Abstract
In prior work we reported that advanced stage, drug-resistant pancreatic cancer cells (the SW1990 line) can be sensitized to the EGFR-targeting tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib by treatment with 1,3,4-O-Bu3ManNAc (Bioorg. Med. Chem. Lett. (2015) 25(6):1223-7). Here we provide mechanistic insights into how this compound inhibits EGFR activity and provides synergy with TKI drugs. First, we showed that the sialylation of the EGFR receptor was at most only modestly enhanced (by ∼20 to 30%) compared to overall ∼2-fold increase in cell surface levels of this sugar. Second, flux-driven sialylation did not alter EGFR dimerization as has been reported for cancer cell lines that experience increased sialylation due to spontaneous mutations. Instead, we present evidence that 1,3,4-O-Bu3ManNAc treatment weakens the galectin lattice, increases the internalization of EGFR, and shifts endosomal trafficking towards non-clathrin mediated (NCM) endocytosis. Finally, by evaluating downstream targets of EGFR signaling, we linked synergy between 1,3,4-O-Bu3ManNAc and existing TKI drugs to a shift from clathrin-coated endocytosis (which allows EGFR signaling to continue after internalization) towards NCM endocytosis, which targets internalized moieties for degradation and thereby rapidly diminishes signaling.
Highlights
In mammals, glycosylation is a ubiquitous co/ post-translational modification of proteins and lipids that modulates the activities of these molecules in many ways that – despite decades of study – often remain poorly understood
This report builds on previous studies where we characterized glycosylation in advanced stage pancreatic cancer SW1990 cells treated with 1,3,4-O-Bu3ManNAc to understand how metabolic flux-driven increases in sialic acid contribute to cancer progression [4, 5]
This premise was supported by our discovery that 1,3,4-O-Bu3ManNAc sensitized SW1990 cells to the epidermal growth factor receptor (EGFR)-targeting tyrosine kinase inhibitors (TKIs) drugs erlotinib and gefitinib [1]
Summary
Glycosylation is a ubiquitous co/ post-translational modification of proteins and lipids that modulates the activities of these molecules in many ways that – despite decades of study – often remain poorly understood. An illustration of a recent, unexpected glycosylation-based result is the ability of 1,3,4-O-Bu3ManNAc to sensitize drug-resistant pancreatic cancer cells to tyrosine kinase inhibitors (TKIs) [1] To elaborate briefly, this compound is a “high flux” N-acetylmannosamine (ManNAc) analog that increases sialylation [2,3,4,5]; it is counterintuitive that such a compound could have anti-cancer potential because sialic acid has generally been regarded as cancerpromoting. Our group found that 1,3,4-O-Bu3ManNAc – a “pro-drug” that is activated by intracellular esterases to generate ManNAc [20] – promotes high levels of flux through the sialic acid biosynthetic pathway [3] and can double cell surface sialylation in human cancer cells [4] This compound had only a modest ( almost negligible) impact on endpoints related to metastasis such as cell motility [4]. Based on the clinical promise of counteracting drug resistance, especially in difficult-to-treat malignancies such as pancreatic cancer, we investigated the impact of 1,3,4-O-Bu3ManNAc on EGFR signaling to gain insight into the underlying molecular mechanisms by which this compound attenuated oncogenic signaling and to understand the unusual synergy between TKIs and increased cellular sialylation supported by this compound
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