Receptor Tyrosine Kinase Signaling Pathways Research Articles

Abstract A31: Regulation of ErbB4 receptor tyrosine kinase by the SUMO system

Abstract The members of the ErbB family (EGFR/ErbB1, ErbB2, ErbB3, and ErbB4) of receptor tyrosine kinases (RTK) regulate key cellular processes including proliferation, differentiation, and survival, and play critical roles in several human malignancies. The alternatively spliced ErbB4 JM-a isoform was the first RTK found to signal by a mechanism involving proteolytic release of an intracellular receptor domain (ErbB4 ICD). The released ErbB4 ICD can translocate into the nucleus, where it functions as a transcriptional coregulator. ErbB4 ICD has been shown to regulate mammary epithelial development in vivo. Furthermore, nuclear ErbB4 immunoreactivity is frequently observed in clinical breast cancer samples. Although these findings suggest that ErbB4 signaling via ICD is biologically significant, little is known about the regulation of ErbB4 ICD functions or cellular localization. We hypothesized that the soluble ErbB4 ICD is likely to be regulated by different mechanisms than the ones controlling conventional RTK signaling. One regulatory mechanism that is often employed by nuclear proteins is the small ubiquitin-like modifier (SUMO) system. SUMO proteins modify protein substrates covalently, and alter their function through changes in activity, stability, or cellular localization. Here, we demonstrate that ErbB4 ICD is modified by SUMO-1 and SUMO-3. Full-length ErbB4 was not sumoylated, indicating that SUMO is a specific regulatory modification for the soluble ICD. By using site-directed mutagenesis followed by sumoylation assays, we mapped the major SUMO acceptor site in ErbB4. No differences were found between wild type and sumoylation deficient mutant of ErbB4 in biochemical assays measuring the tyrosine kinase activity of ErbB4, or activation of canonical RTK signaling pathways downstream of ErbB4. However, we showed that protein inhibitor of activated STAT3 (PIAS3), a SUMO E3 ligase that could stimulate the sumoylation, modulated the transcriptional coregulatory activity of ErbB4 ICD in luciferase reporter assays, as well as the function of ErbB4 ICD in mammary epithelial cell differentiation. Based on these findings we propose that the SUMO system regulates the nuclear signaling of ErbB4 ICD. Citation Format: Anna Korhonen, Maria Sundvall, Katri Vaparanta, Klaus Elenius. Regulation of ErbB4 receptor tyrosine kinase by the SUMO system. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A31.

Switching cell fates in the developing Drosophila eye

The developing Drosophila ommatidium is characterized by two distinct waves of pattern formation. In the first wave, a precluster of five cells is formed by a complex cellular interaction mechanism. In the second wave, cells are systematically recruited to the cluster and directed to their fates by developmental cues presented by differentiating precluster cells. These developmental cues are mediated through the receptor tyrosine kinase (RTK) and Notch (N) signaling pathways and their combined activities are crucial in specifying cell type. The transcription factor Lozenge (Lz) is expressed exclusively in second wave cells. Here, we ectopically supply Lz to precluster cells and concomitantly supply the various RTK/N codes that specify each of three second wave cell fates. We thereby reproduce molecular markers of each of the second wave cell types in precluster cells and draw three inferences. First, we confirm that Lz provides key intrinsic information to second wave cells. We can now combine this with the RTK/N signaling to provide a cell fate specification code that entails both extrinsic and intrinsic information. Second, the reproduction of each second wave cell type in the precluster confirms the accuracy of the RTK/N signaling code. Third, RTK/N signaling and Lz need only be presented to the cells for a short period of time in order to specify their fate.

