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1.  Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms 
Experimental cell research  2008;314(16):2941-2950.
The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen-I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen-I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.
doi:10.1016/j.yexcr.2008.07.021
PMCID: PMC2570357  PMID: 18703050
Ras; integrin; sialic acid; metastasis; collagen; colonocytes
2.  Polypeptide N-acetylgalactosaminyltransferase 2 regulates cellular metastasis-associated behavior in gastric cancer 
Aberrant glycosylation of cell surface glycoprotein due to specific alterations of glycosyltransferase activity is usually associated with invasion and metastasis of cancer, particularly of gastric carcinomas. Polypeptide N-acetylgalactosaminyltransferase 2 (ppGalNAc-T2), which catalyzes initiation of mucin-type O-glycosylation, is also involved in tumor migration and invasion. However, a comprehensive understanding of how ppGalNAc-T2 correlates with the metastasic potential of human gastric cancer is not currently available. In the present study, ppGalNAc-T2 was detected in a variety of human poorly differentiated tumor cells, and expression appeared to be higher in SGC7901 gastric cancer cells. In addition, we investigated the potential effects of ppGalNAc-T2 on growth and metastasis-associated behavior in SGC7901 cells after stable transfection with ppGalNAc-T2 sense and antisense vectors. We found that cell proliferation, adhesion and invasion were decreased in ppGalNAc-T2 overexpressed cells but increased in ppGalNAc-T2 downregulated cells. Therefore, we attempted to clarify the mechanisms underlying the anti-metastatic activities of ppGalNAc-T2. Further investigation indicated that overexpression of ppGalNAc-T2 is involved in the inhibition of matrix metalloproteinase (MMP)-2 expression at both the protein and mRNA levels, which may be associated with ppGalNAc-T2 suppressing the expression of transforming growth factor (TGF)-β1. However, it did not exhibit any apparent correlation with MMP-14 expression levels. Our data show the effect of ppGalNAc-T2 on proliferation, adhesion or invasion of SGC7901 gastric cancer cells, suggesting that ppGalNAc-T2 may exert anti-proliferative and anti-metastatic activity through the decrease of MMP-2 and TGF-β1. These results indicate that ppGalNAc-T2 may be used as a novel therapeutic target for human gastric cancer treatment.
doi:10.3892/ijmm.2012.1130
PMCID: PMC4042861  PMID: 22992780
polypeptide N-acetylgalactosaminyltransferase 2; gastric cancer; metastasis; matrix metalloproteinase-2; transforming growth factor-β1
3.  p70 S6 kinase drives ovarian cancer metastasis through multicellular spheroid-peritoneum interaction and P-cadherin/β1 integrin signaling activation 
Oncotarget  2014;5(19):9133-9149.
Peritoneal dissemination as a manifestation of ovarian cancer is an adverse prognostic factor associated with poor clinical outcome, and is thus a potentially promising target for improved treatment. Sphere forming cells (multicellular spheroids) present in malignant ascites of patients with ovarian cancer represent a major impediment to effective treatment. p70 S6 kinase (p70S6K), which is a downstream effector of mammalian target of rapamycin, is frequently hyperactivated in human ovarian cancer. Here, we identified p70S6K as an important regulator for the seeding and successful colonization of ovarian cancer spheroids on the peritoneum. Furthermore, we provided evidence for the existence of a novel crosstalk between P-cadherin and β1 integrin, which was crucial for the high degree of specificity in cell adhesion. In particular, we demonstrated that the upregulation of mature β1 integrin occurred as a consequence of P-cadherin expression through the induction of the Golgi glycosyltransferase, ST6Gal-I, which mediated β1 integrin hypersialylation. Loss of p70S6K or targeting the P-cadherin/β1-integrin interplay could significantly attenuate the metastatic spread onto the peritoneum in vivo. These findings establish a new role for p70S6K in tumor spheroid-mesothelium communication in ovarian cancer and provide a preclinical rationale for targeting p70S6K as a new avenue for microenvironment-based therapeutic strategy.
PMCID: PMC4253424  PMID: 25193855
p70S6K; P-cadherin; β1 integrin; adhesion; metastasis
4.  Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells 
Background
Previously, we found that β-galactoside α2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi-localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin β1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration.
Methods
We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin β1. After ionizing radiation, migration of cells was measured by in vitro migration assay. α2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an α2, 6 sialyltransferase sandwich ELISA.
Result
We found that ST6Gal I was cleaved by BACE1 (β-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non-cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin β1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin β1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by BACE 1 inhibited integrin β1 sialylation and migration by Golgi-anchored form of ST6Gal I.
Conclusions
Our results suggest that soluble ST6Gal I, possibly in cooperation with the Golgi-bound form, may participate in cancer progression and metastasis prior to being secreted from cancer cells.
doi:10.1186/1748-717X-7-47
PMCID: PMC3351029  PMID: 22449099
BACE1; Migration; Radiation; ST6Gal I
5.  ST6Gal-I sialyltransferase confers cisplatin resistance in ovarian tumor cells 
Background
Platinum drugs, including cisplatin, are a frontline therapeutic in ovarian cancer treatment and acquired resistance to these agents is a major contributor to ovarian cancer morbidity and mortality. In this study a novel glycosylation-dependent mechanism for cisplatin resistance is described. Specifically, cisplatin-induced cell death is blocked by the activity of the ST6Gal-I sialyltransferase. ST6Gal-I modifies specific receptors by adding a negatively charged sialic acid sugar which influences diverse receptor functions. Overexpression of ST6Gal-I is a hallmark of ovarian and other cancers and its expression has been correlated to metastasis and poor prognosis.
