycNacylation promoted migration and invasion by activating IL-6/STAT3 signaling in lung cancer.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Post-translational protein modification by adding O‐linked β-N-acetyl-D-glucosamine (O-GlcNAc) moiety to serine or threonine residues, termed O-GlcNAcylation, is a highly dynamic process conserved throughout eukaryotes. O-GlcNAcylation is reversibly catalyzed by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), and it acts as a fundamental regulator for wide variety of biological processes including gene expression, cell cycle regulation, metabolism, stress response, cellular signaling, epigenetics and proteostasis. O-GlcNAcylation is regulated by various intracellular or extracellular cues such as metabolic status, nutrients availability or stress. Studies over decades have unveiled profound biological significance of this unique protein modification in normal physiology and pathological processes of diverse cell types or tissues. In hematopoiesis, recent studies have shown the essential and pleiotropic roles of O-GlcNAcylation in differentiation, proliferation and function of hematopoietic cells including T cells, B cells, myeloid progenitors and hematopoietic stem and progenitor cells. Moreover, aberrant O-GlcNAcylation is implicated in the development of hematological malignancies with dysregulated epigenetics, metabolism and gene transcription. Thus, it is now recognized that O-GlcNAcylation is one of the key regulators of normal and malignant hematopoiesis.
Glycans play a vital role in modulating protein structure and function from involvement in protein folding, solubility and stability to regulation of tissue distribution, recognition specificity, and biological activity. They can act as both positive and negative regulators of protein function, providing an additional level of control with respect to genetic and environmental conditions.
Due to the complexity of glycosylated protein forms, elucidating structural and functional information has been challenging task for researchers but recent development of chemical biology-based tools and techniques is bridging these knowledge gaps. This book provides a thorough review of the current state of glycoprotein chemical biology, describing the development and application of glycoprotein and glycan synthesis technologies for understanding and manipulating protein glycosylation.
Nuclear and cytoplasmic protein glycosylation is a widespread and reversible posttranslational modification in eukaryotic
cells. Intracellular glycosylation by the addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalyzed by the O-GlcNAc transferase (OGT). This “O-GlcNAcylation”
of intracellular proteins can occur on phosphorylation sites, and has been implicated in controlling gene transcription, neurofilament
assembly, and the emergence of diabetes and neurologic disease. To study OGT function in vivo, we have used gene-targeting approaches in male embryonic stem cells. We find that OGT mutagenesis requires a strategy that
retains an intact OGT gene as accomplished by using Cre-loxP recombination, because a deletion in the OGT gene results in loss of embryonic stem cell viability. A single copy of the OGT gene is present in the male genome and resides on the X chromosome near the centromere in region D in the mouse spanning
markers DxMit41 and DxMit95, and in humans at Xq13, a region associated with neurologic disease. OGT RNA expression in mice is comparably high among
most cell types, with lower levels in the pancreas. Segregation of OGT alleles in the mouse germ line with ZP3-Cre recombination in oocytes reveals that intact OGT alleles are required for completion of embryogenesis. These studies illustrate the necessity of conditional gene-targeting
approaches in the mutagenesis and study of essential sex-linked genes, and indicate that OGT participation in intracellular
glycosylation is essential for embryonic stem cell viability and for mouse ontogeny.
Cancer cells upregulate glycolysis, increasing glucose uptake to meet energy needs. A small fraction of a cell's glucose enters the hexosamine biosynthetic pathway (HBP), which regulates levels of O-linked beta-N-acetylglucosamine (O-GlcNAc), a carbohydrate posttranslational modification of diverse nuclear and cytosolic proteins. We discovered that breast cancer cells upregulate the HBP, including increased O-GlcNAcation and elevated expression of O-GlcNAc transferase (OGT), which is the enzyme catalyzing the addition of O-GlcNAc to proteins. Reduction of O-GlcNAcation through RNA interference of OGT in breast cancer cells leads to inhibition of tumor growth both in vitro and in vivo and is associated with decreased cell-cycle progression and increased expression of the cell-cycle inhibitor p27(Kip1). Elevation of p27(Kip1) was associated with decreased expression and activity of the oncogenic transcription factor FoxM1, a known regulator of p27(Kip1) stability through transcriptional control of Skp2. Reducing O-GlcNAc levels in breast cancer cells decreased levels of FoxM1 protein and caused a decrease in multiple FoxM1-specific targets, including Skp2. Moreover, reducing O-GlcNAcation decreased cancer cell invasion and was associated with the downregulation of matrix metalloproteinase-2, a known FoxM1 target. Finally, pharmacological inhibition of OGT in breast cancer cells had similar anti-growth and anti-invasion effects. These findings identify O-GlcNAc as a novel mechanism through which alterations in glucose metabolism regulate cancer growth and invasion and suggest that OGT may represent novel therapeutic targets for breast cancer.