Structure of native oligomeric Sprouty2 by electron microscopy and its property of electroconductivity

Receptor tyrosine kinases (RTKs) regulate many cellular processes, and Sprouty2 (Spry2) is known as an important regulator of RTK signaling pathways. Therefore, it is worth investigating the properties of Spry2 in more detail. In this study, we found that Spry2 is able to self-assemble into oligomers with a high-affinity KD value of approximately 16nM, as determined through BIAcore surface plasmon resonance analysis. The three-dimensional (3D) structure of Spry2 was resolved using an electron microscopy (EM) single-particle reconstruction approach, which revealed that Spry2 is donut-shaped with two lip-cover domains. Furthermore, the method of energy dispersive spectrum obtained through EM was analyzed to determine the elements carried by Spry2, and the results demonstrated that Spry2 is a silicon- and iron-containing protein. The silicon may contribute to the electroconductivity of Spry2, and this property exhibits a concentration-dependent feature. This study provides the first report of a silicon- and iron-containing protein, and its 3D structure may allow us (1) to study the potential mechanism through the signal transduction is controlled by switching the electronic transfer on or off and (2) to develop a new type of conductor or even semiconductor using biological or half-biological hybrid materials in the future.

Receptor Tyrosine Kinase-Mediated Angiogenesis

The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis.

Identification of ephrinB2 positive vessels in non-Hodgkin lymphoma subtypes.

e19542 Background: Current anti-angiogenic therapies for tumors target signaling pathways such as VEGF. However, the role of alternative vascular pathways in tumor biology in general, and lymphoma biology specifically, are less well understood. Recently, the Eph receptor tyrosine kinase signaling pathway has emerged as a novel target for therapeutic inhibition. The EphB family member EphB4, and its reverse signaling ligand ephrinB2, are indispensible for developmental angiogenesis. Inhibition of ephrinB2 signaling results in impaired tumor growth in pre-clinical models. We therefore sought to determine if ephrinB2 is expressed in the microenvironment of human lymphomas and thereby represents a novel therapeutic target. Methods: We evaluated by immunohistochemistry on paraffin tissue sections expression of ephrinB2, CD34 (endothelial cells) and CD163 (macrophages) in LN biopsies from 12 cases of FL and 11 cases of DLBCL retrieved from the archives of the Department of Pathology at the University of Chicago (2000-2011). Eleven cases of follicular hyperplasia or quiescent LNs were used as controls (Cs). Slides were digitally scanned and examined both semi-quantitatively and by digital image analysis. Stained cells were quantified both in internal areas and at lymphoma-fat interfaces. Results: CD34+ microvessel densities were comparable between DLBCL and C groups but showed a significantly higher trend of interface angiogenic sprouts in FLs. Interface recruited CD163+ cell numbers were significantly higher in DLBCLs than in both FLs and Cs (p<0.0001). EphrinB2-expressing blood vessels were significantly increased in FLs but not in DLBCL. Conclusions: FLs demonstrate an increase in ephrinB2+/CD34+ vessels at the lymphoma-fat interface. In contrast, DLBCLs demonstrate no increase in ephrinB2+ vessels versus controls. However, DLBCLs were notable for increased recruitment of CD163+ macrophages at the tumor/host interface. These data represent the first analysis of ephrinB2 expression in human lymphomas and demonstrate heterogeneity between locations within a lymphoma and between histologic subtypes. Analysis of ephrinB2 in human lymphomas reveals marked heterogeneity in lymphoma microenvironmental domains.

Vandetanib: A novel targeted therapy for the treatment of metastatic or locally advanced medullary thyroid cancer

The pharmacology, pharmacokinetics, efficacy, safety and tolerability, drug and food interactions, cost, and place in therapy of vandetanib are reviewed. Vandetanib is a small-molecule inhibitor of vascular endothelial growth factor receptor, epidermal growth factor receptor, and receptor tyrosine kinase signaling pathways, which are involved in the pathogenesis of medullary thyroid cancer (MTC). Vandetanib is currently approved as an alternative to local therapies for both unresectable and disseminated disease. Vandetanib was evaluated in a randomized, placebo-controlled, double-blind Phase III study comparing vandetanib with placebo in adult patients with unresectable locally advanced or metastatic hereditary or sporadic MTC. Vandetanib demonstrated a statistically significant longer progression-free survival (predicted median of 30.5 months) compared with placebo (median of 19.3 months) (hazard ratio, 0.46; 95% confidence interval, 0.31-0.69; p = 0.0001). The most commonly observed adverse effects of vandetanib include nausea, diarrhea, headache, rash, prolongation of the Q-T interval, and hypertension. Because it can prolong the Q-T interval, vandetanib is contraindicated for use in patients with serious cardiac complications, including congenital long QT syndrome, bradyarrhythmias, uncompensated heart failure, and a history of torsades de pointes. Vandetanib has been shown to be more effective than placebo in the treatment of advanced MTC; however, it has not been compared with radiation, resection, or embolization. Vandetanib also has significant and fairly common cardiac toxicities. The cost, benefits, and risks of vandetanib for patients with MTC should be weighed, as alternative treatments remain an option for most patients.