Methods
Tumor cell viability and apoptotic induction were determined in cell lines with ST6Gal-I overexpression and knockdown. In addition, cell populations with acquired resistance to cisplatin were assayed for endogenous ST6Gal-I expression.
Results
We show that forced expression of ST6Gal-I in OV4 ovarian cancer cells that lack endogenous ST6Gal-I causes reduced activation of caspase 3 and increased cell viability following cisplatin treatment. Conversely, forced ST6Gal-I knockdown in Pa-1 cells with high endogenous ST6Gal-I increases cisplatin-induced caspase activation and cell death. A2780 ovarian cancer cells selected for stable cisplatin resistance display upregulated endogenous ST6Gal-I when compared with parental, cisplatin-sensitive, A2780 cells. Similarly, extended low dose cisplatin treatment of a Pa-1 polyclonal ST6Gal-I shRNA knockdown population led to selection for subclones with elevated ST6Gal-I expression.
Conclusions
Receptor sialylation by ST6Gal-I confers a survival advantage for tumor cells in the presence of cisplatin. These collective findings support a role for ST6Gal-I in chemoresistance and highlight ST6Gal-I as a potential therapeutic target for platinum resistant tumors.
doi:10.1186/1757-2215-6-25
PMCID: PMC3637436  PMID: 23578204
Sialic acid; Cisplatin; Ovarian cancer; Apoptosis; Glycosylation
6.  Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function 
PLoS ONE  2014;9(5):e98595.
In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLex) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2β1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLex and lower α2,6-sialic acid content on the glycans of their α2β1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLex and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2β1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion.
doi:10.1371/journal.pone.0098595
PMCID: PMC4039506  PMID: 24878505
7.  Expression and functions of galectin-7 in ovarian cancer 
Oncotarget  2014;5(17):7705-7721.
There is a critical need to develop effective new strategies for diagnosis and treatment of ovarian cancer. In the present work, we investigated the expression of galectin-7 (gal-7) in epithelial ovarian cancer (EOC) cells and studied its functional relevance. Immunohistochemical analysis of gal-7 expression in tissue microarrays showed that while gal-7 was not detected in normal ovarian tissues, positive cytoplasmic staining of gal-7 was detected in epithelial cells in all EOC histological subtypes but was more frequent in high grade tumors and metastatic samples. Gal-7 expression correlated with a significant difference in the overall survival of patients with ovarian serous cystadenocarcinoma. Furthermore, using human EOC cell lines, we found that gal-7 expression was induced by mutant p53. Mechanistically, Matrigel invasion assays and live cell imaging showed that gal-7 increased the invasive behavior of ovarian cancer cells by inducing MMP-9 and increasing cell motility. EOC cells can also secrete gal-7. Recombinant human gal-7 kills Jurkat T cells and human peripheral T cells, suggesting that gal-7 also has immunosuppressive properties. Taken together, our study validates the clinical significance of gal-7 overexpression in ovarian cancer and provides a rationale for targeting gal-7 to improve the outcome of patients with this disease.
PMCID: PMC4202155  PMID: 25277199
galectin-7; ovarian cancer; immunosupression; p53; MMP-9
8.  Galectin-1 overexpression promotes progression and chemoresistance to cisplatin in epithelial ovarian cancer 
Zhang, P | Zhang, P | Shi, B | Zhou, M | Jiang, H | Zhang, H | Pan, X | Gao, H | Sun, H | Li, Z
Cell Death & Disease  2014;5(1):e991-.
This study was performed to investigate the role of galectin-1 (Gal-1) in epithelial ovarian cancer (EOC) progression and chemoresistance. Tissue samples from patients with EOC were used to examine the correlation between Gal-1 expression and clinical stage of EOC. The role of Gal-1 in EOC progression and chemoresistance was evaluated in vitro by siRNA-mediated knockdown of Gal-1 or lentivirus-mediated overexpression of Gal-1 in EOC cell lines. To elucidate the molecular mechanisms underlying Gal-1-mediated tumor progression and chemoresistance, the expression and activities of some signaling molecules associated with Gal-1 were analyzed. We found overexpression of Gal-1 in advanced stages of EOC. Knockdown of endogenous Gal-1 in EOC cells resulted in the reduction in cell growth, migration, and invasion in vitro, which may be caused by Gal-1's interaction with H-Ras and activation of the Raf/extracellular signal-regulated kinase (ERK) pathway. Additionally, matrix metalloproteinase-9 (MMP-9) and c-Jun were downregulated in Gal-1-knockdown cells. Notably, Gal-1 overexpression could significantly decrease the sensitivities of EOC cells to cisplatin, which might be ascribed to Gal-1-induced activation of the H-Ras/Raf/ERK pathway and upregulation of p21 and Bcl-2. Taken together, the results suggest that Gal-1 contributes to both tumorigenesis and cisplatin resistance in EOC. Thus, Gal-1 is a potential therapeutic target for EOC.
doi:10.1038/cddis.2013.526
PMCID: PMC4040687  PMID: 24407244
galectin-1; epithelial ovarian cancer; progression; chemoresistance
9.  Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells 
PLoS ONE  2013;8(6):e65957.
Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.
doi:10.1371/journal.pone.0065957
PMCID: PMC3688853  PMID: 23824659
10.  ST3Gal.I sialyltransferase relevance in bladder cancer tissues and cell lines 
BMC Cancer  2009;9:357.
Background
The T antigen is a tumor-associated structure whose sialylated form (the sialyl-T antigen) involves the altered expression of sialyltransferases and has been related with worse prognosis. Since little or no information is available on this subject, we investigated the regulation of the sialyltransferases, able to sialylate the T antigen, in bladder cancer progression.
Methods
Matched samples of urothelium and tumor tissue, and four bladder cancer cell lines were screened for: ST3Gal.I, ST3Gal.II and ST3Gal.IV mRNA level by real-time PCR. Sialyl-T antigen was detected by dot blot and flow cytometry using peanut lectin. Sialyltransferase activity was measured against the T antigen in the cell lines.
Results
In nonmuscle-invasive bladder cancers, ST3Gal.I mRNA levels were significantly higher than corresponding urothelium (p < 0.001) and this increase was twice more pronounced in cancers with tendency for recurrence. In muscle-invasive cancers and matching urothelium, ST3Gal.I mRNA levels were as elevated as nonmuscle-invasive cancers. Both non-malignant bladder tumors and corresponding urothelium showed ST3Gal.I mRNA levels lower than all the other specimen groups. A good correlation was observed in bladder cancer cell lines between the ST3Gal.I mRNA level, the ST activity (r = 0.99; p = 0.001) and sialyl-T antigen expression, demonstrating that sialylation of T antigen is attributable to ST3Gal.I. The expression of sialyl-T antigens was found in patients' bladder tumors and urothelium, although without a marked relationship with mRNA level. The two ST3Gal.I transcript variants were also equally expressed, independently of cell phenotype or malignancy.
Conclusion
ST3Gal.I plays the major role in the sialylation of the T antigen in bladder cancer. The overexpression of ST3Gal.I seems to be part of the initial oncogenic transformation of bladder and can be considered when predicting cancer progression and recurrence.
doi:10.1186/1471-2407-9-357
PMCID: PMC2763879  PMID: 19811634
11.  The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading 
Glycobiology  2008;19(1):29-37.
Primary open angle glaucoma (POAG) is a major blindness-causing disease, characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork (TM) lining the aqueous outflow pathway modulates the aqueous outflow facility. TM cell adhesion, cell–matrix interactions, and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. In a recent study, we demonstrated that galectin-8 (Gal8) modulates the adhesion and cytoskeletal arrangement of TM cells and that it does so through binding to β1 integrins and inducing Rho signaling. The current study is aimed at the characterization of the mechanism by which Gal8 mediates TM cell adhesion and spreading. We demonstrate here that TM cells adhere to and spread on Gal8-coated wells but not on galectin-1 (Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TM cells to Gal8-coated wells was abolished by a competing sugar, β-lactose, but not by a noncompeting sugar, sucrose. Also, a trisaccharide, NeuAcα2-3Galβ1-4GlcNAc, which binds specifically to the N-CRD of Gal8, inhibited the spreading of TM cells to Gal8-coated wells. In contrast, NeuAcα2-6Galβ1-4GlcNAc which lacks affinity for Gal8 had no effect. Affinity chromatography of cell extracts on a Gal8-affinity column and binding experiments with plant lectins, Maakia Amurensis and Sambucus Nigra, revealed that α3β1, α5β1, and αvβ1 integrins are major counterreceptors of Gal8 in TM cells and that TM cell β1 integrins carry predominantly α2-3-sialylated glycans, which are high-affinity ligands for Gal8 but not for Gal1 or Gal3. These data lead us to propose that Gal8 modulates TM cell adhesion and spreading, at least in part, by interacting with α2-3-sialylated glycans on β1 integrins.
doi:10.1093/glycob/cwn100
PMCID: PMC2733777  PMID: 18849583
cell adhesion; galectin-8; glaucoma; integrins; trabecular meshwork
12.  Galectin-4 expression is associated with reduced lymph node metastasis and modulation of Wnt/β-catenin signalling in pancreatic adenocarcinoma 
Oncotarget  2014;5(14):5335-5349.
Galectin-4 (Gal-4) has been recently identified as a pivotal factor in the migratory capabilities of a set of defined pancreatic ductal adenocarcinoma (PDAC) cell lines using zebrafish as a model system. Here we evaluated the expression of Gal-4 in PDAC tissues selected according to their lymph node metastatic status (N0 vs. N1), and investigated the therapeutic potential of targeting the cross-link with the Wnt signaling pathway in primary PDAC cells.
Analysis of Gal-4 expression in PDACs showed high expression of Gal-4 in 80% of patients without lymph node metastasis, whereas 70% of patients with lymph node metastases had low Gal-4 expression. Accordingly, in primary PDAC cells high Gal-4 expression was negatively associated with migratory and invasive ability in vitro and in vivo. Knockdown of Gal-4 in primary PDAC cells with high Gal-4 expression resulted in significant increase of invasion (40%) and migration (50%, P<0.05), whereas enforced expression of Gal-4 in primary cells with low Gal-4 expression reduced the migratory and invasive behavior compared to the control cells. Gal-4 markedly reduces β-catenin levels in the cell, counteracting the function of Wnt signaling, as was assessed by down-regulation of survivin and cyclin D1. Furthermore, Gal-4 sensitizes PDAC cells to the Wnt inhibitor ICG-001, which interferes with the interaction between CREB binding protein (CBP) and β-catenin. Collectively, our data suggest that Gal-4 lowers the levels of cytoplasmic β-catenin, which may lead to lowered availability of nuclear β-catenin, and consequently diminished levels of nuclear CBP-β-catenin complex and reduced activation of the Wnt target genes. Our findings provide novel insights into the role of Gal-4 in PDAC migration and invasion, and support the analysis of Gal-4 for rational targeting of Wnt/β-catenin signaling in the treatment of PDAC.