GlcNAcylation, a dynamic posttranslational modification, is involved in a wide range of biological processes and some human diseases. Although there is emerging evidence that some tumor-associated proteins are modified by GlcNAcylation, the role of GlcNAcylation in tumor progression remains unclear. Here, we show that GlcNAcylation enhances the migration/invasion of breast cancer cells in vitro and lung metastasis in vivo. The decrease of cell surface E-cadherin is the molecular mechanism underlying GlcNAcylation-induced breast cancer metastasis. p120 and beta-catenin, but not E-cadherin, are GlcNAcylated; the GlcNAcylation of p120 and beta-catenin might play roles in the decrease of cell surface E-cadherin. Moreover, immunohistochemistry analysis indicated that the global GlcNAcylation level in breast tumor tissues is elevated significantly as compared with that in the corresponding adjacent tissues; further, GlcNAcylation was significantly enhanced in metastatic lymph nodes compared with their corresponding primary tumor tissues. This is the first report to clearly elucidate the roles and mechanisms whereby GlcNAcylation influences the malignant properties of breast cancer cells. These results also suggest that GlcNAcylation might be a potential target for the diagnosis and therapy of breast cancer.
The oncogene EWS-FLI1 encodes a chimeric transcription factor expressed in Ewing's sarcoma family tumors (ESFTs). EWS-FLI1 target gene expression is thought to drive ESFT pathogenesis and, therefore, inhibition of EWS-FLI1 activity holds high therapeutic promise. As the activity of many transcription factors is regulated by post-translational modifications, we studied the presence of modifications on EWS-FLI1. The immuno-purified fusion-protein was recognized by an antibody specific for O-linked beta-N-acetylglucosaminylation, and bound readily to a phosphoprotein-specific dye. Inhibition of Ser/Thr-specific phophatases increased EWS-FLI1 molecular weight and reduced its O-GlcNAc content, suggesting that phosphorylation and O-GlcNAcylation of EWS-FLI1 interact dynamically. By mutation analysis, O-GlcNAcylation was delineated to Ser/Thr residues of the amino-terminal EWS transcriptional-activation domain. Metabolic inhibition of the hexosamine biosynthetic pathway abrogated O-GlcNAcylation of EWS-FLI1 and interfered specifically with transcriptional activation of the EWS-FLI1 target Id2. These results suggest that drugs modulating glycosylation of EWS-FLI1 interfere functionally with its activity and might, therefore, constitute promising additions to the current ESFT chemotherapy.
c-Myc is a helix-loop leucine zipper phosphoprotein that heterodimerizes with Max and regulates gene transcription in cell proliferation, cell differentiation, and programmed cell death. Previously, we demonstrated that c-Myc is modified by O-linked N-acetylglucosamine (O-GlcNAc) within or nearby the N-terminal transcriptional activation domain (Chou, T.-Y., Dang, C.V., and Hart, G.W. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4417-4421). In this paper, we identified the O-GlcNAc attachment site(s) on c-Myc. c-Myc purified from sf9 insect cells was trypsinized, and its GlcNAc moieties were enzymically labeled with [3H]galactose. The [3H]galactose-labeled glycopeptides were isolated by reverse phase high performance liquid chromatography and then subjected to gas-phase sequencing, manual Edman degradation, and laser desorption/ionization mass spectrometry. These analyses show that threonine 58, an in vivo phosphorylation site in the transactivation domain, is the major O-GlcNAc glycosylation site of c-Myc. Mutation of threonine 58, frequently found in retroviral v-Myc proteins and in human Burkitt and AIDS-related lymphomas, is associated with enhanced transforming activity and tumorigenicity. The reciprocal glycosylation and phosphorylation at this biologically significant amino acid residue may play an important role in the regulation of the functions of c-Myc.