MiR‐21 modulates the ERK–MAPK signaling pathway by regulating SPRY2 expression during human mesenchymal stem cell differentiation

The ERK-MAPK signaling pathway plays a pivotal role during mesenchymal stem cell (MSC) differentiation. Studies have demonstrated that ERK-MAPK promotes adipogenesis and osteogenesis through the phosphorylation of differentiation-associated transcription factors and that it is the only active signaling in all three lineages (adipogenic, chondrogenic, and osteogenic) during MSC differentiation. Recent studies pointed to the significant roles of microRNA-21 (miR-21) during several physiological and pathological processes, especially stem cell fate determination. The miR-21 expression pattern is also correlated with ERK-MAPK activity. Here, we found that miR-21 expression is elevated and associated with an increased differentiation potential in MSCs during adipogenesis and osteogenesis. The overexpression of miR-21 elevated the expression level of the differentiation-associated genes PPARγ and Cbfa-1 during MSC differentiation, whereas miR-21 knockdown reduced the expression level of both genes. The ERK-MAPK signaling pathway activity had an increasing tendency to respond to miR-21 upregulation and a decreasing tendency to respond to miR-21 down-regulation during the first 4 days of adipogenesis and osteogenesis. Our data indicate that miR-21 modulated ERK-MAPK signaling activity by repressing SPRY2 expression, a known regulator of the receptor tyrosine kinase (RTK) signaling pathway, to affect the duration and magnitude of ERK-MAPK activity. The ERK-MAPK signaling pathway was regulated by Sprouty2 (SPRY2) expression via a miR-21-mediated mechanism during MSC differentiation.

Abstract 740: Investigating the role of epigenetic mechanisms upon aberrant signaling network activation.

Abstract Growth factor (GF) stimulation of quiescent cells activates critical receptor tyrosine kinase (RTK) signaling pathways as well as waves of downstream gene expression to drive cell proliferation, survival, and migration. Aberrant activation of RTK signaling networks is a root cause of the uncontrolled cell growth and cell survival seen in cancer. In this study, we aim to uncover the role of epigenetic mechanisms in controlling the dynamic gene expression changes that occur as a cell is activated from a quiescent state, in normal and oncogenic cellular contexts. In order to compare controlled RTK signaling with hyperactive RTK signaling cellular states, we created a cell culture system to modulate the duration and intensity of RTK activation. We genetically deleted the Sprouty (Spry) genes, a family of negative feedback inhibitors of RTK signaling that function as putative tumor suppressors. The Spry deficient cells are hypersensitive to several GFs and show significantly enhanced and prolonged activation of several critical RTK signaling pathways, including the Ras-MAPK pathway. Additionally, the Spry deficient cells possess accelerated cell growth and increased tumorigenic potential compared to their wild-type counterparts. To determine whether this deregulated signaling results in aberrant gene expression, quiescent Spry wild-type and deficient cells were stimulated with GFs, and microarrays were used to profile changes in gene expression over time. Deregulated RTK signaling due to the absence of Spry leads to abnormal expression of a large subset of genes. Specifically, many genes display display not only changes in the level of expression, but aberrant patterns of response to GF stimulation. We determined that genes with deregulated activation in Spry deficient cells display significant differences in histone tail modifications at promoter regions and distal regulatory elements. Interestingly, these changes are evident in the quiescent cellular state as well as upon GF-mediated RTK network activation. Our work suggests that dynamic changes in a cell's epigenetic landscape contribute to the deregulated activation of genes upon hyperactive RTK signaling. Our study will provide a comprehensive view of how epigenetic mechanisms contribute to aberrant gene expression in oncogenic cellular contexts. Citation Format: Behnam Nabet, Rosemary Braun, Relja Popovic, Jonathan D. Licht. Investigating the role of epigenetic mechanisms upon aberrant signaling network activation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 740. doi:10.1158/1538-7445.AM2013-740

Abstract LB-318: Reshaping the cancer proteome: therapeutic targeting of signaling and resistance in cancer by antisense manipulation of RNA processing.

