Galectins as modulators of receptor tyrosine kinases signaling in health and disease

Receptor tyrosine kinases (RTKs) constitute a large group of cell surface proteins that mediate communication of cells with extracellular environment. RTKs recognize external signals and transfer information to the cell interior, modulating key cellular activities, like metabolism, proliferation, motility, or death. To ensure balanced stream of signals the activity of RTKs is tightly regulated by numerous mechanisms, including receptor expression and degradation, ligand specificity and availability, engagement of co-receptors, cellular trafficking of the receptors or their post-translational modifications. One of the most widespread post-translational modifications of RTKs is glycosylation of their extracellular domains. The sugar chains attached to RTKs form a new layer of information, so called glyco-code that is read by galectins, carbohydrate binding proteins. Galectins are family of fifteen lectins implicated in immune response, inflammation, cell division, motility and death. The versatility of cellular activities attributed to galectins is a result of their high abundance and diversity of their cellular targets. A various sugar specificity of galectins and the differential ability of galectin family members to form oligomers affect the spatial distribution and the function of their cellular targets. Importantly, galectins and RTKs are tightly linked to the development, progression and metastasis of various cancers. A growing number of studies points on the close cooperation between RTKs and galectins in eliciting specific cellular responses. This review focuses on the identified complexes between galectins and RTK members and discusses their relevance for the cell physiology both in healthy tissues and in cancer.     

Defective downregulation of receptor tyrosine kinases in cancer

Most growth factors control cellular functions by activating specific receptor tyrosine kinases (RTKs). While overactivation of RTK signalling pathways is strongly associated with carcinogenesis, it is becoming increasingly clear that impaired deactivation of RTKs may also be a mechanism in cancer. A major deactivation pathway, receptor downregulation, involves ligand-induced endocytosis of the RTK and subsequent degradation in lysosomes. A complex molecular machinery that uses the small protein ubiquitin as a key regulator assures proper endocytosis and degradation of RTKs. Here we discuss evidence that implicates deregulation of this machinery in cancer.     

Role of Angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis

This review focuses on the signaling system involving the Angiopoietin1/Tie2 receptor, which appears to be involved in the secondary stages of blood vessel formation. Although this system is crucial for blood and lymphatic vessel formation, identifying its precise role in embryonic and adult vascular biology has been a major challenge. The evidence for the key role of the Angiopoietin/Tie system is discussed, and some of the other members of the system (Ang2, Tie1) are mentioned. A comparison is made with the VEGF signaling system, which seems to provide a complementary, and somewhat more tractable, signaling system. Some of the basic unanswered questions concerning Tie/Angiopoietin biology and the secondary stages of blood vessel formation are also highlighted.     

On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling

Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.     

Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.     

Peroxynitrite signaling: receptor tyrosine kinases and activation of stress-responsive pathways

Peroxynitrite, generated for example in inflammatory processes, is capable of nitrating and oxidizing biomolecules, implying a considerable impact on the integrity of cellular structures. Cells respond to stressful conditions by the activation of signaling pathways, including receptor tyrosine kinase-dependent pathways such as mitogen-activated protein kinases and the phosphoinositide-3-kinase/Akt pathway. Peroxynitrite affects signaling pathways by nitration as well as by oxidation: while nitration of tyrosine residues by peroxynitrite modulates signaling processes relying on tyrosine phosphorylation and dephosphorylation, oxidation of phosphotyrosine phosphatases may lead to an alteration in the tyrosine phosphorylation/dephosphorylation balance. The flavanol (-)-epicatechin is a potent inhibitor of tyrosine nitration and may be employed as a tool to distinguish signaling effects due to tyrosine nitration from those that are due to oxidation reactions.     