Heat shock proteins interact with multiple key components of signaling pathways that regulate growth and development. The molecular relationships between heat shock proteins, various signaling proteins and partner proteins appear to be critical for the normal function of signal transduction pathways. The relative levels of these proteins may be important, as too little or too much Hsp70 or Hsp90 can result in aberrant growth control, developmental malformations and cell death. Although the functions of heat shock proteins as molecular chaperones have been well characterized, their complementary role as a 'stress-induced' proteins to monitor changes and alter the biochemical environment of the cell remains elusive. Genetic and molecular interactions between heat shock proteins, their co-chaperones and components of signaling pathways suggest that crosstalk between these proteins can regulate proliferation and development by preventing or enhancing cell growth and cell death as the levels of heat shock proteins vary in response to environmental stress or disease.
Histopathology is insufficient to predict disease progression and clinical outcome in lung adenocarcinoma. Here we show that gene-expression profiles based on microarray analysis can be used to predict patient survival in early-stage lung adenocarcinomas. Genes most related to survival were identified with univariate Cox analysis. Using either two equivalent but independent training and testing sets, or 'leave-one-out' cross-validation analysis with all tumors, a risk index based on the top 50 genes identified low-risk and high-risk stage I lung adenocarcinomas, which differed significantly with respect to survival. This risk index was then validated using an independent sample of lung adenocarcinomas that predicted high- and low-risk groups. This index included genes not previously associated with survival. The identification of a set of genes that predict survival in early-stage lung adenocarcinoma allows delineation of a high-risk group that may benefit from adjuvant therapy.
To observe the gene and protein expression changes of p28GANK in regenerating liver tissues, and to reveal the biological function of p28GANK on the regulation of liver regeneration.
One hundred and thirty two adult male Sprague-Dawley rats were selected, weighing 200-250 g, and divided randomly into sham operation (SO) group and partial hepatectomy (PH) group. Each group had eleven time points: 0, 2, 6, 12, 24, 30, 48, 72, 120, 168 and 240 h, six rats were in each time point. The rats were undergone 70% PH under methoxyflurane anesthesia by resection of the anterior and left lateral lobes of the liver. SO was conducted by laparotomy plus slight mobilization of the liver without resection. Liver specimens were collected at the indicated time points after PH or SO. The expression level of p28GANK mRNA was determined by Northern blot as well as at protein level via immunohistochemical staining. The expressions of p28GANK mRNA in these tissues were analyzed by imaging analysis system of FLA-2000 FUJIFILM and one way analysis of variance. The protein expressions of p28GANK in these tissues were analyzed with Fromowitz' method and Rank sum test.
The expression of p28GANK mRNA in the regenerating liver tissues possessed two transcripts, which were 1.5 kb and 1.0 kb. There was a significantly different expression patterns of p28GANK mRNA between SO and PH groups (P<0.01). The expression of p28GANK mRNA increased 2 h after PH, the peak time was 72 h (SO group: 163.83+/-1.4720; PH group: 510.5+/-17.0499, P<0.01). There was a significant difference in the 1.5 kb transcript, which decreased gradually after 72 hours. The protein expression of p28GANK was mainly in the cytoplasm of regenerating hepatocytes, and increased near the central region 24 h after PH, and became strongly positive at 48 h (+++, vs the other time points P<0.05), but decreased 72 h after PH.
The expression of p28GANK mRNA increases in the early stage of rat liver regeneration, the protein expression of p28GANK is mainly in the cytoplasm of regenerating liver cells. It suggests that the gene of p28GANK may be an important regulatory and controlled factor involved in hepatocyte proliferation during liver regeneration.
The Ogt gene encodes a glycosyltransferase that links N-acetylglucosamine to serine and threonine residues (O-GlcNAc) on nuclear and cytosolic proteins. Efforts to study a mammalian
model of Ogt deficiency have been hindered by the requirement for this X-linked gene in embryonic stem cell viability, necessitating the
use of conditional mutagenesis in vivo. We have extended these observations by segregating Ogt mutation to distinct somatic cell types, including neurons, thymocytes, and fibroblasts, the latter by an approach developed
for inducible Ogt mutagenesis. We show that Ogt mutation results in the loss of O-GlcNAc and causes T-cell apoptosis, neuronal tau hyperphosphorylation, and fibroblast growth
arrest with altered expression of c-Fos, c-Jun, c-Myc, Sp1, and p27. We further segregated the mutant Ogt allele to parental gametes by oocyte- and spermatid-specific Cre-loxP mutagenesis. By this we established an in vivo genetic approach that supports the ontogeny of female heterozygotes bearing
mutant X-linked genes required during embryogenesis. Successful production and characterization of such female heterozygotes
further indicates that mammalian cells commonly require a functional Ogt allele. We find that O-GlcNAc modulates protein phosphorylation and expression among essential and conserved cell signaling
pathways.
