Diabetic women have a 2–3 fold increased risk of developing endometrial cancer, however, the molecular aspects of this risk are not fully understood. This study investigated the alteration of cellular O-GlcNAcylation of proteins as the potential mechanistic connection between these two conditions. The endometrial cancer cell line (Ishikawa) was utilized to study the effect of dysregulation of O-GlcNAcylation on epithelial mesenchymal transition (EMT). Hyper-O-GlcNAcylation (via 1 μM Thiamet-G/ThmG or 25 mM Glucose) enhanced the expression of EMT-associated genes (
WNT5B
and
FOXC2
), and protein expression of the EMT adhesion molecule, N-Cadherin. Reorganization of stress filaments (actin filaments), consistent with EMT, was also noted in ThmG-treated cells. Interestingly, Hypo-O-GlcNAcylation (via 50 μM OSMI-1) also upregulated
WNT5B
, inferring that any disruption to O-GlcNAc cycling impacts EMT. However, Hypo-O-GlcNAcylation reduced overall cellular proliferation/migration and the expression of pro-EMT genes (
AHNAK, TGFB2, FGFBP1, CALD1, TFPI2
). In summary, disruption of O-GlcNAc cycling (i.e., Hyper- or Hypo-O-GlcNAcylation) promoted EMT at both the molecular and cellular levels, but only Hyper-O-GlcNAcylation provoked cellular proliferation/migration, and cytoskeletal reorganization.
Background
O‐N‐acetylglucosaminylation (O‐GlcNAcylation) is unique type of protein glycosylation. The nature of O‐GlcNAcylation and its interplay with O‐phosphorphylation make this modification a regulator of many cellular functions. Hyper‐O‐GlcNAcylation is a general feature of many cancers.
Objective:
To determine the tumorgenic potential of O‐GlcNAcylation in cervical cancer cells through the inhibition of O‐linked N‐acetylglucosamine transferase (OGT).
Methods:
The OGT inhibitor, Alloxan, was used to block global O‐GlcNAcylation of immortalized cervical cancer cells (SiHa cells). Tumorigenic potential was assessed by measurement of cell proliferation and cytokine resistance. Proteomic analysis of control and Alloxan treated samples were compared by LTQ‐Orbitrap.
Results:
Inhibition of O‐GlcNAcylation in SiHa cells inhibited cell growth as assessed by cell proliferation assays. Total number of generations, multiplication rate, and generation time for control and Alloxan‐treated cells was 2.6 vs. 0.68 generations, 0.31 vs. 0.08 doublings per day, and 2.27 vs. 8.76 days, respectively (n=2 independent experiments).The loss of O‐GlcNAcylation also increased the susceptibility of the SiHa cells to cytokine‐induced death (P<0.05, n=3). Initial results of proteomic analysis indicated an up‐regulation of apoptotic proteins (e.g., programmed cell death protein 5), and a down‐regulation of anti‐apoptotic proteins (eukaryotic initiation factor 4) in Alloxan‐treated cells.
Conclusion
Global O‐GlcNAcylation is a critical regulator of cervical cancer cell proliferation, and apoptotic resistance.
Support:
The COLSA Karabelas fund and the NSF Graduate Research Fellowship Program.
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