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.
Ras; integrin; sialic acid; metastasis; collagen; colonocytes
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.
polypeptide N-acetylgalactosaminyltransferase 2; gastric cancer; metastasis; matrix metalloproteinase-2; transforming growth factor-β1
As ovarian cancer stem cells (CSCs) are responsible for tumor initiation, invasion, metastasis, and chemo-resistance, new stratagems that selectively target ovarian CSCs are critically significant. Our previous work have demonstrated that ovarian cancer spheroid cells are tumorigenic and chemo-resistant, and have the properties of ovarian CSCs. Herein, we hypothesized that expressing α-gal epitopes on ovarian spheroid cells may help eliminate CSCs and improve the outcome of therapeutic intervention for ovarian cancer patients.
Lentivirus-mediated transfer of a pig α(1,3)galactosyltransferase [α1,3GT] enzyme gene into human ovarian cell line SKOV3 cells formed α-gal epitope-expressing cells (SKOV3-gal cells), and then these cells were maintained in a serum-free culture system to form SKOV3-gal spheroid cells. Efficacy of this cell vaccine was demonstrated in α1,3GT knockout mice (α1,3GT KO mice).
The antibody titers to α-gal epitopes measured by ELISA were significantly increased in α1,3GT KO mice after immunization with SKOV3-gal spheroid cells. Furthermore, compared with the non-immunized KO mice, the SKOV3 tumors grafted under renal capsules of KO mice immunized with SKOV3-gal spheroid cells grew slower and began to shrink on day 12. Western blot analysis also showed that immunized KO mice can produce effective antibody against certain tumor associated antigens (TAAs) derived from both SKOV3 cells and SKOV3 spheroid cells. The TAAs were further investigated by mass spectrometry and RNA interference (RNAi) technology. The results suggested that antibodies responding to protein c-erbB-2 may be raised in the sera of the mice after immunization with SKOV3-gal spheroid cells. Ultimately, vaccination with SKOV3-gal spheroid cells induced more CD3 + CD4 + T cells in the spleen of immunized mice than non-immunized KO mice.
The results suggest that vaccination using ovarian cancer stem-like cells engineered to express α-gal epitopes may be a novel strategy for treatment of ovarian cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1973-7) contains supplementary material, which is available to authorized users.
Ovarian cancer; α-gal epitopes; Immunotherapy; Ovarian cancer stem-like cells; Protein c-erbB-2
Gastric cancer (GC) is characterized by the excessive deposition of extracellular matrix, which is thought to contribute to this tumor’s malignant behavior. Epithelial-mesenchymal transition (EMT) is regarded as a crucial contributing factor to cancer progression. Galectin-1 (Gal-1), a β-galactoside-binding protein abundantly expressed in activated cancer-associated fibroblasts (CAFs), has been reported to be involved in GC progression and metastasis by binding to β1 integrin, which, in turn, can bind to matrix proteins and activate intracellular cascades that mediate EMT. Increasing evidence suggests that abnormal activation of the hedgehog (Hh) signaling pathway enhances GC cell migration and invasion. The purpose of our study is to explore the role of Gal-1 in the GC progression and metastasis as well as the regulatory mechanism.
We hypothesized that Gal-1 binding to β1 integrin would lead to paracrine signaling between CAFs and GC cells, mediating EMT by upregulating Gli1. Invasion and metastasis effects of the Gal-1 and Gli1 were evaluated using wound healing and invasion assay following transfection with mimics. Additionally, to facilitate the delineation of the role of the Hh signaling in GC, we monitored the expression level of associated proteins. We also evaluated the effects of β1 integrin on these processes. Furthermore, Gal-1 and Gli1 expression in GC patient samples were examined by immunohistochemistry and western blot to determine the correlation between their expression and clinicopathologic characteristics. The Kaplan-Meier method and Cox proportional hazards model were used to analyze the relationship of expression with clinical outcomes.