PMCID: PMC4170638  PMID: 24977327
Pancreatic ductal adenocarcinoma; Galectin-4; migration; lymph node ratio; primary PDAC cells; Wnt/β-catenin pathway
13.  Transient gene silencing of galectin-3 suppresses pancreatic cancer cell migration and invasion through degradation of β-catenin 
Pancreatic cancer is a leading cause of cancer-related mortality and often has a poor prognosis because of its late diagnosis, aggressive local invasion, early metastasis, and poor response to chemotherapy. The chemotherapeutic agent gemcitabine is effective for treating advanced pancreatic cancer, but its efficacy remains less than satisfactory. It is expected that further investigation of pancreatic cancer cell invasion and development of strategies to block this process should improve the disease prognosis. In this study, we tested our hypothesis that galectin-3 (gal-3), a multifunctional member of the β-galactoside-binding protein family, may regulate pancreatic cancer cell motility, and silencing of it inhibit cell motility. Previous studies demonstrated that this protein is associated with tumor cell adhesion, proliferation, differentiation, angiogenesis, apoptosis, and metastasis. Here, we used gal-3 small interfering RNA (siRNA) to silence its expression in various pancreatic cancer cell lines to determine whether gal-3 regulates cell proliferation, migration and invasion in vitro. We found that silencing gal-3 reduced cellular migration and invasion, but failed to affect proliferation. In gal-3 siRNA-transfected cells, we detected a decrease in β-catenin expression, an important signal for cancer cell invasion, which was caused by down-regulation of phosphorylated Akt and GSK-3β. We also found that matrix metalloproteinase (MMP)-2 expression was reduced by gal-3 silencing. These results indicate that gal-3-mediated invasion via MMP-2 regulated by β-catenin degradation is initiated by Akt phosphorylation in pancreatic cancer cells. Our results suggest that gal-3 can be a novel therapeutic target in pancreatic cancer.
doi:10.1002/ijc.25946
PMCID: PMC3833077  PMID: 21448903
β-catenin; Galectin-3; Invasion; MMP-2; Pancreatic cancer; Wnt
14.  Endothelial integrin α3β1 stabilizes carbohydrate-mediated tumor/endothelial cell adhesion and induces macromolecular signaling complex formation at the endothelial cell membrane 
Oncotarget  2014;5(5):1382-1389.
Blood borne metastatic tumor cell adhesion to endothelial cells constitutes a critical rate-limiting step in hematogenous cancer metastasis. Interactions between cancer associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag) and endothelium-expressed galectin-3 (Gal-3) have been identified as the leading molecular mechanism initiating tumor/endothelial cell adhesion in several types of cancer. However, it is unknown how these rather weak and transient carbohydrate/lectin mediated interactions are stabilized. Here, using Western blot and LC tandem mass spectrometry analyses of pull-downs utilizing TF-Ag loaded gold nanoparticles, we identified Gal-3, endothelial integrin α3β1, Src kinase, as well as 5 additional molecules mapping onto focal adhesion pathway as parts of the macromolecular complexes formed at the endothelial cell membranes downstream of TF-Ag/Gal-3 interactions. In a modified parallel flow chamber assay, inhibiting α3β1 integrin greatly reduced the strength of tumor/endothelial cell interactions without affecting the initial cancer cell adhesion. Further, the macromolecular complex induced by TF-Ag/Gal-3/α3β1 interactions activates Src kinase, p38, and ERK1/2, pathways in endothelial cells in a time- and α3β1-dependent manner. We conclude that, following the initial metastatic cell attachment to endothelial cells mediated by TF-Ag/Gal-3 interactions, endothelial integrin α3β1 stabilizes tumor/endothelial cell adhesion and induces the formation of macromolecular signaling complex activating several major signaling pathways in endothelial cells.
PMCID: PMC4012737  PMID: 24675526
tumor metastasis; adhesion; Thomsen-Friedenreich antigen; galectin; integrin
15.  Galectin-4 functions as a tumor suppressor of human colorectal cancer 
Development of CRC involves a series of genetic alterations with altered expression of proteins and cell signaling pathways. Here, we identified that gal-4, a marker of differentiation, was down-regulated in CRC. The goal of this work was to determine the function of gal-4 in CRC. Toward this goal, the human colon biopsies and tissue microarrays containing a gradient of pathology were analyzed for gal-4 expression by immunohistochemistry. Cell proliferation, migration, motility, forced expression, knockdown, cell cycle and apoptosis assays were used to characterize gal-4 function. Immunohistochemistry identified that gal-4 expression was significantly down-regulated in adenomas and was essentially absent in invasive carcinomas. Forced expression of gal-4 in gal-4 -ve cells induced cell cycle arrest and retarded cell migration and motility. Further, gal-4 sensitized the cells to CPT-induced apoptosis. Gal-4 knockdown resulted in increased cell proliferation, migration and motility. Gal-4 was found to be associated with Wnt signaling proteins. Finally, gal-4 expression led to down-regulation of Wnt signaling target genes. This study demonstrates that loss of gal-4 is a common and specific event in CRC. This study also shows that gal-4 exhibits tumor suppressive effects in colorectal cancer cells in vitro. Through its ability to interact with, and down-regulate the functions of Wnt signaling pathway, gal-4 reveals a new dimension in the control of the Wnt signaling pathway. Thus, gal-4 may prove to be an important molecule in understanding the biology of CRC.
