Current Signal Transduction Therapy - Volume 6, Issue 1, 2011

The journal Current Signal Transduction Therapy will start its 6th year in 2011 and in the past five years it has become a very important forum for signaling related pathomechanisms and drug discovery issues. As we have pointed out earlier the general concept of the journal is to describe molecular pathomechanisms and signal transduction therapy with special emphasis on concepts as “targeted therapies”, “network signaling”, “molecular diagnostics”, “multiple targets” and “personalized therapy”. In the last year personalized therapy has become a very hot topic, so CSTT is dedicated to support papers in this field. The aim of personalized therapy is to match the right drug to the right patient and, in some cases, even to design the appropriate treatment for a patient according to his/her genotype. The modern concept of personalized medicine includes pharmacogenomics, pharmacogenetics, pharmacoproteomics, and metabolomics. Pharmacogenomics deals with the application of genomics to drug discovery and development and includes the mechanism of action of drugs on cells as revealed by gene expression patterns. Basic technologies of molecular diagnostics play an important role, particularly mutation/amplification analysis and single nucleotide polymorphism (SNP) genotyping. Molecular diagnostics is integrated with therapy for selection of the treatment as well for monitoring the results. A combination of target-driven and chemistry-driven drug discovery with biomarker identification could be the basis of personalized therapy. Most of the papers in the current issue are related to targeted therapy with possible associated molecular diagnostics in cancer including the role of TGFBeta in tumor metastasis formation, mTOR in heamatologic malignancies, TACC2 as a tumor suppressor for breast cancer working through PLCgamma pathway, CK1, CK2 and GSK3 in Normal and Malignant Haematopoiesis, Dasatinib as a dual Src/Abl kinase inhibitor in the treatment of Chronic Myeloid Leukemia, molecular mechanisms of action of Gas1 and its possible therapeutic applications in cancer, and identification of phosphoproteins and their impact as biomarkers in cancer therapeutics. Two papers describe targeted therapies for other diseases like Dystroglycan, in neuromuscular diseases including muscular dystrophy, and Glycogen Synthase Kinase-3 and Proline-Rich Akt Substrate (PRAS40) in cardiac hypertrophy. One paper describes the role of Leptin signaling in platelets which may be involved in enhanced tendency to thrombosis and atherosclerosis in patients with the metabolic syndrome, and one paper describes the role of p70 S6 kinase and ERK Activity in neuronal development and death.

Treating different cancers is a major challenge nowadays. Several molecules on the cell surface, in the cell particles and genes, can be targets to inhibit the growth of cancerous cells or to stop metastatic processes. Different small molecules are in the focus of newly targeted therapy areas. Cytoplasmatic skeleton structure as well as apparently expressed mitotic regulators can be targets in tumour therapy. Angiogenesis is essential for tumor growth and spreading, therefore NAD(P)H oxidase can also be a target. Ras/Raf/MEK/MAPK pathway can affect multiple cellular functions by stimulating tumor growth. ICMT methylation of prenylated proteins is important for the correct localization and function of a number of proteins ending in cysteine aliphatic residue, therefore ICMT is an enzyme target in anticancer drug discovery. The abnormal activations of several growth factor receptors and their signaling pathways have been implicated in the development and progression of cancerous processes. Activation of JAK/STAT signaling rout occurs with high frequency in humans. The increased activity of STAT in cancerous tissues enhances the level of Bcl-xL and hereby contributes to the drug resistance in chemotherapy. Therefore tyrosine kinase activity can be a therapeutic target as well. Hsp90 participates in many key processes in oncogenesis such as self-sufficiency in growth signals, stabilization of mutant proteins, angiogenesis and metastasis. The inhibitions of histone deacetylase in recent years have an important part of targeted therapy as well. Also selective inhibition of NF-κB signaling is possible in cancer therapy. This review would give an account of the action mechanism of current small molecules and newly developed ones in experimental phase in cancer therapy. The effectiveness of these molecules and their combination with each other and their combination with other therapies will be discussed.

Leptin is a peptide hormone secreted by adipose tissue which is primarily involved in the regulation of food intake and energy expenditure. Apart from the brain, leptin receptors are expressed in many peripheral tissues. Plasma leptin concentration is markedly increased in most patients with obesity/metabolic syndrome reflecting greater amount of adipose tissue and resistance to anorectic effect of this hormone. Recent studies indicate that leptin receptors are also expressed in blood platelets and that leptin stimulates platelet aggregation at least in some experimental conditions. In addition, thrombus formation is impaired in leptin deficient ob/ob mice and leptin supplementation corrects these abnormalities. Clinical studies suggest the link between hyperleptinemia and atherothrombosis independently of body weight. These data indicate that leptin signaling in platelets may be involved in enhanced tendency to thrombosis and atherosclerosis in patients with the metabolic syndrome. In this article I characterize the effect of leptin on platelet function as well as possible therapeutic interventions aimed to target leptin signaling in platelets.

