Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells - PubMed (original) (raw)

Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells

Shingo Hatakeyama et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Cell surfaces of epithelial cancer are covered by complex carbohydrates, whose structures function in malignancy and metastasis. However, the mechanism underlying carbohydrate-dependent cancer metastasis has not been defined. Previously, we identified a carbohydrate-mimicry peptide designated I-peptide, which inhibits carbohydrate-dependent lung colonization of sialyl Lewis X-expressing B16-FTIII-M cells in E/P-selectin doubly-deficient mice. We hypothesized that lung endothelial cells express an unknown carbohydrate receptor, designated as I-peptide receptor (IPR), responsible for lung colonization of B16-FTIII-M cells. Here, we visualized IPR by in vivo biotinylation, which revealed that the major IPR is a group of 35-kDa proteins. IPR proteins isolated by I-peptide affinity chromatography were identified by proteomics as Ser/Arg-rich alternative pre-mRNA splicing factors or Sfrs1, Sfrs2, Sfrs5, and Sfrs7 gene products. Bacterially expressed Sfrs1 protein bound to B16-FTIII-M cells but not to parental B16 cells. Recombinant Sfrs1 protein bound to a series of fucosylated oligosaccharides in glycan array and plate-binding assays. When anti-Sfrs antibodies were injected intravenously into mice, antibodies labeled a subset of lung capillaries. Anti-Sfrs antibodies inhibited homing of I-peptide-displaying phage to the lung colonization of B16-FTIII-M cells in vivo in the mouse. These results strongly suggest that Sfrs proteins are responsible for fucosylated carbohydrate-dependent lung metastasis of epithelial cancers.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Identification of IPRs expressed on the mouse lung endothelial cell surface. (A) Visualization of IPRs by in vivo biotinylation. Mice were injected intravenously either by PBS (lane 1) or a biotinylation reagent (lanes 2–6), followed by i.v. injection of I-peptide displaying phage (lanes 3 and 4) or control phage (lanes 5 and 6). After perfusion with PBS, lungs were isolated, and phage was immunoprecipitated with rabbit anti-phage antibody (lanes 4 and 6) or rabbit IgG (lanes 3 and 5). Biotinylated proteins were resolved by SDS/PAGE and detected by a peroxidase-avidin blot. Lanes 1 and 2 each contain 1/500 of the lung tissue lysate, and lanes 3–6 each contain immunoprecipitated material from1/10 of the lung tissue lysate. (B) SDS/PAGE of affinity-purified IPR proteins visualized by Coomassie blue staining. Microsomal membrane proteins from rat lung were bound to I-peptide agarose beads in the presence of 1 mM CaCl2. Bound materials were eluted in 1 mM EDTA, which was applied to a second aliquot of I-peptide agarose beads. After washing the column with buffer containing an irrelevant peptide (1 mg/mL), the bound materials were eluted by I-peptide (1 mg/mL) and subjected to gel electrophoresis. In A and B, 9% gels were used.

Fig. 2.

Cell surface expression of Sfrs proteins on mouse lung endothelial cells. (A) In vivo biotinylated lung endothelial surface proteins and their immunoprecipitated bands detected by anti-Sfrs antibodies. Total cell lysates (lane 1) were immunoprecipitated with rabbit IgG (lane 2), with rabbit anti-phage antibodies (lane 3), with goat IgG (lane 4), with goat anti-Sfrs antibody E16 (lane 5), and with goat anti-Sfrs antibody P-15 (lane 6). A gradient 6–18% gel was used. Biotinylated cell surface proteins were detected by a peroxidase-avidin blot. Lane 1 contains 1/500 of the lung tissue lysate, and lanes 2–6 each contain immunoprecipitated material from 1/10 of the lung tissue lysate. A 15-kDa band visible in Fig. 1_A_ is not detected, likely because this small protein was not retained on the nitrocellulose filter during electrotransfer. (B) Immunohistochemistry of lung tissue sections from a mouse intravenously injected with FITC-conjugated LEA (tomato) lectin and with irrelevant goat antibody (Upper) or with anti-Sfrs antibody (Lower). Frozen lung sections were stained with Alexa Fluor 560-conjugated F(ab′)2 fragment of anti-goat IgG antibody. (Left) Sfrs (red). (Center) Blood vessels by FITC-LEA (green). (Right) Merged with DAPI nuclear staining (blue). (Scale bar, 200 μm.)

