Regulation Of Tyrosine Kinases Research Articles

Abstract 3453: Autophagic regulation of the Met receptor tyrosine kinase in breast cancer

Abstract The Met receptor tyrosine kinase (RTK) and its ligand, hepatocyte growth factor (HGF), are potent mediators of epithelial-mesenchymal transition and an invasive growth program. The HGF-Met signaling axis plays a significant role in the development or progression of many human cancers, and elevated Met expression is associated with poor prognosis and the basal subtype in breast cancer. Met signaling is regulated temporally and spatially through endocytic trafficking to degradative or recycling compartments, and delayed Met trafficking to the degradative pathway promotes enhanced cell migration, adhesion-independent growth and tumorigenesis. It is now recognized that autophagy, an intracellular degradative process, utilizes the endocytic system in both formation and maturation of double-membraned autophagosomes. Autophagy can have both tumor promoting and tumor suppressor roles in different contexts or stages of disease. We hypothesized that reprogramming of endocytic membrane system to support an autophagic starvation response may alter Met RTK trafficking, and therefore modulate tumorigenic signaling. To test this, we utilized MDA-MB-231 and BT-549 triple-negative breast cancer cell lines, as well as Hela cells, as models to study the trafficking and signaling of the Met RTK under conditions of autophagic stimulation or inhibition. Following HGF stimulation and receptor internalization, Met engages with core autophagic machinery in a starvation-dependent manner. Increased autophagosome formation decreases Met recycling to the plasma membrane, which can be rescued by shRNA-mediated knockdown of genes required for autophagy initiation. We demonstrate that the autophagic process negatively regulates signaling of Met through the PI3K/Akt and MAPK pathways and decreases Met-dependent cellular responses. These findings identify Met RTK regulation as a potential tumor-suppressive function of autophagy. Citation Format: Emily S. Bell, Dongmei Zuo, Morag Park. Autophagic regulation of the Met receptor tyrosine kinase in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3453. doi:10.1158/1538-7445.AM2014-3453

Microparticles Mediate the Intercellular Regulation of microRNA-503 and Proline-Rich Tyrosine Kinase 2 to Alter the Migration and Invasion Capacity of Breast Cancer Cells.

The successful treatment of cancer is hampered by drug resistance and metastasis. While these two obstacles were once considered separately, recent evidence associates resistance with an enhanced metastatic capacity. However, the underlying mechanisms remain undefined. We previously described the intercellular transfer of drug resistance via submicron vesicles called microparticles (MPs). We now propose that MPs derived from drug-resistant cells are also involved in the intercellular transfer of components to enhance the migration and invasion capacity of cells. Thus, MPs may be a conduit between resistance and metastasis. We used microarray analysis to identify regulatory microRNAs (miRNAs), which contribute to the dissemination of metastatic traits. miR-503 was downregulated in recipient cells following co-culture with MPs isolated from drug-resistant cells. miR-503 was inversely associated with metastasis, as demonstrated using wound healing/scratch migration assays and Matrigel®-coated transwell invasion assays. Proline-rich tyrosine kinase 2 (PYK2) was upregulated in recipient cells and associated with increased migration and invasion, with these phenotypes being reversed using a pharmacological inhibitor of PYK2 phosphorylation, tyrphostin A9. However, the MP-mediated promotion of metastatic traits was not due to the presence of these effectors in the MP cargo but rather due to down stream effector molecules in these pathways. This is the first demonstration that the role of MPs in trait acquisition extends beyond the direct transfer of vesicle components and also includes transfer of intermediary regulators that induce down stream mediators following transfer to recipient cells. This implicates an expanding role of MPs in cancer pathogenesis.

The diverse roles of G protein-coupled receptor kinase 2 (GRK2): a focus on regulation of receptor tyrosine kinases (RTKs).

