Aberrant glycosylation promotes lung cancer metastasis through adhesion to galectins in the metastatic niche

Abstract

Metastasis is the leading cause of cancer-associated deaths. Although dissemination of tumor cells likely occurs early in tumorigenesis, the constituents of the microenvironment play essential rate-limiting roles in determining whether these cells will form clinically relevant tumors. Recent studies have uncovered many molecular factors that contribute to the establishment of a protumorigenic metastatic niche. Here, we demonstrate that galectin-3, whose expression has clinical associations with advanced malignancy and poor outcome, contributes to metastatic niche formation by binding to carbohydrates on metastatic cells. We show that galectin-3 is expressed early during tumorigenesis by both CD11b(+)Gr-1(+) and CD11b(+)Ly-6C(hi) leukocytes. Tumors mobilize these myeloid populations through secretion of soluble factors, including IL6. We find that metastatic cancer cells exhibit elevated presentation of the oncofetal galectin-3 carbohydrate ligand, the Thomsen-Friedenreich antigen, on their surfaces as a result of altered C2GnT2 and St6GalNAc4 glycosyltransferase activity that inhibits further glycosylation of this carbohydrate motif and promotes metastasis.

Significance: Although clinical observations of elevated serum galectin-3 levels and altered glycosylation have been associated with malignancy, we identify novel roles for glycosyltransferases in promoting adhesion to galectins in the metastatic niche. This identification of a cytokine-leukocyte-glycosylation axis in metastasis provides mechanistic explanations for clinical associations between malignancy and aberrant glycosylation.

Figure 1. Mice bearing tumors have accumulation of the adhesion ligand, galectin-3, in the early metastatic niche

(A) Cell lines are generated from autochthonous tumors in Kras^LSL-G12D/+;p53^flox/flox mice following tumor development. The lines, which represent discrete stages of metastasis, are derived from primary tumors that did not metastasize (T_nonMet), primary tumors that gave rise to metastases (T_Met), lymph node metastases (N, not shown), and distant metastases (M). Lines derived from increasingly metastatic tumors exhibit increased adhesion to galectin-3 and galectin-8. (B) Mice bearing the no tumors, T_nonMet, and M tumors were analyzed for the presence of galectin-3 in their livers prior to the detection of overt metastases. Mice were perfused with saline prior to homogenization of liver tissue and analysis by western blot. (C) Left: Immunostaining of livers from mice bearing M tumors with no detectable overt metastases in their livers for the presence of macrophages (F4/80, green) and galectin-3 (pink). Nuclei (blue) are stained with Hoechst. Dashed boxes in the top row highlight the area shown in the bottom row. Scale bars in (C) are 50μm (top) and 25μm (bottom). Right: Quantification of galectin-3^posF4/80^pos cells in the livers of mice without tumors, T_nonMet tumors, or M tumors. Error bars are s.e.m. P-values in (C) were determined by One-way ANOVA with Tukey’s Multiple Comparison Test. * P < 0.05; *** P < 0.001.

Figure 2. Tumor-derived soluble factors induce the mobilization of galectin-3⁺ myeloid cells into peripheral circulation early in tumorigenesis

Wild-type mice or mice bearing T_nonMet (802T4) or M (393M1) tumors were analyzed for the presence of galectin-3⁺ leukocytes in their peripheral blood by flow cytometry. (A) Analysis of staining for galectin-3 and CD11b on all leukocytes from mice without tumors (NT), T_nonMet tumors, or M tumors. Numbers represent percentages of all leukocytes that were double positive. (B) Percentage of all leukocytes that were galectin-3⁺. (C) Percentage of all cells that were CD11b⁺. (D) Percentage of all cells that were CD11b⁺galectin-3⁺. (E) Percentage of all leukocytes that were Gr-1⁺. (F) Ratio of CD11b⁺Ly-6C^hi cells to CD11b⁺Ly-6C^lo cells. (G) Galectin-3 expression on Ly-6C^lo and Ly-6C^hi cells. P-values in (B-F) were determined by One-way ANOVA with Tukey’s Multiple Comparison Test. P-values in (G) were determined by Two-way ANOVA with Bonferroni post-test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 3. Secretion of IL-6 by tumors induces the rapid mobilization of CD11b⁺galectin-3⁺ leukocytes

(A) Analysis of CD11b⁺galectin-3⁺ myeloid cell mobilization to peripheral blood following injections of control medium (white), M line conditioned medium (red), or recombinant murine galectin-3 supplemented control medium (gray). (B) Analysis of CD11b⁺galectin-3⁺ myeloid cell mobilization following injections of conditioned media from M lines containing either a control hairpin (shLuc, black) or galectin-3 hairpins (red). (C) Luminex cytokine levels of the conditioned medium. (D) Gene expression microarray analysis of Il6 expression in all cell lines from the four classes. (E) IL6 exhibits a gain of copy number in human lung adenocarcinomas compared to normal lung tissue or blood (P = 5.04 × 10⁻²⁷) in the “Lung adenocarcinoma” data set available from The Cancer Genome Atlas website (see Methods). (F,G) Analysis of CD11b⁺galectin-3⁺ cell mobilization to peripheral blood following injections of control medium or medium supplemented with recombinant murine IL-6. (H) Mobilization of CD11b⁺ leukocytes in wild-type and galectin-3 knockout (Lgals3^−/−) mice following IL-6 injections. Error bars in (C) are s.e.m. ‘N.D.’: not detected. P-values in (A) and (H) were calculated by One-way ANOVA with Tukey’s Multiple Comparison Test. P-value in (G) determined by Student’s t-test. *** P < 0.001; ‘n.s.’ not significant.

