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The article discusses research which examined the role and regulation of microRNAs targeting Bruton tyrosine kinase (BTK) in the survival of acute myelogenous leukemia (AML) cells. The study showed that BTK is a relevant therapeutic target in AML cells and the key signature of microRNA that regulated BTK. The flow cytometry analysis confirmed that BTK protein expression was downregulated by each microRNA. Also, ibrutinib exposure inhibited the kinase activity of BTK in OCI-AML3 cells.

Sprouty (Spry) and Spred proteins have been identified as closely related negative regulators of the receptor tyrosine kinase (RTK)-mediated MAPK pathway, inhibiting cellular proliferation, migration and differentiation in many systems. As the different members of this antagonist family are strongly expressed in the lens epithelium in overlapping patterns, in this study we used lens epithelial explants to examine the impact of these different antagonists on the morphologic and molecular changes associated with fibroblast growth factor (FGF)-induced lens fiber differentiation. Cells in lens epithelial explants were transfected using different approaches to overexpress the different Spry (Spry1, Spry2) and Spred (Spred1, Spred2, Spred3) members, and we compared their ability to undergo FGF-induced fiber differentiation. In cells overexpressing any of the antagonists, the propensity for FGF-induced cell elongation was significantly reduced, indicative of a block to lens fiber differentiation. Of these antagonists, Spry1 and Spred2 appeared to be the most potent among their respective family members, demonstrating the greatest block in FGF-induced fiber differentiation based on the percentage of cells that failed to elongate. Consistent with the reported activity of Spry and Spred, we show that overexpression of Spry2 was able to suppress FGF-induced ERK1/2 phosphorylation in lens cells, as well as the ERK1/2-dependent fiber-specific marker Prox1, but not the accumulation of β-crystallins. Taken together, Spry and Spred proteins that are predominantly expressed in the lens epithelium in situ, appear to have overlapping effects on negatively regulating ERK1/2-signaling associated with FGF-induced lens epithelial cell elongation leading to fiber differentiation. This highlights the important regulatory role for these RTK antagonists in establishing and maintaining the distinct architecture and polarity of the lens. [ABSTRACT FROM AUTHOR]

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+- permeable channel of the transient receptor potential (TRP) superfamily activated by diverse stimuli, including warm temperature, mechanical forces and lipid mediators such as arachidonic acid (AA) and its metabolites. This activation is tightly regulated by protein phosphorylation carried out by various serine/ threonine or tyrosine kinases. It remains poorly understood how phosphorylation differentially regulates TRPV4 activation in response to different stimuli. We investigated how TRPV4 activation by AA, an important signaling process in the dilation of coronary arterioles, is affected by protein kinase A (PKA)- mediated phosphorylation at Ser-824. Wildtype and mutant TRPV4 channels were expressed in human coronary artery endothelial cells (HCAECs). AA-induced TRPV4 activation was blunted in the S824A mutant but was enhanced in the phosphomimetic S824E mutant, whereas the channel activation by the synthetic agonist GSK1016790A was not affected. The low level of basal phosphorylation at Ser-824 was robustly increased by the redox signaling molecule hydrogen peroxide (H2O2). The H2O2-induced phosphorylation was accompanied by an enhanced channel activation by AA and this enhanced response was largely abolished by PKA inhibition or S824A mutation.We further identified a potential structural context dependence of Ser-824 phosphorylation-mediated TRPV4 regulation involving an interplay betweenAAbinding and the possible phosphorylation- induced rearrangements of the C-terminal helix bearing Ser-824. These results provide insight into how phosphorylation specifically regulates TRPV4 activation. Redox-mediated TRPV4 phosphorylation may contribute to pathologies associated with enhanced TRPV4 activity in endothelial and other systems. [ABSTRACT FROM AUTHOR]

The neurotrophic factors neuregulins (NRGs) and their receptors, ErbB tyrosine kinases, regulate neurotransmission, synaptic plasticity and cognitive functions and their alterations have been associated to different neuropsychiatric disorders. Group 1 metabotropic glutamate receptors (mGluRI)-dependent mechanisms are also altered in animal models of neuropsychiatric diseases, especially mGluRI-induced glutamatergic long-term depression (mGluRI-LTD), a form of synaptic plasticity critically involved in learning and memory. Despite this evidence, a potential link between NRGs/ErbB signalling and mGluRI-LTD has never been considered. Here, we aimed to test the hypothesis that NRGs/ErbB signalling regulates mGluRI functions in the hippocampus, thus controlling CA1 pyramidal neurons excitability and synaptic plasticity as well as mGluRI-dependent behaviors. We investigated the functional interaction between NRG1/ErbB signalling and mGluRI in hippocampal CA1 pyramidal neurons, by analyzing the effect of a pharmacological modulation of NRG1/ErbB signalling on the excitation of pyramidal neurons and on the LTD at CA3-CA1 synapses induced by an mGluRI agonist. Furthermore, we verified the involvement of ErbB signalling in mGluRI-dependent learning processes, by evaluating the consequence of an intrahippocampal in vivo injection of a pan-ErbB inhibitor in the object recognition test in mice, a learning task dependent on hippocampal mGluRI. We found that NRG1 potentiates mGluRI-dependent functions on pyramidal neurons excitability and synaptic plasticity at CA3-CA1 synapses. Further, endogenous ErbB signalling per se regulates, through mGluRI, neuronal excitability and LTD in CA1 pyramidal neurons, since ErbB inhibition reduces mGluRI-induced neuronal excitation and mGluRI-LTD. In vivo intrahippocampal injection of the ErbB inhibitor, PD158780, impairs mGluRI-LTD at CA3-CA1 synapses and affects the exploratory behavior in the object recognition test. Thus, our results identify a key role for NRG1/ErbB signalling in the regulation of hippocampal mGluRI-dependent synaptic and cognitive functions, whose alteration might contribute to the pathogenesis of different brain diseases. [ABSTRACT FROM AUTHOR]