Abstract Activation of the receptor tyrosine kinase (RTK) signaling pathways represents a key aspect of tumorigenesis in a broad range of human cancers. Thus, targeting of pathogenic RTKs, mainly with small molecule inhibitors and antibodies, has become an attractive therapeutic approach for cancer therapy. However, resistance by multiple mechanisms, including mutational re-activation of RTK signaling, activation of alternative RTK pathways, EMT transition and others, inevitably occurs over time. We recently identified a number of novel secreted soluble decoy RTKs isoforms (sdRTKs) generated by a U1-snRNP-dependent alternative intronic polyadenylation mechanism. We therefore developed a novel antisense-based method to effectively activate the expression of these sdRTKs, both in vitro and in vivo. This leads to the generation of endogenous dominant negative variants of RTKs, which can specifically affect the signaling of pathogenic RTKs and therefore provides a novel cancer therapy approach. Employing specific antisense compounds designed to activate intronic polyA sites, we are able to induce natural truncated EGFR variants that encode its extracellular ligand-binding domain but lack the intracellular signaling domain. These compounds thus can inhibit EGFR signaling by a three fold-mechanism: 1) removal of FL receptor, 2) sequestration of ligands and 3) non-productive dimerization with residual receptors. They are therefore designed to affect signaling both of targeted cells and of bystanders. Here we will show that, using this approach, we can target EGFR signaling both in vitro and in tumors. Importantly, with this method we can bypass the emergence of resistance typically seen with targeted drug treatments. Further development of this methodology will therefore provide the basis for an exciting novel approach to targeting and inhibiting oncogenic EGFR signaling and other pathways in a variety of cancers. Citation Format: Luca Cartegni, lee Spraggon. Reshaping the cancer proteome: therapeutic targeting of signaling and resistance in cancer by antisense manipulation of RNA processing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-318. doi:10.1158/1538-7445.AM2013-LB-318

Reduction of the ST6 β-Galactosamide α-2,6-Sialyltransferase 1 (ST6GAL1)-catalyzed Sialylation of Nectin-like Molecule 2/Cell Adhesion Molecule 1 and Enhancement of ErbB2/ErbB3 Signaling by MicroRNA-199a

Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 β-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

Kaposi's Sarcoma-Associated Herpesvirus Induces Rapid Release of Angiopoietin-2 from Endothelial Cells

Kaposi sarcoma-associated herpesvirus (KSHV) stimulates proliferation, angiogenesis, and inflammation to promote Kaposi sarcoma (KS) tumor growth, which involves various growth factors and cytokines. Previously, we found that KSHV infection of human umbilical vein endothelial cells (HUVECs) induces a transcriptional induction of the proangiogenic and proinflammatory cytokine angiopoietin-2 (Ang-2). Here, we report that KSHV induces rapid release of Ang-2 that is presynthesized and stored in the Weibel-Palade bodies (WPB) of endothelial cells upon binding to its integrin receptors. Blocking viral binding to integrins inhibits Ang-2 release. KSHV binding activates the integrin tyrosine kinase receptor signaling pathways, leading to tyrosine phosphorylation of focal adhesion kinase (FAK), the tyrosine kinase Src, and the Calα2 subunit of the l-type calcium channel to trigger rapid calcium (Ca(2+)) influx. Pretreatment of endothelial cells with specific inhibitors of protein tyrosine kinases inhibits KSHV-induced Ca(2+) influx and Ang-2 release. Inhibition of Ca(2+) mobilization with calcium channel blockers also inhibits Ang-2 release. Thus, the interaction between KSHV and its integrin receptors plays a key role in regulating rapid Ang-2 release from endothelial cells. This finding highlights a novel mechanism of viral induction of angiogenesis and inflammation, which might play important roles in the early event of KS tumor development.

