Inhibition of N-linked glycosylation disrupts receptor tyrosine kinase signaling in tumor cells

Abstract

Receptor tyrosine kinases (RTK) are therapeutic targets for the treatment of malignancy. However, tumor cells develop resistance to targeted therapies through the activation of parallel signaling cascades. Recent evidence has shown that redundant or compensatory survival signals responsible for resistance are initiated by nontargeted glycoprotein RTKs coexpressed by the cell. We hypothesized that disrupting specific functions of the posttranslational machinery of the secretory pathway would be an effective strategy to target both primary and redundant RTK signaling. Using the N-linked glycosylation inhibitor, tunicamycin, we show that expression levels of several RTKS (EGFR, ErbB2, ErbB3, and IGF-IR) are exquisitely sensitive to inhibition of N-linked glycosylation. Disrupting this synthetic process reduces both cellular protein levels and receptor activity in tumor cells through retention of the receptors in the endoplasmic reticulum/Golgi compartments. Using U251 glioma and BXPC3 pancreatic adenocarcinoma cell lines, two cell lines resistant to epidermal growth factor receptor-targeted therapies, we show that inhibiting N-linked glycosylation markedly reduces RTK signaling through Akt and radiosensitizes tumor cells. In comparison, experiments in nontransformed cells showed neither a reduction in RTK-dependent signaling nor an enhancement in radiosensitivity, suggesting the potential for a therapeutic ratio between tumors and normal tissues. This study provides evidence that enzymatic steps regulating N-linked glycosylation are novel targets for developing approaches to sensitize tumor cells to cytotoxic therapies.

Figure 1

Tunicamycin reduces RTK protein and phosphorylation levels. A, BXPC3 and U251 cell lines were exposed to increasing concentrations of tunicamycin (T) for 18 h. Western blot analysis was performed for EGFR, ErbB2, ErbB3, and IGF-IR, as well as phosphorylated forms of EGFR (Y1173) and ErbB2 (Y1248). Blots were also probed for Hsp70 to ensure equal protein loading. B and C, parallel experiments performed with swainsonine (S) and castanospermine (C) in U251 cells.

Figure 2

Mechanism of reduced RTK protein production. A, Western blot analysis showing the time course of reduced RTK protein levels in BXPC3 cells following treatment with 500 nmol/L of tunicamycin. B, cells were labeled with ³⁵S-Met for 6 h during 500 nmol/L tunicamycin treatment (top) or tunicamycin (T) was added after labeling was complete (bottom), and cultures were harvested at 8-h and 16-h time points after removal of the ³⁵S-Met label. EGFR was immunoprecipitated, separated by SDS-PAGE, and gels were exposed to film for 18 h. C, quantitative real-time PCR of EGFR mRNA after 500 nmol/L of tunicamycin treatment. Relative RNA represents the ratio of EGFR RNA/GAPDH RNA. Columns, averages of two experiments performed in triplicate; bars, SE.

Figure 3

Live-cell EGFR imaging. BXPC3 cells with stable expression of an EGFR-GFP vector were generated as detailed in Materials and Methods. A, time-lapse images of EGFR localization in cells treated or untreated with tunicamycin and maintained at 37°C with 5% CO₂. Images were acquired every hour for 4 h. B, fluorescence micrograph of BXPC3-EGFR-GFP cells treated with or without tunicamycin for 6 h. Cells were counterstained with ER tracker to visualize ER and Golgi bodies. Arrows, Golgi bodies (GB); arrowheads, membrane staining (M) or the endoplasmic reticulum (ER); N, nucleus. C, BXPC3-EGFR-GFP cells overexpress the EGFR-EGFP fusion protein. This fusion protein is activated by 10 ng/mL of epidermal growth factor, as shown by Y1173 phosphorylation, and is inhibited by 1-h pretreatment with 5 μmol/L of erlotinib.

Figure 4

Radiosensitization of BXPC3 and U251 cells. A, clonogenic dose-response survival of BXPC3 cells pretreated for 18 h with 500 nmol/L of tunicamycin. Points, averages of three independent experiments performed in triplicate; bars, SE. B, clonogenic survival of U251 cells; conditions were identical to those in A. The dose enhancement ratio (DER) reports the comparative surviving fractions at 2 Gy.

Figure 5

Differential effect of tunicamycin on normal fibroblasts. A, Western blot analysis of EGFR and phosphorylated EGFR (Y1173) in primary fibroblasts exposed to increasing doses of tunicamycin (T). B, time course of EGFR protein inhibition in primary fibroblasts following an 18-h treatment with 500 nmol/L of tunicamycin. Blots were also probed for Hsp70 to ensure equal loading. C, clonogenic survival of primary fibroblasts pretreated for 18 h with 500 nmol/L of tunicamycin. Points, averages of two independent experiments performed in triplicate.

Figure 6

Differential effect of tunicamycin on Akt phosphorylation in tumor cells and fibroblasts. BXPC3, U251, and primary fibroblasts were treated for 18 h with increasing doses of tunicamycin. Western blot analysis was performed to determine the phosphorylation status of Akt (S473) compared with total Akt protein levels.