The G protein-coupled receptor (GPCR) kinases (GRKs) are a family of seven serine/threonine kinases that are recruited to and activated by almost all agonist-occupied GPCRs. Upon recruitment and activation, GRK2 inhibits G protein-dependent signaling downstream of GPCRs both directly, for example by sequestering activated G proteins, and indirectly, by promoting the recruitment of β-arrestins, which sterically hinder further G protein activation and drive receptor internalization. In addition to switching off G protein signaling, GRK2 also initiates a number of G protein-independent signaling pathways downstream of activated GPCRs by recruiting scaffolding proteins or by phosphorylating non-GPCR substrates. Furthermore, it has recently become clear that the GRKs regulate signaling downstream of receptors that belong to families other than the GPCRs, including the transforming growth factor β (TGFβ) receptor and the toll-like receptor TLR4. Here we focus on recent studies demonstrating an important role for GRK2 in regulating signaling in both positive and negative ways downstream of various receptor tyrosine kinases (RTKs), including platelet-derived growth factor (PDGF) receptors, epidermal growth factor (EGF) receptors and insulin-like growth factor 1 (IGF-1) receptors.

C-terminal Src Kinase (Csk)-mediated Phosphorylation of Eukaryotic Elongation Factor 2 (eEF2) Promotes Proteolytic Cleavage and Nuclear Translocation of eEF2

Protein-tyrosine kinase C-terminal Src kinase (Csk) was originally purified as a kinase for phosphorylating Src and other Src family kinases. The phosphorylation of a C-terminal tyrosine residue of Src family kinases suppresses their kinase activity. Therefore, most physiological studies regarding Csk function have been focused on Csk as a negative regulator of Src family tyrosine kinases and as a potential tumor suppressor. Paradoxically, the protein levels of Csk were elevated in some human carcinomas. In this report, we show that eukaryotic elongation factor 2 (eEF2) is a new protein substrate of Csk and could locate in the nucleus. We demonstrate that Csk-mediated phosphorylation of eEF2 has no effect on its cytoplasmic function in regulating protein translation. However, phosphorylation of eEF2 enhances its proteolytic cleavage and the nuclear translocation of the cleaved eEF2 through a SUMOylation-regulated process. Furthermore, we show that cleaved fragments of eEF2 can induce nuclear morphological changes and aneuploidy similar to those in cancer cells, suggesting that there is an additional mechanism for Csk in tumorigenesis through regulation of eEF2 subcellular localization.

Recurrent PTPRB and PLCG1 mutations in angiosarcoma.

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionising radiation or chronic lymphoedema1. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signalling genes, as a key driver of angiosarcoma1. Here, we employed whole genome, exome, and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harboured predominantly truncating mutations in 10/39 (26%) tumours. PLCG1, a signal transducer of tyrosine kinases, presented with a recurrent, likely activating R707Q missense variant in 3/34 cases (9%). Overall, 15/39 (38%) tumours harboured at least one driver mutation in angiogenesis signalling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signalling in angiosarcoma.

Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer

BackgroundNon-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC.MethodsUsing IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay.ResultsRelative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0–3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability.ConclusionPAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

Enigma Prevents Cbl-c-Mediated Ubiquitination and Degradation of RETMEN2A

The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation.

Regulation of Src trafficking and activation by the endocytic regulatory proteins MICAL-L1 and EHD1