Gal-1 was found to induce EMT, GC cell migration and invasion. Further data showed that Gal-1 up-regulated Gli1 expression. β1 integrin was responsible for Gal-1-induced Gli1 expression and EMT. In clinical GC tissue, it confirmed a positive relationship between Gal-1 and Gli1 expression. Importantly, their high expression is correlated to poor prognosis.
Gal-1 from CAFs binds to a carbohydrate structure in β1 integrin and plays an important role in the development of GC by inducing GC metastasis and EMT through targeting Gli1. This study highlights the potential therapeutic value of Gal-1 for suppression of GC metastasis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13046-016-0449-1) contains supplementary material, which is available to authorized users.
Galectin-1; Cancer-associated fibroblasts; Epithelial–mesenchymal transition; Glioma-associated oncogene 1; β1 integrin; Hedgehog; Gastric cancer
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.
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.
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.
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.
BACE1; Migration; Radiation; ST6Gal I
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.
p70S6K; P-cadherin; β1 integrin; adhesion; metastasis
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.
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.
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.
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.
Sialic acid; Cisplatin; Ovarian cancer; Apoptosis; Glycosylation
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.
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.
galectin-7; ovarian cancer; immunosupression; p53; MMP-9
Metastatic clear cell renal cell carcinoma (ccRCC) patients have <9% 5-year survival rate, do not respond well to targeted therapy and eventually develop resistance. A better understanding of molecular pathways of RCC metastasis is the basis for the discovery of novel prognostic markers and targeted therapies.
We investigated the biological impact of galectin-1 (Gal-1) in RCC cell lines by migration and invasion assays. Effect of Gal-1 expression on the mitogen-activated protein kinase pathway was assessed by proteome array.
Increased expression of Gal-1 increased cell migration while knocking down Gal-1 expression by siRNA resulted in reduced cellular migration (P<0.001) and invasion (P<0.05). Gal-1 overexpression increased phosphorylation of Akt, mTOR and p70 kinase. Upon hypoxia and increased HIF-1α, Gal-1 increased in a dose-dependent manner. We also found miR-22 overexpression resulted in decreased Gal-1 and HIF-1α. Immunohistochemistry analysis showed that high Gal-1 protein expression was associated with larger size tumor (P=0.034), grades III/IV tumors (P<0.001) and shorter disease-free survival (P=0.0013). Using the Cancer Genome Atlas data set, we found that high Gal-1 mRNA expression was associated with shorter overall survival (41 vs 78 months; P<0.01).
Our data suggest Gal-1 mediates migration and invasion through the HIF-1α–mTOR signaling axis and is a potential prognostic marker and therapeutic target.
HIF-1α; galectin-1; renal cell carcinoma; miR-22; tumor markers
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.
galectin-1; epithelial ovarian cancer; progression; chemoresistance
Excessive ST3Gal-I levels predict a poor outcome for patients with several types of tumors. This study aims to investigate the role of ST3Gal-I in determining the invasive and metastatic potential of human hepatocellular carcinoma (HCC) and clinical prognosis for patients with HCC.
We compared the expression of ST3Gal-I in various HCC cell lines and in 20 pairs of tumor and peritumor tissue samples using Western blot analysis. Changes in the degree of invasiveness and migration were determined before and after small interfering RNA-induced knockdown of ST3Gal-I using a Transwell matrigel invasion assay and scratch wound assay. The correlation between ST3Gal-I expression and prognosis was determined in a large HCC patient cohort (n=273).
ST3Gal-I expression was higher in metastatic HCCLM3 cells and tumor tissue compared with normal adjacent tissue. Following the ST3Gal-I knockdown, the invasiveness and migration of HCCLM3 cells were markedly reduced. ST3Gal-I expression in HCC correlated closely with tumor thrombus (P<0.001), tumor size (>5.0 cm, P=0.032), tumor node metastasis stages II–III (P=0.002), and Barcelona Clinic Liver Cancer stages B–C (P<0.001). Cox regression analysis demonstrated that ST3Gal-I is an independent predictor of prognosis in patients with HCC, and related to disease-free survival (hazard ratio =1.464, P=0.037) and overall survival (hazard ratio =1.662, P=0.012).