Proliferation-Independent Control of Tumor Glycolysis by PDGFR-Mediated AKT Activation

The differences in glucose metabolism that distinguish most malignant and normal tissues have called attention to the importance of understanding the molecular mechanisms by which tumor energy metabolism is regulated. Receptor tyrosine kinase (RTK) pathways that are implicated in proliferation and transformation have been linked to several aspects of tumor glucose metabolism. However, the regulation of glycolysis has invariably been examined under conditions in which proliferation is concomitantly altered. To determine whether RTKs directly regulate glycolysis without prerequisite growth modulation, we first identified a specific RTK signaling pathway, platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) that regulates glycolysis in glioma-derived tumor stem-like cells from a novel mouse model. We determined that PDGF-regulated glycolysis occurs independent of PDGF-regulated proliferation but requires the activation of AKT, a known metabolic regulator in tumor. Our findings identifying a key characteristic of brain tumors, aerobic glycolysis, mediated by a pathway with multiple therapeutic targets suggests the possibility of inhibiting tumor energy metabolism while also treating with agents that target other pathways of pathologic significance.

Phosphotyrosine Profiling of NSCLC Cells in Response to EGF and HGF Reveals Network Specific Mediators of Invasion

Growth factor signaling is deregulated in cancer and often leads to invasion, yet receptor tyrosine kinase signaling pathways driving invasion under different growth factor conditions are not well understood. To identify specific signaling molecules regulating invasion of A549 non-small cell lung carcinoma (NSCLC) cells downstream of the epidermal growth factor receptor (EGFR) and Met, quantitative site-specific mass spectrometric analysis of tyrosine phosphorylation was performed following epidermal growth factor (EGF) or hepatocyte growth factor (HGF) stimulation, at three different growth factor concentrations and at two time points. Through this analysis, the temporal and concentration dependent phosphorylation profiles were obtained for 131 and 139 sites downstream of EGF and HGF stimulation, respectively. To characterize the effect of these signaling network alterations, we quantified 3D cell migration/invasion through Matrigel. Partial least-squares regression (PLSR) analysis was performed to identify the tyrosine phosphorylation sites most strongly correlated with EGF and/or HGF mediated invasion. Potential common and specific signaling events required for driving invasion downstream of EGFR and Met were identified using either a combined or two independent PLSR models, based on the quantitative EGF or HGF data. Our data highlight the integration and compartmentalization of signaling required for invasion in cancer.

Transcriptional regulation of Profilin during wound closure in Drosophila larvae

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. Here, we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

A Kinome-Wide RNAi Screen in Drosophila Glia Reveals That the RIO Kinases Mediate Cell Proliferation and Survival through TORC2-Akt Signaling in Glioblastoma

Glioblastoma, the most common primary malignant brain tumor, is incurable with current therapies. Genetic and molecular analyses demonstrate that glioblastomas frequently display mutations that activate receptor tyrosine kinase (RTK) and Pi-3 kinase (PI3K) signaling pathways. In Drosophila melanogaster, activation of RTK and PI3K pathways in glial progenitor cells creates malignant neoplastic glial tumors that display many features of human glioblastoma. In both human and Drosophila, activation of the RTK and PI3K pathways stimulates Akt signaling along with other as-yet-unknown changes that drive oncogenesis. We used this Drosophila glioblastoma model to perform a kinome-wide genetic screen for new genes required for RTK- and PI3K-dependent neoplastic transformation. Human orthologs of novel kinases uncovered by these screens were functionally assessed in mammalian glioblastoma models and human tumors. Our results revealed that the atypical kinases RIOK1 and RIOK2 are overexpressed in glioblastoma cells in an Akt-dependent manner. Moreover, we found that overexpressed RIOK2 formed a complex with RIOK1, mTor, and mTor-complex-2 components, and that overexpressed RIOK2 upregulated Akt signaling and promoted tumorigenesis in murine astrocytes. Conversely, reduced expression of RIOK1 or RIOK2 disrupted Akt signaling and caused cell cycle exit, apoptosis, and chemosensitivity in glioblastoma cells by inducing p53 activity through the RpL11-dependent ribosomal stress checkpoint. These results imply that, in glioblastoma cells, constitutive Akt signaling drives RIO kinase overexpression, which creates a feedforward loop that promotes and maintains oncogenic Akt activity through stimulation of mTor signaling. Further study of the RIO kinases as well as other kinases identified in our Drosophila screen may reveal new insights into defects underlying glioblastoma and related cancers and may reveal new therapeutic opportunities for these cancers.