Localization of the non-receptor tyrosine kinase Src to the cell periphery is required for its activation and to mediate focal adhesion turnover, cell spreading and migration. Inactive Src localizes to a perinuclear compartment and the movement of Src to the plasma membrane is mediated by endocytic transport. However, the precise pathways and regulatory proteins that are responsible for SRC transport are incompletely understood. Here, we demonstrate that Src partially colocalizes with the endocytic regulatory protein MICAL-L1 (molecule interacting with CasL-like protein 1) in mammalian cells. Furthermore, MICAL-L1 is required for growth-factor- and integrin-induced Src activation and transport to the cell periphery in HeLa cells and human fibroblasts. Accordingly, MICAL-L1 depletion impairs focal adhesion turnover, cell spreading and cell migration. Interestingly, we find that the MICAL-L1 interaction partner EHD1 (EH domain-containing protein 1) is also required for Src activation and transport. Moreover, the MICAL-L1-mediated recruitment of EHD1 to Src-containing recycling endosomes is required for the release of Src from the perinuclear endocytic recycling compartment in response to growth factor stimulation. Our study sheds new light on the mechanism by which Src is transported to the plasma membrane and activated, and provides a new function for MICAL-L1 and EHD1 in the regulation of intracellular non-receptor tyrosine kinases.

Acute Myeloid Leukemia With Isolated Trisomy 13 Is a Genetically Homogenous Entity With a High Frequency Of Mutations In Genes Encoding Components Of The Splicing Machinery and Extremely Poor Prognosis

Acute myeloid leukemia (AML) with isolated trisomy 13 (AML+13) is rare and frequently associated with FAB M0 morphology. The clinical course is not well characterized but according to the ELN classification of intermediate prognosis. Eighty to one-hundred percent of patients (pts) with AML+13 carry mutations in the RUNX1 gene. Over-expression of FLT3 (located on chromosome 13 [chr 13]) due to the additional gene copy on the third chr 13 was proposed as a mechanism of leukemogenesis in AML+13 (gene dosage hypothesis).We set out to characterize the clinical course of AML+13 pts and elucidate their molecular background using whole exome sequencing, targeted resequencing and gene expression profiling. We identified 23 pts with AML+13 enrolled in a multicenter trial of the German AML Cooperative Group (AMLCG-1999) and compared this group to 386 pts without +13 who were classified in the ELN Intermediate-II genetic category. All pts received intensive induction chemotherapy. There was no significant difference in age, white blood cell or platelet count between the two groups. However, LDH levels were significantly (p=.01) lower in the AML+13 group while bone marrow blast percentage was significantly higher (p=.04). Twelve AML+13 pts (52%) reached complete remission, but all relapsed. Relapse-free and overall survival were inferior in the AML+13 group compared to other ELN Intermediate-II pts (median RFS, 9 vs 15 months, p=.01; median OS, 7 vs. 13 months, p=.03).Remission samples from two AML+13 pts were available as normal control for exome sequencing. Using SureSelect human all exon target enrichment (Agilent) followed by 80bp paired-end sequencing on an Illumina GAIIx platform, we were able to identify non-synonymous leukemia-specific mutations affecting, among others, RUNX1, ASXL1, PTPN11 and CEBPZ.Genes identified by exome sequencing and a panel of genes recurringly mutated in AML were studied by targeted amplicon resequencing in all AML+13 pts with available material (16/23; Figure). As described before, a high incidence of RUNX1 mutations (75%) was identified. In addition, we detected mutations in spliceosome components in 14/16 (88%) of AML+13 pts, including SRSF2 codon 95 mutations in 13/16 pts (81%). One patient without SRSF2 mutation showed a mutation in SF3B1. Moreover, recurring mutations were found in ASXL1 (44%) and BCOR (25%), and were associated with RUNX1 and SRSF2 mutations. Interestingly, both pts without mutations in the splicing machinery had mutations in DNMT3A, which were also mutually exclusive with mutations in RUNX1 or ASXL1. Two pts carried mutations in CEBPZ suggesting that CEBPZ is a novel recurringly mutated gene in AML. [Display omitted] To further characterize this genetically homogenous subgroup, we compared gene expression profiles of 9 pts with AML+13 with 509 AML pts without +13. We identified 678 (up-regulated 492; down-regulated 186) probe sets as significantly deregulated. Only 59 (8.7%) of these probe sets were localized on chr 13, but of those, 55 were up-regulated and only 4 were down-regulated. Up-regulated probe sets on chr 13 included FOXO1, FLT3 and RB1. The strongest down-regulated probe set on chr 13 belonged to the tumor suppressor gene SPRY2, which is a negative regulator of receptor tyrosine kinases. Gene set enrichment analysis showed significant deregulation of gene sets associated with regulation of transcription and nuclear transport.In summary, our study is the first to show that AML+13 is significantly associated with inferior OS and RFS compared to other intermediate-risk cytogenetic abnormalities in a homogeneously treated cohort. Furthermore, we present evidence that AML+13 leukemias are a genetically quite homogenous subgroup. AML+13 is not only associated with a high rate of RUNX1 mutations but also with mutations in SRFS2, ASXL1 and BCOR. The incidence of mutations in SRSF2 in AML+13 is the highest of any AML subgroup reported so far. In addition, our gene expression data show a homogenous expression profile associated with AML+13. The striking association of a few recurring mutations in AML+13 suggests a biological relationship with synergistic lesions during leukemogenesis. While mutations in RUNX1, ASXL1 and up-regulation of FLT3 were previously reported as markers of poor prognosis in AML, the combination of these lesions might be responsible for the extremely poor outcome of AML+13. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrsflox/flox;mb1cre/+:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