Cellular response to environmental, physiological, or chemical stress is key to survival following injury or disease. Here
we describe a unique signaling mechanism by which cells detect and respond to stress in order to survive. A wide variety of
stress stimuli rapidly increase nucleocytoplasmic protein modification by O-linked β-N-acetylglucosamine (O-GlcNAc), an essential post-translational modification of Ser and Thr residues of metazoans. Blocking this post-translational
modification, or reducing it, renders cells more sensitive to stress and results in decreased cell survival; and increasing
O-GlcNAc levels protects cells. O-GlcNAc regulates both the rates and extent of the stress-induced induction of heat shock proteins, providing a molecular basis
for these findings.
Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to p53 is known to occur, but the site of O-GlcNAcylation and its effects on p53 are not understood. Here, we show that Ser 149 of p53 is O-GlcNAcylated and that this modification is associated with decreased phosphorylation of p53 at Thr 155, which is a site that is targeted by the COP9 signalosome, resulting in decreased p53 ubiquitination. Accordingly, O-GlcNAcylation at Ser 149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Our results indicate that the dynamic interplay between O-GlcNAc and O-phosphate modifications coordinately regulate p53 stability and activity.
Full-grown Xenopus oocytes are arrested at the prophase of the first meiotic division in a G2-like state. Progesterone triggers meiotic resumption also called the G2/M transition. This event is characterized by germinal vesicle breakdown (GVBD) and by a burst in phosphorylation level that
reflects activation of M-phase-promoting factor (MPF) and MAPK pathways. Besides phosphorylation and ubiquitin pathways, increasing
evidence has suggested that the cytosolic and nucleus-specific O-GlcNAc glycosylation also contributes to cell cycle regulation. To investigate the relationship between O-GlcNAc and cell cycle, Xenopus oocyte, in which most of the M-phase regulators have been discovered, was used. Alloxan, an O-GlcNAc transferase inhibitor, blocked G2/M transition in a concentration-dependent manner. Alloxan prevented GVBD and both MPF and MAPK activations, either triggered
by progesterone or by egg cytoplasm injection. The addition of detoxifying enzymes (SOD and catalase) did not rescue GVBD,
indicating that the alloxan effect did not occur through reactive oxygen species production. These results were strengthened
by the use of a benzoxazolinone derivative (XI), a new O-GlcNAc transferase inhibitor. Conversely, injection of O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, an O-GlcNAcase inhibitor, accelerated the maturation process. Glutamine:fructose-6-phosphate amidotransferase inhibitors, azaserine
and 6-diazo-5-oxonorleucine, failed to prevent GVBD. Such a strategy appeared to be inefficient; indeed, UDP-GlcNAc assays
in mature and immature oocytes revealed a constant pool of the nucleotide sugar. Finally, we observed that cyclin B2, the
MPF regulatory subunit, was associated with an unknown O-GlcNAc partner. The present work underlines a crucial role for O-GlcNAc in G2/M transition and strongly suggests that its function is required for cell cycle regulation.
The development of microarrays permits us to monitor transcriptomes on a genome-wide scale. To validate microarray measurements, quantitative-real time-reverse transcription PCR (Q-RT-PCR) is one of the most robust and commonly used approaches. The new challenge in gene quantification analysis is how to explicitly incorporate statistical estimation in such studies. In the realm of statistical analysis, the various available methods of the probe level normalization for microarray analysis may result in distinctly different target selections and variation in the scores for the correlation between microarray and Q-RT-PCR. Moreover, it remains a major challenge to identify a proper internal control for Q-RT-PCR when confirming microarray measurements.
