Victor Hogan

Galectin-3 induces endothelial cell morphogenesis and angiogenesis

Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis. However, conflicting results on their function in the regulation of cell proliferation and differentiation during angiogenesis have been reported. We have examined the role of galectin-3 in the regulation of human umbilical vein endothelial cell proliferation, differentiation, migration, and neovascularization. Galectin-3, a carbohydrate-binding protein, with specificity for type 1 and 11 ABH blood group epitopes and polylactosamine glycan containing cell surface glycoproteins, is the major nonintegrin cellular laminin-binding protein. Because galectin-3 expression was shown to be associated in some tumor systems with metastasis, we questioned whether it induces endothelial cell morphogenesis. Here we show that galectin-3 affects chemotaxis and morphology and stimulates capillary tube formation of HUV-EC-C in vitro and angiogenesis in vivo. Endothelial cell morphogenesis is a carbohydrate-dependent process, as it is neutralized by specific sugars and antibodies. These findings demonstrate that endothelial cell surface carbohydrate recognition event(s) can induce a signaling cascade leading to the differentiation and angiogenesis of endothelial cells.

Galectin-3, a Novel Binding Partner of β-Catenin

Galectin-3 (gal-3), a pleiotrophic protein, is an important regulator of tumor metastasis, which like β-catenin shuttles between the nucleus and the cytosol in a phosphorylation-dependent manner. We report herein that β-catenin stimulation of cyclin D1 and c-myc expression is gal-3 dependent. Gal-3 binds to β-catenin/Tcf complex, colocalizes with β-catenin in the nucleus, and induces the transcriptional activity of Tcf-4 as determined by the TOP/FOPFLASH reporter system. We have identified the β-catenin–gal-3–binding sequences, which are in the NH2 and COOH termini of the proteins encompassing amino acid residues 1 to 131 and 143 to 250, respectively. These data indicate that gal-3 is a novel binding partner for β-catenin involved in the regulation of Wnt/β-catenin signaling pathway.

Carbohydrate-binding proteins in cancer, and their ligands as therapeutic agents

Experimental evidence directly implicates complex carbohydrates in recognition processes, including adhesion between cells, adhesion of cells to the extracellular matrix, and specific recognition of cells by one another. In addition, carbohydrates are recognized as differentiation markers and as antigenic determinants. Lectins are nonenzymatic proteins present in plants and animals, which preferentially bind to specific carbohydrate structures and play an important role in cell recognition. Modified carbohydrates and oligosaccharides have the ability to interfere with carbohydrate-protein interactions and therefore, inhibit the cell-cell recognition and adhesion processes, which play an important role in cancer growth and progression. Carbohydrate ligands therefore, are candidates to play important roles in cancer therapeutics.

Implication of Galectin-3 in Wnt Signaling

Galectin-3 (gal-3), a member of the beta-galactoside-binding proteins family, was identified as a binding partner of beta-catenin. Analysis of the human gal-3 sequence reveled a structural similarity to beta-catenin as it also contains the consensus sequence (S92XXXS96) for glycogen synthase kinase-3beta (GSK-3beta) phosphorylation and can serve as its substrate. In addition, Axin, a regulator protein of Wnt that complexes with beta-catenin, also binds gal-3 using the same sequence motif identified here by a deletion mutant analysis. The data presented here give credence to the suggestion that gal-3 is a key regulator in the Wnt/beta-catenin signaling pathway and highlight the functional similarities between gal-3 and beta-catenin.

Decreased Galectin-3 Expression in Prostate Cancer

Galectin‐3 is a carbohydrate‐binding protein whose level of expression has been shown to be correlated with metastatic potential in a number of different tumor types. The purpose of this investigation was to examine galectin‐3 expression in several tumorigenic and nontumorigenic prostate cell lines and prostate tissue samples.

Galectin-3 inhibits tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by activating Akt in human bladder carcinoma cells

The antiapoptotic molecule galectin-3 was previously shown to regulate CD95, a member of the tumor necrosis factor (TNF) family of proteins in the apoptotic signaling pathway. Here, we question the generality of the phenomenon by studying a different member of this family of proteins [e.g., TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in a wide variety of cancer cells]. Overexpression of galectin-3 in J82 human bladder carcinoma cells rendered them resistant to TRAIL-induced apoptosis, whereas phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY-294002) blocked the galectin-3 protecting effect. Because Akt is a major downstream PI3K target reported to play a role in TRAIL-induced apoptosis, we questioned the possible relationship between galectin-3 and Akt. Parental J82 and the control vector-transfected J82 cells (barely detectable galectin-3) exhibit low level of constitutively active Akt, resulting in sensitivity to TRAIL. On the other hand, J82 cells overexpressing galectin-3 cells expressed a high level of constitutively active Akt and were resistant to TRAIL. Moreover, the blockage of TRAIL-induced apoptosis in J82 cells seemed to be mediated by Akt through the inhibition of BID cleavage. These results suggest that galectin-3 involves Akt as a modulator molecule in protecting bladder carcinoma cells from TRAIL-induced apoptosis.