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.

Tyrosine Kinase Signal Modulation: A Matter of H2O2 Membrane Permeability?

Abstract 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.

Sprouty genes regulate proliferation and survival of human embryonic stem cells

Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling, which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs, suggesting that they may influence self-renewal. To test this hypothesis, we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation, whereas SPRY4 KD enhanced survival. In both cases, after KD the cells were able to differentiate into cells of the three germ layers, although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage, explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs.

Leucine-rich Repeat and Immunoglobulin Domain-containing Protein-1 (Lrig1) Negative Regulatory Action toward ErbB Receptor Tyrosine Kinases Is Opposed by Leucine-rich Repeat and Immunoglobulin Domain-containing Protein 3 (Lrig3)

Lrig1 is the founding member of the Lrig family of transmembrane leucine-rich repeat proteins, which also includes Lrig2 and Lrig3. Lrig1 is a negative regulator of oncogenic receptor tyrosine kinases, including ErbB and Met receptors, and promotes receptor degradation. Lrig1 has recently emerged as both a tumor suppressor and a key regulator of epidermal and epithelial stem cell quiescence. Despite this, little is known of the mechanisms by which Lrig1 is regulated. Lrig3 was recently reported to increase ErbB receptor expression suggesting that it may function in a manner opposite to Lrig1. In this study, we explore the interaction between Lrig1 and Lrig3 and demonstrate that Lrig1 and Lrig3 functionally oppose one another. Lrig3 opposes Lrig1 negative regulatory activity and stabilizes ErbB receptors. Conversely, Lrig1 destabilizes Lrig3, limiting Lrig3's positive effects on receptors and identifying Lrig3 as a new target of Lrig1. These studies provide new insight into the regulation of Lrig1 and uncover a complex cross-talk between Lrig1 and Lrig3.

Molecular mechanisms of fibroblast growth factor signaling in physiology and pathology.

Fibroblast growth factors (FGFs) signal in a paracrine or endocrine fashion to mediate a myriad of biological activities, ranging from issuing developmental cues, maintaining tissue homeostasis, and regulating metabolic processes. FGFs carry out their diverse functions by binding and dimerizing FGF receptors (FGFRs) in a heparan sulfate (HS) cofactor- or Klotho coreceptor-assisted manner. The accumulated wealth of structural and biophysical data in the past decade has transformed our understanding of the mechanism of FGF signaling in human health and development, and has provided novel concepts in receptor tyrosine kinase (RTK) signaling. Among these contributions are the elucidation of HS-assisted receptor dimerization, delineation of the molecular determinants of ligand-receptor specificity, tyrosine kinase regulation, receptor cis-autoinhibition, and tyrosine trans-autophosphorylation. These structural studies have also revealed how disease-associated mutations highjack the physiological mechanisms of FGFR regulation to contribute to human diseases. In this paper, we will discuss the structurally and biophysically derived mechanisms of FGF signaling, and how the insights gained may guide the development of therapies for treatment of a diverse array of human diseases.