Sixty-six Affymetrix microarray slides using lung adenocarcinoma tissue RNAs were analyzed by a statistical re-sampling method in order to detect genes with minimal variation in gene expression. By this approach, we identified DDX5 as a novel internal control for Q-RT-PCR. Twenty-three genes, which were differentially expressed between adjacent normal and tumor samples, were selected and analyzed using 24 paired lung adenocarcinoma samples by Q-RT-PCR using two internal controls, DDX5 and GAPDH. The percentage correlation between Q-RT-PCR and microarray were 70% and 48% by using DDX5 and GAPDH as internal controls, respectively.
Together, these quantification strategies for Q-RT-PCR data processing procedure, which focused on minimal variation, ought to significantly facilitate internal control evaluation and selection for Q-RT-PCR when corroborating microarray data.
The expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5.
We report here that 39 colon tumors from four independent mouse models and 100 human CRCs encompassing all clinical stages shared a striking recapitulation of embryonic colon gene expression. Compared to normal adult colon, all mouse and human tumors over-expressed a large cluster of genes highly enriched for functional association to the control of cell cycle progression, proliferation, and migration, including those encoding MYC, AKT2, PLK1 and SPARC. Mouse tumors positive for nuclear beta-catenin shifted the shared embryonic pattern to that of early development. Human and mouse tumors differed from normal embryonic colon by their loss of expression modules enriched for tumor suppressors (EDNRB, HSPE, KIT and LSP1). Human CRC adenocarcinomas lost an additional suppressor module (IGFBP4, MAP4K1, PDGFRA, STAB1 and WNT4). Many human tumor samples also gained expression of a coordinately regulated module associated with advanced malignancy (ABCC1, FOXO3A, LIF, PIK3R1, PRNP, TNC, TIMP3 and VEGF).
Cross-species, developmental, and multi-model gene expression patterning comparisons provide an integrated and versatile framework for definition of transcriptional programs associated with oncogenesis. This approach also provides a general method for identifying pattern-specific biomarkers and therapeutic targets. This delineation and categorization of developmental and non-developmental activator and suppressor gene modules can thus facilitate the formulation of sophisticated hypotheses to evaluate potential synergistic effects of targeting within- and between-modules for next-generation combinatorial therapeutics and improved mouse models.
Gangliosides are sialic acid containing glycosphingolipids that are ubiquitously distributed on vertebrate plasma membranes. GM3, a precursor for most of the more complex ganglioside species, is synthesized by GM3 synthase. Although total ganglioside levels are significantly higher in breast tumor tissue than in normal mammary tissue, the roles played by gangliosides in breast cancer formation and metastasis are not clear.
To investigate the roles of gangliosides in breast tumor development, GM3 synthase was silenced in the highly metastatic 4T1 cells and over-expressed in the non-metastatic 67NR cells. The behavior of breast cancer cells was examined in vitro using migration assay, invasion assay, and soft agar assay. Tumor formation and metastasis in vivo were examined using a well established mouse mammary tumor model.
GM3 synthase silencing in 4T1 cells significantly inhibited cell migration, invasion and anchorage-independent growth in vitro, and lung metastasis in vivo. In addition, over-expression of GM3 synthase in nonmetastatic 67NR cells significantly induced cell migration and anchorage-independent growth. Further studies indicated that activation of the phosphoinositide-3 kinase/Akt pathway, and consequently inhibition of nuclear factor of activated T cell (NFAT)1 expression, could be the mechanism underlying the suppression of breast cancer migration/invasion induced by GM3 synthase silencing.
Our findings indicate that GM3 synthase silencing suppressed lung metastasis in murine breast cancer cells. The molecular mechanism that underlies GM3 synthase mediated migration and invasion was inhibition of the phosphoinositide-3 kinase/Akt pathway. The findings suggest that GM3 synthase may be of value as a therapeutic target in breast cancer.
Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.
O-linked β-N-acetylglucosamine (O-GlcNAc) is a sugar attachment to serine or threonine hydroxyl moieties on nuclear and cytoplasmic proteins. In many ways, O-GlcNAcylation is similar to phosphorylation because both post-translational modifications cycle rapidly in response to internal or environmental cues. O-GlcNAcylated proteins are involved in transcription, translation, cytoskeletal assembly, signal transduction, and many other cellular functions. O-GlcNAc signaling is intertwined with cellular metabolism; indeed, the donor sugar for O-GlcNAcylation (UDP-GlcNAc) is synthesized from glucose, glutamine, and UTP via the hexosamine biosynthetic pathway. Emerging research indicates that O-GlcNAc signaling and its crosstalk with phosphorylation are altered in metabolic diseases, such as diabetes and cancer.