Transforming growth factor-β (TGF-β) is a cytokine involved in cell proliferation, apoptosis, differentiation, angiogenesis, cell adhesion, migration, extracellular matrix deposition, wound healing and immune regulation. The cellular response to TGF-β depends on the cell type and cell microenvironment. TGF-β supresses tumor growth in the early phase of neoplasia, while promotes tumor progression and metastasis in later phases. Thus, many malignant cells produce large amounts of TGF-β, but are resistant to its growth inhibitory effects. TGF-β produced by tumors depresses antitumor immune responses and diminishes cancer immunotherapy. TGF-β initiates the epithelial-to-mesenchymal transition (EMT) associated specifically with tumor invasiveness and metastasis and also with the generation of fibroblasts associated with accumulation of extracellular matrix in chronic fibrotic disorders. There are several possibilities for the disruption of TGF-β signaling: 1) targeting the expression and function of TGF-β by a small interfering RNA strategy, by a neutralizing TGF-β monoclonal antibody or by decreasing the enzymatic activity of furin, a TGF-β activating protease, 2) inhibiting TGF-β receptor kinase 1, named also activin receptor-like kinase (ALK5), activity by pyridinylimidazoles (SB- 431542, SB-505124, SB-525334, A-83-01), by 2,4-disubstituted pteridine (SD-208) or by a quinazoline-derived inhibitor (SD-093) which all interact with the ATP-binding site of ALK5; 3) inhibiting of Smad 2 and Smad 3 signaling by overexpression of their physiological inhibitor (Smad 7) or by using thioredoxin as an Smad anchor disambling Smad from activation; 4) inducing an immune response by administration of TGF-β-resistant cytotoxic T-lymphocytes or by the treatment with a small-molecule ALK5 inhibitor.

The mammalian target of rapamycin (mTOR) is a downstream mediator of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway which regulates cell survival and proliferation. mTOR is a highly conserved serine/threonine protein kinase involved in the contol of cell growth, protein synthesis, and cell cycle progression. The importance of the mTOR pathway has stimulated the development of pharmacological inhibitors of this pathway. The best studied mTOR inhibitor is rapamycin (sirolimus), a natural antibiotic produced by soil bacterium Streptomyces hygroscopicus with antifungal, immunosuppressive and antipoliferative properties. Rapamycin analogues such as CCI-779 (temsirolimus), RAD001 (everolimus) and AP23573 (deforolimus) have a potent antineoplastic effect in many solid tumor models and recent data have also shown their antiproliferative properties in hematological malignancies. Therapeutic trials have been completed or are ongoing for the treatment of acute myeloid leukemia, chronic myeloid leukemia, acute and chronic lymphoid leukemia, lymphoma including mantle cell lymphoma, and multiple myeloma. With an increasing understanding of the feedback loops existing in the PI3K/Akt/mTOR pathway, it has been recognized that inhibition on one output costs at the expense of activation on the other output. Therefore, it is better to inhibit two or more components in this pathway by NVP-BEZ235 (a dual pan-PI3K/mTOR inhibitor) and SF-1126 (inhibitor of all isoforms of PI3K). S9, a novel anticancer agent, a hybrid of α-methylene-γ-lactone and 2-phenyl indole compound which interferes with both PI3K/Akt/mTOR signaling and microtubule cytoskeleton, induces rapid apoptosis and has potent antiproliferative activity in tumor cells. As PI3K inhibitors could impact insulin signaling, diabetes, obesity, and aging, unwanted side effects should be considered.

TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Recent work has shown that members of this family, including TACC2, may be involved in the progression of certain solid tumours. The aim of the current study was to identify the role of TACC2 in the TACC2-knockdown breast cancer cell lines. TACC2 expression was examined in human cancer cell lines. TACC2 was experimentally reduced expression in human breast cancer cells. The influence of TACC2 on the biological behavior of breast cancer cells was then investigated in in vitro studies. TACC2 expression showed moderate or high levels in breast cancer cell lines. The reduced expression of TACC2 significantly increased (p < 0.05) the invasive, migratory, growth and adherence properties of the human breast cancer cell lines. Furthermore, PLCγ was expressed at higher levels in TACC2 knockdown breast cancer cell lines. The increased motile pheonotype could be inhibited by the PLCγ inhibitor, which suggested TACC2 could suppress the aggressiveness of breast cancer cells through the PLCγ Pathway. This study shows that TACC2 demonstrates the ability to play a role in governing the metastatic nature of breast cancer cells. The increased motile properties could be regulated via PLCγ signaling pathway in the TACC2 knockdown breast cancer cells. This suggests that TACC2 may function as a tumor suppressor for breast cancer. The findings also suggest that TACC2 may be a potential therapeutic target.