Figure 4. Elevated Thomsen-Friedenreich Antigen presentation promotes increased galectin-3 adhesion in metastatic populations

(A) Potential carbohydrate ligands for the galectin-3 carbohydrate recognition domain (CRD). The T-Antigen (Galβ1-3GalNac-α1-O-S/T) is specific for the CRD of galectin-3 and -8. N-Acetyllactosamine (LacNAc) binds the CRD of all galectins and exhibits increased affinities when in a polymeric form. The A- B- Type 2 blood group antigens have reported affinities for a variety of galectins, including galectin-3, by glycan array analysis (35). (B) On normal cells binding of galectin-3 to the T-Antigen on glycoproteins is occluded as the epitope is masked by further glycan extension. On malignant cells, abberant expression of the disaccharide without further glycosylation permits galectin-3 binding. (C) Peanut agglutinin (PNA) labeling of the T-Antigen on representative cell lines from the T_nonMet (blue), T_Met (green), and M (red) classes as determined by flow cytometry. (D) Binding of galectin-3 fluorophore conjugates in the presence of a glycan competitor for galectin-3, N-acetyllactosamine (LacNAc), or control disaccharide (sucrose). (E–F) Human NSCLC tissue microarrays were analyzed for surface T-Antigen presentation by PNA staining. (E) Sample tissue staining in lung and lymph node tissue. Examples of tissues scored as PNA^neg and PNA^pos are shown. (F) Quantification of TMA spots for PNA staining: ‘N’: non-cancerous tissue; ‘C’: cancer tissue. (G) PNA labeling of human NSCLC cell lines as determined by flow cytometry. P-values in (F) determined by Fisher’s Exact Test. Scale bars in (E) are 400μm (100μm for insets).

Figure 5. Metastatic cells downregulateGcnt3 and upregulate St6galnac4

(A) Lectin blot of surface proteins. Cell surface proteins were isolated from representative T_nonMet (802T4), T_Met (393T5), and M (393M1) lines and run on SDS-PAGE gels. Membranes were blotted for the T-Antigen with PNA. (B) Gene expression microarray analysis of all glycosyltansferases. The average expression for all primary tumor-derived lines (ordinate) is plotted against the average expression for all metastatic lines (abscissa). Size of points represents the absolute difference in expression between the clonally-related T_Met and M lines (393T5 and 393M1, respectively). The color represents statistical significance of the differences between any two adjacent cell line classes (i.e. T_nonMet vs. T_Met, T_Met vs. N, N vs. M) as determined by Student’s t-test. The dashed line represents equivalent expression between the metastatic and primary tumor-derived lines. (C) qRT-PCR analysis of transferase gene expression. Top: gene expression of Gcnt3 and St6galnac4 in representative T_nonMet, T_Met, and M cell lines. Bottom: C2GnT2 (Gcnt3) can transfer a GlcNAc to the core GalNAc of T-Antigen by a β1-6 linkage. St6galnac4 can transfer a NeuAc to the core GalNAc of the sialyl-T-Antigen. (D) GCNT3 exhibits a loss of copy number in human lung adenocarcinomas compared to normal lung tissue or blood (P = 1.44 × 10⁻¹¹) in the “Lung adenocarcinoma” data set available from The Cancer Genome Atlas website (54). P-values in (C) were determined by One-way ANOVA with Tukey’s Multiple Comparison Test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 6. Knockdown of St6galnac4 reduces galectin-3 binding and prevents metastasis in vivo

(A) Analysis of galectin-3 binding to the M line 393M1 following knockdown of St6galnac4 by retroviral transduction of short hairpins targeting St6galnac4 or a control gene (Luc, firefly luciferase) by flow cytometry. (E,F) Wild-type mice were transplanted with GFP⁺ M cells bearing the short hairpin targeting St6galnac4 (393M1-shSt6galnac4) or control hairpin (393M1-shLuc) by intrasplenic injection. Two weeks following implantation, the livers of mice were excised and imaged. (B) Representative livers as visualized by fluorescence (top), brightfield (middle), and hematoxylin and eosin staining (bottom). (C) Quantification of the number of surface nodules on the livers. Scale bars in (B) are 2mm. P-value in (C) was determined by Mann-Whitney test. ** P < 0.01.

Figure 7. T-Antigen presentation promotes metastasis through interactions with galectins in the metastatic niche

Schematic model of galectin-glycan interactions in lung cancer metastasis. Primary lung tumors have high expression of branching glycosyltransferases such as C2GnT2 (Gcnt3) and low expression sialyltransferases such as St6GalNAcIV that act to promote O-glycan branching and elongation. Both the primary and metastatic tumors secrete tumor-derived soluble factors, such as IL-6, which act to mobilize CD11b⁺galectin-3⁺ myeloid cells into the peripheral blood. Galectin-3 levels become elevated in the liver early metastatic niche where it is presented on macrophages. Metastatic cells adhere to galectin-3 and -8 through increased T-Antigen presentation, which is mediated by aberrant glycosyltansferase activity. Decreased expression of the branching transferases and increased expression of sialyltransferases prevent glycan elongation and promote preservation of the T-Antigen, which in turn, mediates galectin-3 adhesion.