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.

A galectin-3 sequence polymorphism confers TRAIL sensitivity to human breast cancer cells.

BACKGROUND: A common polymorphism, rs4644, coding for Pro64 or His64 of the carbohydrate-binding protein galectin-3, influences the susceptibility of galectin-3 to cleavage by matrix metalloproteinases and is associated with breast cancer incidence. Because forced expression of galectin-3 in a galectin-3 null breast cancer cell line confers sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the authors sought to determine whether the His64/Pro64 polymorphism of galectin-3 affects the sensitivity to TRAIL. METHODS: Genomic DNA of breast cell lines was analyzed for the single nucleotide polymorphism rs4644, and cytotoxicity was determined with the MTT assay. RESULTS: When a collection of 9 breast cancer cell lines that express galectin-3 was examined for lectin, galactoside-binding, soluble, 3 (LGALS3) genotype and sensitivity to doxorubicin and TRAIL, doxorubicin sensitivity was not found to be related to LGALS3 genotype. In contrast, none of the 5 cell lines that were homozygous for Pro64 galectin-3 were found to be sensitive to TRAIL, but 2 of 2 homozygous His64 cell lines and 1 of 2 heterozygous His64 cell lines were sensitive to TRAIL. Forced expression of galectin-3 of defined genotype in galectin-3 null cells was used to more directly test the effect of the Pro64His mutation on TRAIL sensitivity. High levels of expression of His64 galectin-3 rendered BT549 cells sensitive to TRAIL and resistant to doxorubicin, but cells expressing Pro64 galectin-3 remained resistant to TRAIL and sensitive to doxorubicin. CONCLUSIONS: The results of the current study indicate that the naturally occurring Pro64His mutation in galectin-3 increases sensitivity to death receptor-mediated apoptosis. This finding could be relevant to disparities in breast cancer outcomes across population groups, and could guide the design of future clinical trials of TRAIL-based therapies.

Phosphorylation of galectin-3 contributes to malignant transformation of human epithelial cells via modulation of unique sets of genes.

Galectin-3 is a multifunctional beta-galactoside-binding protein implicated in apoptosis, malignant transformation, and tumor progression. The mechanisms by which galectin-3 contributes to malignant progression are not fully understood. In this study, we found that the introduction of wild-type galectin-3 into nontumorigenic, galectin-3-null BT549 human breast epithelial cells conferred tumorigenicity and metastatic potential in nude mice, and that galectin-3 expressed by the cells was phosphorylated. In contrast, BT549 cells expressing galectin-3 incapable of being phosphorylated (Ser6-->Glu Ser6-->Ala) were nontumorigenic. A microarray analysis of 10,000 human genes, comparing BT549 transfectants expressing wild-type and those expressing phosphomutant galectin-3, identified 188 genes that were differentially expressed (>2.5-fold). Genes affected by introduction of wild-type phosphorylated but not phosphomutant galectin-3 included those involved in oxidative stress, a novel noncaspase lysosomal apoptotic pathway, cell cycle regulation, transcriptional activation, cytoskeleton remodeling, cell adhesion, and tumor invasion. The reliability of the microarray data was validated by real-time reverse transcription-PCR (RT-PCR) and by Western blot analysis, and clinical relevance was evaluated by real-time RT-PCR screening of a panel of matched pairs of breast tumors. Differentially regulated genes in breast cancers that are also predicted to be associated with phospho-galectin-3 in transformed BT549 cells include C-type lectin 2, insulin-like growth factor-binding protein 5, cathepsins L2, and cyclin D1. These data show the functional diversity of galectin-3 and suggest that phosphorylation of the protein is necessary for regulation (directly or indirectly) of unique sets of genes that play a role in malignant transformation.

Cell surface galectin-3 defines a subset of chemoresistant gastrointestinal tumor-initiating cancer cells with heightened stem cell characteristics.

