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. 2004 Dec;165(6):1931-41.
doi: 10.1016/S0002-9440(10)63245-2.

Alterations in galectin-3 Expression and Distribution Correlate With Breast Cancer Progression: Functional Analysis of galectin-3 in Breast Epithelial-Endothelial Interactions

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Free PMC article

Alterations in galectin-3 Expression and Distribution Correlate With Breast Cancer Progression: Functional Analysis of galectin-3 in Breast Epithelial-Endothelial Interactions

Malathy P V Shekhar et al. Am J Pathol. .
Free PMC article

Abstract

To define the role of galectin-3 in breast cancer progression, we have used a novel three-dimensional co-culture system that recapitulates in vivo reciprocal functional breast epithelial-endothelial cell-cell and cell-matrix interactions, and examined the expression of galectin-3 mRNA and protein in human breast tumors and xenografts. Galectin-3 is required for the stabilization of epithelial-endothelial interaction networks because immunoneutralization with galectin-3 antibodies abolishes the interactions in a dose-dependent manner. Co-culture of epithelial cells with endothelial cells results in increase in levels of secreted galectin-3 and presence of proteolytically processed form of galectin-3 in the conditioned media. In contrast, intracellular galectin-3 predominantly exists in the intact form. This difference in sensitivity to proteolytic processing of secreted versus intracellular galectin-3 probably arises from differences in accessibility of protease-sensitive sites, levels, and/or type of activated protease(s), and may be indicative of different functional roles for intact and processed galectin-3. To determine whether the proteolytically cleaved galectin-3 retains its ability to bind to endothelial cells, binding assays were performed with the full-length and matrix metallopeoteinase-2-cleaved recombinant galectin-3. Although a dose-dependent increase in binding to human umbilical vein endothelial cells was observed with both full-length and cleaved galectin-3, proteolytically cleaved galectin-3 displayed approximately 20-fold higher affinity for human umbilical vein endothelial cells as compared to the full-length protein. Examination of galectin-3 expression in breast tumors and xenografts revealed elevated levels of galectin-3 mRNA and protein in the luminal epithelial cells of normal and benign ducts, down-regulation in early grades of ductal carcinoma in situ (DCIS), and re-expression in peripheral tumor cells as DCIS lesions progressed to comedo-DCIS and invasive carcinomas. These data suggest that galectin-3 expression is associated with specific morphological precursor subtypes of breast cancer and undergoes a transitional shift in expression from luminal to peripheral cells as tumors progressed to comedo-DCIS or invasive carcinomas. Such a localized expression of galectin-3 in cancer cells proximal to the stroma could lead to increased invasive potential by inducing novel or better interactions with the stromal counterparts.