PTPRB is a transmembrane protein tyrosine phosphatase known to regulate blood vessel remodelling and angiogenesis. Here, we demonstrate that PTPRB negatively regulates branching morphogenesis in the mouse mammary epithelium. We show that Ptprb is highly expressed in adult mammary stem cells and also, although at lower levels, in oestrogen receptor-positive luminal cells. During mammary development, Ptprb expression is downregulated during puberty, a period of extensive ductal outgrowth and branching. In vivo shRNA knockdown of Ptprb in the cleared mammary fat pad transplant assay resulted in smaller epithelial outgrowths with an increased branching density and also increased branching in an in vitro organoid assay. Organoid branching was dependent on stimulation by FGF2, and Ptprb knockdown in mammary epithelial cells resulted in a higher level of fibroblast growth factor receptor (FGFR) activation and ERK1/2 phosphorylation, both at baseline and following FGF2 stimulation. Therefore, PTPRB regulates branching morphogenesis in the mammary epithelium by modulating the response of the FGFR signalling pathway to FGF stimulation. Considering the importance of branching morphogenesis in multiple taxa, our findings have general importance outside mammary developmental biology. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinase (RTK) signaling cascades coordinate intracellular signaling in response to growth factors, chemokines, and other extracellular stimuli to control fundamental biological processes such as cellular proliferation, metabolism, and survival. Hyperactivation of pathways associated with growth factor signaling (e.g., RTK and downstream effectors including Ras, PI3K/AKT, and Raf) is a frequent event in human cancers, which uncouples ligand-mediated activation with signal transduction. While the contributions of direct genomic events are well understood as causative agents of hyperactive signal transduction, other non-heritable genomic modifications promote the activation of growth factor-associated signaling cascades. In this review, we highlight epigenomic mechanisms by which hyperactivation of RTK-associated signaling cascades occurs and may contribute to cancer. [ABSTRACT FROM AUTHOR]

Drug resistance is an obstacle in the treatment of chronic myelogenous leukemia (CML), and is a common reason for treatment failure or disease progression. However, the underlying mechanisms of cyclophosphamide resistance remain poorly defined. In the present study, microarray data concerning cyclophosphamide-sensitive and -resistant chronic myelogenous leukemia cell lines were analyzed. A total of 258 differentially-expressed genes (DEGs) were identified between these two groups, from which 139 DEGs were upregulated and 119 were downregulated. Several candidate genes that were associated with cyclophosphamide resistance were also identified. These DEGs were subsequently classified using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis. A total of 487 biological processes and 17 KEGG pathways were revealed to be enriched. Furthermore, an interaction network was established to identify the core genes that regulated cyclophosphamide resistance. Signal transducer and activator of transcription 5A (STAT5A), FYN proto-oncogene, Src family tyrosine kinase and spleen associated tyrosine kinase were revealed to be the hub genes in multiple enriched biological processes and signaling pathways, indicating that these were involved in mediating cyclophosphamide sensitivity in CML cells. The expression levels of 5 DEGs were also confirmed in two human CML cell lines (K-562 and KU812) by reverse transcription-quantitative polymerase chain reaction. Furthermore, selective knockdown of STAT5A and S100 calcium binding protein A4 (S100A4) recovered cyclophosphamide sensitivity in K-562 cells, suggesting their involvement in drug resistance. The present study identified several potential genes and pathways contributing to cyclophosphamide resistance, and confirmed the involvement of STAT5A and S100A4 in drug resistance. These results enable improved understanding of the mechanisms underlying drug resistance in CML cells. [ABSTRACT FROM AUTHOR]

Inhibitors of the receptor tyrosine kinase (RTK) MET have been ineffective at treating cancer, possibly because of lack of knowledge that would allow selection of tumors likely to respond to this treatment. In contrast, specific epidermal growth factor receptor (EGFR) inhibitors have been used successfully against lung tumors displaying activating mutations in the kinase domain of EGFR. Recent publications describe a set of mutations causing MET exon 14 skipping, and importantly, several case reports describe objective responses to MET-targeting tyrosine kinase inhibitors in patients with such mutations. These observations suggest a novel therapeutic strategy for fighting cancer, especially in the lung. Exon 14 encodes the MET juxtamembrane domain targeted by mechanisms that negatively regulate receptor stability and activity. In this review, we describe the molecular mechanisms leading first to exon 14 skipping and then to activation of the MET receptor and how this process differs from that triggered by classical RTK-activating mutations in the kinase domain. We detail the clinical characteristics of patients carrying these mutations and the sensitivity of their tumors to MET inhibitors. Lastly, we discuss future challenges related to MET mutations in cancers, including patient screening and anticipating resistance to MET inhibitors. [ABSTRACT FROM AUTHOR]

In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9 H -pyrimido[4,5- b ]indole-2,4-diamines 3 – 7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9 H -pyrimido[4,5- b ]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3 , 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed. [ABSTRACT FROM AUTHOR]

The structural mechanisms by which receptor tyrosine kinases (RTKs) regulate catalytic activity are diverse and often based on subtle changes in conformational dynamics. The regulatory mechanism of one such RTK, fibroblast growth factor receptor 2 (FGFR2) kinase, is still unknown, as the numerous crystal structures of the unphosphorylated and phosphorylated forms of the kinase domains show no apparent structural change that could explain how phosphorylation could enable catalytic activity. In this study, we use several enhanced sampling molecular dynamics (MD) methods to elucidate the structural changes to the kinase’s activation loop that occur upon phosphorylation. We show that phosphorylation favors inward motion of Arg664, while simultaneously favoring outward motion of Leu665 and Pro666. The latter structural change enables the substrate to bind leading to its resultant phosphorylation. Inward motion of Arg664 allows it to interact with the γ-phosphate of ATP as well as the substrate tyrosine. We show that this stabilizes the tyrosine and primes it for the catalytic phosphotransfer, and it may lower the activation barrier of the phosphotransfer reaction. Our work demonstrates the value of including dynamic information gleaned from computer simulation in deciphering RTK regulatory function. [ABSTRACT FROM AUTHOR]

The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2Y393F) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes. [ABSTRACT FROM AUTHOR]

Therapeutic angiogenesis has been implicated in ischemic diseases and wound healing. Ginsenoside-Rg 1 (Rg 1 ), one of the most abundant active components of ginseng, has been demonstrated as an angiogenesis-stimulating compound in different models. There is increasing evidence implicating microRNAs (miRNAs), a group of non-coding RNAs, as important regulators of angiogenesis, but the role of microRNAs in Rg 1 -induced angiogenesis has not been fully explored. In this report, we found that stimulating endothelial cells with Rg 1 could reduce miR-23a expression. In silico experiments predicted hepatocyte growth factor receptor (MET), a well-established mediator of angiogenesis, as the target of miR-23a. Transfection of the miR-23a precursor or inhibitor oligonucleotides validated the inverse relationship of miR-23a and MET expression. Luciferase reporter assays further confirmed the interaction between miR-23a and the MET mRNA 3′-UTR. Intriguingly, ginsenoside-Rg 1 was found to increase MET protein expression in a time-dependent manner. We further demonstrated that ginsenoside-Rg 1 -induced angiogenic activities were indeed mediated through the down-regulation of miR-23a and subsequent up-regulation of MET protein expression, as confirmed by gain- and loss-of-function angiogenic experiments. In summary, our results demonstrated that ginsenoside-Rg 1 could induce angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a. This study has broadened our understanding of the non-genomic effects of ginsenoside-Rg 1, and provided molecular evidence that warrant further development of natural compound as novel angiogenesis-promoting therapy. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 2A (LMP2A) is an EBV latency-associated protein regularly expressed in nasopharyngeal carcinoma (NPC). In B cells, LMP2A activity resembles that of a constitutively activated antigen receptor, which recruits the Syk tyrosine kinase to activate a set of downstream signaling pathways. LMP2A also downregulates cellular Syk levels. In the present study, we demonstrate that Syk interacts with the integrin β4 subunit (ITGβ4) of integrin α6β4 in epithelial cells and that concurrent LMP2A expression interferes with this interaction by competitive binding to Syk. We find that both Syk and LMP2A have an effect on ITGβ4 cell surface expression. However, in LMP2A expressing cells, ITGβ4 remains concentrated at the cellular protrusions, an expression pattern characteristic of motile cells, including NPC-derived epithelial cells. This effect of LMP2A on ITGβ4 localization is associated with a greater propensity for migration and invasion in-vitro, and may contribute to the invasive property of LMP2A-expressing NPC. [ABSTRACT FROM AUTHOR]