doi:10.1002/ijc.25750
PMCID: PMC3071872  PMID: 21064109
Colorectal cancer; galectins; Wnt signaling; tumor suppressor
16.  Expression patterns of α2,3-Sialyltransferase I and α2,6-Sialyltransferase I in human cutaneous epithelial lesions 
Skin tumors have become one of the most common cancers in the world and their carcinogenesis is frequently associated with altered glycosylation patterns. The aberrant sialylation, a type of glycosylation, can mediate pathophysiological key events during various stages of tumor progression, including invasion and metastasis. Sialyltransferases play a key role in a variety of biological processes, including cell-cell communication, cell-matrix interaction, adhesion, and protein targeting. In this study, it was evaluated the expression of ST3Gal I and ST6Gal I in cutaneous epithelial lesions that include actinic keratosis (n=15), keratoacanthoma (n=9), squamous cell carcinoma (n=22) and basal cell carcinoma (n=28) in order to evaluate if sialyltransferases expression is different in premalignant and in malignant tumors. The expression of ST3Gal I was observed in actinic keratosis (53%), keratoacanthoma (78%), squamous cell carcinoma (73%) and basal cell carcinoma (32%) with statistic differences between basal cell carcinoma and keratoacanthoma (P=0.0239) and basal cell carcinoma and squamous cell carcinoma (P=0.0096); for ST6Gal I, cytoplasmic expression was noted in actinic keratosis (40%), heterogeneous and cytoplasmic expression was noted in keratoacanthoma (67%), squamous cell carcinoma (41%) and basal cell carcinoma (7%) with statistic differences between basal cell carcinoma and squamous cell carcinoma (P=0.0061) and basal cell carcinoma and keratoacanthoma (P=0.0008). In summary, our results showed that the high expression of ST3Gal I and ST6Gal I, in skin tumors, is associated with tumors with greater potential for invasion and metastasis, as in the case of squamous cell carcinoma, and this may be related to their behavior.
doi:10.4081/ejh.2013.e7
PMCID: PMC3683614  PMID: 23549466
sialic acid; α2,3-sialyltransferases; α2,6-sialyltransferases; basal cell carcinoma; squamous cell carcinoma; actinic keratosis; keratoacanthoma
17.  Hepatitis B virus X protein specially regulates the sialyl lewis a synthesis among glycosylation events for metastasis 
Molecular Cancer  2014;13(1):222.
Background
The metastasis of hematogenous cancer cells is associated with abnormal glycosylation such as sialyl lewis antigens. Although the hepatitis B virus X protein (HBx) plays important role in liver disease, the precise function of HBx on aberrant glycosylation for metastasis remains unclear.
Methods
The human hepatocellular carcinoma tissues, HBx transgenic mice and HBx-transfected cells were used to check the correlation of expressions between HBx and Sialyl lewis antigen for cancer metastasis. To investigate whether expression levels of glycosyltransferases induced in HBx-transfected cells are specifically associated with sialyl lewis A (SLA) synthesis, which enhances metastasis by interaction of liver cancer cells with endothelial cells, ShRNA and siRNAs targeting specific glycosyltransferases were used.
Results
HBx expression in liver cancer region of HCC is associated with the specific synthesis of SLA. Furthermore, the SLA was specifically induced both in liver tissues from HBx-transgenic mice and in in vitro HBx-transfected cells. HBx increased transcription levels and activities of α2-3 sialyltransferases (ST3Gal III), α1-3/4 fucosyltransferases III and VII (FUT III and VII) genes, which were specific for SLA synthesis, allowing dramatic cell-cell adhesion for metastatic potential. Interestingly, HBx specifically induced expression of N-acetylglucosamine-β1-3 galactosyltransferase V (β1-3GalT 5) gene associated with the initial synthesis of sialyl lewis A, but not β1-4GalT I. The β1-3GalT 5 shRNA suppressed SLA expression by HBx, blocking the adhesion of HBx-transfected cells to the endothelial cells. Moreover, β1-3GalT 5 silencing suppressed lung metastasis of HBx-transfected cells in in vivo lung metastasis system.
Conclusion
HBx targets the specific glycosyltransferases for the SLA synthesis and this process regulates hematogenous cancer cell adhesion to endothelial cells for cancer metastasis.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-222) contains supplementary material, which is available to authorized users.
doi:10.1186/1476-4598-13-222
PMCID: PMC4190352  PMID: 25255877
Hepatocellular carcinoma; Hepatitis B virus; Sialyl lewis antigen; E-selectin; Endothelial cells
18.  Decreased Galectin-9 and Increased Tim-3 Expression Are Related to Poor Prognosis in Gastric Cancer 
PLoS ONE  2013;8(12):e81799.
