O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome

Yehezkel, Galit; Cohen, Liz; Kliger, Adi; Manor, Esther; Khalaila, Isam

doi:10.1074/jbc.m112.345546

Cited by 78 publications

(65 citation statements)

References 42 publications

(49 reference statements)

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“…Different from our in vivo observation, we found that OGA knockdown in C2C12 cells could decrease OGT protein content, which is consistent with observations in the intestines of OGA knockdown mice by Yang et al [38]. However, OGA knockdown in cancer cells had no effect on the OGT level [39], suggesting a complex relationship between OGA and OGT, and how OGA activity regulates OGT in different contexts remains to be elucidated.…”

Section: Discussionsupporting

confidence: 90%

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis

et al. 2016

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Aims/hypothesis O-GlcNAcylation is implicated in modulating mitochondrial function, which is closely involved in regulating muscle metabolism. The presence of O-GlcNAcase (OGA), the enzyme involved in the removal of O-GlcNAc, in mitochondria was recently confirmed in rats. In the present study, we investigated the regulation of myogenesis and muscle insulin sensitivity to OGA in mice, with a focus on mitochondria. Methods C57BL/6J mice fed a high-fat diet for 4 months were used to observe mitochondrial density, activity and O-GlcNAcylation in muscle. Small interfering RNA and overexpression vectors were used to modulate protein content in vitro. Results High-fat feeding decreased the OGA level and largely increased mitochondrial O-GlcNAcylation in mouse skeletal muscle that was accompanied by decreased levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), decreased mitochondrial density and disrupted mitochondrial complex activities. Knockdown of OGA in C2C12 myoblasts promoted PGC-1α degradation, resulting in the suppression of mitochondrial biogenesis and myogenesis, whereas neither knockdown of O-GlcNAc transferase nor overexpression of OGA had significant effects on myogenesis. Mitochondrial dysfunction as evidenced by decreased ATP content and increased reactive oxygen species production, and increased lipid and protein oxidation was observed in both myoblasts and myotubes after OGA knockdown. Meanwhile, elevated O-GlcNAcylation through either OGA knockdown or treatment with the OGA inhibitor PUGNAc and the O-GlcNAc transferase substrate D-GlcNAc suppressed myotube insulin signalling transduction and glucose uptake. OGA overexpression had no significant effect on insulin sensitivity but sufficiently improved the insulin resistance induced by D-GlcNAc treatment. Conclusions/interpretation These data suggest that OGA can modulate mitochondrial density via PGC-1α and mitochondrial function via protein O-GlcNAcylation. In this manner, OGA appears to play a key role in myogenesis and the development of muscle insulin resistance.

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Section: Discussionsupporting

confidence: 90%

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis

et al. 2016

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“…21 Interestingly, OGA knockdowns in colorectal cancer cells also had an increase in polyploidy. 25 Our data corroborates the mitotic defects seen in either OGA knockout mice or knockdown cells. Clearly, OGA has an important role in maintaining the fidelity of mitotic progression.…”

Section: Discussionsupporting

confidence: 86%

“…These data corroborate previous experiments performed in colorectal carcinoma cells in which OGA knockdown caused an increase in polyploidy. 25 Next, we measured the length and width of the condensed DNA in metaphase spindles. Both OGA 040 and 877 showed a significant reduction in the length and width of the condensed chromatid DNA compared to control cells ( Fig.…”

Section: Oga Knockdown Shortens Spindle Chromatids and Increases Multmentioning

confidence: 99%

“…RNA was converted into cDNA using random primers and according to the manufacturer's instructions. Briefly, samples were incubated in a thermal cycler (Model 2720, Applied Biosystems) using the following protocol: priming 5 min at 25 C, RT 30 min at 42 C, and RT inactivation 5 min at 85 C. cDNA products were diluted with 100 ml of nuclease free water and analyzed by Qpcr. 24 qPCR assay cDNA samples were analyzed by qPCR using SsoAdvanced Universal SYBR Green Supermix (172-5271, Bio-Rad) according to manufacturer's instruction.…”

Section: Total Rna Isolation and Rt-pcrmentioning

confidence: 99%

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Reduced O-GlcNAcase expression promotes mitotic errors and spindle defects

et al. 2016

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Alterations in O-GlcNAc cycling, the addition and removal of O-GlcNAc, lead to mitotic defects and increased aneuploidy. Herein, we generated stable O-GlcNAcase (OGA, the enzyme that removes OGlcNAc) knockdown HeLa cell lines and characterized the effect of the reduction in OGA activity on cell cycle progression. After release from G 1 /S, the OGA knockdown cells progressed normally through S phase but demonstrated mitotic exit defects. Cyclin A was increased in the knockdown cells while Cyclin B and D expression was reduced. Retinoblastoma protein (RB) phosphorylation was also increased in the knockdown compared to control. At M phase, the knockdown cells showed more compact spindle chromatids than control cells and had a greater percentage of cells with multipolar spindles. Furthermore, the timing of the inhibitory tyrosine phosphorylation of Cyclin Dependent Kinase 1 (CDK1) was altered in the OGA knockdown cells. Although expression and localization of the chromosomal passenger protein complex (CPC) was unchanged, histone H3 threonine 3 phosphorylation was decreased in one of the OGA knockdown cell lines. The Ewing Sarcoma Breakpoint Region 1 Protein (EWS) participates in organizing the CPC at the spindle and is a known substrate for O-GlcNAc transferase (OGT, the enzyme that adds OGlcNAc). EWS O-GlcNAcylation was significantly increased in the OGA knockdown cells promoting uneven localization of the mitotic midzone. Our data suggests that O-GlcNAc cycling is an essential mechanism for proper mitotic signaling and spindle formation, and alterations in the rate of O-GlcNAc cycling produces aberrant spindles and promotes aneuploidy.

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“…O-GlcNAc modification of proteins is linked to cancer development and progression in various tissues, including lung, breast, prostate, colon, liver, and pancreas [9][10][11][12][13]. In lung and colon cancer, immunohistochemical studies have indicated that O-GlcNAcylation and OGT expression are significantly higher in cancer tissues compared to corresponding adjacent tissues.…”

Section: Discussionmentioning

confidence: 99%