Low expression of galectin-3 is associated with poor survival in node-positive breast cancers and mesenchymal phenotype in breast cancer stem cells

Background Galectin-3 (Gal3) plays diverse roles in cancer initiation, progression, and drug resistance depending on tumor type characteristics that are also associated with cancer stem cells (CSCs). Recurrence of breast carcinomas may be attributed to the presence of breast CSCs (BCSCs). BCSCs exist in mesenchymal-like or epithelial-like states and the transition between these states endows BCSCs with the capacity for tumor progression. The discovery of a feedback loop with galectins during epithelial-to-mesenchymal transition (EMT) prompted us to investigate its role in breast cancer stemness. Method To elucidate the role of Gal3 in BCSCs, we performed various in vitro and in vivo studies such as sphere-formation assays, Western blotting, flow cytometric apoptosis assays, and limited dilution xenotransplant models. Histological staining for Gal3 in tissue microarrays of breast cancer patients was performed to analyze the relationship of clinical outcome and Gal3 expression. Results Here, we show in a cohort of 87 node-positive breast cancer patients treated with doxorubicin-based chemotherapy that low Gal3 was associated with increased lymphovascular invasion and reduced overall survival. Analysis of in vitro BCSC models demonstrated that Gal3 knockdown by small hairpin RNA (shRNA) interference in epithelial-like mammary spheres leads to EMT, increased sphere-formation ability, drug-resistance, and heightened aldefluor activity. Furthermore, Gal3negative BCSCs were associated with enhanced tumorigenicity in orthotopic mouse models. Conclusions Thus, in at least some breast cancers, loss of Gal3 might be associated with EMT and cancer stemness-associated traits, predicts poor response to chemotherapy, and poor prognosis

Overexpressed Galectin-3 in Pancreatic Cancer Induces Cell Proliferation and Invasion by Binding Ras and Activating Ras Signaling

<div><p>Pancreatic cancer (PDAC) is a lethal disease with a five-year survival of 3–5%. Mutations in K-Ras are found in nearly all cases, but K-Ras mutations alone are not sufficient for the development of PDAC. Additional factors contribute to activation of Ras signaling and lead to tumor formation. Galectin-3 (Gal-3), a multifunctional β-galactoside-binding protein, is highly expressed in PDAC. We therefore investigated the functional role of Gal-3 in pancreatic cancer progression and its relationship to Ras signaling. Expression of Gal-3 was determined by immunohistochemistry, Q-PCR and immunoblot. Functional studies were performed using pancreatic cell lines genetically engineered to express high or low levels of Gal-3. Ras activity was examined by Raf pull-down assays. Co-immunoprecipitation and immunofluorescence were used to assess protein-protein interactions. In this study, we demonstrate that Gal-3 was highly up-regulated in human tumors and in a mutant K-Ras mouse model of PDAC. Down-regulation of Gal-3 by lentivirus shRNA decreased PDAC cell proliferation and invasion in vitro and reduced tumor volume and size in an orthotopic mouse model. Gal-3 bound Ras and maintained Ras activity; down-regulation of Gal-3 decreased Ras activity as well as Ras down-stream signaling including phosphorylation of ERK and AKT and Ral A activity. Transfection of Gal-3 cDNA into PDAC cells with low-level Gal-3 augmented Ras activity and its down-stream signaling. These results suggest that Gal-3 contributes to pancreatic cancer progression, in part, by binding Ras and activating Ras signaling. Gal-3 may therefore be a potential novel target for this deadly disease.</p> </div

Gal-3 expression in human pancreatic tumor tissues.

<p>A, Tissue microarray slides consisting of 125 pancreatic ductal adenocarcinomas and paired non-neoplastic pancreatic tissue samples were immunohistochemically stained using a monoclonal Gal-3 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. Gal-3 expression was increased along the disease sequence-normal (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#pone-0042699-g001" target="_blank">Figure 1A, a,b</a>), pancreatitis (c,d) and pancreatic ductal adenocarcinoma (e,f). B. Summary of Gal-3 IHC of the PDAC tissue microarrays. The tumors were categorized into Gal3-low (combined scores ≤3) and Gal3-high (combined score ≥4) based on the percentage of Gal-3 staining in tumor cells (0, no staining; 1, ≤10%; 2, 10–50% and 3, >50%) and the staining intensity (0-negative, 1-weak, 2-moderate and 3- strong).</p

Gal-3 expression in pancreatic tumor tissues and cells from a K-RAS mutant mouse model.