The serine/threonine kinase Akt (PKB) is activated in response to growth factors, cytokines and other growth promoting stimuli and is involved in the regulation of a number of cellular processes including metabolism, cell growth, proliferation and survival. To study intracellular metabolic effects of left ventricular hypertrophy (LVH) induced by chronic aortic banding in a recently established swine model, protein arrays can be used to analyze changes in cytosolic protein phosphorylation levels such as cell signal transduction and protein kinase pathways. Miniaturized sandwich immunoassays allow the simultaneous analysis of several parameters in a single experiment. Bead-based protein array systems or suspension microarrays are well-established multiplex sandwich immunoassay formats. Cytosolic proteins were extracted from pig hearts. Using the bead-based Luminex system, multiplexed sandwich immunoassays have been developed to analyze the phosphorylated proteins IR[pYpY1162/1163], IGF-1R[pYpY1135 /1136], IRS-1[pS312], Akt[pS473], PRAS40[pT246], p70S6K[pTpS421/424], and GSK-3β[pS9], which were measured simultaneously in extracted myocardial tissue homogenates with the Akt Pathway Phospho 7-Plex Panel using a Luminex 100 IS system. Using this assay, tissue homogenates derived from myocardial tissue samples from 28 individual porcine in vivo model experiments in normal hearts (n=17) and LVH hearts (n=11) were analyzed for phosphorylated protein concentrations. Results obtained allowed grouping of myocardial tissue samples into a LVH and a normal group. Two candidate molecules: PRAS40 (normal vs. LVH two-tailed probability P= 0.0022) and GSK-3β (P=0.003), had lower concentrations in LVH pigs than in normal animals, suggesting loss of GSK-3β inhibitory activity and contributing to speculation of altered insulin sensitivity associated with LVH.

Dystroglycan (DG) is a ubiquitous membrane-spanning cell adhesion molecule and forms a crucial link between the extracellular matrix (ECM) and actin cytoskeleton. DG is composed of two proteins α- and β- DG which are encoded by a single gene. It was originally identified as the extracellular and transmembrane constituents of a large oligomeric complex of sarcolemmal proteins associated with dystrophin, the protein product of the Duchenne muscular dystrophy gene. Mutations of the components of this complex are associated with several forms of severe muscular dystrophies. DG is also expressed in the neuromuscular junction and non-muscle tissues such as brain, peripheral nerve, retina and kidney. With a broad expression pattern and multiple interacting proteins, DG is now thought to be important not only as a structural molecule but also as a cell signaling molecule, playing roles in cytoskeletal reorganization such as early development, myelinogenesis, synaptogenesis, epithelial morphogenesis and angiogenesis. Defective glycosylation of α-DG caused by mutations of several glycosyltransferases leads to the disruption of its linkage to the ECM. This aberrant glycosylation of α-DG is now considered as a universal pathogenic mechanism of neuromuscular diseases, especially congenital muscular dystrophies. Recently, we reported that the extracellular domain of β-DG is cleaved by matrix metalloproteinase (MMP)-2 and MMP-9, raising a possibility of therapy for muscular dystrophy by blocking MMP activity. We also demonstrated that the N-terminal domain of extracellular α-DG (α-DG-N) is cleaved by a proprotein convertase and secreted into body fluid in the physiological state. These findings imply that the cleaved α-DG fragment may play an important biological role in various tissues. Here, we review the current understanding of diverging roles of DG and its implications in therapy for neuromuscular diseases.

In order to evaluate the role of translational regulator pathways between p70 S6 kinase (S6K), S6 ribosomal protein (S6) and eIF4E binding protein 1 (4E-BP1) in the development of neurons, we analyzed their phosphorylated status in embryo, fetus and adult murine tissues, and compared neurons with the other types of cells. Phosphorylated 4EBP1 was abundantly present in all kinds of tissues during the entire course of development. S6K and S6 activities were highly maintained in the neurons from embryo to adult, while they were decreasing in the other types of cells from prenatal 17-19dpc fetus to mature adults. Assessing the differentiation in Neuro-2a neuronal and C2C12 muscle cells, we found that extracellular signal-regulated kinase (ERK) activity contributed to the conserved activities of S6K-S6, neurite growth and differentiation in the developing neurons. The translational balance between S6K-S6 and 4E-BP1 plays important roles in the developing embryo and fetus, and the highly preserved S6K-S6 activities are required for adequate development of neuronal cells and probably for glucose homeostasis in the neuron. Impaired ERK activity causes disturbances in the neurite growth and the differentiation of developing neurons, and may deregulate the control of whether neurons die or stay alive.

