Investigation of the Effects of Inhibiting N-glycosylation in Cancer

Glycosylation, the addition of sugar moieties to nascent proteins, is one of the most common posttranslational modifications. Glycosylation regulates protein structure, function and localization. Most cell surface proteins and secreted proteins are glycosylated by the addition of Asparagine(N)-linked glycans (N-glycans). Aberrant N-glycosylation is a well-accepted feature of malignancy and is a potential prognostic marker for some types of cancer. For example, increased expression of complex N-glycans has been detected in cancers of breast, colon and has been correlated with reduced survival of the patients. Therefore, understanding the role of N-glycosylation in malignancy could be beneficial for developing novel therapeutic and prognostic strategies. To examine the role of N-glycosylation in malignancy, we applied chemical biology and genetic approaches. First, we conducted a high throughput screen to identify compounds that could block L-PHA-induced cell death. Our screen identified the cardiac glycoside Na+/K+-ATPase inhibitors as novel inhibitors of N-glycosylation. Further analysis of N-glycans consistently confirmed that inhibition of Na+/K+-ATPase impairs the N-glycosylation, as well as migration and invasion. Interestingly, other studies have shown antimetastatic effects of cardiac glycosides in patients. Thus, our high throughput screen identified Na+/K+-ATPase inhibition as a novel strategy to target the N-glycosylation pathway. In addition, we used a genetic approach to investigate the role of N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) in malignancy. Knockdown of GlcNAc-TI decreased the cell-surface expression of complex N-glycans. By confocal microscopy, knockdown of GlcNAc-TI decreased cell surface expression of β1 integrins and increased their localization around the nucleus. Moreover, GlcNAc-TI knockdown decreased the migration and invasion of malignant cells. Next, we investigated the effect of GlcNAc-TI in an orthotopic xenograft mouse model of metastasis. GlcNAc-TI knockdown significantly decreased the lung colony formation of the highly metastatic PC3N7 human prostate cancer cell line in mice. Our results suggest an important role for GlcNAc-TI in tumor metastasis. Interestingly, breast cancer patients with lower expression levels of Mgat1 had lower risk of disease relapse after therapy. Thus, GlcNAc-TI plays an important role in cancer progression and metastasis and GlcNAc-TI inhibitors could have therapeutic benefits for cancer patients. Moreover, expression levels of GlcNAc-TI could be used as a prognostic marker in patients with cancer.Ph