Signalling networks that cause cancer

Cancer is caused by the stepwise accumulation of mutations that affect growth control, differentiation and survival. The view that mutations affect discrete signalling pathways, each contributing to a specific aspect of the full malignant phenotype, has proved to be too simplistic. We now know that oncogenes and tumour suppressors depend on one another for their selective advantage, and that they affect multiple pathways that intersect and overlap. The interactive nature of each genetic change has important implications for cancer therapy and for the stepwise model of carcinogenesis.     

Signalling networks that cause cancer

Cancer is caused by the stepwise accumulation of mutations that affect growth control, differentiation and survival. The view that mutations affect discrete signalling pathways, each contributing to a specific aspect of the full malignant phenotype, has proved to be too simplistic. We now know that oncogenes and tumour suppressors depend on one another for their selective advantage, and that they affect multiple pathways that intersect and overlap. The interactive nature of each genetic change has important implications for cancer therapy and for the stepwise model of carcinogenesis.     

Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization.

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity.     

11beta-hydroxysteroid dehydrogenases,cell proliferation and malignancy

The enzymes 11β-hydroxysteroid dehydrogenase type 1 and 2 (11β-HSD1 and 2) have well-defined roles in the tissue-specific metabolism of glucocorticoids which underpin key endocrine mechanisms such as adipocyte differentiation (11β-HSD1) and mineralocorticoid action (11β-HSD2). However, in recent studies we have shown that the effects of 11β-HSD1 and 2 are not restricted to distinct tissue-specific hormonal functions. Studies of normal fetal and adult tissues, as well as their tumor equivalents, have shown a further dichotomy in 11β-HSD expression and activity. Specifically, most normal glucocorticoid receptor (GR)-rich tissues such as adipose tissue, bone, and pituitary cells express 11β-HSD1, whereas their fetal equivalents and tumors express 11β-HSD2. We have therefore postulated that the ability of 11β-HSD1 to generate cortisol acts as an autocrine anti-proliferative, pro-differentiation stimulus in normal adult tissues. In contrast, the cortisol-inactivating properties of 11β-HSD2 lead to pro-proliferative effects, particularly in tumors. This proposal is supported by experiments in vitro which have demonstrated divergent effects of 11β-HSD1 and 2 on cell proliferation. Current studies are aimed at (1) characterizing the underlying mechanisms for a ‘switch’ in 11β-HSD isozyme expression in tumors; (2) defining the molecular targets for glucocorticoids as regulators of cell proliferation; (3) evaluating the potential for targeting glucocorticoid metabolism as therapy for some cancers. These and other issues are discussed in the present review.     

Differentially expressed genes in L6 rat skeletal muscle myoblasts after incubation with inflammatory cytokines

OBJECTIVE: The mechanism underlying exercise intolerance in chronic heart failure is still unclear. An increased concentration of inflammatory cytokines could be detected in the serum of patients with chronic heart failure (CHF) exhibiting a correlation with the severity of the disease. The variety of molecular alterations triggered by these cytokines in the skeletal muscle is almost unknown. The study was designed to analyze the differential gene expression in skeletal muscle myoblasts after stimulation with inflammatory cytokines. METHODS: L6 rat skeletal muscle myoblasts were incubated for 24 h with a combination of IL-1beta/IFN-gamma and the differential gene expression profile was determined by a PCR-based subtractive hybridization method. RESULTS: Out of 173 picked clones 141 different sequences could be identified. By comparison with Genebank, the identity of 73 genes (51.7%) could be confirmed, whereas the rest did not show a homology to any known gene. Some of the identified genes are known to be altered in patients with CHF. CONCLUSION: In summary, the results of this study provide information about changes in gene expression after exposure of skeletal muscle cells to inflammatory cytokines. This information may yield a new gene pool, worthwhile to be analyzed in skeletal muscle of patients with chronic heart failure.     

Caffeine inhibits cell proliferation and regulates PKA/GSK3β pathways in U87MG human glioma cells

Caffeine is the most commonly ingested methylxanthine and has anti-cancer effects in several types of cancer. In this study, we examined the anti-cancer effects of caffeine on gliomas, both in vitro and in vivo. In vitro, caffeine treatment reduced glioma cell proliferation through G(0)/G(1)-phase cell cycle arrest by suppressing Rb phosphorylation. In addition, caffeine induced apoptosis through caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Caffeine also phosphorylated serine 9 of glycogen synthase kinase 3 beta (GSK3β). Pretreatment with H89, a pharmacological inhibitor of protein kinase A (PKA), was able to antagonize caffeine-induced GSK3β(ser9) phosphorylation, suggesting that the mechanism might involve a cAMP-dependent PKA-dependent pathway. In vivo, caffeine-treated tumors exhibited reduced proliferation and increased apoptosis compared with vehicle-treated tumors. These results suggest that caffeine induces cell cycle arrest and caspase-dependent cell death in glioma cells, supporting its potential use in chemotherapeutic options for malignant gliomas.     

Purification of GSK-3 by affinity chromatography on immobilized axin

Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of beta-catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ss, beta-catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419-672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These "Axin-His6" matrices were found to selectively bind recombinant rat GSK-3 beta and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes.     