Cellular protein kinases (PKs) have critical roles in regulating the hallmarks of cancer (autonomous cell growth, resistance to antigrowth signals, unlimited replication, neoangiogenesis, tissue invasion and metastasis). Consequently, a strong rationale drives those therapeutic strategies aimed at targeting PKs to achieve tumour regression. Haematological malignancies have been at the forefront in this field of investigation, as major successes with the use of PKs inhibitors have been obtained in the therapy of some types of leukaemia. We will herein review the established and potential roles of three serine-threonine PKs, CK1, CK2 and GSK3 in normal and malignant haematopoiesis. These PKs regulate a multitude of cellular processes, are either over-expressed or hyper-activated in leukaemia, multiple myeloma and lymphoma cells and are essential for cell survival and resistance to apoptosis through their interaction with critical signal transduction pathways. However, the knowledge of their function in normal and malignant haematopoiesis is only at the beginning. Consequently, a more precise description of these “druggable” kinases specifically in blood cells, which is a prerequisite to rationally develop targeted therapeutic approaches, will necessarily be achieved through the use of molecular, genetic and animal experimental models.

The recognition that the BCR-ABL gene and corresponding protein with deregulated tyrosine kinase (TK) activity is crucial for malignant transformation in chronic myeloid leukemia (CML), led to the synthesis of the smallmolecule drugs designed to interfere with BCR-ABL TK activation. The first tyrosine kinase inhibitor (TKI) was imatinib mesylate, introduced into clinical practice in 1998, which became the first choice drug in chronic phase CML. However, approximately 20-25% of patients initially successfully treated with imatinib will need alternative therapy because of unsatisfactory therapeutic results due to drug resistance. The risk of resistance is even higher in patients in advanced phases of CML. Resistance to imatinib is in about 35%-45% attributed to selection of clones expressing mutant forms of BCRABL which impair imatinib binding but preserve the kinase activity. The availability of second-generation TKIs has provided a new therapeutic option for patients with imatinib resistance. Among them, dasatinib is the first to be approved in the US and European Union for CML patients resistant or intolerant to imatinib. This drug is a dual SRC/ABL kinase inhibitor, 325-fold more potent than imatinib against cells expressing wild-type BCR-ABL and active in most clinically relevant BCR-ABL mutations, except highly resistant T315I. Several clinical trials have demonstrated that dasatinib is effective and generally well tolerated in imatinib resistant or intolerant CML and represents a promising therapeutic option for these patients.

Growth Arrest Specific1 (Gas1) is a protein expressed during development and when cells arrest their growth. The potential of Gas1 as an adjuvant in the treatment of cancer, and its role as a tumor suppressor have also been proposed. We had previously demonstrated the structural relationship between Gas1 and the α receptors for the Glial-cell line-Derived Neurotrophic Factor (GDNF) family of ligands, and showed that Gas1 acts by inhibiting the intracellular signaling induced by GDNF, reducing both the pattern of autophosphorylation of Ret and the activation of AKT, thus inducing cell arrest and apoptosis. On the other hand, it has also been proposed that Gas1 positively interacts with Sonic Hedgehog (Shh) during embryonic development. In this review we will critically evaluate the data regarding the molecular mechanisms of action of Gas1, and discuss that the potential therapeutic effects of Gas1 treating cancer are related with its capacity inhibiting the intracellular signaling cascade induced by GDNF.

Many cellular processes are regulated by the reversible reaction of protein phosphorylation on serine, threonine and tyrosine residues. Deregulation of the signal transduction cascade upsets this well-balanced system and has been implicated in many diseases, including cancer. Identification of the proteins and post-translational modifications (PTMs) involved in the breakdown of certain cell signaling pathways provide unique insight into the disease's pathology. The importance of phosphorylation on a molecular level has been implicated specifically within signaling pathways involved in the pathogenesis of cancer. Emerging phosphoproteomic technologies have proven to be valuable tools in identifying new biomarkers, which presents an opportunity to probe phosphoproteins in specific cancer related pathways for both diagnosis and therapeutic exploitation. This review will discuss the development and impact of current phosphoproteomic identification and quantification methodologies and the utility of these technologies to assist in the determination of phosphoproteins as potential biomarkers and/or drug targets in cancer.