Nachman Mazurek

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.

Galectin-3 Mediates Nuclear β-Catenin Accumulation and Wnt Signaling in Human Colon Cancer Cells by Regulation of Glycogen Synthase Kinase-3β 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.

Cell-surface galectin-3 confers resistance to TRAIL by impeding trafficking of death receptors in metastatic colon adenocarcinoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors, resulting in the internalization of ligand/receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms, we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL/death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL resistance. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL sensitivity and promotes TRAIL-mediated endocytosis of TRAIL/death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand/receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal.

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.

A Galectin-3 Sequence Polymorphism Confers TRAIL Sensitivity to Human Breast Cancer Cells

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.

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.

Abstract 1930: Low surface Gal3 expression in breast cancer stem cells is associated with chemoresistance, tumorigenesis, and decreased overall patient survival

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The development of therapeutic approaches that target specific breast cancer subtypes has improved overall survival, but some patients nevertheless become resistant to treatment and develop metastatic disease. Recent insights suggest multiple hallmarks of cancer - especially drug-resistance, unlimited self-renewal, as well as invasion and metastasis - that can be attributed to a small subpopulation frequently designated as cancer stem cells (CSC). CSCs are rare, self-renewing cells that initiate and maintain tumor growth. Galectin-3 (Gal3), a multifunctional oncogenic beta-galactoside binding lectin, was implicated in the metastatic cascade by aiding the homing of breast cancer circulating tumor cells to the metastatic niche. In addition, it was proposed that Gal3 protects breast cancer cells from mitochondria-dependent apoptosis induced by chemotherapeutic agents while concurrently promoting sensitivity to cell death ligands. Recently, we have found that in a subset of metastatic breast cancer cells (GI-LM2), cell-surface Gal3 is highly expressed compared to its non-metastatic counterpart (GI-101). Breast CSCs have been characterized by CD24-/CD44+/EpCAM+ surface marker expression in flowcytometry analysis. These subpopulations of GI-LM2 exhibit an enhanced self-renewal capability in sphere-formation assays (SFA) when compared to a similar subpopulation of GI-101. Surprisingly, depletion of Gal3 from this subset of GI-LM2 resulted in epithelial-mesenchymal-transition (EMT) - a mechanism linked to cancer stemness - as evidenced by Immunoblot and -fluorescence, resistance to the adjuvant FAC regimen (5-Fluorouracil, Doxorubicin, Cyclophosphamide), and markedly increased tumorigenicity in in vivo limited dilution assays. These findings are in keeping with results obtained from a stage 2 and 3a cohort of breast cancer patients (n=87) with axillary lymph node metastases. Gal3 expression in tissue microarrays from these patients was found to be significantly associated with the absence of lymphovascular invasion (p=0.015) as well as increased locoregional-free, disease-specific and overall survival (p=0.054, p=0.18, and p=0.019, respectively). Taken together, we present evidence in vitro, in vivo, and in clinico that lack of surface Gal3 in a subset of breast CSCs is associated with chemoresistance. This contrasts with earlier reports that Gal3 renders cancer cells chemoresistant, enhances tumorigenesis, and is associated with poor overall survival. Further studies will determine whether these finding can be applied to therapeutic stratification of breast cancer patients, or as a diagnostic tool for detection of circulating tumor cells which are frequently described as early indicators of dormant metastatic breast cancer. Citation Format: Matthias Illmer, Nachman Mazurek, James Byrd, Jody Vykoukal, Robert Scott Bresalier. Low surface Gal3 expression in breast cancer stem cells is associated with chemoresistance, tumorigenesis, and decreased overall patient survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1930. doi:10.1158/1538-7445.AM2014-1930