Introduction
Galectin-9 (Gal-9) induces adhesion and aggregation of certain cell types and inhibits the metastasis of tumor cells. T-cell immunoglobulin–and mucin domain-3–containing molecule 3 (TIM-3) plays a pivotal role in immune regulation. The aim of this study is to investigate Gal-9 and TIM-3 alterations in gastric cancer and their prognostic values.
Methods
Gal-9 and Tim-3 expression was evaluated using a tissue microarray immunohistochemistry method in 305 gastric cancers, of which 84 had paired adjacent normal samples. Cell lines SGC-7901, BGC-823, MGC-803, MKN45 and GES-1 were also stained. Correlations were analyzed between expression levels of Gal-9 and Tim-3 protein and tumor parameters or clinical outcomes.
Results
Gal-9 and Tim-3 stained positive on tumor cells in 86.2% (263/305), and 60.0% (183/305) patients with gastric cancer, respectively. Gal-9 expression was significantly higher in cancer than in normal mucosa (P<0.001). Reduced Gal-9 expression was associated with lymph-vascular invasion, lymph node metastasis, distant metastasis and worse TNM staging (P = 0.034, P = 0.009, P = 0.002 and P = 0.043, respectively). In contrast, Tim-3 expression was significantly lower in cancer than in control mucosa (P<0.001). Patients with lymph-vascular invasion had higher expression levels of Tim-3 (P<0.001). Moreover, multivariate analysis shows that both high Gal-9 expression and low Tim-3 expression were significantly associated with long overall survival (P = 0.002, P = 0.010, respectively); the combination of Gal-9 and Tim-3 expression was an independent prognostic predictor for patients with gastric cancer (RR: 0.43; 95%CI: 0.20–0.93). H.pylori infection status was not associated with Gal-9 and Tim-3 expression (P = 0.102, P = 0.565).
Conclusion
The results suggest that expression of Gal-9 and Tim-3 in tumor cells may be a potential, independent prognostic factor for patients with gastric cancer. Gal-9 and TIM-3 may play an important part in the gastric carcinogenesis.
doi:10.1371/journal.pone.0081799
PMCID: PMC3858245  PMID: 24339967
19.  Correlation of CD44v6 expression with ovarian cancer progression and recurrence 
BMC Cancer  2013;13:182.
Background
Previously some groups demonstrated that CD44 variant 6 (CD44v6) is correlated with progression and metastasis of ovarian cancer. However, a number of other groups failed to find such an association. Moreover, epithelial ovarian cancer is known to easily metastasize to distinct sites such as the pelvic and abdominal cavities, but the potential association of CD44v6 expression with site-specific metastasis of ovarian cancer has not been explored. This study sought to evaluate the expression of CD44 standard (CD44s) and CD44v6 in primary, metastatic and recurrent epithelial ovarian cancer to explore the potential association of CD44s and CD44v6 with tumor progression and recurrence.
Methods
Tumor specimens were procured from patients with advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma. CD44s and CD44v6 expression in the tumor tissues was evaluated by real-time RT-PCR and Western blot. Moreover, serum soluble CD44s or CD44v6 concentrations of early stage (FIGO I, G1), advanced (FIGO III, G3) and recurrent ovarian serous adenocarcinoma patients were determined by enzyme-linked immunosorbent assays (ELISA). CD44v6 expression in a different set of tumor samples on an ovarian cancer tissue chip was evaluated by immunohistochemistry (IHC) and the correlation of CD44v6 expression with clinicopathologic features was analyzed. Finally, the effects of knockdown of CD44v6 in SKOV3 cells on cell adhesion, invasion and migration were assessed.
Results
The expression of CD44v6, but not CD44s, is up-regulated in recurrent ovarian serous cancer compared to advanced primary tumor. CD44v6 expression is also preferentially increased in the tumor at the abdominal cavity metastasis site of advanced diseases. Consistently, serum soluble CD44v6 levels of recurrent ovarian cancer were higher than those of early stage and advanced primary diseases. The IHC data demonstrate that CD44v6 expression is correlated with clinicopathologic features and tumor progression. Lastly, knockdown of CD44v6 decreases the adhesion and migration but not invasion capacities of SKOV3 cells.
Conclusions
CD44v6 expression levels are associated with epithelial ovarian cancer progression, metastasis and relapse. Moreover, serum soluble CD44v6 may be used as a potential marker for identifying tumor relapse. Finally, CD44v6 may play a role in ovarian cancer metastasis by mediating tumor cell adhesion and migration.
doi:10.1186/1471-2407-13-182
PMCID: PMC3635997  PMID: 23565736
Ovarian cancer; CD44v6; Tumor progression; Abdominal cavity metastasis; Recurrence
20.  Tumor galectin-1 mediates tumor growth and metastasis through regulation of T-cell apoptosis 
Cancer research  2011;71(13):4423-4431.
Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced by hypoxia, promotes tumor aggressiveness by promoting angiogenesis and T cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is undefined. Here we offer evidence that implicates tumor Gal-1 and its modulation of T cell immunity in progression. Comparing Gal-1 deficient mice as hosts for Lewis lung carcinoma cells where Gal-1 levels were preserved or knocked down, we found that tumor Gal-1 was more critical than host Gal-1 in promoting tumor growth and spontaneous metastasis. Enhanced growth and metastasis associated with Gal-1 related to its immunomodulatory function, insofar as the benefits of Gal-1 expression to Lewis lung carcinoma growth were abolished in immune-deficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between tumors with divergent Gal-1 levels when examined at a comparable size. Our findings establish that tumor rather than host Gal-1 is responsible for mediating tumor progression through intratumoral immune modulation, with broad implications in developing novel targeting strategies for Gal-1 in cancer.
doi:10.1158/0008-5472.CAN-10-4157
PMCID: PMC3129397  PMID: 21546572
Galectin-1; Lung cancer; immune-modulation; apoptosis; hypoxia
21.  Galectin-3 gene silencing inhibits migration and invasion of human tongue cancer cells in vitro via downregulating β-catenin 
Acta Pharmacologica Sinica  2012;34(1):176-184.
Aim:
Galectin-3 (Gal-3) is a member of the carbohydrate-binding protein family that contributes to neoplastic transformation, tumor survival, angiogenesis, and metastasis. The aim of this study is to investigate the role of Gal-3 in human tongue cancer progression.
Methods:
Human tongue cancer cell lines (SCC-4 and CAL27) were transfected with a small-interfering RNA against Gal-3 (Gal-3-siRNA). The migration and invasion of the cells were examined using a scratch assay and BD BioCoat Matrigel Invasion Chamber, respectively. The mRNA and protein levels of β-catenin, Akt/pAkt, GSK-3β/pGSK-3β, MMP-9 in the cells were measured using RT-PCR and Western blotting, respectively.
Results:
Transient silencing of Gal-3 gene for 48 h significantly suppressed the migration and invasion of both SCC-4 and CAL27 cells. Silencing of Gal-3 gene significantly decreased the protein level of β-catenin, leaving the mRNA level of β-catenin unaffected. Furthermore, silencing Gal-3 gene significantly decreased the levels of phosphorylated Akt and GSK-3β, and suppressed the mRNA and protein levels of MMP-9 in the cells.
Conclusion:
Our data suggest that Gal-3 mediates the migration and invasion of tongue cancer cells in vitro via regulating the Wnt/β-catenin signaling pathway and Akt phosphorylation.
doi:10.1038/aps.2012.150
PMCID: PMC4086502  PMID: 23103626
tongue cancer; galectin-3; β-catenin; Wnt signaling pathway; MMP-9; RNA interference; tumor invasion; metastasis
22.  Targeting the Urokinase Plasminogen Activator Receptor Inhibits Ovarian Cancer Metastasis 
Purpose
To understand the functional and preclinical efficacy of targeting urokinase plasminogen activator receptor (u-PAR) in ovarian cancer.
Experimental Design
Expression of u-PAR was studied in 162 epithelial ovarian cancers, including 77 pairs of corresponding primary and metastatic tumors. The effect of an antibody against u-PAR (ATN-658) on proliferation, adhesion, invasion, apoptosis, and migration was assessed in three (SKOV3ip1, HeyA8, and CaOV3) ovarian cancer cell lines. The impact of the u-PAR antibody on tumor weight, number, and survival was examined in corresponding ovarian cancer xenograft models and the mechanism by which ATN-658 blocks metastasis was explored.
Results
Only 8% of all ovarian tumors were negative for u-PAR expression. Treatment of SKOV3ip1, HeyA8, and CaOV3 ovarian cancer cells with the u-PAR antibody inhibited cell invasion, migration and adhesion. In vivo, anti-u-PAR treatment reduced the number of tumors and tumor weight in CaOV3 and SKOV3ip1 xenografts, and reduced tumor weight and increased survival in HeyA8 xenografts. Immunostaining of CaOV3 xenograft tumors and ovarian cancer cell lines showed an increase in active-caspase 3 and TUNEL staining. Treatment with u-PAR antibody inhibited α5-integrin and u-PAR colocalization on primary human omental ECM. Anti-u-PAR treatment also decreased the expression of urokinase, u-PAR, β3-integrin and fibroblast growth factor receptor-1 both in vitro and in vivo.
Conclusions
This study shows that an antibody against u-PAR reduces metastasis, induces apoptosis, and reduces the interaction between u-PAR and α5-integrin. This provides a rationale for targeting the u-PAR pathway in patients with ovarian cancer and for further testing of ATN-658 in this indication.
doi:10.1158/1078-0432.CCR-10-2258
PMCID: PMC3073583  PMID: 21149615
urokinase; urokinase receptor; ovarian cancer; metastasis
23.  α2,3-Sialyltransferase ST3Gal III Modulates Pancreatic Cancer Cell Motility and Adhesion In Vitro and Enhances Its Metastatic Potential In Vivo 
PLoS ONE  2010;5(9):e12524.
Background
Cell surface sialylation is emerging as an important feature of cancer cell metastasis. Sialyltransferase expression has been reported to be altered in tumours and may account for the formation of sialylated tumour antigens. We have focused on the influence of alpha-2,3-sialyltransferase ST3Gal III in key steps of the pancreatic tumorigenic process.
Methodology/Principal Findings
ST3Gal III overexpressing pancreatic adenocarcinoma cell lines Capan-1 and MDAPanc-28 were generated. They showed an increase of the tumour associated antigen sialyl-Lewisx. The transfectants' E-selectin binding capacity was proportional to cell surface sialyl-Lewisx levels. Cellular migration positively correlated with ST3Gal III and sialyl-Lewisx levels. Moreover, intrasplenic injection of the ST3Gal III transfected cells into athymic nude mice showed a decrease in survival and higher metastasis formation when compared to the mock cells.