Background: Focal adhesion kinase (FAK) mediates survival of normal pancreatic islets through activation of AKT. Upon malignant transformation of islet cells into pancreatic neuroendocrine tumors (PanNETs), AKT is frequently overexpressed and mutations in the AKT/mTOR pathway are detected. Because mTOR inhibitors rarely induce PanNET tumor regression, partly because of feedback activation of AKT, novel combination strategies are needed to target FAK/AKT/mTOR signaling. Methods: We characterized the activation of FAK in PanNETs using immunohistochemistry and Western blot analysis and tested the FAK inhibitor PF-04554878 in human PanNET cells in vitro and in vivo (at least three mice per group). In addition, we evaluated the effect of combined FAK and mTOR inhibition on PanNET viability and apoptosis. All statistical tests were two-sided. Results: We found that FAK is overexpressed and hyperphosphorylated in human PanNETs and that PF-04554878 strongly inhibited FAK (Tyr397) autophosphorylation in a dose-dependent manner. We found that PF-04554878 inhibited cell proliferation and clonogenicity and induced apoptosis in PanNET cells. Moreover, oral administration of PF-04554878 statistically significantly reduced tumor growth in a patient-derived xenograft model of PanNET (P = .02) and in a human PanNET xenograft model of peritoneal carcinomatosis (P = .03). Importantly, PF-04554878 synergized with the mTOR inhibitor everolimus by preventing feedback AKT activation. Conclusions: We demonstrate for the first time that FAK is overexpressed in PanNETs and that inhibition of FAK activity induces apoptosis and inhibits PanNET proliferation. We found that the novel FAK inhibitor PF-04554878 synergizes with everolimus, a US Food and Drug Administration-approved agent for PanNETs. Our findings warrant the clinical investigation of combined FAK and mTOR inhibition in PanNETs. [ABSTRACT FROM AUTHOR]

Neurons are particularly vulnerable to perturbations in endo-lysosomal transport, as several neurological disorders are caused by a primary deficit in this pathway. In this report, we used positional cloning to show that the spontaneously occurring neurological mutation teetering (tn) is a single nucleotide substitution in hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). The tn mice exhibit hypokenesis, muscle weakness, reduced muscle size and early perinatal lethality by 5-weeks of age. Although HGS has been suggested to be essential for the sorting of ubiquitinated membrane proteins to the lysosome, there were no alterations in receptor tyrosine kinase levels in the central nervous system, and only a modest decrease in tropomyosin receptor kinase B (TrkB) in the sciatic nerves of the tn mice. Instead, loss of HGS resulted in structural alterations at the neuromuscular junction (NMJ), including swellings and ultra-terminal sprouting at motor axon terminals and an increase in the number of endosomes and multivesicular bodies. These structural changes were accompanied by a reduction in spontaneous and evoked release of acetylcholine, indicating a deficit in neurotransmitter release at the NMJ. These deficits in synaptic transmission were associated with elevated levels of ubiquitinated proteins in the synaptosome fraction. In addition to the deficits in neuronal function, mutation of Hgs resulted in both hypermyelinated and dysmyelinated axons in the tn mice, which supports a growing body of evidence that ESCRTs are required for proper myelination of peripheral nerves. Our results indicate that HGS has multiple roles in the nervous system and demonstrate a previously unanticipated requirement for ESCRTs in the maintenance of synaptic transmission. [ABSTRACT FROM AUTHOR]

Aims The present study showed that the expression of PTPN12 is epigenetically regulated. 5-Azacytidine (5-Azac), a DNA hypomethylating agent, significantly increased the expression of PTPN12 at low concentrations (1 μM and 2.5 μM) and decreased the expression of PTPN12 at 5 μM in the MDA-MB-231 and BT-549 triple-negative breast cancer cell lines. Main methods Human MCF-7, MDA-MB-231 and BT-549 cells were exposed to different concentrations of 5-Azac for 24 and 48 h. RT-PCR was performed to determine the mRNA expression of PTPN12, E-cadherin and miRNA-124. Western blotting was performed to assess the protein expression of various proteins, including PTPN12, E-cadherin, DNMT and PARP. Key findings 5-Azac, a DNA hypomethylating agent, significantly increased the expression of PTPN12 at low concentrations (1 μM and 2.5 μM) and decreased PTPN12 expression at 5 μM. We provide the first evidence that PTPN12 expression is epigenetically regulated and that it is up-regulated at a lower dose of a DNMT1 inhibitor in MDA-MB-231 and BT-549 cells. Interestingly, the levels of miRNA-124 were increased only at 5 μM, the concentration at which PTPN12 expression was suppressed. Significance To the best of our knowledge, this is the first report that highlights the therapeutic potential of low-dose 5-Azac for the treatment of TNBC. Therefore, 5-Azac, an agent that has already been tested in acute myeloid leukemia, may be more effective at lower doses for the treatment of triple-negative breast cancer. [ABSTRACT FROM AUTHOR]

