The Significance of Cell Surface N-Glycosylation for Internalization and Potency of Cytotoxic Conjugates Targeting Receptor Tyrosine Kinases

Abstract

Precise anticancer therapies employing cytotoxic conjugates constitute a side-effect-limited, highly attractive alternative to commonly used cancer treatment modalities, such as conventional chemotherapy, radiotherapy or surgical interventions. Receptor tyrosine kinases are a large family of N-glycoproteins intensively studied as molecular targets for cytotoxic conjugates in various cancers. At the cell surface, these receptors are embedded in a dense carbohydrate layer formed by numerous plasma membrane glycoproteins. The complexity of the cell surface architecture is further increased by galectins, secreted lectins capable of recognizing and clustering glycoconjugates, affecting their motility and activity. Cell surface N-glycosylation is intensively remodeled by cancer cells; however, the contribution of this phenomenon to the efficiency of treatment with cytotoxic conjugates is largely unknown. Here, we evaluated the significance of N-glycosylation for the internalization and toxicity of conjugates targeting two model receptor tyrosine kinases strongly implicated in cancer: HER2 and FGFR1. We employed three conjugates of distinct molecular architecture and specificity: Affibody_HER2-vcMMAE (targeting HER2), vcMMAE-KCK-FGF1.E and T-Fc-vcMMAE (recognizing different epitopes within FGFR1). We demonstrated that inhibition of N-glycosylation reduced the cellular uptake of all conjugates tested and provided evidence for a role of the galectin network in conjugate internalization. In vitro binding studies revealed that the reduced uptake of conjugates is not due to impaired HER2 and FGFR1 binding. Importantly, we demonstrated that alteration of N-glycosylation can affect the cytotoxic potential of conjugates. Our data implicate a key role for cell surface N-glycosylation in the delivery of cytotoxic conjugates into cancer cells.

Keywords: N-glycosylation; RTK; cancer therapy; cytotoxic conjugates; endocytosis; galectins.

Figure 1

The effect of N-glycosylation and galectins on the internalization of CCs. (A) Hypothetical model of FGFR1 and HER2 domain organization and membrane topology with putative N-glycosylation sites marked. The scheme of cytotoxic conjugates targeting FGFR1: vcMMAE-FGF1.E and T-Fc-vcMMAE and HER2: Affibody_HER2-vcMMAE are shown. (B) The efficiency of vcMMAE conjugation to Affibody_HER2, KCK-FGF1.E and T-Fc and purity of targeting molecules and conjugates Affibody_HER2-vcMMAE, vcMMAE-KCK-FGF1.E and T-Fc-vcMMAE analyzed by SDS-PAGE. (C) FGFR1 and HER2 expression levels in the studied cell lines were analyzed by Western blotting using anti-FGFR1 and anti-HER2 antibodies. Tubulin level assessed with anti-tubulin antibody served as a loading control. (D,E) The efficiency and selectivity of T-Fc, Affibody_HER2 and KCK-FGF1.E internalization under different conditions were studied by flow cytometry. Internalization was analyzed in serum-starved SKBR3 (D) and U2OSR1 cells (E). Cells were incubated with tunicamycin 24 h before the experiment or lactose 15 min before the experiment. Then cells were treated with T-Fc or Affibody_HER2 or KCK-FGF1.E labeled with DyLight550. After 30 min incubation on ice, cells were transferred to 37 °C for 20 min, the cell surface was extensively washed to remove cell-bound, non-internalized proteins, and then cells were subsequently analyzed by flow cytometry. Results presented are mean values of three experiments ± SEM. The t-test was used to assess the statistical significance of measured differences in internalization; * p < 0.05, ** p < 0.01, n.s.—not significant.

Figure 2

The impact of galectin-1 and -3 on the FGFR1-mediated uptake of KCK-FGF1.E. (A) Purity of recombinant galectin-1 and galectin-3 determined by SDS-PAGE (CBB) (left panel) and Western blotting using anti-His antibody (right panel). (B) Efficiency of KCK-FGF1.E internalization into U2OSR1 cells upon administration of recombinant galectins. Cells were treated with lactose for 15 min and then treated with KCK-FGF1.E labeled with DyLight550 in the presence of galectin-1 or galectin-3. After 30 min incubation on ice, cells were transferred to 37 °C for 20 min, washed to remove cell surface-bound, non-internalized proteins and then analyzed by flow cytometry. Results presented are mean values from three experiments ± SEM. The t-test was used to assess the statistical significance of measured differences in internalization; * p < 0.05, n.s.—not significant.

Figure 3

The significance of N-glycosylation for the interaction of CCs with RTKs. (A) SDS-PAGE analysis of enzymatic deglycosylation of HER2-Fc and FGFR1-Fc using PNGase F. (B–D) BLI-determined kinetic parameters of targeting protein interactions with FGFR1-Fc, HER2-Fc and their de-glycosylated variants. HER2-Fc or FGFR1-Fc and deglycosylated receptors were immobilized on BLI sensors and incubated with various concentrations of Affibody_HER2, T-Fc and KCK-FGF1.E. K_D, k_on and k_off were calculated using ForteBio Data Analysis 11.0 software (Pall ForteBio, San Jose, CA, USA).

Figure 4

The role of cell surface N-glycosylation in the cytotoxicity of CCs. Cytotoxicity of CCs against FGFR1- or HER2-overproducing cells lacking N-glycosylation. The cytotoxic potential of Affibody_HER2-vcMMAE (A) was measured using SKBR3 cell line, and cytotoxic potential of T-Fc-vcMMAE (B) and vcMMAE-KCK-FGFE.1 (C) were measured using U2OSR1 cell line. Cells were incubated with tunicamycin 24 h before the experiment and treated with the indicated agents at various concentrations for 96 h, and cell viability was assessed in the Presto Blue assay. Results are mean values from three independent experiments ± SD. Statistical significance: * p < 0.05, *** p < 0.0001, n.s.—not significant.