Pratima Nangia-Makker

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.

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.

Galectin-3 Regulates Mitochondrial Stability and Antiapoptotic Function in Response to Anticancer Drug in Prostate Cancer

Prostate cancer is one of the malignant tumors which exhibit resistance to anticancer drugs, at least in part due to enhanced antiapoptotic mechanisms. Therefore, the understanding of such mechanisms should improve the design of chemotherapy against prostate cancer. Galectin-3 (Gal-3), a multifunctional oncogenic protein involved in the regulation of tumor proliferation, angiogenesis, and apoptosis has shown antiapoptotic effects in certain cell types. Here, we show that the expression of exogenous Gal-3 in human prostate cancer LNCaP cells, which do not express Gal-3 constitutively, inhibits anticancer drug-induced apoptosis by stabilizing the mitochondria. Thus, Gal-3-negative cells showed 66.31% apoptosis after treatment with 50 micromol/L cis-diammine-dichloroplatinum for 48 hours, whereas two clones of Gal-3-expressing cells show only 2.92% and 1.42% apoptotic cells. Similarly, Gal-3-negative cells showed 43.8% apoptosis after treatment with 300 micromol/L etoposide for 48 hours, whereas only 15.38% and 14.51% of Gal-3-expressing LNCaP cells were apoptotic. The expression of Gal-3 stimulated the phosphorylation of Ser(112) of Bcl-2-associated death (Bad) protein and down-regulated Bad expression after treatment with cis-diammine-dichloroplatinum. Gal-3 also inhibited mitochondrial depolarization and damage after translocation from the nuclei to the cytoplasm, resulting in inhibition of cytochrome c release and caspase-3 activation. These findings indicate that Gal-3 inhibits anticancer drug-induced apoptosis through regulation of Bad protein and suppression of the mitochondrial apoptosis pathway. Therefore, targeting Gal-3 could improve the efficacy of anticancer drug chemotherapy in prostate cancer.

Regulation of Tumor Progression by Extracellular Galectin-3

The relationship between a tumor cell and its microenvironment is bi-directional. The proteins expressed by the tumor cells alter the signatures on the seemingly normal stromal cells within the microenvironment, while the tumor cell signatures reflect the changes that occur as these cells interact with the host microenvironment. Galectin-3 is a carbohydrate-binding protein that is over-expressed in a variety of tumors and immune cells in response to various stimuli. Ever since its discovery, it has been associated with cell and extracellular matrix interactions. However, in the last decade, an extensive accumulation of data has changed the perspective of this multifunctional protein. The unique structure of this protein, consisting of a carbohydrate-binding domain and a matrix metalloproteinase cleavable domain, enables it to interact with a plethora of ligands in a carbohydrate-dependent or independent manner. It is now becoming evident that galectin-3 is involved with a variety of extracellular functions like cell adhesion, migration, invasion, angiogenesis, immune functions, apoptosis and endocytosis. Galectin-3 is a substrate for matrix metalloproteinases and its cleavage plays an important role in tumor progression and can be used as a surrogate diagnostic marker for in vivo MMP activity.

Alterations in Galectin-3 Expression and Distribution Correlate with Breast Cancer Progression : Functional Analysis of Galectin-3 in Breast Epithelial-Endothelial Interactions

To define the role of galectin-3 in breast cancer progression, we have used a novel three-dimensional co-culture system that recapitulates in vivo reciprocal functional breast epithelial-endothelial cell-cell and cell-matrix interactions, and examined the expression of galectin-3 mRNA and protein in human breast tumors and xenografts. Galectin-3 is required for the stabilization of epithelial-endothelial interaction networks because immunoneutralization with galectin-3 antibodies abolishes the interactions in a dose-dependent manner. Co-culture of epithelial cells with endothelial cells results in increase in levels of secreted galectin-3 and presence of proteolytically processed form of galectin-3 in the conditioned media. In contrast, intracellular galectin-3 predominantly exists in the intact form. This difference in sensitivity to proteolytic processing of secreted versus intracellular galectin-3 probably arises from differences in accessibility of protease-sensitive sites, levels, and/or type of activated protease(s), and may be indicative of different functional roles for intact and processed galectin-3. To determine whether the proteolytically cleaved galectin-3 retains its ability to bind to endothelial cells, binding assays were performed with the full-length and matrix metallopeoteinase-2-cleaved recombinant galectin-3. Although a dose-dependent increase in binding to human umbilical vein endothelial cells was observed with both full-length and cleaved galectin-3, proteolytically cleaved galectin-3 displayed approximately 20-fold higher affinity for human umbilical vein endothelial cells as compared to the full-length protein. Examination of galectin-3 expression in breast tumors and xenografts revealed elevated levels of galectin-3 mRNA and protein in the luminal epithelial cells of normal and benign ducts, down-regulation in early grades of ductal carcinoma in situ (DCIS), and re-expression in peripheral tumor cells as DCIS lesions progressed to comedo-DCIS and invasive carcinomas. These data suggest that galectin-3 expression is associated with specific morphological precursor subtypes of breast cancer and undergoes a transitional shift in expression from luminal to peripheral cells as tumors progressed to comedo-DCIS or invasive carcinomas. Such a localized expression of galectin-3 in cancer cells proximal to the stroma could lead to increased invasive potential by inducing novel or better interactions with the stromal counterparts.