Recurrence of gastrointestinal adenocarcinomas after surgery and chemotherapy may be attributed, in part, to the presence of a small population of tumor-initiating cancer stem cells (CSC). The expression of galectin-3 (Gal3), a multifunctional oncolectin, has been associated with biological behaviors associated with CSC. We examined the ability of Gal3 to characterize the CSC phenotype, and to identify a clinically important gastrointestinal cancer CSC population. Human colorectal and pancreatic cancer cell lines were sorted to identify subpopulations expressing commonly used CSC markers, and Gal3-positive CSC subpopulations. The association of Gal3 with the stem cell properties and alterations of these phenotypes by manipulation of Gal3 expression was examined. Gastrointestinal cancer cell lines contain both Gal3-positive and Gal3-negative subpopulations. Gal3-positive CSCs are characterized by high ALDH activity, enhanced self-renewal ability in vitro (sphere formation) and tumor forming ability in vivo, and resistance to chemotherapeutic agents and death-receptor-mediated apoptosis compared to Gal3-negative CSCs. Silencing Gal3 modifies this behavior. Cell surface Gal3 expression identifies a subset of CSCs in gastrointestinal cancers with high levels of stem cell characteristics, including chemoresistance. This may provide a platform for developing treatment strategies that target CSC.

Galectin-3 mediates nuclear beta-catenin accumulation and Wnt signaling in human colon cancer cells by regulation of glycogen synthase kinase-3beta activity.

Wnt/beta-catenin signaling plays an essential role in colon carcinogenesis. Galectin-3, a beta-galactoside-binding protein, has been implicated in Wnt signaling, but the precise mechanisms by which galectin-3 modulates the Wnt pathway are unknown. In the present study, we determined the effects of galectin-3 on the Wnt/beta-catenin pathway in colon cancer cells, as well as the mechanisms involved. Galectin-3 levels were manipulated in human colon cancer cells by stable transfection of galectin-3 antisense, short hairpin RNA, or full-length galectin-3 cDNA, and effects on beta-catenin levels, subcellular distribution, and Wnt signaling were determined. Galectin-3 levels correlated with beta-catenin levels in a variety of colon cancer cell lines. Down-regulation of galectin-3 resulted in decreased beta-catenin protein levels but no change in beta-catenin mRNA levels, suggesting that galectin-3 modulates beta-catenin by another mechanism. Reduction of galectin-3 led to reduced nuclear beta-catenin with a concomitant decrease in TCF4 transcriptional activity and expression of its target genes. Conversely, transfection of galectin-3 cDNA into colon cancer cells increased beta-catenin expression and TCF4 transcriptional activity. Down-regulation of galectin-3 resulted in AKT and glycogen synthase kinase-3beta (GSK-3beta) dephosphorylation and increased GSK activity, increasing beta-catenin phosphorylation and degradation. Ly294002, an inhibitor of phosphatidylinositol 3-kinase, and dominant-negative AKT, suppressed TCF4 transcriptional activity induced by galectin-3 whereas LiCl, a GSK-3beta inhibitor, increased TCF4 activity, mimicking the effects of galectin-3. These results suggest that galectin-3 mediates Wnt signaling, at least in part, by regulating GSK-3beta phosphorylation and activity via the phosphatidylinositol 3-kinase/AKT pathway, and, thus, the degradation of beta-catenin in colon cancer cells.

Phosphorylated galectin-3 mediates tumor necrosis factor-related apoptosis-inducing ligand signaling by regulating phosphatase and tensin homologue deleted on chromosome 10 in human breast carcinoma cells.

Galectin-3 (GAL3), a beta-galactoside-binding lectin, confers chemoresistance to a wide variety of cancer cell types. It may exhibit anti- or pro-apoptotic activity depending on the nature of the stimulus. We report here that introducing phosphorylated galectin-3 (P-GAL3) into GAL3-null, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant human breast carcinoma cells promotes TRAIL-induced apoptotic cell death by stimulating the phosphorylation/inactivation of the pro-apoptotic molecule Bad resulting in the inhibition of mitochondrial depolarization and the release of cytochrome c. Exposure of the transfectant cells to TRAIL leads to the recruitment of the initiator capase-8 followed by activation of the effector caspase-9, independent of cytochrome c, and subsequently the processing of the executioner caspase-3. P-GAL3 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) were coordinately expressed, with concomitant dephosphorylation of Akt in TRAIL-sensitive cells. In contrast, overexpression of phospho-mutant GAL3 (incapable of phosphorylation) failed to elicit similar responses. Depletion of PTEN using small interference RNAs reinstated Akt phosphorylation and conferred TRAIL resistance. In addition phosphatidylinositol 3-kinase inhibitors rendered the phospho-mutant GAL3-resistant cells sensitive to TRAIL. These findings suggest a pivotal role for P-GAL3 in promoting TRAIL sensitivity through activation of a nonclassic apoptotic pathway and identify P-GAL3 as a novel regulator of PTEN.

