Suppression of FUT1 attenuates cell proliferation in the HER2-overexpressing cancer cell line NCI-N87

Abstract

Lewis Y (LeY) antigen is an oligosaccharide that is highly expressed at the cell surface in various human cancers. Increased LeY expression activates epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) and promotes cell proliferation in EGFR-overexpressing cells. However, the effect of downregulation of LeY expression on cell proliferation in HER2-overexpressing cells remains unknown. FUT1 encodes α1,2-fucosyltransferase, a key enzyme for LeY synthesis. We knocked down FUT1 by short interfering RNA (siRNA) in four HER2-overexpressing human cancer cell lines, including NCI-N87, MKN7, SKBr3 and BT474. We investigated whether downregulation of LeY and alteration in the glycosylation status of these cells affect cell proliferation and HER2 activation. Knocking down FUT1 expression markedly inhibited proliferation of NCI-N87, which highly expressed EGFR and was sensitive to EGFR deprivation. Furthermore, FUT1 siRNA downregulated the total amount of HER2 protein, phosphorylation of HER2 and EGFR, and phosphorylation of extracellular signal-regulated kinase (ERK) in this cell line. Moreover, the marked downregulation of phosphorylation of HER2 and ERK was observed following short-time EGF-stimulation. These effects were not observed in the other three cell lines. Our results suggest that knockdown of FUT1 downregulates HER2 signaling via EGFR downregulation. FUT1 may serve as a new molecular target for HER2-overexpressing human cancers with activated EGFR signaling.

Figure 1

(A) Suppression of FUT1 mRNA expression after transfection of siRNAs was analyzed by real-time PCR. The level of FUT1 mRNA expression was determined and plotted as fold change relative to the control. Data were normalized to the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. (B) Alteration of LeY expression after transfection of siRNAs in each whole cell lysate. GAPDH is shown as a loading control. Lane 1, reagent; lane 2, control; lane 3, FUT1-1; lane 4, FUT1-2; and lane 5, FUT1-3. A molecular weight standard is shown at the right side.

Figure 2

The growth curves after transfection with FUT1 siRNA. Cells were assayed for growth 0, 72 and 120 h after transfection. The absorbance at 450 nm at 0 h was normalized to one. Absorbance at subsequent time points was plotted relative to the initial value.

Figure 3

Analysis of the proportion of cells in each cell cycle phase after transfection with each FUT1 siRNA in NCI-N87 cells. Values shown are average ± SE (n=3). Values of the sub-G1, G1, S and G2/M fractions are portions of the total population. ^*P<0.01.

Figure 4

The effect of downregulating FUT1 on protein expression and tyrosine phosphorylation of HER2 and ERK1/2 in normal culture condition. The bar chart shows the relative expression ratio of each protein by western blotting. Each value is calculated as the ratio of signal intensity compared to that of control. Data were normalized to signal intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lane 1, reagent; lane 2, control; lane 3, FUT1-1; lane 4, FUT1-2; and lane 5, FUT1-3. (A) NCI-N87, (B) MKN7, (C) SKBr3 and (D) BT474 cells. ^**P<0.05.

Figure 5

The effect of downregulating FUT1 on protein expression and tyrosine phosphorylation of HER2 and ERK1/2 under EGF-stimulated condition. The bar chart shows the relative expression ratio of each protein found by western blotting. Each value is calculated as the ratio of signal intensity compared to that of control. Data were normalized to signal intensity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Lane 1, reagent; lane 2, control; lane 3, FUT1-1; lane 4, FUT1-2; and lane 5, FUT1-3. (A) NCI-N87, (B) MKN7, (C) SKBr3 and (D) BT474 cells. ^**P<0.05.

Figure 6

(A) HER2 and EGFR expression in each cell line was analyzed by western blotting. A431 is shown as a positive control for EGFR-overexpression and VMRC-LCD is shown as a negative control. (B) The effect of downregulating FUT1 on protein expression and tyrosine phosphorylation of EGFR under normal cultural condition in NCI-N87 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as the loading control. (C) The effect of downregulating FUT1 on protein expression and tyrosine phosphorylation of EGFR under EGF-stimulated condition in NCI-N87 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as the loading control. Lane 1, reagent; lane 2, control; lane 3, FUT1-1; lane 4, FUT1-2; and lane 5, FUT1-3.

Figure 7

The growth curves after transfection with EGFR siRNA-1 and EGFR siRNA-2. Cells were assayed for growth 0, 72 and 120 h after transfection. The absorbance at 450 nm at 0 h was normalized to one. Absorbance at subsequent time points was plotted relative to the initial value.

Figure 8

The effect of EGFR knockdown on protein expression and tyrosine phosphorylation of HER2 and ERK1/2 in NCI-N87 cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as the loading control.