Subject(s)
Oligosaccharides/chemistry , Sialic Acids/chemistry , Animals , HEK293 Cells , Hexosamines/chemistry , Humans , ZebrafishABSTRACT
Early invasive growth and metastasis are features of pancreatic cancer that rely on its resistance to anoikis, an apoptosis program activated on loss of matrix anchorage. How anoikis is regulated is unclear. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine-kinase (GNE) was silenced, or p16 was overexpressed, in human pancreatic carcinoma cells. Gene expression profiling, enzymatic assays, Western blotting, and cell cycle analysis were conducted. Silencing of GNE, the key enzyme of sialic acid biosynthesis, sensitizes pancreatic cancer cells to anoikis. Accordingly, we observed a loss of GNE enzyme activity in cells, which became anoikis susceptible after transfection with the tumor suppressor p16. Similarly, studies of another cell line with low GNE activity revealed strong anoikis susceptibility, confirming the association of low GNE activity and anoikis susceptibility. Gene expression profiling demonstrated that the loss of GNE triggered the transcriptional activation of the ATF4-ATF3-CHOP pathway, leading to apoptosis in the framework of the unfolded protein response. In silico analysis showed that GNE up-regulation occurred predominantly in pancreatic cancer but also in other malignancies. Delineation of GNE-dependent signaling pathways may provide targets that control anchorage dependence and/or restore drug efficacy, which is of utmost relevance for the treatment of pancreatic cancer.
Subject(s)
Apoptosis/physiology , Multienzyme Complexes/deficiency , Pancreatic Neoplasms/enzymology , Anoikis/genetics , Anoikis/physiology , Apoptosis/genetics , Base Sequence , Cell Line, Tumor , Endoplasmic Reticulum Stress , Gene Knockdown Techniques , Genes, p16 , Humans , Integrin alpha5beta1/metabolism , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , RNA Interference , RNA, Small Interfering/genetics , Sialic Acids/metabolism , Up-RegulationABSTRACT
Improving the accessibility and functions of therapeutic and diagnostic glycoproteins is one of the major goals of glycobiotechnology. Here we present that stable knock-down of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the sialic acid biosynthetic pathway, dramatically increases incorporation of N-acetylmannosamine analogues into glycoproteins of HEK293 cells. By means of these GNE-deficient cells highly sialylated glycoproteins can efficiently be decorated with reactive functional groups, which can be employed in bioorthogonal functionalization strategies for fluorescence labelling or biotinylation.