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.

Galectin-3 in angiogenesis and metastasis

Galectin-3 is a member of the family of β-galactoside-binding lectins characterized by evolutionarily conserved sequences defined by structural similarities in their carbohydrate-recognition domains. Galectin-3 is a unique, chimeric protein consisting of three distinct structural motifs: (i) a short NH2 terminal domain containing a serine phosphorylation site; (ii) a repetitive proline-rich collagen-α-like sequence cleavable by matrix metalloproteases; and (iii) a globular COOH-terminal domain containing a carbohydrate-binding motif and an NWGR anti-death motif. It is ubiquitously expressed and has diverse biological functions depending on its subcellular localization. Galectin-3 is mainly found in the cytoplasm, also seen in the nucleus and can be secreted by non-classical, secretory pathways. In general, secreted galectin-3 mediates cell migration, cell adhesion and cell-cell interactions through the binding with high affinity to galactose-containing glycoproteins on the cell surface. Cytoplasmic galectin-3 exhibits anti-apoptotic activity and regulates several signal transduction pathways, whereas nuclear galectin-3 has been associated with pre-mRNA splicing and gene expression. Its unique chimeric structure enables it to interact with a plethora of ligands and modulate diverse functions such as cell growth, adhesion, migration, invasion, angiogenesis, immune function, apoptosis and endocytosis emphasizing its significance in the process of tumor progression. In this review, we have focused on the role of galectin-3 in tumor metastasis with special emphasis on angiogenesis.

Metformin: a potential therapeutic agent for recurrent colon cancer

Accumulating evidence suggests that metformin, a biguanide class of anti-diabetic drugs, possesses anti-cancer properties. However, most of the studies to evaluate therapeutic efficacy of metformin have been on primary cancer. No information is available whether metformin could be effectively used for recurrent cancer, specifically colorectal cancer (CRC) that affects up to 50% of patients treated by conventional chemotherapies. Although the reasons for recurrence are not fully understood, it is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs/CSLCs). Therefore, development of non-toxic treatment strategies targeting CSCs would be of significant therapeutic benefit. In the current investigation, we have examined the effectiveness of metformin, in combination with 5-fluorouracil and oxaliplatin (FuOx), the mainstay of colon cancer therapeutics, on survival of chemo-resistant colon cancer cells that are highly enriched in CSCs/CSLCs. Our data show that metformin acts synergistically with FuOx to (a) induce cell death in chemo resistant (CR) HT-29 and HCT-116 colon cancer cells, (b) inhibit colonospheres formation and (c) enhance colonospheres disintegration. In vitro cell culture studies have further demonstrated that the combinatorial treatment inhibits migration of CR colon cancer cells. These changes were associated with increased miRNA 145 and reduction in miRNA 21. Wnt/β-catenin signaling pathway was also down-regulated indicating its pivotal role in regulating the growth of CR colon cancer cells. Data from SCID mice xenograft model of CR HCT-116 and CR HT-29 cells show that the combination of metformin and FuOX is highly effective in inhibiting the growth of colon tumors as evidenced by ∼50% inhibition in growth following 5 weeks of combination treatment, when compared with the vehicle treated controls. Our current data suggest that metformin together with conventional chemotherapy could be an effective treatment regimen for recurring colorectal cancer (CRC).