<p>A. Gal-3 expression was analyzed in cells derived from normal pancreas and pancreatic tumors from K-Ras mutant mice by western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. B. Real-time quantitative PCR for Gal-3 RNA expression using Gal-3-specific primers after total RNA extraction from normal pancreas, pancreatitis tissues and pancreatic tumors and from isolated cells from different tumors arising in a K-Ras mutant mouse model as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. Fold change from determinations performed in triplicate.</p

Gal-3 binds to Ras and mediates Ras activity in PDAC cells.

<p>A. Co-Immunoprecipitation was performed in Panc-1 and MPanc96 with knock down Gal-3 using either anti-Ras or anti-Gal-3 antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. B. Equal amounts of protein from Panc-1 and Mpanc96 GN10 control cells or shRNA A3 cells were incubated with agarose beads coated with Raf1-RBD, and active Ras-GTP was detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. C. Active Ras GTP activity was determined in BXPC-3 L-gal3 cells and control cells (V) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. D. Cell plasma membrane and Cytoplasmic fractions of GN10 and A3 cells from both Panc-1 and MPan96 were subjected to SDS-PAGE and then immunoblotted with anti-Ras antibody. E. Indirect immunofluorescence was performed on BXPC-3-V and BXPC-3 L-gal3 cells using anti-Gal-3 antibody (TIB166 1∶100, red) and anti-Ras antibody (1∶100, green), followed by DAPI counterstaining (blue). The merge of Gal-3 (red) and Ras (green) with DAPI (blue) is also shown.</p

Effects of Gal-3 on PDAC cell colony formation and the growth of PDAC tumor in vivo.

<p>A. Colony formation assays were performed in MPanc96 GN10 control cells and Gal-3 shRNA knock down A3 cells (top panel) or BXPC-3 V control cells and BXPC-3 Gal3 cDNA overexpressed L-gal3 cells (lower panel) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>. MPanc96-A3 cells formed smaller and fewer colonies compared with the vector transfected control cells (GN10). In contrast, BXPC-3 L-gal3 cells formed larger and a greater number of colonies than control V cells. B. Bar graph in the right panel demonstrates the mean colony numbers after plating either MPanc96 GN10 control cells and shRNA knock down A3 cells (top) or BXPC-3 V and BXPC-3 L-gal3 (lower); p<0.001. C. Representative bioluminescence images of athymic mice 3 weeks after orthotopic implantation of GN10 and A3 pancreatic cancer cells orthotopically into the pancreas of athymic mice. D. Measurements of photons/s/cm²/steridian depicting bioluminescence area at 10% peak margin (mean ± SE) at week 3 using Xenogen IVIS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a> (<i>n</i> = <i>5</i>). E. Tumor weights from control mice (GN10, n = 5) and Gal3 knockdown group (A3, n = 5) were weighted after mice were sacrifice at week four. F. Mouse tumor tissues from control (GN10) and Gal-3 knockdown group (A3) were immunohistochemically stained using Gal-3, Ras and phospho-ERK antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042699#s2" target="_blank">Materials and Methods</a>.</p

Additional file 2: Figure S1. of Low expression of galectin-3 is associated with poor survival in node-positive breast cancers and mesenchymal phenotype in breast cancer stem cells

The Cancer Genome Atlas (TCGA) data show the gene expression of Gal3 (LGALS3) in normal (no value), ductal breast carcinoma in situ and invasive ductal breast carcinoma (A) or normal (no value), primary site and metastatic site of human breast cancer samples (B). (C) Western blot analysis of whole cell lysates of GI-101A and its derivatives (GI-LM2, GI-LM2C, GI-LM2G) on estrogen receptor (ER) expression. Figure S2 (A) Immunofluorescence staining of GI-LM2C (upper row) and GI-LM2G spheres (lower row) for Gal3 (red), E-cadherin (CDH1, green), and vimentin (gray). (B) Immunofluorescence staining of the same cell lines for cytokeratin 18 (red) and vimentin (green). Counterstaining with DAPI (blue) was used to visualize cell nuclei. Figure S3 (A) Flow cytometric analysis shows that Gal3-positive populations (in red) of the same cell line consistently contain a lower BCSC pool than Gal3-negative populations (in green). (B) Correlation of Gal3 with CD24 and EpCAM expression is listed in a table. Figure S4 (A) Brightfield pictures of spheres in low magnification. Figure is related with Fig. 3a. (B) Sphere-formation assay and its quantification of GI-101A, GI-101A after knockout of Gal3 (GI-101A-G) as well as derivatives GI-LM2C and GI-LM2G. (C) Western blot of whole cell lysates of GI-LM2C and GI-LM2G for Wnt targets Axin2 and Tcf4. Loading control β-actin was used. The same membrane is used in Fig. S1C. (PPTX 2636 kb