Binding of the sialic acid-binding lectin, Siglec-9, to the membrane mucin, MUC1, induces recruitment of β-catenin and subsequent cell growth

Abstract

Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, β-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of β-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited β-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.

Keywords: Lectin; Mucins; Signaling; Tumor Microenvironment; β-Catenin.

FIGURE 1.

Binding of soluble recombinant Siglec-9 to MUC1. A, expression of MUC1 in 3T3/mock and 3T3/MUC1 cells was analyzed by flow cytometry after incubation with mouse anti-MUC1-ND antibodies (solid line) or control antibodies (broken line) and then with FITC-labeled goat anti-mouse IgG antibodies. B, recombinant Fc-tagged Siglec-3, -5, and -9, or His-tagged Siglec-1 was added to a lysate of 3T3/MUC1 cells. Siglec-binding proteins were pulled down using protein G-Sepharose (lanes a–d) or nickel-nitrilotriacetic acid-agarose (lanes e and f) and then subjected to SDS-PAGE followed by Western blotting (IB). MUC1-ND was detected as described under “Materials and Methods.” Lane a, control (protein G-Sepharose); lane b, Siglec-9; lane c, Siglec-3; lane d, Siglec-5; lane e, control (nickel-nitrilotriacetic acid-agarose); lane f, Siglec-1. C, MUC1-coated plate was prepared as described under “Materials and Methods.” The plate assay was performed in duplicate using Fc-tagged Siglec-9.

FIGURE 2.

Distribution of Siglec-9-positive cells in tumor tissues. Sections of paraffin-embedded human pancreas (a), breast (b), and colon (c) tumor tissues, and nonmalignant colon tissues (d) were stained with H&E, DAPI (blue), and the combinations of rabbit anti-Siglec-9 antibodies and Alexa Fluor 594-labeled goat anti-rabbit IgG antibodies (red), and mouse anti-MUC1-ND antibodies and Alexa Fluor 488-labeled goat anti-mouse IgG antibodies (green) and then observed by microscopy. A magnified image in colon tumor tissues is shown in e.

FIGURE 3.

Analyses of proteins co-immunoprecipitated with MUC1. A, expression of FGFR-3 and EGFR was analyzed by flow cytometry as described under “Materials and Methods.” Broken line, control IgG; solid line, rabbit anti-FGFR-3 antibodies or rabbit anti-EGFR antibodies. B, MUC1 (lanes a and b) and FGFR-3 (lanes c and d) were immunoprecipitated (IP) with mouse anti-MUC1-ND antibodies and rabbit anti-FGFR-3 antibodies, respectively, from a lysate of 3T3/MUC1 cells, and the immunoprecipitate was subjected to SDS-PAGE, followed by Western blotting and detection with anti-MUC1-ND antibodies (lanes a and c) and anti-FGFR-3 antibodies (lanes b and d). C and D, after treatment of 3T3/MUC1 cells with FGF (C) or sSiglec-9 (D), the same procedure was performed as described in B. Each lane contained a sample treated similarly to that in B. E, MUC1 (lanes a and b) and EGFR (lanes c and d) were immunoprecipitated with mouse anti-MUC1-ND antibodies and rabbit anti-EGFR antibodies, respectively, from a lysate of HCT116/MUC1 cells, and then subjected to SDS-PAGE. After Western blotting, MUC1-ND (lanes a and c) and EGFR (lanes b and d) were detected with the same antibodies as described above.

FIGURE 4.

Recruitment of β-catenin to MUC1 on stimulation with sSiglec-9. A, 3T3/mock (lanes a and b), 3T3/MUC1 (lanes c–f), HCT116/MUC1 (lanes g and h), and A549 (lanes i and j) cells (1 × 10⁶ cells) were stimulated with or without sSiglec-9 for 20 min, and then MUC1-CD (lanes a–d, g–j) and β-catenin (lanes e and f) were immunoprecipitated (IP) with Armenian hamster anti-MUC1 Ab5 antibodies and mouse anti-β-catenin antibodies, respectively, from the cell lysates. MUC1-CD and β-catenin were detected with the same antibodies as described above after SDS-PAGE and Western blotting (IB). B, MUC1-CD was immunoprecipitated as described above from a lysate of 3T3/MUC1 cells (1 × 10⁶ cells) stimulated with sSiglec-9 for 0–40 min. After SDS-PAGE and Western blotting, co-immunoprecipitated β-catenin was detected. C, 3T3/MUC1 cells (1 × 10⁶ cells) were stimulated with sSiglec-9 (0–5 μg of protein/ml) for 20 min, and the same procedure was performed as described in B. D, the intensity of the band of β-catenin in B was estimated with ImageJ software. The level of β-catenin relative to that of MUC1-CD was compared. The value obtained in the experiment in which 3T3/MUC1 cells were treated with sSiglec-9 for 0 min was taken as 1 (mean ± S.D. (error bars), n = 5; *, p < 0.05). E, the level of β-catenin in C was estimated as described in D, and the intensity of β-catenin relative to that of MUC1-CD was compared. The value obtained in the experiment in which 3T3/MUC1 cells were treated without sSiglec-9 for 20 min was taken as 1. F, a lysate of 3T3/MUC1 cells (lane a) and immunoprecipitates with Armenian hamster anti-MUC1 Ab5 antibodies from lysates of 3T3/MUC1 cells (1 × 10⁶ cells) treated with (lane c) or without (lane b) sSiglec-9 for 20 min were subjected to SDS-PAGE, followed by Western blotting and detection with rabbit anti-c-Src, anti-Lck, and anti-Lyn antibodies. G, 3T3/MUC1 cells (1 × 10⁶ cells) were stimulated with sSiglec-9 in the presence (lane b) or absence (lane a) of PP2 (10 μm) for 20 min, and β-catenin co-immunoprecipitated with MUC1 was detected as described in A. H, the intensity of β-catenin in H was estimated as described in D, and the level of β-catenin relative to that of MUC1-CD was compared. The value obtained in the experiment in which 3T3/MUC1 cells were treated with sSiglec-9 in the absence of PP2 (10 μm) for 20 min was taken as 1 (mean ± S.D., n = 3; *, p < 0.01).