Figures

Figure 1
Figure 1
Regulation of three-dimensional growth of EIII8-HUVEC co-cultures by galectin-3. Control cultures were untreated (control) or 24 hours after seeding treated with 0.5 or 1.0 μg of HL-31 or TIB166, or 1 μg of corresponding rat or rabbit nonimmune IgG. a: Phase-contrast morphology of heterotypic three-dimensional EIII8-HUVEC co-cultures. A and B: Co-cultures that were untreated or treated with 1.0 μg of normal rabbit IgG, respectively. C and D: Co-cultures that were treated with 1.0 μg of TIB166 or HL-31 galectin-3 antibody, respectively. Arrows indicate ductal alveolar outgrowths that co-localize with endothelial enriched regions. b: Cultures were treated with dispase to solubilize Matrigel- and trypan blue-excluded cells were counted by hemocytometer. Results obtained from three independent experiments performed in triplicate are expressed as mean ± SE. *, P < 0.001 and **, P < 0.005 are amounts of antibody that inhibited cell number significantly over corresponding control. Scale bar, 40 μm (a).
Figure 2
Figure 2
Three-dimensional EIII8-HUVEC co-cultures show higher steady-state levels of secreted galectin-3 and differences in protease sensitivity between secreted and intracellular galectin-3. Proteins, 20 or 50 μg, present in culture media and corresponding matrix fractions, respectively, at day 5 were analyzed by Western blotting. A and B: Galectin-3 levels in conditioned media of EIII8 or EIII8-HUVEC co-cultures that were untreated (control) or treated with 0.01 or 0.1% CP or MCP. A’ and B’: Galectin-3 detected in lysates of untreated, CP- or MCP-treated EIII8-HUVEC co-cultures, and EIII8 homotypic cultures. A and A’: Galectin-3 detected with polyclonal HL-31. B and B’: Detection with monoclonal TIB-166 galectin-3 antibody. A“: Short exposure of A’. C: Coomassie blue R250 staining of proteins in conditioned media; C’: Steady-state levels of β-actin in lysate samples. Arrows indicate positions of 62 (A)-, 27 (A)-, and 22 (A’)-kd bands.
Figure 3
Figure 3
MMP-2 cleaved galectin-3 displays greater affinity for endothelial cells. HUVECs were incubated with indicated amounts of radioiodinated full-length or MMP-2-cleaved recombinant galectin-3 protein in the absence or presence of 50 mmol/L lactose. A and B: Full-length galectin-3 in the absence (A) or presence (B) of lactose. C and D: Cleaved galectin-3 in the absence (C) or presence (D) of lactose.
Figure 4
Figure 4
Formalin-fixed, paraffin-embedded sections of EIII8-HUVEC three-dimensional co-cultures were either stained with H&E (A and B) or with antibodies to cd31 (C), factor VIII (D), cytokeratins (E), proliferating cell nuclear antigen (F), or galectin-3 monoclonal TIB-166 antibody (G and H). Note the widespread immunoreactivity to cytokeratins in the branching end buds as opposed to the localized cd31 and factor VIII8-expressing endothelial cells. Also note the presence of numerous proliferating cells in branching end buds invading into the surrounding ECM. Note that galectin-3 staining is exclusively localized in epithelial buds with higher TIB-166 immunoreactivities in epithelial cells that are adjacent to endothelial cells (arrows, G and H). Original magnifications: ×25 (A–F); ×40 (H); ×10 (G).
Figure 5
Figure 5
Galectin-3 mRNA synthesis occurs only in the epithelial compartment of EIII8-HUVEC co-cultures. A and B: In situ hybridization analysis with anti-sense and sense DIG-labeled galectin-3 RNA probes, respectively. Note the presence of galectin-3 mRNA signals detected only with anti-sense galectin-3 probe and its exclusive presence in the epithelial compartment. Original magnifications, ×40.
Figure 6
Figure 6
Galectin-3 mRNA and protein expression in premalignant lesions produced by MCF10AT1 xenografts. A and B: In situ hybridization with DIG-labeled anti-sense and sense galectin-3 RNA probes, respectively. C: Galectin-3 immunoreactivity to TIB-166 anti-galectin-3 antibody. Note the presence of strong galectin-3 protein expression in normal (thin arrow) and hyperplastic (block arrow) ducts. Also, note the presence of galectin-3 staining in the lumens of normal and hyperplastic ducts. Original magnifications, ×4.
Figure 7
Figure 7
Galectin-3 mRNA and protein expression in human breast tumors. A–C and A’–C’: In situ hybridization with anti-sense and sense galectin-3 RNA probes, respectively. A”–C”: Galectin-3 protein staining with TIB-166 anti-galectin-3 antibody. A, A’, and A”: Normal areas of breast cancer tissue. B, B’, and B”: Papillary hyperplasias. C, C’, and C”: Infiltrating carcinoma cells. Note the mRNA signals detected only with anti-sense galectin-3 probe (A–C). Also note the presence of intense galectin-3 protein immunoreactivity in the lumens of normal and hyperplastic ducts and absence of staining in the adjacent stroma (A” and B”). In contrast, note the presence of very strong galectin-3 protein staining in invasive cancer cells and in fibroblasts and extracellular matrix surrounding them (C”). Original magnifications: ×25 (A, B, A’, B’, A”, B”); ×4 (C, C’, C”).
Figure 8
Figure 8
Galectin-3 mRNA and protein expression exhibit alterations that are coincident with progression to comedo-DCIS of DCIS.com-derived xenografts. A and D: DCIS lesions harvested at 20 days. B, C, and F: Lesions harvested at 60 days. E: Lesions harvested at 40 days. A–C: In situ hybridization analysis with anti-sense galectin-3 RNA probe. D–F: Galectin-3 protein detected with TIB-166 galectin-3 antibody. Note the absence of detectable galectin-3 mRNA signals in early DCIS lesions, ie, before manifestation of comedo necrotic core (A). However, on progression to comedo-type, note the expression of galectin-3 mRNA in tumor cells away from the central necrotic core (B and C). Arrows in B and C indicate the comedo necrotic cores. Similarly, note that only very few tumor cells express galectin-3 protein in early DCIS lesions (D, arrow). By day 40, galectin-3 protein-staining peripheral cells are prevalent (E), and on manifestation of central comedo-necrosis, focally intense galectin-3-immunoreactive signals are observed in the tumor cells that are in close proximity to the stroma (F). Original magnifications: ×10 (A, CE); ×4 (B, F).

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