Galectin-3 modulates MUC2 mucin expression in human colon cancer cells at the level of transcription via AP-1 activation.

BACKGROUND & AIMS: Galectin-3 and MUC2 intestinal mucin each have been correlated with the malignant behavior of colon cancer cells. Galectin-3 modulates expression of MUC2 protein, but the specific regulatory mechanisms are unknown. This study sought to determine how galectin-3 increases MUC2 expression. METHODS: Galectin-3 levels in human colon cancer cells of high and low metastatic ability were manipulated via expression of galectin-3 complementary DNA in sense or antisense orientation. Galectin-3 and MUC2 protein expression were determined by Western analysis and immunocytochemistry. Transient transfections of promoter reporter constructs were used to monitor MUC2 transcription and AP-1 activity. Electrophoretic mobility shift assays, site-directed mutagenesis, and chromatin immunoprecipitation were used to monitor the participation of AP-1 in MUC2 transcription. RESULTS: Alterations in galectin-3 levels correlated with both MUC2 protein expression and transcription. By using MUC2 promoter constructs of different lengths, galectin-3 responsiveness was found between 1500 and 2186 bp upstream of the translation start site, a region that contains 1 consensus AP-1 binding site. AP-1 activity paralleled MUC2 transcription in the different cell lines. Mutation in the AP-1 site markedly decreased MUC2 promoter activity, and MUC2 transcription was inhibited by cotransfection with a dominant-negative AP-1 vector. Electrophoretic mobility shift assays, co-immunoprecipitation, and chromatin immunoprecipitation analyses suggested an association between galectin-3, c-Jun, and Fra-1 in forming a complex at the AP-1 site on the MUC2 promoter. CONCLUSIONS: Galectin-3 up-regulation of MUC2 transcription occurs at the level of transcription through AP-1 activation. This may have important implications for understanding the role of galectin-3 and MUC2 in colon cancer metastasis.

A circulating ligand for galectin-3 is a haptoglobin-related glycoprotein elevated in individuals with colon cancer.

BACKGROUND & AIMS: Galectin-3 is a beta-galactoside-binding protein implicated in tumor progression and metastasis of colorectal cancers. To determine whether circulating galectin-3 ligands are related to the presence of colon cancer, we sought to identify and quantify ligands in serum that bind to galectin-3. METHODS: Sera from patients with colon cancer, adenomas, and normal individuals were desialylated, reduced, and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and blots probed with biotinylated galectin-3. RESULTS: In colon cancer sera, the major galectin-3 ligand was a 40-kilodalton band distinct from mucin, carcinoembryonic antigen, and Mac-2 binding protein. Serum 40-kilodalton ligand was 10- to 30-fold higher in patients with colon cancer than in healthy subjects. Ligand was purified by gel filtration, affinity precipitation on galectin-3/agarose, and SDS-PAGE. When tryptic peptides were analyzed by matrix-assisted laser-desorption ionization mass spectrometry and protein database searching, the 40-kilodalton ligand was identified as haptoglobin beta subunit. In confirmation of this finding, depletion of haptoglobin by immunoprecipitation also eliminated the 40-kilodalton ligand. Colon cancer sera had only a modest increase in total haptoglobin as compared with healthy subjects, suggesting that the structure rather than the amount of haptoglobin is altered in patients with colon cancer. Immunohistochemical staining confirmed the absence of haptoglobin in normal colon and the ectopic expression of haptoglobin in colon cancers and adenomatous polyps. CONCLUSIONS: A major circulating ligand for galectin-3, which is elevated in the sera of patients with colon cancer, is a cancer-associated glycoform of haptoglobin.