FIGURE 5.

MUC1-mediated signaling in 3T3/MUC1 and HCT116/MUC1 cells co-incubated with Siglec-9-expressing cells and effect of neuraminidase treatment of MUC1-expressing cells. A, expression of Siglec-9 in HEK293/Siglec-9 and HEK293/mock cells was examined with a flow cyotmeter. Solid trace, mouse anti-Siglec-9 antibodies; broken trace, control antibodies. B, binding of MAM to the surface of 3T3/MUC1 and HCT116/MUC1 cells was analyzed before (thick solid trace) and after (thin solid trace) neuraminidase treatment with a flow cytometer as described under “Materials and Methods.” Control analysis (broken trace) was performed using intact cells without MAM. C, lysates of 3T3/MUC1 and HCT116/MUC1 cells (1 × 10⁶ cells) treated with or without neuraminidase were subjected to SDS-PAGE, followed by Western blotting and detection with mouse anti-MUC1-ND antibodies. D, binding of sSiglec-9 to 3T3/MUC1 and HCT116/MUC1 cells (1 × 10⁶ cells) was examined before (thick solid trace) after (thin solid trace) neuraminidase treatment. Control analysis (broken trace) was performed using intact cells without sSiglec-9. E, 3T3/MUC1 and HCT116/MUC1 cells (1 × 10⁶ cells) treated with or without neuraminidase were co-incubated with HEK293/Siglec-9 or HEK293/mock cells, and then recruitment of β-catenin to MUC1-CD was examined as described above. IP, immunoprecipitation; IB, Western blotting. F, the intensity of β-catenin in E was estimated as described in Fig. 4D, and the level of β-catenin relative to that of MUC1-CD was compared. The value obtained in the experiment in which 3T3/MUC1 and HCT116/MUC1 cells were stimulated with sSiglec-9 before neuraminidase treatment was taken as 1 (mean ± S.D. (error bars), n = 3; *, p < 0.05).

FIGURE 6.

Down-regulation of β-catenin phosphorylation and enhancement of nuclear transport of β-catenin by treatment with sSiglec-9. A, 3T3/mock and 3T3/MUC1 cells (1 × 10⁶ cells) were stimulated with or without sSiglec-9 for 40 min. Phosphorylated β-catenin, β-catenin, and β-actin were detected with mouse anti-phosphorylated β-catenin, anti-β-catenin, and anti-β-actin antibodies, respectively, after SDS-PAGE and Western blotting (IB). B, the intensity of phosphorylated β-catenin in A was estimated as described in Fig. 4D. The level of phosphorylated β-catenin relative to that of β-catenin was compared. The value obtained in the experiment in which 3T3/MUC1 cells were not treated with sSiglec-9 was taken as 1 (mean ± S.D. (error bars), n = 4; *, p < 0.001). C, 3T3/MUC1 cells (1 × 10⁶ cells) were cultured in the presence or absence of sSiglec-9 for 1 h. The cells were immunostained with mouse anti-β-catenin antibodies as described under “Materials and Methods.” Red, propidium iodide; green, β-catenin. D, 3T3/MUC1 cells (1 × 10⁶ cells) were treated as described in C, and cell fractionation was performed as described under “Materials and Methods.” Cytosol and nuclear fractions were subjected to SDS-PAGE, followed by Western blotting and detection with mouse anti-histone 2B antibodies and anti-IκB-α antibodies. E, 3T3/MUC1 cells (1 × 10⁶ cells) were treated as described in A, and cell fractionation was performed. β-Catenin was immunoprecipitated (IP) from the nuclear fraction obtained from 3T3/MUC1 cells cultured in the presence (lane b) or absence (lane a) of sSiglec-9, and β-catenin and MUC1-CD were detected after SDS-PAGE and Western blotting as described above in Fig. 4A. F, distribution of MUC1-CD and β-catenin in the nucleus of 3T3/MUC1 cells treated with or without sSiglec-9 as described in C was observed using the combinations of mouse anti-β-catenin antibodies and Alexa Fluor 488-labeled goat anti-mouse IgG antibodies (green) and Armenian hamster anti-MUC1 Ab5 antibodies and DyLight 594 labeled goat anti-hamster IgG antibodies (red), respectively. G, 3T3/MUC1 cells (1 × 10⁶ cells) were stimulated with or without sSiglec-9 for 20 min and then cultured for 24 h. The cell lysates were subjected to SDS-PAGE, followed by Western blotting and detection with anti-c-myc antibodies. H, 3T3/MUC1 cells (2 × 10³ cells) were stimulated with (■) or without (♦) sSiglec-9 for 20 min and then cultured for 72 h. The level of cell proliferation was determined by means of the MTT assay (mean ± S.D., n = 5; *, p < 0.01).