Endogenous Galectin-3 Determines the Routing of CD95 Apoptotic Signaling Pathways

Studies of CD95 (APO-1/Fas), a member of the death receptor family, have revealed that it is involved in two primary CD95 apoptotic signaling pathways, one regulated by the large amount of active caspase-8 (type I) formed at the death-inducing signaling complex and the other by the apoptogenic activity of mitochondria (type II). To date, it is still unclear which pathway will be activated in response to an apoptotic insult. Here, we demonstrate that the antiapoptotic molecule galectin-3, which contains the four amino acid-anti-death-motif (NWGR) conserved in the BH1 domain of the Bcl-2 member proteins, is expressed only in type I cells. Transfection of galectin-3 cDNA into galectin-3 null cells (type II) resulted converting them to type I apoptotic phenotype. In addition, we show that galectin-3 is complexed with CD95 in vivo identifying galectin-3 as a novel CD95-binding partner that determines which of the CD95 apoptotic signaling pathways the cell will select.

Regulation of Prostate Cancer Progression by Galectin-3

Galectin-3, a beta-galactoside-binding protein, has been implicated in a variety of biological functions including cell proliferation, apoptosis, angiogenesis, tumor progression, and metastasis. The present study was undertaken to understand the role of galectin-3 in the progression of prostate cancer. Immunohistochemical analysis of galectin-3 expression revealed that galectin-3 was cleaved during the progression of prostate cancer. Galectin-3 knockdown by small interfering RNA (siRNA) was associated with reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in the prostates of nude mice. Galectin-3 knockdown in human prostate cancer PC3 cells led to cell-cycle arrest at G(1) phase, up-regulation of nuclear p21, and hypophosphorylation of the retinoblastoma tumor suppressor protein (pRb), with no effect on cyclin D1, cyclin E, cyclin-dependent kinases (CDK2 and CDK4), and p27 protein expression levels. The data obtained here implicate galectin-3 in prostate cancer progression and suggest that galectin-3 may serve as both a diagnostic marker and therapeutic target for future disease treatments.

Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer

Galectin‐3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin‐3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)‐2/‐9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin‐3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin‐3 in induction of angiogenesis and (iii) determination of the galectin‐3 domain responsible for induction of angiogenic response. Galectin‐3 null breast cancer cells BT‐459 were transfected with either cleavable full‐length galectin‐3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial‐endothelial cell interactions and angiogenesis were compared to noncleavable galectin‐3. BT‐549‐H64 cells harboring cleavable galectin‐3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT‐549‐P64 cells harboring noncleavable galectin‐3. BT‐549‐H64 cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT‐549 cells transfected with galectin‐3 peptides indicate that amino acids 1–62 and 33–250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin‐3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin‐3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression.

Galectin-3 Cleavage: A Novel Surrogate Marker for Matrix Metalloproteinase Activity in Growing Breast Cancers

Failed therapies directed against matrix metalloproteinases (MMP) in cancer patients may be attributed, in part, to lack of diagnostic tools to differentiate between pro-MMPs and active MMPs, which indicate whether a treatment is efficacious or not. Because galectin-3 is cleavable in vitro by MMPs, we have developed differential antibodies recognizing its cleaved and noncleaved forms and tested their clinical utilization as a surrogate diagnostic marker for the presence of active MMPs in growing breast cancers. Wild-type and cleavage-resistant galectin-3 were constructed and expressed in galectin-3-null human breast carcinoma cells (BT-549). Tumorigenic and angiogenic potential of the clones was studied by injections into nude mice. MMP-2, MMP-9, full-length, and cleaved galectin-3 were localized in the xenografts by immunohistochemical analysis of paraffin-embedded sections using specific antibodies. Activities of MMP-2/9 were corroborated by in situ zymography on frozen tissue sections. Galectin-3 cleavage was shown in vivo by differential antibody staining and colocalized with predicted active MMPs both in mouse xenografts and human breast cancer specimens. In situ zymography validated these results. In addition, BT-549 cells harboring noncleavable galectin-3 showed reduced tumor growth and angiogenesis compared with the wild-type. We conclude that galectin-3 cleavage is an active process during tumor progression and could be used as a simple, rapid, and reliable surrogate marker for the activities of MMPs in growing breast cancers.