Fig. 3.

Carbohydrate-binding activity of Sfrs proteins. (A) Binding of Qdot-conjugated Sfrs1 to B16-FTIII-M (a) in the presence of 1 mM I-peptide (Inset) and mock-transfected B16 (b) cells. (B) Binding of synthetic biotinylated PAA-conjugated carbohydrates to GST-His-Sfrs1. Binding of each carbohydrate was determined by peroxidase avidin and a peroxidase color reaction. PAA carbohydrates added to GST-His-coated plates showed absorbance of <0.1. LacNAc (1); Type 1H (2); Type 2H (3); Lea (4); Lex (5); sLea (6); sLex (7); LeY (8); LeB (9); biotinylated (10) PAA without carbohydrate. (C) Glycan arrays by GST-His-Sfrs1. Glycan structures listed starting from the strongest binder are 13: α-L-Rhα–Sp8; 76: Fucα1–3GlcNAcβ-Sp8; 58: Fucα1–2Galβ1–3GalNAcα–Sp8; 68: Fucα1–2Galβ1–4(Fucα1–3)GlcNAcβ–Sp8; 67: Fucα1–2Galβ1–4(Fucα1–3)GlcNAcβ–Sp8; 74: Fucα1–2Galβ–Sp8; 66: Fucα1–2Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ-Sp0; 65: Fucα1–2Galβ1–4(Fucα1–3)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ-Sp0; 73: Fucα1–2Galβ1–4Glcβ–Sp0; 72: Fucα1–2Galβ1–4GlcNAcβ–Sp8; 70:. Fucα1–2Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ-Sp0; and 69:. Fucα1–2Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc–Sp0.

Fig. 4.

Inhibition of I-phage binding and B16-FTIII-M cell colonization to lung vasculature by anti-Sfrs antibody. (A) Effect of anti-Sfrs antibody on I-phage targeting to mouse lung. A wild type mouse was intravenously injected with normal goat IgG (pink) or with anti-Sfrs antibody (blue), followed by i.v. injection of I-peptide displaying phage. I-phage targeting lung or liver was determined by counting transformant bacterial colonies. Graph shows results obtained from triplicate analysis. (B) Effect of anti-Sfrs antibody on lung colonization of B16-FTIII-M cells in vivo. Mice were injected with normal goat IgG (1) or with goat anti-Sfrs antibodies (2) through the tail vein, followed by i.v. injection with B16-FTIII-M cells. Two weeks later, numbers of melanoma foci (mean ± SE) formed in the lungs were determined. Numbers of foci in 1 are 307 ± 34, n = 6, and those in 2 are 32 ± 9, n = 6. Differences indicated by asterisks are statistically significant (P < 0.0001). Photo at the left shows representative images of control lung (Upper) and that from anti-Sfrs antibody -treated mice (Lower). Swelling of the lung with many melanoma foci (Upper) is likely caused by the large amount of cancer cells colonized to this organ.

Fig. 5.

I-peptide-targeted apoptosis of lung endothelial cells and blocked lung colonization of B16-FTIII-M cells. (A) Mice were injected with liposomes containing irrelevant palmitoyl peptide plus GD3 ganglioside (a) or with palmitoyl I-peptide plus GD3 (b). One day later, lung tissue sections were prepared and subjected to a TUNEL assay. Green fluorescence indicates apoptotic nuclei. (B) Numbers of melanoma foci in mouse lung. Mice (n = 6 for each group) were treated with irrelevant peptide/GD3 (column 1) or with I-peptide/GD3 (column 2) as described above. One day later, B16-FTIII-M cells were injected through the tail vein. Numbers of melanoma foci in lungs 2 weeks after injection were determined. Differences indicated by asterisks are statistically significant (P = 0.0007). Photo at the left shows representative images of control (Upper) and that from I-peptide/GD3-treated mice (Lower).

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Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells - PubMed (original) (raw)