In the foreign body response, infiltrating PMNs exocytose granule subsets to influence subsequent downstream inflammatory and wound healing events. In previous studies, we found that PMNs cultured on poly(ethylene glycol) (PEG)-containing hydrogels (i.e., PEG and gelatin + PEG hydrogels) had enhanced primary granule release, yet similar tertiary granule release compared with PMNs cultured on polydimethylsiloxane or tissue culture polystyrene. PMN primary granules contain microbicidal proteins and proteases, which can potentially injure bystander cells, degrade the extracellular matrix, and promote inflammation. Here, we sought to understand the mechanism of the enhanced primary granule release from PMNs on PEG hydrogels. We found that primary granule release from PMNs on PEG hydrogels was adhesion mediated and involved Src family kinases and PI3K-γ. The addition of gelatin to PEG hydrogels did not further enhance PMN primary granule release. Using stable-isotope dimethyl labeling-based shotgun proteomics, we identified many serum proteins – including Ig gamma constant chain region proteins and alpha-1-acid glycoprotein 1 – that were absorbed/adsorbed in higher quantities on PEG hydrogels than on TCPS, and may be involved in mediating PMN primary granule release. Ultimately, this mechanistic knowledge can be used to direct inflammation and wound healing following biomaterial implantation to promote a more favorable healing response. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR) is a member of the transmembrane receptor tyrosine kinase family. In normal and malignant cells, activation of the EGFR cascade is involved in the regulation of various cellular activities. The objective of this study was to identify and assess associations of positive EGFR expression and K-RAS mutations with various clinicopathological parameters and survival of colorectal carcinoma patients. EGFR of colorectal cancer (CRC) tissue specimens was subjected to immunohistochemical analysis, polymerase chain reaction, and DNA sequencing. Immunohistochemical staining was performed using monoclonal antibodies against EGFR antigens and examination of mutations was performed to detect mutations in codons 12 and 13 of the K-RAS . Statistical analysis was performed using SPSS version 16.0. The results of this study showed that of the 40 study participants, 62.5% (25/40) showed positive EGFR overexpression. Of the patients showing positive EGFR expressions, 52% had mutations in the K-RAS . Mutations were spread in codon 12 (64.3%) and codon 13 (35.7%) and there was one sample with mutations in codons 12 and 13 at the same time. A statistically significant association was found between the presence of metastasis and EGFR overexpression and survival of CRC patients. In addition, a significant association was found between K-RAS mutations and metastasis and survival of CRC patients. In conclusion, EGFR overexpression and K-RAS mutations were found in CRC patients. Both factors are known to be associated with poor prognosis of cancer patients in terms of patient survival. Early detection of K-RAS mutations in CRC patients is a crucial component in the determination of the type of therapy and treatment for the patient. [ABSTRACT FROM AUTHOR]

The Hippo signaling pathway is involved in regulating tissue size by inhibiting cell proliferation and promoting apoptosis. Aberrant Hippo pathway function is often detected in human cancers and correlates with poor prognosis. The Drosophila C-terminal Src kinase ( d-Csk ) is a genetic modifier of warts ( wts ), a tumor-suppressor gene in the Hippo pathway, and interacts with the Src oncogene. Reduction in d-Csk expression and the consequent activation of Src are frequently seen in several cancers including hepatocellular and colorectal tumors. Previous studies show that d-Csk regulates cell proliferation and tissue size during development. Given the similarity in the loss-of-function phenotypes of d-Csk and wts , we have investigated the interactions of d-Csk with the Hippo pathway. Here we present multiple lines of evidence suggesting that d-Csk regulates growth via the Hippo signaling pathway. We show that loss of dCsk caused increased Yki activity, and our genetic epistasis places dCsk downstream of Dachs. Furthermore, dCsk requires Yki for its growth regulatory functions, suggesting that dCsk is another upstream member of the network of genes that interact to regulate Wts and its effector Yki in the Hippo signaling pathway. [ABSTRACT FROM AUTHOR]

C1-Ten is a member of the tensin family of focal adhesion molecules but recent studies suggest it plays a more active role in many biological processes because of its potential association with diabetes and cancers. However, relatively little is known about the regulation of C1-Ten, such as changes in its protein level or cellular localization. The cellular localization of C1-Ten is unique because it is expressed in cytoplasmic puncta but nothing is known about these puncta. Here, we show that p62 sequestrates C1-Ten into puncta, making C1-Ten diffuse into the cytoplasm upon p62 depletion. More importantly, p62-mediated C1-Ten sequestration promoted C1-Ten ubiquitination and proteasomal degradation. p62-mediated protein reduction was specific to C1-Ten, and not other tensins such as tensin1 and tensin3. Thus, our results link cellular localization of C1-Ten to an off-switch site for C1-Ten. Additionally, p62 expression increased but C1-Ten protein decreased during muscle differentiation, supporting a role for p62 as a physiological regulator of C1-Ten. [ABSTRACT FROM AUTHOR]

Sensing of nucleic acids by TLRs is crucial in the host defense against viruses and bacteria. Unc-93 hontolog B1 (UNC93B1) regulates the trafficking of nucleic acid-sensing TLRs from the endoplasmic reticulum to endolysosomes, where the TLRs encounter their respective ligands and become activated. In this article, we show that a carboxyl-terminal tyrosine-based sorting motif (YxxΦ) in UNC93B1 differentially regulates human nucleic acid-sensing TLRs in a receptor- and ligandspecific manner. Destruction of YxxΦ abolished TLR7, TLR8, and TLR9 activity toward nucleic acids in human B cells and monocytes, whereas TLR8 responses toward small molecules remained intact. YxxΦ in UNC93B1 influenced the subcellular localization of human UNC93B1 via both adapter protein complex (AP)l- and AP2-dependent trafficking pathways. However, loss of AP function was not causal for altered TLR responses, suggesting AP-independent functions of YxxΦ in UNC93B1. [ABSTRACT FROM AUTHOR]

The activation of receptor tyrosine kinases in response to extracellular signals is a principal component of metazoan signaling. Structural analysis of the extracellular and intracellular domains of these receptors has shed substantial light on the mechanisms underlying their activation. A remaining challenge is to understand how these domains operate together in the context of the full-length receptors. With a focus on the epidermal growth factor (EGF) receptor, this review highlights recent advances towards this goal. Although receptor tyrosine kinases are divergent in terms of the details of how they operate, these studies reveal common mechanisms that ensure activation in the proper context. Understanding these mechanisms provides insights into the vulnerabilities of these receptors to disease-causing mutations. [ABSTRACT FROM AUTHOR]

Tumor cells secrete factors that modulate macrophage activation and polarization into M2 type tumor-associated macrophages, which promote tumor growth, progression, and metastasis. The mechanisms that mediate this polarization are not clear. Macrophages are phagocytic cells that participate in the clearance of apoptotic cells, a process known as efferocytosis. Milk fat globule- EGF factor 8 (MFG-E8) is a bridge protein that facilitates efferocytosis and is associated with suppression of proinflammatory responses. This study investigated the hypothesis that MFG-E8-mediated efferocytosis promotes M2 polarization. Tissue and serum exosomes from prostate cancer patients presented higher levels of MFG-E8 compared with controls, a novel finding in human prostate cancer. Coculture of macrophages with apoptotic cancer cells increased efferocytosis, elevated MFG-E8 protein expression levels, and induced macrophage polarization into an alternatively activated M2 phenotype. Administration of antibody against MFG-E8 significantly attenuated the increase in M2 polarization. Inhibition of STAT3 phosphorylation using the inhibitor Stattic decreased efferocytosis and M2 macrophage polarization in vitro, with a correlating increase in SOCS3 protein expression. Moreover, MFG-E8 knockdown tumor cells cultured with wild-type or MFG-E8-deficient macrophages resulted in increased SOCS3 expression with decreased STAT3 activation. This suggests that SOCS3and phospho-STAT3 act in an inversely dependent manner when stimulated by MFG-E8 and efferocytosis. These results uncover a unique role of efferocytosis via MFG-E8 as a mechanism for macrophage polarization into tumor-promoting M2 cells. [ABSTRACT FROM AUTHOR]