The FOXO4 transcription factor plays an important role in cell survival in response to oxidative stress. The regulation of FOXO4 is orchestrated by post-translational modifications including phosphorylation, acetylation, and ubiquitination. Here, we demonstrate that O-GlcNAcylation also contributes to the FOXO4-dependent oxidative stress response. We show that hydrogen peroxide treatment of HEK293 cells increases FOXO4 association with OGT, the enzyme that adds O-GlcNAc to proteins, causing FOXO4 O-GlcNAcylation and enhanced transcriptional activity under acute oxidative stress. O-GlcNAcylation is known to be protective for cells under stress conditions, including oxidative stress. Our data provide a mechanism of FOXO4 anti-oxidative protection through O-GlcNAcylation.
The dynamic post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), termed O-GlcNAcylation, is an important mechanism for modulating cellular signaling pathways. O-GlcNAcylation impacts transcription, translation, organelle trafficking, proteasomal degradation and apoptosis. O-GlcNAcylation has been implicated in the etiology of several human diseases including type-2 diabetes and neurodegeneration. This review describes the pair of enzymes responsible for the cycling of this post-translational modification: O-GlcNAc transferase (OGT) and beta-N-acetylglucosaminidase (OGA), with a focus on the function of their structural domains. We will also highlight the important processes and substrates regulated by these enzymes, with an emphasis on the role of O-GlcNAc as a nutrient sensor impacting insulin signaling and the cellular stress response. Finally, we will focus attention on the many ways by which O-GlcNAc cycling may affect the cellular machinery in the neuroendocrine and central nervous systems.
The differential expression of the ras oncogene product p21 in the primary tumor, regional nodes, and distant metastatic sites in patients with disseminated breast cancer was examined to define the biologic and clinical significance of the ras oncogene in the progression of breast cancer. The avidin-biotin peroxidase complex method was used on formalin-fixed, paraffin-embedded tissues from 16 patients with metastatic disease. The primary antibody used in this protocol was RAP-5, an anti-p21 murine monoclonal IgG2a. p21 antigen staining was similar in the primary tumor and regional nodes from the same patient (P less than 0.05), but the staining of distant metastases was more variable. Expression of ras p21 was consistently increased in invasive components of the primary tumor as compared with intraductal tumor. In addition, a high level of p21 expression was seen in tumor emboli in lymphatics and blood vessels as compared with contiguous tumor in parenchymal tissue. Although p21 staining is present in aggressive primary breast cancers and most metastatic sites, our findings indicate that markedly enhanced p21 expression is associated with the earlier stages (invasion and dissemination) of aggressive breast cancers.
Glucose is an important regulator of cell growth and metabolism. Thus, it is likely that some of the adverse effects of hyperglycemia are reflections of normal regulation by abnormal concentrations of glucose. How the cell senses glucose, however, is still incompletely understood. Evidence has been presented that the hexosamine biosynthesis pathway serves this function for regulation of aspects of glucose uptake, glycogen synthesis, glycolysis, and synthesis of growth factors. Excess hexosamine flux causes insulin resistance in cultured cells, tissues, and intact animals. Further evidence for the possible role of this pathway in normal glucose homeostasis and disease is that the level of activity of the rate-limiting enzyme in hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase, is correlated with glucose disposal rates (GDRs) in normal humans and transgenic mice.
Protein O-GlcNAcylation is proceeded by O-linked GlcNAc transferase (OGT) in nucleocytoplasm and is involved in many biological processes although its physiological role is not clearly defined. To identify the functional significance of O-GlcNAcylation, we investigated heat stress effects on protein O-GlcNAcylation. Here, we found that protein O-GlcNAcylation was significantly increased in vivo during acute heat stress in mammalian cells and simultaneously, the enhanced protein O-GlcNAcylation was closely associated with cell survival in hyperthermia. Our results demonstrate that hyperthermal cytotoxicity may considerably be facilitated under the condition of insufficient level of protein O-GlcNAcylation inside cells. Furthermore, OGT reaction might be crucial for triggering thermotolerance to recover hyperthermal sensitivity without particular induction of heat shock proteins (hsps). Thus, we propose that OGT can respond rapidly to heat stress through the enhancement of nucleocytoplasmic protein O-GlcNAcylation for a rescue from the early phase of hyperthermal cytotoxicity.
Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.
A dynamic cycle of addition and removal of O-linked N-acetylglucosamine (O-GlcNAc) at serine and threonine residues is emerging as a key regulator of nuclear and cytoplasmic protein activity. Like phosphorylation, protein O-GlcNAcylation dramatically alters the posttranslational fate and function of target proteins. Indeed, O-GlcNAcylation may compete with phosphorylation for certain Ser/Thr target sites. Like kinases and phosphatases, the enzymes of O-GlcNAc metabolism are highly compartmentalized and regulated. Yet, O-GlcNAc addition is subject to an additional and unique level of metabolic control. O-GlcNAc transfer is the terminal step in a "hexosamine signaling pathway" (HSP). In the HSP, levels of uridine 5'-diphosphate (UDP)-GlcNAc respond to nutrient excess to activate O-GlcNAcylation. Removal of O-GlcNAc may also be under similar metabolic regulation. Differentially targeted isoforms of the enzymes of O-GlcNAc metabolism allow the participation of O-GlcNAc in diverse intracellular functions. O-GlcNAc addition and removal are key to histone remodeling, transcription, proliferation, apoptosis, and proteasomal degradation. This nutrient-responsive signaling pathway also modulates important cellular pathways, including the insulin signaling cascade in animals and the gibberellin signaling pathway in plants. Alterations in O-GlcNAc metabolism are associated with various human diseases including diabetes mellitus and neurodegeneration. This review will focus on current approaches to deciphering the "O-GlcNAc code" in order to elucidate how O-GlcNAc participates in its diverse functions. This ongoing effort requires analysis of the enzymes of O-GlcNAc metabolism, their many targets, and how the O-GlcNAc modification may be regulated.
Despite Otto Warburg's 1931 Nobel Prize for his work affirming the role of metabolism in carcinogenesis, there has been little further interest in this association between metabolism and cancer. Disinterest has, in part, been attributable to the notion that Warburg's description of a relation between a shift to glycolysis in carcinogenesis may be an epiphenomenon rather than a mechanistic determinant. By studying the critical cellular energy sensor AMP-activated protein kinase (AMPK), I postulate that the association between intermediary metabolism and tumours varies over time. Through accumulation of carbohydrates and pan-inhibition of AMPK, premalignant tumours may gain a replicative advantage through the repression of senescence. Conversely, malignant tumours, with a defective tumour suppressor contingent, undergo a "glycolytic switch", in part by tolerating a degree of AMPK activation, to mitigate substrate limitation. I contend that this Janus-faced relation with intermediary metabolism contributes to carcinogenesis; if proven, this finding would have important implications for public health, in that it would lend support to the idea that prevention of obesity, and caloric restriction and exercise could reduce the predisposition to cancer.
How tumors spread and kill their host organism remains an enigma, but not for lack of attention. For more than a century, cancer biologists have postulated that metastasis results from the interplay of wandering tumor cells with permissive target tissues. Yet, decades of scrutiny into the molecular bases of cancer have largely focused on what causes oncogenic transformation and the incipient emergence of tumors. By comparison, the study of how tumor cells take steps toward metastasis (that is, by altering their microenvironment, entering the circulation, and colonizing a distant organ) has received less attention. Progressively, however, the idea has emerged that tumors are more than just a mass of transformed cells. A renewed focus on the problem of metastasis is now apparent, and for good reason—metastasis remains the cause of 90% of deaths from solid tumors.
Tumor cells display increased metabolic autonomy in comparison to non-transformed cells, taking up nutrients and metabolizing them in pathways that support growth and proliferation. Classical work in tumor cell metabolism focused on bioenergetics, particularly enhanced glycolysis and suppressed oxidative phosphorylation (the 'Warburg effect'). But the biosynthetic activities required to create daughter cells are equally important for tumor growth, and recent studies are now bringing these pathways into focus. In this review, we discuss how tumor cells achieve high rates of nucleotide and fatty acid synthesis, how oncogenes and tumor suppressors influence these activities, and how glutamine metabolism enables macromolecular synthesis in proliferating cells.
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