The Imprinted Gene PEG3 Inhibits Wnt Signaling and Regulates Glioma Growth

The imprinted gene PEG3 confers parenting and sexual behaviors, alters growth and development, and regulates apoptosis. However, a molecular mechanism that can account for the diverse functions of Peg3/Pw1 is not known. To elucidate Peg3-regulated pathways, we performed a functional screen in zebrafish. Enforced overexpression of PEG3 mRNA during zebrafish embryogenesis decreased β-catenin protein expression and inhibited Wnt-dependent tail development. Peg3/Pw1 also inhibited Wnt signaling in human cells by binding to β-catenin and promoting its degradation via a p53/Siah1-dependent, GSK3β-independent proteasomal pathway. The inhibition of the Wnt pathway by Peg3/Pw1 suggested a role in tumor suppression. Hypermethylation of the PEG3 promoter in primary human gliomas led to a loss of imprinting and decreased PEG3 mRNA expression that correlated with tumor grade. The decrease in Peg3/Pw1 protein expression increased β-catenin, promoted proliferation, and inhibited p53-dependent apoptosis in human CD133⁺ glioma stem cells. Thus, mammalian imprinting utilizes Peg3/Pw1 to co-opt the Wnt pathway, thereby regulating development and glioma growth.     

Role of C/EBP-β, p38 MAPK, and MKK6 in IL-1β-mediated C3 gene regulation in astrocytes

Complement component C3, the central player in the complement cascade and the pro-inflammatory cytokine IL-1β is expressed by activated glial cells and may contribute to neurodegeneration. This study examines the regulation of the expression of C3 by IL-1β in astroglial cells focusing on the role of the upstream kinase MKK6, p38-α MAPK, and C/EBP-β isoforms (LAP1, LAP2, or LIP) in astroglial cells. Activation of human astroglial cell line, U373 with IL-1β, led to the induction of C3 mRNA and protein expression as determined by real-time RT-PCR and Western blot analysis, respectively. This induction was suppressed by the pharmacological inhibitor of p38 MAPK (i.e., SB202190-HCl), suggesting the involvement of p38 MAPK in C3 gene expression. IL-1β also induced C3 promoter activity in U373 cells in a MAP kinase- and C/EBP-β-dependent manner. Cotransfection of C3 luciferase reporter construct with constitutively active form of the upstream kinase in the MAP kinase cascade, that is, MKK6 (the immediate upstream activator of p38 kinase) resulted in marked stimulation of the promoter activity, whereas overexpression of a dominant negative forms of MKK6 and p38α MAPK inhibited C3 promoter activity. Furthermore, a mutant form of C/EBP-β, LAP(T235A) showed reduction in IL-1β-mediated C3 promoter activation. These results suggest that the p38α, MAPK, and MKK6 play prominent roles in IL-1β and C/EBP-β-mediated C3 gene expression in astrocytes.     

The Intracellular Domain of Cadherin-11 is not Required for the Induction of Cell Aggregation, Adhesion or Gap-Junction Formation

The cadherin family of cell adhesion molecules demonstrates calcium-dependent hemophilic binding, leading to cellular recognition and adhesion. The adhesion mediated by the classical type 1 cadherins is strengthened through catenin-mediated coupling of the cytoplasmic domain to the cytoskeleton. This cytoskeletal interaction may not be essential for the adhesion promoted by all cadherins, several of which lack cytosolic catenin-binding sequences. Cadherin-11, a classical cadherin, possesses a cytoplasmic domain that interacts with catenins, but may also occur as a variant form expressing a truncated cytoplasmic domain. To study the role of the cytoplasmic sequence in cadherin-11 mediated adhesion we have constructed and expressed a truncated cadherin-11 protein lacking the cytoplasmic domain and unable to bind β-catenin. Expression of the truncated cadherin-11 in MDA-MB-435S human mammary carcinoma cells reduced their motility and promoted calcium-dependent cell aggregation, frequent cell contacts, and functional gap-junctions. We conclude that the intracellular catenin-binding domain of cadherin-11, and by inference cytoskeletal interaction, is not required for the initiation and formation of cell adhesion.     

Gene expression and cell growth are modified by silencing SUMO2 and SUMO3 expression

Small ubiquitin-like modifier (SUMO) is a group of proteins binding to lysine residues of target proteins and thereby modifying their stability, activity and subcellular localization. Here we report that blocking SUMO2 and SUMO3 conjugation by silencing their expression markedly modifies gene expression. A microRNA-based RNAi system was used to specifically silence SUMO2 and SUMO3 expression simultaneously and stably transfected neuroblastoma B35 cells expressing dual SUMO2/3 microRNA were created. In cells stably expressing SUMO2/3 microRNA, mRNA levels of 105 and 58 known genes were significantly up- and down-regulated, respectively. About 20% of differentially regulated genes were associated with pathways involved in cell growth and differentiation. Cell division was significantly suppressed in SUMO2/3 miRNA expressing cells. Elucidating what effect the silencing of SUMO2/3 expression has on gene expression will help to identify the impact of SUMO2/3 conjugation on the various cellular pathways.     

Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.