The Met receptor tyrosine kinase is an attractive target for cancer therapy as it promotes invasive tumor growth. SAIT301 is a novel anti-Met antibody, which induces LRIG1-mediated Met degradation and inhibits tumor growth. However, detailed downstream mechanism by which LRIG1 mediates target protein down-regulation is unknown. In the present study, we discovered that SAIT301 induces ubiquitination of LRIG1, which in turn promotes recruitment of Met and LRIG1 complex to the lysosome through its interaction with Hrs, resulting in concomitant degradation of both LRIG1 and Met.Wealso identified USP8 as a LRIG1-specific deubiquitinating enzyme, reporting the interaction between USP8 and LRIG1 for the first time. SAIT301 triggers degradation of LRIG1 by inhibiting the interaction of LRIG1 and USP8, which regulates ubiquitin modification and stability of RIG. In summary, SAIT301 employs ubiquitination of LRIG1 for its highly effective Met degradation. This unique feature of SAIT301 enables it to function as a fully antagonistic antibody without Met activation. We found that USP8 is involved in deubiquitination of LRIG1, influencing the efficiency of Met degradation. The relation of Met, LRIG1 and USP8 strongly supports the potential clinical benefit of a combination treatment of a USP8 inhibitor and a Met inhibitor, such as SAIT301. [ABSTRACT FROM AUTHOR]

Objective: To test the hypothesis that the risk of preeclampsia in nulliparous women may be due to an anti-angiogenic state. Methods: Maternal serum samples obtained in the third trimester from nulliparous ( n = 86) and multiparous ( n = 165) singleton uncomplicated pregnancies were analyzed for levels of angiogenic factors - soluble fms like tyrosine kinase 1 (sFlt1) and placental growth factor (PlGF) by enzyme-linked immunosorbent assay (ELISA). Results: For nulliparous and multiparous pregnancies, serum sFlt1 levels were 12 732 ± 832 and 10 162 ± 666 ( p = 0.020), serum PlGF levels were 215 ± 15 and 249 ± 14 ( p = 0.093) (all reported as mean SD in pg/ml) and mean ratios of sFlt1/PlGF were 93 ± 12 and 62 ± 5 ( p = 0.023), respectively. Adjustment for maternal age and fetal birth weight did not alter the results. Conclusions: Nulliparous pregnancies had higher circulating sFlt1 levels and sFlt1/PlGF ratios than multiparous pregnancies, suggesting an association with an angiogenic imbalance. Taken together with the pathogenic role of anti-angiogenic factors in preeclampsia, our data may be one explanation for the epidemiological observation that nulliparity is a risk factor for the development of preeclampsia. [ABSTRACT FROM AUTHOR]

We have previously identified 1 241 regions of somatic copy number alterations (CNAs) in hepatocellular carcinoma (HCC). In the present study, we found that a novel recurrent focal amplicon, 1q24.1-24.2, targets the MPZL1 gene in HCC. Notably, there is a positive correlation between the expression levels of MPZL1 and intrahepatic metastasis of the HCC specimens. MPZL1 can significantly enhance the migratory and metastatic potential of the HCC cells. Moreover, we found that one of the mechanisms by which MPZL1 promotes HCC cell migration is by inducing the phosphorylation and activation of the pro-metastatic protein, cortactin. Additionally, we found that Src kinase mediates the phosphorylation and activation of cortactin induced by MPZL1 overexpression. Taken together, these findings suggest that MPZL1 is a novel pro-metastatic gene targeted by a recurrent region of copy number amplification at 1q24.1-24.2 in HCC. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinases (RTKs) regulate a wide range of important biological activities, including cell proliferation, differentiation, migration, and apoptosis. Abnormalities in RTKs are involved in numerous diseases, including cancer and other proliferative disorders. AXL belongs to the TAM (Tyso3, AXL, and Mer) family of RTKs. The AXL signaling pathway represents an attractive target for the treatment of diseases, such as cancer. Using phospho-AKT as readout, a high-throughput 384-well cell-based assay was established in the NCI-H1299 human non-small cell lung carcinoma cell line to evaluate compound potency in inhibiting AXL pathway activation. In addition, a counter screen assay was established in the same cellular background to differentiate AXL kinase inhibitors from AXL receptor antagonists, which block the interaction of AXL and its natural ligand GAS6. These cell-based functional assays are useful tools in the identification and optimization of small molecules and biological reagents for potential therapeutics for the treatment of GAS6/AXL-related diseases. [ABSTRACT FROM AUTHOR]

H2O2 produced by extracellular NADPH oxidases regulates tyrosine kinase signaling inhibiting phosphatases. How does it cross the membrane to reach its cytosolic targets? Silencing aquaporin-8 ( AQP8), but not AQP3 or AQP4, inhibited H2O2 entry into HeLa cells. Re-expression of AQP8 with silencing-resistant vectors rescued H2O2 transport, whereas a C173A-AQP8 mutant failed to do so. Lowering AQP8 levels affected H2O2 entry into the endoplasmic reticulum, but not into mitochondria. AQP8 silencing also inhibited the H2O2 spikes and phosphorylation of downstream proteins induced by epidermal growth factor. These observations lead to the hypothesis that H2O2 does not freely diffuse across the plasma membrane and AQP8 and other H2O2 transporters are potential targets for manipulating key signaling pathways in cancer and degenerative diseases. Antioxid. Redox Signal. 19, 1447-1451. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the expression and significance of a disintegrin and metalloproteinase 10 (ADAM10), epidermal growth factor receptor (EGFR) and E-cadherin protein in hepatocellular carcinomas. The expression levels of ADAM10, EGFR and E-cadherin were analyzed in 40 cases of hepatocellular carcinoma using immunohistochemistry and quantitative polymerase chain reaction (qPCR). The expression levels of ADAM10, EGFR and E-cadherin were significantly correlated with portal vein thrombosis, intrahepatic metastasis, differentiation degree and tumor size (P<0.05). In hepatocellular carcinoma, the expression levels of ADAM10 and EGFR were increased and the levels of E-cadherin were decreased compared with those in the adjacent tissues. The elevated expression levels of ADAM10 and EGFR may be associated with the malignancy of the tumors. E-cadherin expression is negatively correlated with the degree of malignancy. The detection of ADAM10, EGFR and E-cadherin expression levels may contribute to an understanding of the oncogenesis and development of hepatocellular carcinomas. [ABSTRACT FROM AUTHOR]