ST3Gal-I might contribute to the invasiveness and metastatic nature of HCC and, thus, could be an independent predictor of recurrence and a suitable pharmaceutical target in patients with HCC.
ST3Gal-I; hepatocellular carcinoma; sialyltransferase; prognosis; tumor progression
GalNAc-T3 catalyzes initial glycosylation of mucin-type O-linked protein involved in proliferation, adhesion, and migration of tumor cells. This study was performed to explore the relationships of the expression of GalNAc-T3 in small peripheral lung adenocarcinoma, especially as an indicator of prognosis.
Materials and methods
A retrospective analysis of the patients with small peripheral lung lesions, including 106 adenocarcinoma and two precancerous lesions (atypical adenomatous hyperplasia and adenocarcinoma in situ) after complete surgical resection, was launched. Expression of GalNAc-T3 was examined using immunohistochemistry staining on primary tumor specimens, and the tumors were reclassified in light of the IASLC/ATS/ERS (International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society) adenocarcinoma classifications followed by grading and scoring. Moreover, reverse transcription polymerase chain reaction and Western blot were used to study the expression of GalNAc-T3 in vivo.
The low expression of GalNAc-T3 was found in the cytoplasm of tumor cells in 56 of 108 patients (51.9%) and was associated with IASLC/ATS/ERS classification of high risk groups (P=0.007), high Sica score (P=0.036), poorly differentiated tumor (P=0.023), poor tumor-node-metastasis (TNM) stage (P=0.007), pleural invasion (P=0.007), and vascular invasion (P<0.001) by Pearson’s chi-squared test, but not with sex, age, smoking status, concentration of carcinoembryonic antigen, and lymph node metastasis. In logistic regression analysis, low GalNAc-T3 expression was only correlated with high-ranking TNM stage (odds ratio [OR] =8.975, 95% confidence interval [CI]: 1.797–44.661), vascular invasion (OR =5.668, 95% CI: 1.827–17.578), and the higher risk grade (low risk grade: OR =0.141, 95% CI: 0.027–0.719; moderate risk grade: OR =0.122, 95% CI: 0.017–40.871). The low expression of the GalNAc-T3 usually in adenocarcinoma cell lines was compared with normal bronchial epithelium cell line. Based on the univariate and multivariate analysis, poor TNM stage (P<0.001), pleural invasion (hazard ratio [HR]: 7.958, P=0.021), vascular invasion (HR: 2.403, P=0.040), and low GalNAc-T3 expression (HR: 3.317, P=0.016) were shown to be independently associated with an unfavorable prognosis. However, IASLC/ATS/ERS classification of risk groups and Sica score (P=0.034 and P=0.032, respectively) was correlated with overall survival on Kaplan–Meier method but not Cox regression model.
GalNAc-T3 expression was correlated with the IASLC/ATS/ERS classification and also associated with prognosis of patients with completely resected small (≤2 cm) peripheral lung adenocarcinoma.
GalNAc-T3; classification; lung adenocarcinoma; prognosis
Cell surface sialylation is associated with tumor cell invasiveness in many cancers. Glioblastoma is the most malignant primary brain tumor and is highly infiltrative. ST3GAL1 sialyltransferase gene is amplified in a subclass of glioblastomas, and its role in tumor cell self-renewal remains unexplored.
Self-renewal of patient glioma cells was evaluated using clonogenic, viability, and invasiveness assays. ST3GAL1 was identified from differentially expressed genes in Peanut Agglutinin–stained cells and validated in REMBRANDT (n = 390) and Gravendeel (n = 276) clinical databases. Gene set enrichment analysis revealed upstream processes. TGFβ signaling on ST3GAL1 transcription was assessed using chromatin immunoprecipitation. Transcriptome analysis of ST3GAL1 knockdown cells was done to identify downstream pathways. A constitutively active FoxM1 mutant lacking critical anaphase-promoting complex/cyclosome ([APC/C]-Cdh1) binding sites was used to evaluate ST3Gal1-mediated regulation of FoxM1 protein. Finally, the prognostic role of ST3Gal1 was determined using an orthotopic xenograft model (3 mice groups comprising nontargeting and 2 clones of ST3GAL1 knockdown in NNI-11 [8 per group] and NNI-21 [6 per group]), and the correlation with patient clinical information. All statistical tests on patients’ data were two-sided; other P values below are one-sided.
High ST3GAL1 expression defines an invasive subfraction with self-renewal capacity; its loss of function prolongs survival in a mouse model established from mesenchymal NNI-11 (P < .001; groups of 8 in 3 arms: nontargeting, C1, and C2 clones of ST3GAL1 knockdown). ST3GAL1 transcriptomic program stratifies patient survival (hazard ratio [HR] = 2.47, 95% confidence interval [CI] = 1.72 to 3.55, REMBRANDT P = 1.92x10-8; HR = 2.89, 95% CI = 1.94 to 4.30, Gravendeel P = 1.05x10-11), independent of age and histology, and associates with higher tumor grade and T2 volume (P = 1.46x10-4). TGFβ signaling, elevated in mesenchymal patients, correlates with high ST3GAL1 (REMBRANDT gliomacor = 0.31, P = 2.29x10-10; Gravendeel gliomacor = 0.50, P = 3.63x10-20). The transcriptomic program upon ST3GAL1 knockdown enriches for mitotic cell cycle processes. FoxM1 was identified as a statistically significantly modulated gene (P = 2.25x10-5) and mediates ST3Gal1 signaling via the (APC/C)-Cdh1 complex.
The ST3GAL1-associated transcriptomic program portends poor prognosis in glioma patients and enriches for higher tumor grades of the mesenchymal molecular classification. We show that ST3Gal1-regulated self-renewal traits are crucial to the sustenance of glioblastoma multiforme growth.
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.
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.
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.
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.
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.
cell adhesion; galectin-8; glaucoma; integrins; trabecular meshwork
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.
Increased expression of galectin-1 (Gal-1) in carcinoma-associated fibroblasts (CAFs) has been reported to correlate with progression and prognosis in many cancers. However, rarely have reports sought to determine whether high Gal-1 expression in CAFs in gastric cancer is involved in the tumor process, and the specific mechanism by which it promotes the evolution of gastric cancer is still unknown. In this study, we cultured gastric cancer CAFs, which showed strong expression of Gal-1, and established a co-culture system of CAFs with gastric cancer cells. Specific siRNA and in vitro migration and invasion assays were used to explore the effects of the interaction between Gal-1 expression of CAFs and gastric cancer cells on cell migration and invasion. We found that the overexpression of Gal-1 in CAFs enhanced gastric cancer cell migration and invasion, and these stimulatory effects could be blocked by specific siRNA which reduced the Gal-1 expression level. A set of cancer invasion-associated genes were then chosen to identify the possible mechanism of Gal-1-induced cell invasion. Among these genes, integrin β1 expression in cancer cells was considered to be associated with Gal-1 expression. Pre-blocking of the integrin β1 expression in gastric cancer cells with siRNA could interrupt the invasion-promoting effect of CAFs with high Gal-1 expression. Furthermore, immunohistochemical assay confirmed a positive correlation between Gal-1 and integrin β1 expression. Our results showed that high expression of Gal-1 in CAFs might facilitate gastric cancer cell migration and invasion by upregulating integrin β1 expression in gastric cancer.
Carcinoma-associated fibroblasts; Gal-1; gastric cancer; integrin β1; invasion
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.
Pancreatic ductal adenocarcinoma; Galectin-4; migration; lymph node ratio; primary PDAC cells; Wnt/β-catenin pathway
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.
β-catenin; Galectin-3; Invasion; MMP-2; Pancreatic cancer; Wnt
Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model.
We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS.
Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin–glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30–40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels.
Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.
Galectin-8; Glioblastoma; Cancer; Cell cycle; Apoptosis; Proliferation; Migration
Radiotherapy (RT) can result in lymphopenia, which has been linked to poorer survival. Here, we test the hypothesis that RT-induced lymphopenia is mediated by a tumor-secreted factor, Galectin-1 (Gal-1), which possesses T-cell pro-apoptotic activities.
Matched Gal-1 wildtype or null mice were implanted with Lewis Lung carcinoma (LLC-1) that either expressed Gal-1 or had Gal-1 stably down-regulated. Tumors were irradiated locally and circulating Gal-1 and T-cells were measured. Tumor growth, lung metastasis, intratumoral T-cell apoptosis, and microvessel density count were quantified. Thiodigalatoside (TDG), a Gal-1 inhibitor, was used to inhibit Gal-1 function in another group of mice to validate the observations noted with Gal-1 down-regulation. Lymphocyte counts, survival and plasma Gal-1 were analyzed in cohorts of RT-treated lung (NSCLC) and head and neck cancer patients.
LLC irradiation increased Gal-1 secretion and decreased circulating T-cells in mice, regardless of host Gal-1 expression. Inhibition of tumor Gal-1 with either shRNA or TDG ablated RT-induced lymphopenia. Irradiated shGal-1 tumors showed significantly less intratumoral CD8+ T-cell apoptosis and microvessel density, which led to marked tumor growth delay and reduced lung metastasis compared to controls. Similar observations were made after TDG treatment. RT-induced lymphopenia was associated with poorer overall survival in NSCLC patients treated with hypofractionated RT. Plasma Gal-1 increased while T-cell decreased after radiation in another group of patients.
RT-related systemic lymphopenia appeared to be mediated by RT-induced tumor Gal-1 secretion that could lead to tumor progression through intratumoral immune suppression and enhanced angiogenesis.
NSCLC; Galectin-1; radiation; lymphopenia; tumorgenesis; HNC
Galectin-1 (Gal-1)-binding to Gal-1 ligands on immune and endothelial cells can influence melanoma development through dampening anti-tumor immune responses and promoting angiogenesis. However, whether Gal-1 ligands are functionally expressed on melanoma cells to help control intrinsic malignant features remains poorly understood. Here, we analyzed expression, identity and function of Gal-1 ligands in melanoma progression. Immunofluorescent analysis of benign and malignant human melanocytic neoplasms revealed that Gal-1 ligands were abundant in severely-dysplastic nevi as well as in primary and metastatic melanomas. Biochemical assessments indicated that melanoma cell adhesion molecule (MCAM) was a major Gal-1 ligand on melanoma cells that was largely dependent on its N-glycans. Other melanoma cell Gal-1 ligand activity conferred by O-glycans was negatively regulated by α2,6 sialyltransferase ST6GalNAc2. In Gal-1-deficient mice, MCAM-silenced (MCAMKD) or ST6GalNAc2-overexpressing (ST6O/E) melanoma cells exhibited slower growth rates, underscoring a key role for melanoma cell Gal-1 ligands and host Gal-1 in melanoma growth. Further analysis of MCAMKD or ST6O/E melanoma cells in cell migration assays indicated that Gal-1 ligand-dependent melanoma cell migration was severely inhibited. These findings provide a refined perspective on Gal-1 – melanoma cell Gal-1 ligand interactions as contributors to melanoma malignancy.
sialyltransferase; cell surface carbohydrates; tumor cell adhesion; galectin; melanoma
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.
tumor metastasis; adhesion; Thomsen-Friedenreich antigen; galectin; integrin
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.
Colorectal cancer; galectins; Wnt signaling; tumor suppressor