Proteoglycans and their roles in brain cancer

Glioblastoma, a malignant brain cancer, is characterized by abnormal activation of receptor tyrosine kinase signalling pathways and a poor prognosis. Extracellular proteoglycans, including heparan sulfate and chondroitin sulfate, play critical roles in the regulation of cell signalling and migration via interactions with extracellular ligands, growth factor receptors and extracellular matrix components, as well as intracellular enzymes and structural proteins. In cancer, proteoglycans help drive multiple oncogenic pathways in tumour cells and promote critical tumour-microenvironment interactions. In the present review, we summarize the evidence for proteoglycan function in gliomagenesis and examine the expression of proteoglycans and their modifying enzymes in human glioblastoma using data obtained from The Cancer Genome Atlas (http://cancergenome.nih.gov/). Furthermore, we demonstrate an association between specific proteoglycan alterations and changes in receptor tyrosine kinases. Based on these data, we propose a model in which proteoglycans and their modifying enzymes promote receptor tyrosine kinase signalling and progression in glioblastoma, and we suggest that cancer-associated proteoglycans are promising biomarkers for disease and therapeutic targets.

Synthesis and biological activity of 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic agents

Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropanamide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and semaxinib.

Correction: Corrigendum: A novel potent tumour promoter aberrantly overexpressed in most human cancers

The complexity and heterogeneity of tumours have hindered efforts to identify commonalities among different cancers. Furthermore, because we have limited information on the prevalence and nature of ubiquitous molecular events that occur in neoplasms, it is unfeasible to implement molecular-targeted cancer screening and prevention. Here, we found that the FEAT protein is overexpressed in most human cancers, but weakly expressed in normal tissues including the testis, brain and liver. Transgenic mice that ectopically expressed FEAT in the thymus, spleen, liver and lung spontaneously developed invasive malignant lymphoma (48%, 19/40) and lung-metastasizing liver cancer (hepatocellular carcinoma) (35%, 14/40) that models human hepatocarcinogenesis, indicating the FEAT protein potently drives tumorigenesis in vivo. Gene expression profiling suggested that FEAT drives receptor tyrosine kinase and hedgehog signalling pathways. These findings demonstrate that integrated efforts to identify FEAT-like ubiquitous oncoproteins are useful and may provide promising approaches for cost-effective cancer screening and prevention.

Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

The drosophila Arf GEF steppke controls MAPK activation in EGFR signaling

Guanine nucleotide exchange factors (GEFs) of the cytohesin protein family are regulators of GDP/GTP exchange for members of the ADP ribosylation factor (Arf) of small GTPases. They have been identified as modulators of various receptor tyrosine kinase signaling pathways including the insulin, the vascular epidermal growth factor (VEGF) and the epidermal growth factor (EGF) pathways. These pathways control many cellular functions, including cell proliferation and differentiation, and their misregulation is often associated with cancerogenesis. In vivo studies on cytohesins using genetic loss of function alleles are lacking, however, since knockout mouse models are not available yet. We have recently identified mutants for the single cytohesin Steppke (Step) in Drosophila and we could demonstrate an essential role of Step in the insulin signaling cascade. In the present study, we provide in vivo evidence for a role of Step in EGFR signaling during wing and eye development. By analyzing step mutants, transgenic RNA interference (RNAi) and overexpression lines for tissue specific as well as clonal analysis, we found that Step acts downstream of the EGFR and is required for the activation of mitogen-activated protein kinase (MAPK) and the induction of EGFR target genes. We further demonstrate that step transcription is induced by EGFR signaling whereas it is negatively regulated by insulin signaling. Furthermore, genetic studies and biochemical analysis show that Step interacts with the Connector Enhancer of KSR (CNK). We propose that Step may be part of a larger signaling scaffold coordinating receptor tyrosine kinase-dependent MAPK activation.