Protein O-GlcNAcylation is a dynamic post-translational modification involving the attachment of N-acetylglucosamine (GlcNAc) to the hydroxyl groups of Ser/Thr residues on numerous nucleocytoplasmic proteins. Two enzymes are responsible for O-GlcNAc cycling on substrate proteins: O-GlcNAc transferase (OGT) catalyzes the addition while O-GlcNAcase (OGA) helps the removal of GlcNAc. O-GlcNAcylation modifies protein functions; therefore, dysregulation of O-GlcNAcylation affects cell physiology and contributes to pathogenesis. To maintain homeostasis of cellular O-GlcNAcylation, there exists feedback regulation of OGT and OGA expression responding to fluctuations of O-GlcNAc levels; yet, little is known about the molecular mechanisms involved. In this study, we investigated the O-GlcNAc-feedback regulation of OGT and OGA expression in lung cancer cells. Results suggest that, upon alterations in O-GlcNAcylation, the regulation of OGA expression occurs at the mRNA level and likely involves epigenetic mechanisms, while modulation of OGT expression is through translation control. Further analyses revealed that the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) contributes to the downregulation of OGT induced by hyper-O-GlcNAcylation; the S5A/S6A O-GlcNAcylation-site mutant of 4E-BP1 cannot support this regulation, suggesting an important role of O-GlcNAcylation. The results provide additional insight into the molecular mechanisms through which cells may fine-tune intracellular O-GlcNAc levels to maintain homeostasis.

High O -linked N -acetylglucosamine transferase expression predicts poor survival in patients with early stage lung adenocarcinoma High O-linked N-acetylglucosamine transferase expression predicts poor survival in patients with early stage lung adenocarcinoma

O-GlcNAcylation is a reversible and dynamic post-translational protein modification catalyzed by O-GlcNAc transferase (OGT). Despite the reported association of O-GlcNAcylation with cancer metastasis, the O-GlcNAc proteome profile for cancer aggressiveness remains largely uncharacterized. Here, we report our comparative O-GlcNAc proteome profiling of two differentially invasive lung adenocarcinoma cell lines, which identified 158 down-regulated and 106 up-regulated candidates in highly invasive cells. Among these differential proteins, a nuclear RNA-binding protein, SAM68 (SRC associated in mitosis of 68 kDa), was further investigated. Results showed that SAM68 is O-GlcNAcylated and may interact with OGT in the nucleus. Eleven O-GlcNAcylation sites were identified, and data from mutant analysis suggested that multiple serine residues in the N-terminal region are important for O-GlcNAcylation and the function of SAM68 in modulating cancer cell migration and invasion. Analysis of clinical specimens found that high SAM68 expression was associated with late cancer stages, and patients with high-OGT/high-SAM68 expression in their tumors had poorer overall survival compared to those with low-OGT/low-SAM68 expression. Our study revealed an invasiveness-associated O-GlcNAc proteome profile and connected O-GlcNAcylated SAM68 to lung cancer aggressiveness.

The use of liquid biopsy to detect epidermal growth factor receptor (EGFR) T790M mutation in non-small cell lung cancer (NSCLC) is a promising method to screen patients eligible for third-generation EGFR inhibitors. Proficiency testing (PT) programs involving liquid biopsy are currently lacking. In this study, we conducted a PT program to assess the quality assurance of liquid biopsy tests for detecting EGFR T790M mutation in molecular pathology laboratories in Taiwan. Whole blood samples (2 mL) with various concentrations of the EGFR T790M mutation were prepared and analyzed in six participating laboratories using their clinically validated assays. For circulating cell-free DNA (cfDNA) isolation, three of the six participating laboratories used the cobas® cfDNA Sample Preparation Kit, and three used the QIAamp Circulating Nucleic Acid Kit. For testing platforms, two of the six participating laboratories used mass spectrometry, three used the cobas® EGFR mutation test, and one used a laboratory-developed test. There was 100% concordance in detection of all the given concentrations of EGFR T790M mutation between the participating laboratories and different testing platforms. The testing platforms used by all participating laboratories could successfully detect EGFR T790M mutation to an expected frequency of 1%. In this first PT program using liquid biopsy in Taiwan, local clinical laboratories were suitably equipped and proficient in the use of cfDNA to test for the EGFR T790M mutation. Establishing a routine PT system to ensure the reliability and accuracy of liquid biopsy in clinical practice in Taiwan would be helpful. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).