Mesenchymal-epithelial transition (MET) is a member of a distinct subfamily of heterodimeric receptor tyrosine kinase receptors that specifically binds the hepatocyte growth factor (HGF). Binding to HGF leads to receptor dimerization/multimerization and phosphorylation, resulting in its catalytic activation. MET activation drives the malignant progression of several tumor types, including colorectal cancer (CRC), by promoting signaling cascades that mainly result in alterations of cell motility, survival, and proliferation. MET is aberrantly activated in many human cancers through various mechanisms, including point mutations, gene amplification, transcriptional up-regulation, or ligand autocrine loops. MET promotes cell scattering, invasion, and protection from apoptosis, thereby acting as an adjuvant pro-metastatic gene for many tumor types. In CRC, MET expression confers more aggressiveness and worse clinical prognosis. With all of this rationale, inhibitors that target the HGF/MET axis with different types of response have been developed. HGF and MET are new promising targets to understand the pathogenesis of CRC and for the development of new, targeted therapies. [ABSTRACT FROM AUTHOR]

The discovery of the highly prevalent activating JAK (Janus kinase) 2 V617F mutation in myeloproliferative neoplasms, and of other pseudokinase domain-activating mutations in JAK2, JAK1 and JAK3 in blood cancers, prompted great interest in understanding how pseudokinase domains regulate kinase domains in JAKs. Recent functional and mutagenesis studies identified residues required for the V617F mutation to induce activation. Several X-ray crystal structures of either kinase or pseudokinase domains including the V617F mutant of JAK2 pseudokinase domains are now available, and a picture has emerged whereby the V617F mutation induces a defined conformational change around helix C of JH (JAK homology) 2. Effects of mutations on JAK2 can be extrapolated to JAK1 and TYK2 (tyrosine kinase 2), whereas JAK3 appears to be different. More structural information of the full-length JAK coupled to cytokine receptors might be required in order to define the structural basis of JH1 activation by JH2 mutants and eventually obtain mutant-specific inhibitors. [ABSTRACT FROM AUTHOR]

JAKs (Janus kinases) are essential mediators of almost all biological signalling events initiated by haemopoietic and immune cytokines. However, aberrant and/or prolonged JAK-induced signalling is detrimental and can give rise to a number of inflammatory and proliferative pathologies. For this reason, the tyrosine kinase activity of the JAKs is carefully regulated at a number of different levels. Primarily, this is achieved by: (i) ensuring that the catalytic domain is 'switched off' under basal conditions, and (ii) inhibiting the activity of JAK after it has been switched on. Whereas the first mode of inhibition is mediated by JAK's own pseudokinase domain as well as the action of phosphatases, the second is achieved by the action of the SOCS (suppressor of cytokine signalling) proteins, negative-feedback inhibitors of JAK-mediated signalling. The present review focuses on the mode of action of SOCS1 and SOCS3, the two most potent JAK inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is considered as an attractive target for small molecule drug discovery. In this study, a series of indazoles were designed, synthesized and evaluated as novel c-Met inhibitors. The results showed that the majority of the compounds exhibited significant inhibition on c-Met and compound 4d showed highest activity against c-Met with IC50 value of 0.17 μM in TR-FRET-based assay and IC50 value of 5.45 μM in cell-based assay as compared to other tested compounds. Molecular docking experiments verified the results and explained the molecular mechanism of pretty activities to c-Met. [Copyright &y& Elsevier]

Metastasis is responsible for 90% of cancer-related deaths. Strategies are needed that can inhibit the capacity of cancer cells to migrate across the anatomic barriers and colonize distant organs. Here, we show an association between metastasis and expression of a type I receptor tyrosine kinase-like orphan receptor, ROR1, which is expressed during embryogenesis and by various cancers, but not by normal postpartum tissues. We found that expression of ROR1 associates with the epithelial-mesenchymal transition (EMT), which occurs during embryogenesis and cancer metastasis. Breast adenocarcinomas expressing high levels of ROR1 were more likely to have gene expression signatures associated with EMT and had higher rates of relapse and metastasis than breast adenocarcinomas expressing low levels of ROR1. Suppressing expression of ROR1 in metastasis-prone breast cancer cell lines, MDA-MB-231, HS-578T, or BT549, attenuated expression of proteins associated with EMT (e.g., vimentin, SNAIL-1/2, and ZEB1), enhanced expression of E-cadherin, epithelial cytokeratins (e.g., CK-19), and tight junction proteins (e.g., ZO-1), and impaired their migration/invasion capacity in vitro and the metastatic potential of MDA-MB-231 cells in immunodeficient mice. Conversely, transfection of MCF-7 cells to express ROR1 reduced expression of E-cadherin and CK-19, but enhanced the expression of SNAIL-1/2 and vimentin. Treatment of MDA-MB-231 with a monoclonal antibody specific for ROR1 induced downmodulation of vimentin and inhibited cancer cell migration and invasion in vitro and tumor metastasis in vivo. Collectively, this study indicates that ROR1 may regulate EMT and metastasis and that antibodies targeting ROR1 can inhibit cancer progression and metastasis. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinases (RTKs) exist in equilibrium between tyrosyl-phosphorylated and dephosphorylated states. Despite a detailed understanding of how RTKs become tyrosyl phosphorylated, much less is known about RTK tyrosyl dephosphorylation. Receptor protein tyrosine phosphatases (RPTPs) can play essential roles in the dephosphorylation of RTKs. However, a complete understanding of the involvement of the RPTP subfamily in RTK tyrosyl dephosphorylation has not been established. In this study, we have employed a small interfering RNA (siRNA) screen to identify RPTPs in the human genome that serve as RTK phosphatases. We observed that each RPTP induced a unique fingerprint of tyrosyl phosphorylation among 42 RTKs. We identified EphA2 as a novel LAR substrate. LAR dephosphorylated EphA2 at phosphotyrosyl 930, uncoupling Nck1 from EphA2 and thereby attenuating EphA2-mediated cell migration. These results demonstrate that each RPTP exerts a unique regulatory fingerprint of RTK tyrosyl dephosphorylation and suggest a complex signaling interplay between RTKs and RPTPs. Furthermore, we observed that LAR modulates cell migration through EphA2 site-specific dephosphorylation. [ABSTRACT FROM AUTHOR]

Deregulation of the receptor tyrosine kinase Axl has been implicated in the progression of several human cancers. However, the role of Axl in prostate cancer remains poorly understood, and the therapeutic efficacy of Axl targeting remains untested. In this report we identified Axl as a new therapeutic target for prostate cancer. Axl is consistently overexpressed in prostate cancer cell lines and human prostate tumors. Interestingly, the blockage of Axl gene expression strongly inhibits proliferation, migration, invasion and tumor growth. Furthermore, inhibition of Axl expression by small interfering RNA regulates a transcriptional program of genes involved in cell survival, strikingly all connected to the nuclear factor-κB pathway. Additionally, blockage of Axl expression leads to inhibition of Akt, IKKα and IκBα phosphorylation, increasing IκBα expression and stability. Furthermore, induction of Akt phosphorylation by insulin-like growth factor 1 in Axl knockdown cells restores Akt activity and proliferation. Taken together, our results establish an unambiguous role for Axl in prostate cancer tumorigenesis with implications for prostate cancer treatment. [ABSTRACT FROM AUTHOR]

The development of a functional organ requires coordinated programs of cell fate specification, terminal differentiation and morphogenesis. Whereas signaling mechanisms that specify individual cell fates are well documented, little is known about the pathways and molecules that maintain these fates stably as normal development proceeds or how their dysregulation may contribute to altered cell states in diseases such as cancer. In Drosophila, the tyrosine kinase Abelson (Abl) interfaces with multiple signaling pathways to direct epithelial and neuronal morphogenesis during embryonic and retinal development. Here we show that Abl is required for photoreceptor cell fate maintenance, as Abl mutant photoreceptors lose neuronal markers during late pupal stages but do not re-enter a proliferative state or undergo apoptosis. Failure to maintain the differentiated state correlates with impaired trafficking of the Notch receptor and ectopic Notch signaling, and can be suppressed by reducing the genetic dose of Notch or of its downstream transcriptional effector Suppressor of Hairless. Together, these data reveal a novel mechanism for maintaining the terminally differentiated state of Drosophila photoreceptors and suggest that neuronal fates in the fly retina retain plasticity late into development. Given the general evolutionary conservation of developmental signaling mechanisms, Abl-mediated regulation of Notch could be broadly relevant to cell fate maintenance and reprogramming during normal development, regeneration and oncogenic transformation. [ABSTRACT FROM AUTHOR]

Abstract: Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell–cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant “activated PTK-selective” ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease. [Copyright &y& Elsevier]

Abstract: Ten new halogenated alkaloids named purpuroines A–J (1–10), and a known analogue (11), were isolated from the marine sponge Iotrochota purpurea. Their structures were elucidated by extensive spectroscopic (IR, MS, 1D and 2D NMR) data analyses. The inhibitory activity of some compounds against a panel of human disease related fungi and bacteria are evaluated. Bioassay for the regulation of tyrosine kinases revealed compounds 1 and 4 possessing selective inhibition against the kinase LCK. Primary structure–activity relationship is discussed. [Copyright &y& Elsevier]

Abstract: Previously we demonstrated that systemically administered neuregulin-1-β1, a nerve growth and differentiation factor, passed the blood–brain barrier and accumulated in brain areas with expression of its receptor ErbB4. In substantia nigra (SN), neuregulin-1-β1 phosphorylated ErbB4 and protected dopaminergic neurons in a toxin-based mouse model of Parkinson disease (PD). We studied ErbB4 in the context of human midbrain dopaminergic degeneration in vivo and in vitro. Post-mortem ventral midbrain tissue sections of neuropsychiatric healthy individuals and PD patients (matched for age, gender and post-mortem delay) were immunostained for ErbB4. Cultured Lund human mesencephalic (LUHMES) post-mitotic dopaminergic neurons were treated with dopaminergic toxins and analyzed for ErbB4 expression. In control individuals, 85.0±5.0% of dopaminergic neurons, containing cytoplasmic neuromelanin, expressed ErbB4 in the SN. In PD cases, the percentage of ErbB4-positive nigral dopaminergic neurons was increased to 94.9±2.5%. The mean ErbB4 immunoreactivity of melanized neurons was higher in PD than controls. LUHMES neurons upregulated ErbB4 when exposed to toxins 1-methyl-4-phenylpyridinium and 6-hydroxydopamine. Increased rate of ErbB4-positive dopaminergic neurons in PD may either reflect a better survival of ErbB4-positive neurons or an increased expression of ErbB4 by remaining neurons to seek trophic support. Enhanced ErbB4 expression in human in vitro toxin-based PD models supports the latter interpretation. Thus, dopaminergic neurons in SN might be susceptible to neuregulin-1 treatment in PD. [Copyright &y& Elsevier]

The B cell receptor (BCR) pathway plays a crucial role in the survival, proliferation and trafficking of chronic lymphocytic leukemia (CLL) cells. Inhibitors of the key kinases in this pathway, including spleen tyrosine kinase (SYK), mammalian target of rapamycin (mTOR), phosphoinositide 3'-kinase (PI3K) and Bruton's tyrosine kinase (BTK), have been found in preclinical models to decrease CLL cell viability both directly and indirectly through modulation of the microenvironment. Recently, oral agents targeting each of these kinases have been explored in early phase clinical trials in patients with CLL. BCR pathway antagonists appear to be highly active in relapsed/refractory CLL, independent of high-risk disease markers such as del(17p). These agents have shown a unique pattern of inducing early transient lymphocytosis, which typically is associated with nodal response. Here, we review the biology of the BCR, the kinases within this pathway and their interaction with the CLL microenvironment. We also discuss data from recent and ongoing clinical trials of BCR antagonists. We address the development of potential biomarkers for response to these agents such as ZAP-70, IGHV status and CCL3, and discuss where these exciting new drugs may fit in the evolving landscape of CLL therapy. [ABSTRACT FROM AUTHOR]

Abstract: In the past decade, substantial progress has been made in understanding how Src family kinases regulate the formation and function of invadosomes. Invadosomes are organized actin-rich structures that contain an F-actin core surrounded by an adhesive ring and mediate invasive migration. Src kinases orchestrate, either directly or indirectly, each phase of the invadosome life cycle including invadosome assembly, maturation and matrix degradation and disassembly. Complex arrays of Src effector proteins are involved at different stages of invadosome maturation and their spatiotemporal activity must be tightly regulated to achieve effective invasive migration. In this review, we highlight some recent progress and the challenges of understanding how Src is regulated temporally and spatially to orchestrate the dynamics of invadosomes and mediate cell invasion. [Copyright &y& Elsevier]

