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

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

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF

Symphyseal fixation in open book injuries cannot fully compensate anterior SI joint injury-A biomechanical study in a two-leg alternating load model.

In open book injuries type Tile B1.1 or B1.2 also classified as APC II (anteroposterior compression), it remains controversial, if a fixation of the anterior ring provides sufficient stability or a fixation of the posterior ring should be included. Therefore the relative motion at the sacroiliac joint was quantified in a two-leg alternating load biomechanical pelvis model in the intact, the injured and the restored pelvis.Fresh-frozen intact (I) pelvises (n = 6) were subjected to a non-destructive cyclic test under sinosuidal axial two-leg alternating load with progressively increasing amplitude. Afterwards an open book injury (J) including the anterior ligament complex of the left sacroiliac joint, the sacrospinal and sacrotuberal ligaments (Tile B1.1) was created and the specimens were retested. Finally, the symphysis was stabilized with a modular fixation system (1-, 2- or 4-rod configuration) (R) and specimens were cyclically retested. Relative motion at the sacroiliac joint was captured at both sacroiliac joints by motion tracking system at two load levels of 170 N and 340 N during all tests.Relative sacroiliac joint movements at both load levels were significantly higher in the J-state compared to the I-state, excluding superoinferior translational movement. With exception of the anteroposterior translational movement at 340N, the relative sacroiliac joint movements after each of the three reconstructions (1-, 2-, 4-rod fixation) were significantly smaller compared to the J-state and did not differ significantly to the I-state, but stayed above the values of the latter. Relative movements did not differ significantly in a direct comparison between the 1-rod, 2-rod and 4-rod fixations.Symphyseal locked plating significantly reduces relative movement of the sacroiliac joint in open book injuries type Tile B1.1 or B1.2 (APC II) but cannot fully restore the situation of the intact sacroiliac joint