Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-κB Activity in Pancreatic Cancer Cells

Zhiyuan Ma,David J Vocadlo,Keith Vosseller

doi:10.1074/jbc.m113.470047

Abstract

Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of glutamine for increased biosynthetic needs. This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). The HBP end product UDP-N-acetylglucosamine (UDP-GlcNAc) is used in enzymatic post-translational modification of many cytosolic and nuclear proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). Here, we observed increased HBP flux and hyper-O-GlcNAcylation in human pancreatic ductal adenocarcinoma (PDAC). PDAC hyper-O-GlcNAcylation was associated with elevation of OGT and reduction of the enzyme that removes O-GlcNAc (OGA). Reducing hyper-O-GlcNAcylation had no effect on non-transformed pancreatic epithelial cell growth, but inhibited PDAC cell proliferation, anchorage-independent growth, orthotopic tumor growth, and triggered apoptosis. PDAC is supported by oncogenic NF-κB transcriptional activity. The NF-κB p65 subunit and upstream kinases IKKα/IKKβ were O-GlcNAcylated in PDAC. Reducing hyper-O-GlcNAcylation decreased PDAC cell p65 activating phosphorylation (S536), nuclear translocation, NF-κB transcriptional activity, and target gene expression. Conversely, mimicking PDAC hyper-O-GlcNAcylation through pharmacological inhibition of OGA suppressed suspension culture-induced apoptosis and increased IKKα and p65 O-GlcNAcylation, accompanied by activation of NF-κB signaling. Finally, reducing p65 O-GlcNAcylation specifically by mutating two p65 O-GlcNAc sites (T322A and T352A) attenuated the induction of PDAC cell anchorage-independent growth. Our data indicate that hyper-O-GlcNAcylation is anti-apoptotic and contributes to NF-κB oncogenic activation in PDAC.

Highlights

Cancer cells rely on energy metabolism that requires increased glucose uptake and constitutive NF-␬B activity for survival
Hyper-O-GlcNAcylation Occurs in Pancreatic Cancer—To determine whether O-GlcNAcylation is elevated in pancreatic cancer, we first examined O-GlcNAc modification levels in human pancreatic ductal adenocarcinoma (PDAC) cell lines compared with non-transformed human pancreatic epithelial human pancreatic duct epithelial cells (HPDE) cells
The concentration of UDP-HexNAc was almost 2-fold increased in MiaPaCa-2 PDAC cells compared with HPDE cells (Fig. 1B), and correlated with MiaPaCa-2 cells displaying the highest increase in O-GlcNAc levels among PDAC cell lines (Fig. 1A)

Summary

Background

Cancer cells rely on energy metabolism that requires increased glucose uptake and constitutive NF-␬B activity for survival. Cancer cell metabolic reprogramming includes a shift in energy production from oxidative phosphorylation to less efficient glycolysis even in the presence of oxygen (Warburg effect) and use of glutamine for increased biosynthetic needs This necessitates greatly increased glucose and glutamine uptake, both of which enter the hexosamine biosynthetic pathway (HBP). In response to stimuli such as TNF␣, the IKK complex composed of IKK␣, IKK␤ and IKK␥ phosphorylates I␬B␣ at S32 and S36 and p65 at S536 leading to I␬B␣ proteasome-mediated degradation, release of the NF-␬B dimer from sequestration, nuclear translocation of NF-␬B, and transcription of target genes including cyclin D1, Vimentin, Bcl-xL, IL-8, VEGF, GLUT3 involved in cell cycle progression, anti-apoptosis, angiogenesis, and glycolysis [10, 15, 16]. Reduction of PDAC hyper-O-GlcNAcylation inhibited constitutive NF-␬B activity, while elevation of O-GlcNAc activated NF-␬B and suppressed apoptosis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION