Role of glycosylation in TGF-β signaling and epithelial-to-mesenchymal transition in cancer

Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions.Aberrant glycosylation can lead to uncontrolled cell proliferation,cell-matrix interactions,migration and differentiation,and has been shown to be involved in cancer and other diseases.The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream.This cellular transformation process,which is associated by morphological change,loss of epithelial traits and gain of mesenchymal markers,is triggered by the secreted cytokine transforming growth factor-β(TGF-β).TGF-βbioactivity is carefully regulated,and its effects on cells are mediated by its receptors on the cell surface.In this review,we first provide a brief overview of major types of glycans,namely,N-glycans,O-glycans,glycosphingolipids and glycosaminoglycans that are involved in cancer progression.Thereafter,we summarize studies on how the glycosylation of TGF-βsignaling components regulates TGF-βsecretion,bioavailability and TGF-βreceptor function.Then,we review glycosylation changes associated with TGF-β-induced epithelial-to-mesenchymal transition in cancer.Identifying and understanding the mechanisms by which glycosylation affects TGF-βsignaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.     

SPARC expression in tumor microenvironment induces partial epithelial-to-mesenchymal transition of esophageal adenocarcinoma cells via cooperating with TGF-β signaling

Secreted protein, acidic and rich in cysteine (SPARC) has been characterized as an oncoprotein in esophageal squamous cell carcinoma (ESCC), but its involvement in the pathological development of esophageal adenocarcinoma (ESAD) remains poorly understood. In this study, we aimed to explore the sources of SPARC in the tumor microenvironment (TME) and its functional role in ESAD. Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA)-esophageal cancer (ESCA) and Genotype-Tissue Expression (GTEx). ESAD tumor cell line OE33 and OE19 cells were used as in vitro cell models. Results showed that SPARC upregulation was associated with unfavorable disease-specific survival (DSS) in ESAD. ESAD tumor cells (OE33 and OE19) had no detectable SPARC protein expression. In contrast, IHC staining in ESAD tumor tissues suggested that peritumoral stromal cells (tumor-associated fibroblasts and macrophages) were the dominant SPARC source in TME. Exogenous SPARC induced partial epithelial-to-mesenchymal transition of ESAD cells, reflected by reduced CDH1 and elevated ZEB1/VIM expression at both mRNA and protein levels. Besides, exogenous SPARC enhanced tumor cell invasion. When TGFBR2 expression was inhibited, the activation of TGF-β signaling induced by exogenous SPARC was impaired. However, the activating effects were rescued by overexpressing mutant TGFBR2 resistant to the shRNA sequence. Copresence of exogenous SPARC and TGF-β1 induced higher expression of mesenchymal markers and enhanced the invading capability of ESAD cells than TGF-β1 alone. In conclusion, this study suggests a potential cross-talk between ESAD tumor stromal cells and cancer cells via a SPARC-TGF-β1 paracrine network.     

Helicobacter pylori promotes epithelial-to-mesenchymal transition by downregulating CK2β in gastric cancer cells

Strains of Helicobacter pylori that are positive for the oncoprotein CagA (cytotoxin-associated gene A) are associated with gastric cancer and might be related to the epithelial-to-mesenchymal transition (EMT). Casein kinase 2 (CK2) is a serine/threonine protein kinase that plays a major role in tumorigenesis through signaling pathways related to the EMT. However, the role played by the interaction between CagA and CK2 in gastric carcinogenesis is poorly understood. Although CK2α protein expression remained unchanged during H. pylori infection, we found that CK2α kinase activity was increased in gastric epithelial cells. We also found that the CK2β protein level decreased in H. pylori-infected gastric cancer cells in CagA-dependent manner and demonstrated that CagA induced CK2β degradation via HDM2 (human double minute 2; its murine equivalent is MDM2). We observed that CagA induced HDM2 protein phosphorylation and that p53 levels were decreased in H. pylori-infected gastric cancer cells. In addition, downregulation of CK2β induced AKT Ser473 phosphorylation and decreased the AKT Ser129 phosphorylation level in gastric cancer cells. We also found that the downregulation of CK2β triggered the upregulation of Snail levels in gastric cancer cells. Furthermore, our in vivo experiments and functional assays of migration and colony formation suggest that CK2β downregulation is a major factor responsible for the EMT in gastric cancer. Therefore, CK2 could be a key mediator of the EMT in H. pylori-infected gastric cancer and could serve as a molecular target for gastric cancer treatment.     

Glucocorticoid induces mesenchymal-to-epithelial transition and inhibits TGF-β1-induced epithelial-to-mesenchymal transition and cell migration

Epithelial-to-mesenchymal transition (EMT) has been implicated in various physiological and pathological events. In this study, we found that the synthetic glucocorticoid dexamethasone (Dex) can inhibit transforming growth factor-beta1-induced EMT and cell migration. We also demonstrated that Dex inhibits EMT through a mechanism involving the suppression of ROS generation. Surprisingly, Dex alone induced mesenchymal-to-epithelial transition (MET). Dexamethasone treatment abolished Snail1 binding to the E-cadherin promoter, suggesting that suppression of Snail1 contributes to the above roles of Dex. Our findings demonstrate that Dex functions as both a suppressor of EMT and as an inducer of MET and therefore may be implicated in certain pathophysiological events.     

Genetic variants in TGF-β pathway are associated with ovarian cancer risk

The transforming growth factor-β (TGF-β) signaling pathway is involved in a diverse array of cellular processes responsible for tumorigenesis. In this case-control study, we applied a pathway-based approach to evaluate single-nucleotide polymorphisms (SNPs) in the TGF-β signaling pathway as predictors of ovarian cancer risk. We systematically genotyped 218 SNPs from 21 genes in the TGF-β signaling pathway in 417 ovarian cancer cases and 417 matched control subjects. We analyzed the associations of these SNPs with ovarian cancer risk, performed haplotype analysis and identified potential cumulative effects of genetic variants. We also performed analysis to identify higher-order gene-gene interactions influencing ovarian cancer risk. Individual SNP analysis showed that the most significant SNP was SMAD6: rs4147407, with an adjusted odds ratio (OR) of 1.60 (95% confidence interval [CI], 1.14–2.24, P = 0.0066). Cumulative genotype analysis of 13 SNPs with significant main effects exhibited a clear dose-response trend of escalating risk with increasing number of unfavorable genotypes. In gene-based analysis, SMAD6 was identified as the most significant gene associated with ovarian cancer risk. Haplotype analysis further revealed that two haplotype blocks within SMAD6 were significantly associated with decreased ovarian cancer risk, as compared to the most common haplotype. Gene-gene interaction analysis further categorized the study population into subgroups with different ovarian cancer risk. Our findings suggest that genetic variants in the TGF-β signaling pathway are associated with ovarian cancer risk and may facilitate the identification of high-risk subgroups in the general population.     

Free radical generation induces epithelial-to-mesenchymal transition in lung epithelium via a TGF-β1-dependent mechanism

Fibrotic remodelling of lung parenchymal and airway compartments is the major contributor to life-threatening organ dysfunction in chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD). Since transforming growth factor-β1 (TGF-β1) is believed to play a key role in disease pathogenesis and markers of oxidative stress are also commonly detected in bronchoalveolar lavage (BAL) from such patients we sought to investigate whether both factors might be interrelated. Here we investigated the hypothesis that oxidative stress to the lung epithelium promotes fibrotic repair by driving epithelial-to-mesenchymal transition (EMT) via the augmentation of TGF-β1. We show that in response to 400μM hydrogen peroxide (H(2)O(2)) A549 cells, used a model for alveolar epithelium, and human primary bronchial epithelial cells (PBECs) undergo EMT displaying morphology changes, decreased expression of epithelial markers (E-cadherin and ZO-1), increased expression of mesenchymal markers (vimentin and α-smooth muscle actin) as well as increased secretion of extracelluar matrix components. The same oxidative stress also promotes expression of TGF-β1. Inhibition of TGF-β1 signalling as well as treatment with antioxidants such as phenyl tert-butylnitrone (PBN) and superoxide dismutase 3 (SOD3) prevent the oxidative stress driven EMT-like changes described above. Interventions also inhibited EMT-like changes. This study identifies a link between oxidative stress, TGF-β1 and EMT in lung epithelium and highlights the potential for antioxidant therapies to limit EMT and its potential contribution to chronic lung disease.     

Dynamics of TGF-β induced epithelial-to-mesenchymal transition monitored by electric cell-substrate impedance sensing

The epithelial-to-mesenchymal transition (EMT) is a program of cellular development associated with loss of cell-cell contacts, a decreased cell adhesion and substantial morphological changes. Besides its importance for numerous developmental processes, EMT has also been held responsible for the development and progression of tumors and formation of metastases. The influence of the cytokine transforming growth factor β1 (TGF-β1) induced EMT on structure, migration, cytoskeletal dynamics and long-term correlations of the mammalian epithelial cell lines NMuMG, A549 and MDA-MB231 was investigated with time-resolved impedance analysis. The three cell lines show important differences in concentration dependency, cellular morphology and dynamics upon their response to TGF-β1. A549 cells and the non-tumor mouse epithelial cell line NMuMG show a substantial change in morphology mirrored in stepwise changes of their phenotype upon cytokine treatment. Impedance based measurements of micromotility reveal a complex dynamic response to TGF-β1 exposure which leads to a transient increase in fluctuation amplitude and long-term correlation. These changes in fluctuation amplitude are also detectable for MDA-MB231 cells, whereas the long-term correlation remains unvaried. We were able to distinguish three time domains during EMT. Initially, all cell lines display an increase in micromotion lasting 4 to 9h termed transitional state I. This regime is followed by transitional state II lasting approximately 20 h, where cellular dynamics are diminished and, in case of the NMuMG cell line, a loss of cell-cell contacts occurs. Finally, the transformation into the mesenchymal-like phenotype occurs 24-30 h after exposure to TGF-β1.     

Regulation of the TGF-β pathway by deubiquitinases in cancer

The transforming growth factor-β (TGF-β) pathway regulates diverse cellular processes. It signals via serine/threonine kinase receptors and intracellular Smad and non-Smad effector proteins. In cancer cells, aberrant TGF-β signalling can lead to loss of growth inhibition and an increase in invasion, epithelial-to-mesenchymal transition (EMT) and metastasis. Therapeutic targeting of the pro-oncogenic TGF-β responses is currently being explored as a potential therapy against certain invasive and metastatic cancer types. The ubiquitin post-translational regulation system is emerging as a key regulatory mechanism for the control of TGF-β pathway components. In this review, we focus on the role of deubiquitinases (DUBs), which counteract the activity of E3 ubiquitin ligases. We will discuss the mechanisms by which specific DUBs control Smad and non-Smad TGF-β signalling routes, and how perturbation of the expression and function of DUBs contributes to misregulation of TGF-β signalling in cancer.     

TGF-β family signaling in stem cells

Background

The diversity of cell types and tissue types that originate throughout development derives from the differentiation potential of embryonic stem cells and somatic stem cells. While the former are pluripotent, and thus can give rise to a full differentiation spectrum, the latter have limited differentiation potential but drive tissue remodeling. Additionally cancer tissues also have a small population of self-renewing cells with stem cell properties. These cancer stem cells may arise through dedifferentiation from non-stem cells in cancer tissues, illustrating their plasticity, and may greatly contribute to the resistance of cancers to chemotherapies.

Scope of review

The capacity of the different types of stem cells for self-renewal, the establishment and maintenance of their differentiation potential, and the selection of differentiation programs are greatly defined by the interplay of signaling molecules provided by both the stem cells themselves, and their microenvironment, the niche. Here we discuss common and divergent roles of TGF-β family signaling in the regulation of embryonic, reprogrammed pluripotent, somatic, and cancer stem cells.

Major conclusions

Increasing evidence highlights the similarities between responses of normal and cancer stem cells to signaling molecules, provided or activated by their microenvironment. While TGF-β family signaling regulates stemness of normal and cancer stem cells, its effects are diverse and depend on the cell types and physiological state of the cells.

General significance

Further mechanistic studies will provide a better understanding of the roles of TGF-β family signaling in the regulation of stem cells. These basic studies may lead to the development of a new therapeutic or prognostic strategies for the treatment of cancers. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Glycogen synthase kinase-3β positively regulates the proliferation of human ovarian cancer cells

Although glycogen synthase kinase-3 （GSK-3） might act as a tumor suppressor since its inhibition is expected to mimic the activation of Wnt-signaling pathway, GSK-3β may contribute to NF-κB activation in cancer cells leading to increased cancer cell proliferation and survival. Here we report that GSK-3β activity was involved in the proliferation of human ovarian cancer cell both in vitro and in vivo. Inhibition of GSK-3 activity by pharmacological inhibitors suppressed proliferation of the ovarian cancer cells. Overexpressing constitutively active form of GSK-3β induced entry into the S phase, increased cyclin D1 expression and facilitated the proliferation of ovarian cancer cells. Furthermore, GSK-3 inhibition prevented the formation of the tumor in nude mice generated by the inoculation of human ovarian cancer cells. Our findings thus suggest that GSK-3β activity is important for the proliferation of ovarian cancer cells, implicating this kinase as a potential therapeutic target in ovarian cancer.     

Genes suppressed by DNA methylation in non-small cell lung cancer reveal the epigenetics of epithelial–mesenchymal transition

Background

DNA methylation is associated with aberrant gene expression in cancer, and has been shown to correlate with therapeutic response and disease prognosis in some types of cancer. We sought to investigate the biological significance of DNA methylation in lung cancer.

Results

We integrated the gene expression profiles and data of gene promoter methylation for a large panel of non-small cell lung cancer cell lines, and identified 578 candidate genes with expression levels that were inversely correlated to the degree of DNA methylation. We found these candidate genes to be differentially methylated in normal lung tissue versus non-small cell lung cancer tumors, and segregated by histologic and tumor subtypes. We used gene set enrichment analysis of the genes ranked by the degree of correlation between gene expression and DNA methylation to identify gene sets involved in cellular migration and metastasis. Our unsupervised hierarchical clustering of the candidate genes segregated cell lines according to the epithelial-to-mesenchymal transition phenotype. Genes related to the epithelial-to-mesenchymal transition, such as AXL, ESRP1, HoxB4, and SPINT1/2, were among the nearly 20% of the candidate genes that were differentially methylated between epithelial and mesenchymal cells. Greater numbers of genes were methylated in the mesenchymal cells and their expressions were upregulated by 5-azacytidine treatment. Methylation of the candidate genes was associated with erlotinib resistance in wild-type EGFR cell lines. The expression profiles of the candidate genes were associated with 8-week disease control in patients with wild-type EGFR who had unresectable non-small cell lung cancer treated with erlotinib, but not in patients treated with sorafenib.

Conclusions

Our results demonstrate that the underlying biology of genes regulated by DNA methylation may have predictive value in lung cancer that can be exploited therapeutically.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1079) contains supplementary material, which is available to authorized users.     

ALG2 inhibits the epithelial-to-mesenchymal transition and stemness of ovarian granulosa cells through the Wnt/β-catenin signaling pathway in polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects fertility in women of reproductive age, and a leading cause of anovulatory infertility. Ovarian granulosa cells are a major functional cell type in the ovary that undergo epithelial-to-mesenchymal transition (EMT) to initiate ovulation. Protein glycosylation, catalyzed by specific glycosyltransferases, has been implicated in reproductive events, such as embryo implantation, endometrial receptivity, and decidualization, etc. However, the relationship between glycosylation and EMT-mediated ovulation in PCOS is not well understood. To clarify the role of cobalt chloride (CoCl₂) and α-1,3/1,6-mannosyltransferase (ALG2) in PCOS, transwell assay, Real-time PCR, Western blot, immunofluorescence, and sphere formation assay were applied to assess cell migration, invasion, EMT, and stemness of ovarian granulosa cells. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum level of ALG2 in PCOS patients. We found that CoCl₂ promoted the migration, invasion, EMT, and stemness of ovarian granulosa cells by downregulating the expression of ALG2. Upregulation of ALG2 rescued the effects of CoCl₂ partially, and inhibited the EMT and stemness of ovarian granulosa cells by inactivating the Wnt/β-catenin signaling pathway. Moreover, the serum level of ALG2 was increased in patients with PCOS. Elevated ALG2, in combination with testosterone, represented better diagnostic value for PCOS as a multimarker than ALG2 or testosterone alone as a single marker. Thus, ALG2, downregulated by CoCl₂, was increased in PCOS patients which inhibited the EMT and stemness of ovarian granulosa cells by deactivating the Wnt/β-catenin signaling pathway. The combination of ALG2 and testosterone might thus act as a novel but promising biomarker for PCOS detection.     

Roles for the type III TGF-β receptor in human cancer

Transforming growth factor β (TGF-β) superfamily ligands have important roles in regulating cellular homeostasis, embryonic development, differentiation, proliferation, immune surveillance, angiogenesis, motility, and apoptosis in a cell type and context specific manner. TGF-β superfamily signaling pathways also have diverse roles in human cancer, functioning to either suppress or promote cancer progression. The TGF-β superfamily co-receptor, the type III TGF-β receptor (TβRIII, also known as betaglycan) mediates TGF-β superfamily ligand dependent as well as ligand independent signaling to both Smad and non-Smad signaling pathways. Loss of TβRIII expression during cancer progression and direct effects of TβRIII on regulating cell migration, invasion, proliferation, and angiogenesis support a role for TβRIII as a suppressor of cancer progression and/or as a metastasis suppressor. Defining the physiological function and mechanism of TβRIII action and alterations in TβRIII function during cancer progression should enable more effective targeting of TβRIII and TβRIII mediated functions for the diagnosis and treatment of human cancer.     

γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells

γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0444-0) contains supplementary material, which is available to authorized users.