Defects in stem cell renewal or progenitor cell expansion underlie ageing-related diseases such as osteoporosis. Yet much remains unclear about the mechanisms regulating progenitor expansion. Here we show that the tyrosine kinase c-Abl plays an important role in osteoprogenitor expansion. c-Abl interacts with and phosphorylates BMPRIA and the phosphorylation differentially influences the interaction of BMPRIA with BMPRII and the Tab1-Tak1 complex, leading to uneven activation of Smad1/5/8 and Erk1/2, the canonical and non-canonical BMP pathways that direct the expression of p16INK4a. c-Abl deficiency shunts BMP signalling from Smad1/5/8 to Erk1/2, leading to p16INK4a upregulation and osteoblast senescence. Mouse genetic studies revealed that p16INK4a controls mesenchymal stem cell maintenance and osteoblast expansion and mediates the effects of c-Abl deficiency on osteoblast expansion and bone formation. These findings identify c-Abl as a regulator of BMP signalling pathways and uncover a role for c-Abl in p16INK4a expression and osteoprogenitor expansion. [ABSTRACT FROM AUTHOR]

Breast tumor kinase (Brk) was originally isolated from a human metastatic breast tumor, but also is found expressed in other epithelial tumors and in a subset of normal epithelia. Brk is a tyrosine kinase and its expression in breast carcinoma has been linked to tumor progression. The signal transducer and activator of transcription 3 (STAT3) is one of the substrate targets of Brk, and elevated tyrosine phosphorylation of STAT3 is known to contribute to oncogenesis. Conventional activation of STAT3 occurs in response to cytokine stimulation of Janus tyrosine kinases (JAK). One of the negative regulators discovered in cytokine signaling of the JAK-STAT pathway is the suppressor of cytokine signaling 3 (SOCS3). In this report we describe the finding that SOCS3 can also inhibit the unconventional target, Brk. Investigation of the mechanism by which SOCS3 inhibits Brk reveals the SOCS3 protein binds to Brk primarily via its SH2 domain, and its main inhibitory effect is mediated by the SOCS3 kinase inhibitory region (KIR). SOCS3 has only a modest effect on promoting Brk degradation, and this requires the C-terminal SOCS box domain. SOCS3 is the only known inhibitor of Brk, and knowledge of the mechanisms by which SOCS3 inhibits Brk may lead to methods that block Brk in cancer progression. [ABSTRACT FROM AUTHOR]

Abstract: SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27Kip1 expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27Kip1 expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer. [Copyright &y& Elsevier]

During development and tissue homeostasis, patterns of cellular organization, proliferation and movement are highly choreographed. Receptor tyrosine kinases (RTKs) have a crucial role in establishing these patterns. Individual cells and tissues exhibit tight spatial control of the RTKs that they express, enabling tissue morphogenesis and function, while preventing unwarranted cell division and migration that can contribute to tumorigenesis. Indeed, RTKs are deregulated in most human cancers and are a major focus of targeted therapeutics. A growing appreciation of the essential role of spatial RTK regulation during development prompts the realization that spatial deregulation of RTKs is likely to contribute broadly to cancer development and may affect the sensitivity and resistance of cancer to pharmacological RTK inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na+, K+, vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src. [Copyright &y& Elsevier]

SUMMARY The pathogenesis of eosinophilic esophagitis (EoE) is incompletely understood. In certain eosinophilic diseases, activation of tyrosine kinase after fusion of the Fip1-like-1 and platelet-derived growth factor receptor-α genes (F-P fusion gene) mediates eosinophilia via downstream effectors such as extracellular-regulated kinase (ERK1/2) and signal transducers and activators of transcription (STAT5). This mechanism has not been examined in EoE. Our aim was to detect the F-P fusion gene, pERK1/2, and pSTAT5 in esophageal tissue from patients with EoE, gastresophageal reflux disease (GERD), and normal controls. We performed a cross-sectional pilot study comparing patients with steroid-responsive and steroid-refractory EoE, to GERD patients and normal controls. EoE cases were defined by consensus guidelines. Fluorescence in situ hybridization (FISH) was performed to detect the F-P fusion gene and immunohistochemistry (IHC) was performed to detect pERK1/2 and pSTAT5 in esophageal biopsies. Twenty-nine subjects (median age 30 years [range 1-59]; 16 males; 24 Caucasians) were included: eight normal, six GERD, and 15 EoE (five steroid-refractory). On FISH, 98%, 99%, and 99% of the nuclei in the normal, GERD, and EoE groups, respectively, were normal ( P= 0.42). On IHC, a median of 250, 277, and 479 nuclei/mm2 stained for pERK 1/2 in the normal, GERD, and EoE groups, respectively ( P= 0.07); the refractory EoE patients had the highest degree pERK 1/2 staining (846 nuclei/mm2; P= 0.07). No trend was seen for pSTAT5. In conclusion, the F-P fusion gene was not detected with increased frequency in EoE. Patients with EoE had a trend toward higher levels of pERK 1/2, but not STAT5, in the esophageal epithelium, with highest levels in steroid-refractory EoE patients. [ABSTRACT FROM AUTHOR]

Sprouty (Spry) proteins modulate the actions of receptor tyrosine kinases during development and tumorigenesis. Decreases in cellular levels of Spry, especially Sprouty2 (Spry2), have been implicated in the growth and progression of tumors of the breast, prostate, lung, and liver. During development and tumor growth, cells experience hypoxia. Therefore, we investigated how hypoxia modulates the levels of Spry proteins. Hypoxia elevated the levels of all four expressed Spry isoforms in HeLa cells. Amounts of endogenous Spry2 in LS147T and HEP3B cells were also elevated by hypoxia. Using Spry2 as a prototype, we demonstrate that silencing and expression of prolyl hydroxylase domain proteins (PHD1-3) increase and decrease, respectively, the cellular content of Spry2. Spry2 also preferentially interacted with PHD1-3 and von Hippel-Lindau protein (pVHL) during normoxia but not in hypoxia. Additionally, Spry2 is hydroxylated on Pro residues 18, 144, and 160, and substitution of these residues with Ala enhanced stability of Spry2 and abrogated its interactions with pVHL. Silencing of pVHL increased levels of Spry2 by decreasing its ubiquitylation and degradation and thereby augmented the ability of Spry2 to inhibit FGF-elicited activation of ERK1/2. Thus, prolyl hydroxylase mediated hydroxylation and subsequent pVHL-elicited ubiquitylation of Spry2 target it for degradation and, consequently, provide a novel mechanism of regulating growth factor signaling. [ABSTRACT FROM AUTHOR]

The article discusses the possible clinical effects of epidermal growth factor receptor (EGFR) and its increased expression in thyroid tumor progression. The author observes the common event of EGFR's overexpression in tumor progression. He contends the importance of further investigation in the increased EGFR levels in thyroid cancer progression.