Increased glucose utilization is a feature of cancer cells to support cell survival, proliferation, and metastasis. An association between diabetes mellitus and cancer progression was previously demonstrated in cancers including cholangiocarcinoma (CCA). This study was aimed to determine the effects of high glucose on protein O-GlcNAcylation and metastatic potentials of CCA cells. Two pairs each of the parental low metastatic and highly metastatic CCA sublines were cultured in normal (5.6 mM) or high (25 mM) glucose media. The migration and invasion abilities were determined and underlying mechanisms were explored. Results revealed that high glucose promoted migration and invasion of CCA cells that were more pronounced in the highly metastatic sublines. Concomitantly, high glucose increased global O-GlcNAcylated proteins, the expressions of vimentin, hexokinase, glucosamine-fructose-6-phosphate amidotransferase (GFAT) and O-GlcNAc transferase of CCA cells. The glucose level that promoted migration/invasion was shown to be potentiated by the induction of GFAT, O-GlcNAcylation and an increase of O-GlcNAcylated vimentin and vimentin expression. Treatment with a GFAT inhibitor reduced global O-GlcNAcylated proteins, vimentin expression, and alleviated cell migration. Altogether, these results suggested the role of high glucose enhanced CCA metastasis via modulation of O-GlcNAcylation, through the expressions of GFAT and vimentin.
O-GlcNAcylation, an O-linked protein glycosylation with a single molecule of N-acetylglucosamine (GlcNAc), is reversibly controlled by O-GlcNAc transferase (OGT) and N-acetyl D-glucosaminidase (OGA). Aberrant O-GlcNAcylation contributes an important role in initiation and progression of many human cancers. Elevation of O-GlcNAcylation in tumor tissues and poor prognosis of cholangiocarcinoma (CCA) patients have been reported. In this study, the role of O-GlcNAcylation in promoting tumor progression was further investigated in CCA cell lines. Suppression of O-GlcNAcylation using small interfering RNAs of OGT (siOGT) significantly reduced cell migration and invasion of CCA cells whereas siOGA treated cells exhibited opposite effects. Manipulating levels of O-GlcNAcylation did affect the nuclear translocation of NF-κB and Akt-phosphorylation together with expression of matrix-metalloproteinases (MMPs). O-GlcNAcylation and nuclear translocation of NF-κB, the upstream signaling cascade of MMP activation were shown to be important for MMP activation. Immunoprecipitation revealed the elevation of O-GlcNAc-modified NF-κB with increased cellular O-GlcNAcylation. Involvement of O-GlcNAcylation in MMP-mediated migration and invasion of CCA cells was shown to be via O-GlcNAcylation and nuclear translocation of NF-κB. This information indicates the significance of O-GlcNAcylation in controlling the metastatic ability of CCA cells, hence, O-GlcNAcylation and its products may be new targets for treatment of metastatic CCA.
Cholangiocarcinoma (CCA) is a bile duct cancer with a very poor prognosis. Currently, there is no effective pharmacological treatment available for it. We showed that CCA ubiquitously relies on cyclin-dependent kinases 4 and 6 (CDK4/6) activity to proliferate. Primary CCA tissues express high levels of cyclin D1 and the specific marker of CDK4/6 activity, phospho-RB Ser780. Treatment of a 15-CCA cell line collection by pharmacological CDK4/6 inhibitors leads to reduced numbers of cells in the S-phase and senescence in most of the CCA cell lines. We found that expression of retinoblastoma protein (pRB) is required for activity of the CDK4/6 inhibitor, and that loss of pRB conferred CDK4/6 inhibitor-drug resistance. We also identified that sensitivity of CCA to CDK4/6 inhibition is associated with the activated KRAS signature. Effectiveness of CDK4/6 inhibition for CCA was confirmed in the three-dimensional spheroid-, xenograft-, and patient-derived xenograft models. Last, we identified a list of genes whose expressions can be used to predict response to the CDK4/6 inhibitor. Conclusion: We investigated a ubiquitous dependency of CCA on CDK4/6 activity and the universal response to CDK4/6 inhibition. We propose that the CDK4/6-pRB pathway is a suitable therapeutic target for CCA treatment.
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