A mathematical model of the effects of hypoxia on the cell-cycle of normal and cancer cells

The evolution of the cell-cycle is known to be influenced by environmental conditions, including lack of extracellular oxygen (hypoxia). Notably, hypoxia appears to have different effects on normal and cancer cells. Whereas both experience hypoxia-induced arrest of the G1 phase of the cell-cycle (i.e. delay in the transition through the restriction point), experimental evidence suggests that only cancer cells undergo hypoxia-induced quiescence (i.e. the transition of the cell to a latent state in which most of the cell functions, including proliferation, are suspended). Here, we extend a model for the cell-cycle due to Tyson and Novak (J. Theor. Biol. 210 (2001) 249) to account for the action of the protein p27. This protein, whose expression is upregulated under hypoxia, inhibits the activation of the cyclin dependent kinases (CDKs), thus preventing DNA synthesis and delaying the normal progression through the cell-cycle. We use a combination of numerical and analytic techniques to study our model. We show that it reproduces many features of the response to hypoxia of normal and cancer cells, as well as generating experimentally testable predictions. For example our model predicts that cancer cells can undergo quiescence by increasing their levels of p27, whereas for normal cells p27 expression decreases when the cellular growth rate increases.     

Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding

Epidermal growth factor receptors (ErbB1-4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100-1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts their mechanisms of action.     

The breast cancer cells response to chronic hypoxia involves the opposite regulation of NF-kB and estrogen receptor signaling

The progression of cancer is associated with tumor's ability to outgrow the existing vasculature resulting in chronic hypoxic pressure, however the molecular mechanism of cancer cell response to chronic hypoxia is poorly understood. In this study we have analyzed the reorganization of estrogen receptor (ER) signaling in breast cancer cells under chronic hypoxia and examined the role of interrelations between ER and NF-kB signaling in cell adaptation to hypoxia. Using long-term culturing of MCF-7 breast cancer cells in hypoxia-mimetic conditions (cobalt chloride) we have established a hypoxia-tolerant subline characterized by HIF-1 hyperexpression that retained the tolerance to hypoxia even when the cells were returned to normoxic conditions.The hypoxia-tolerant cells were characterized by non-affected ER signaling, irreversible suppression of NF-kB activity, and increased sensitivity to cytokine-induced apoptosis. Estradiol treatment suppressed the NF-kB activity in both parent and hypoxia-tolerant MCF-7 cells. In contrast to MCF-7 cells, the exposure of estrogen-independent MCF-7/T2 subline to chronic hypoxia was not accompanied by noticeable changes in NF-kB activity or cell sensitivity to cytokines. Taken together, the results presented demonstrate the importance of interrelations between ER and NF-kB signaling in the response of estrogen-dependent breast cancer cells to chronic hypoxia.     

Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor

Heterogeneous receptor-receptor interactions may play a role in intracellular signaling. Accordingly, the interaction of two dissimilar tyrosine kinase receptors, Ron and epidermal growth factor receptor (EGFR) was investigated. The functional interaction of Ron and EGFR in cell scatter and oncogenic transformation was investigated in vivo. Transfection of a dominant negative form of EGFR into human embryonic kidney cells stably expressing Ron (293-Ron) dramatically reduced the scatter response induced by the Ron ligand hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL). The scatter response of the 293-Ron cells was also attenuated by treatment of the cells with the specific EGFR inhibitor AG 1478. Co-transfection of Ron and dominant-negative EGFR, or co-transfection of EGFR and a dominant-negative form of Ron reduced focus formation in NIH/3T3 cells. Western analysis of NIH/3T3 cells overexpressing murine Ron and expressing endogenous levels of EGFR was used to demonstrate that Ron and EGFR co-immunoprecipitate. Stimulation of the cells in vitro with the Ron ligand HGFL or with the EGFR ligand epidermal growth factor (EGF) appeared to induce phosphorylation of both receptors. Co-immunoprecipitation and phosphorylation of phosphatidyl inositol 3-kinase (PI3-K) was also observed. This novel finding of a functional and biochemical interaction between Ron and EGFR suggests that heterologous tyrosine kinase receptor interactions may play a role in cellular processes such as scatter and transformation.     

Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack

The tolerance of cancer cells to hypoxia depends on the combination of different factors – from increase of glycolysis (Warburg Effect) to activation of intracellular growth/apoptotic pathways. Less is known about the influence of epithelial–mesenchymal transition (EMT) and EMT-associated pathways on the cell sensitivity to hypoxia. The aim of this study was to explore the role of Snail signaling, one of the key EMT pathways, in the mediating of hypoxia response and regulation of cell sensitivity to hypoxia, using as a model in vitro cultured breast cancer cells. Earlier we have shown that estrogen-independent HBL-100 breast cancer cells differ from estrogen-dependent MCF-7 cells with increased expression of Snail1, and demonstrated Snail1 involvement into formation of hormone-resistant phenotype. Because Snail1 belongs to hypoxia-activated proteins, here we studied the influence of Snail1 signaling on the cell tolerance to hypoxia. We found that Snail1-enriched HBL-100 cells were less sensitive to hypoxia-induced growth suppression if compared with MCF-7 line (31% MCF-7 vs. 71% HBL-100 cell viability after 1% O₂ atmosphere for 3 days). Snail1 knock-down enhanced the hypoxia-induced inhibition of cell proliferation giving the direct evidence of Snail1 involvement into cell protection from hypoxia attack. The protective effect of Snail1 was shown to be mediated, at least in a part, via beta-catenin which positively regulated expression of HIF-1-dependent genes. Finally, we found that cell tolerance to hypoxia was accompanied with the failure in the phosphorylation of AMPK – the key energy sensor, and demonstrated an inverse relationship between AMPK and Snail/beta-catenin signaling.Totally, our data show that Snail1 and beta-catenin, besides association with loss of hormone dependence, protect cancer cells from hypoxia and may serve as an important target in the treatment of breast cancer. Moreover, we suggest that the level of these proteins as well the level of AMPK phosphorylation may be considered as predictors of the tumor sensitivity to anti-angiogenic drugs.     

Visfatin in adipocytes is upregulated by hypoxia through HIF1alpha-dependent mechanism

Obesity is associated with metabolic disorders, such as insulin resistance. Visfatin is an adipose-derived secretory factor to exert insulin-mimetic effects. Plasma visfatin levels and mRNA levels of visfatin in adipose tissues are increased in obesity. However, the mechanism that mediates induction of visfatin mRNA in adipose tissue of obesity remains unknown. Recent studies have reported that fat tissue is hypoxia in obesity. In this study, we investigated the effects of hypoxia on mRNA expression of visfatin in adipocytes. Hypoxia increased visfatin mRNA expression. Desferoxamine and Cobaltous chloride, two hypoxia mimetic compounds, also increased visfatin mRNA levels. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1alpha (HIF1alpha), mimicked the hypoxia-mediated upregulation of visfatin, and YC1, an inhibitor of HIF1 cancelled the hypoxia-induced upregulation of visfatin mRNA. We identified two functional hypoxia responsive elements (HRE) in mouse visfatin promoter. Hypoxic treatment and overexpression of HIF1alpha increased the promoter activity, and mutation of the HRE blunted hypoxia-induced activation of visfatin promoter. Our results suggest that visfatin mRNA expression is upregulated in the fat tissue of obesity through the activation of HIF1alpha pathway due to hypoxia.     

Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor

Hypoxia sensing is the generic term for pO₂-sensing in humans and other higher organisms. These cellular responses to pO₂ are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O₂-activation to the hydroxylation of the hypoxia inducible factor α (HIFα). Uncoupled O₂-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (αKG + Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λ_max = 583 nm) arising from enzyme auto-hydroxylation of Trp²⁹⁶, forming an Fe(III)-O-Trp²⁹⁶ chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met²⁷⁵. The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however, product yield was saturable with increasing [αKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate + Fe)FIH-1, or by glycerol addition to (αKG + Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (αKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O₂-activation by loss of an H₂O ligand from the metal center.     

Calpain-2 regulates hypoxia/HIF-induced plasticity toward amoeboid cancer cell migration and metastasis

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