Conclusion
In summary, the overexpression of ST3Gal III in these pancreatic adenocarcinoma cell lines underlines the role of this enzyme and its product in key steps of tumour progression such as adhesion, migration and metastasis formation.
doi:10.1371/journal.pone.0012524
PMCID: PMC2931708  PMID: 20824144
24.  Galectin-3 Mediates Cross-Talk between K-Ras and Let-7c Tumor Suppressor microRNA 
PLoS ONE  2011;6(11):e27490.
Background
Galectin-3 (Gal-3) and active (GTP-bound) K-Ras contribute to the malignant phenotype of many human tumors by increasing the rate of cell proliferation, survival, and migration. These Gal-3-mediated effects result from a selective binding to K-Ras.GTP, causing increased nanoclustering in the cell membrane and leading to robust Ras signaling. Regulation of the interactions between Gal-3 and active K-Ras is not fully understood.
Methods and Findings
To gain a better understanding of what regulates the critical interactions between these two proteins, we examined the role of Gal-3 in the regulation of K-Ras by using Gal-3-knockout mouse embryonic-fibroblasts (Gal-3-/- MEFs) and/or Gal-3/Gal-1 double-knockout MEFs. We found that knockout of Gal-3 induced strong downregulation (∼60%) of K-Ras and K-Ras.GTP. The downregulation was somewhat more marked in the double-knockout MEFs, in which we also detected robust inhibition(∼50%) of ERK and Akt activation. These additional effects are probably attributable to inhibition of the weak interactions of K-Ras.GTP with Gal-1. Re-expression of Gal-3 reversed the phenotype of the Gal-3-/- MEFs and dramatically reduced the disappearance of K-Ras in the presence of cycloheximide to the levels seen in wild-type MEFs. Furthermore, phosphorylation of Gal-3 by casein kinase-1 (CK-1) induced translocation of Gal-3 from the nucleus to the cytoplasm and the plasma membrane, leading to K-Ras stabilization accompanied by downregulation of the tumor suppressor miRNA let-7c, known to negatively control K-Ras transcription.
Conclusions
Our results suggest a novel cross-talk between Gal-3-mediated downregulation of let 7c microRNA (which in turn negatively regulates K-Ras transcription) and elucidates the association among Gal-3 let-7c and K-Ras transcription/translation, cellular compartmentalization and activity.
doi:10.1371/journal.pone.0027490
PMCID: PMC3216962  PMID: 22102901
25.  PRL-3 suppresses c-Fos and integrin α2 expression in ovarian cancer cells 
BMC Cancer  2013;13:80.
Background
Phosphatase of regenerating liver-3 (PRL-3), a protein tyrosine phosphatase, is highly expressed in multiple human cancers and strongly implicated in tumor progression and cancer metastasis. However, the mechanisms by which PRL-3 promotes cancer cell migration, invasion, and metastasis are not very well understood. In this study, we investigated the contribution and molecular mechanisms of PRL-3 in ovarian cancer progression.
Methods
PRL-3 protein expression was detected on ovarian cancer tissue microarrays using immunohistochemistry. Stable PRL-3 depleted cell lines were generated using short hairpin RNA (shRNA) constructs. The migration and invasion potential of these cells were analyzed using Transwell and Matrigel assays, respectively. Immunoblotting and immunofluorescence were used to detect protein levels and distribution in PRL-3-ablated cells and the control cells. Cell morphology was observed with hematoxylin-eosin staining and transmission electron microscopy. Finally, PRL-3-ablated and control cells were injected into nude mice for xenograft tumorigenicity assays.
Results
Elevated PRL-3 expression was detected in 19% (26 out of 135) of human ovarian cancer patient samples, but not in normal ovary tissues (0 out of 14). Stable depletion of PRL-3 in A2780 ovarian cancer cells resulted in decreased migration ability and invasion activity compared with control parental A2780 cells. In addition, PRL-3-ablated cells also exhibited flattened morphology and extended lamellipodia. To address the possible molecular basis for the altered phenotypes associated with PRL-3 down-regulation, we assessed the expression profiles of various proteins involved in cell-matrix adhesion. Depletion of PRL-3 dramatically enhanced both RNA and protein levels of the cell surface receptor integrin α2, but not its heterologous binding partner integrin β1. Inhibition of PRL-3 also correlated with elevated expression and phosphorylation of paxillin. A pronounced increase in the expression and activation of c-fos, a transcriptional activator of integrin α2, was observed in these PRL-3 knock-down cells. Moreover, forced expression of EGFP-PRL-3 resulted in the suppression of both integrin α2 and c-fos expression in A2780 cells. Significantly, using a xenograft tumor model, we observed a greatly reduced tumorigenicity of A2780 PRL-3 knock-down cells in vivo.
Conclusions
These results suggest that PRL-3 plays a critical role in ovarian cancer tumorigenicity and maintaining the malignant phenotype. PRL-3 may inhibit c-fos transcriptional regulation of integrin α2 signaling. Our results strongly support a role for PRL-3 as a promising therapeutic target and potential early biomarker in ovarian cancer progression.
doi:10.1186/1471-2407-13-80
PMCID: PMC3620920  PMID: 23418787
PRL-3 phosphatase; Cancer metastasis; Integrin α2; c-fos transcription factor; Adhesion molecule; Cell migration

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