Profiling mRNA of the Graying Human Hair Follicle Constitutes a Promising State-of-the-Art Tool to Assess Its Aging: An Exemplary Report

Determining hitherto uninvestigated and safe targets to halt the aging process is important in our aging society. Graying is a hallmark of the aging process and may be used to identify aging tissue for comparative analysis. Here we analyzed differential gene expressions between pigmented, gray, and white human scalp skin hair follicles (HFs) from identical donors. Forming intersections between five donors identified 194/192 downregulated and 186/177 upregulated genes in gray/white HFs. These included melanogenesis (tyrosinase; tyrosinase-related protein 1)- and melanosome structure (Melan-A; Pmel17)-associated genes and regulation of melanocyte relevant tyrosine kinases. Alongside these expected changes, regulated genes included nonmelanocyte-related genes associated with aging as well as nonaging-related genes associated with melanocytes. Intriguingly, among them, genes associated with energy metabolism (i.e., glutaminase) and axon guidance (plexin C1) were altered. These results were reflected by pathway analysis and exemplarily confirmed by PCR and immunohistochemical studies. Supplementing cultured HFs with glutamine or plexin C1 revealed biological relevance and pharmacointerventional potential of these microarray results in altering the HF aging process. Together, we present intriguing data obtained from intra-individual sample comparison that suggest the graying HF to be a valid aging model and a promising target for testing therapeutic interventions.

Abstract 3068: MicroRNA 4723-5p is novel tumor suppressor microRNA in prostate cancer that directly regulates Abelson family of nonreceptor protein tyrosine kinases.

Abstract The Abelson (c-Abl) proto-oncogene encodes a highly conserved nonreceptor protein tyrosine kinase that has been implicated in many cellular processes including regulation of cell growth and survival, oxidative stress and DNA-damage responses, actin dynamics and cell migration. Aberrant activity of this tyrosine kinase has been implicated in the stimulation of cancer growth and progression. Abl tyrosine kinase represents a target for specific anti-cancer therapy in prostate cancer and Abl inhibitors are being tested clinically for the treatment of prostate cancer bone metastasis. Here we report that Abl kinases are regulated by a novel microRNA- miR-4723-5p in prostate cancer. Expression profiling of miR-4723-5p expression in a cohort of prostate cancer clinical specimens showed that miR-4723-5p expression is widely attenuated in prostate cancer. Low miR-4723-5p expression was significantly correlated with poor survival outcome and tumor progression and recurrence in prostate cancer. Further, our analyses suggest that miR-4723-5p has significant potential as a disease biomarker for diagnosis and prognosis in prostate cancer. To evaluate the functional significance of decreased miR-4723-5p expression in prostate cancer, miR-4723-5p was reintroduced in prostate cancer cell lines followed by functional assays. The over expression of miR-4723-5p led to significant decreases in cell growth, clonability, invasion and migration. Importantly, miR-4723-5p reexpression led to dramatic induction of apoptosis in prostate cancer cell lines suggesting that miR-4723-5p is a pro-apoptotic microRNA that regulates prostate carcinogenesis. Analysis of putative miR-4723-5p targets showed that miR-4723-5p targets Integrin alpha 3 (ITGA3) and Methyl CpG binding protein (MeCP2) in addition to Abl1 and Abl2 kinases. In conclusion, we have identified a novel microRNA mediated regulation of Abl tyrosine kinases in prostate cancer. This study suggests that miR-4723-5p may be an attractive target for therapeutic intervention in prostate cancer. Citation Format: Sumit Arora, Sharanjot Saini, Shahana Majid, Varahram Shahryari, Soichiro Yamamura, Takeshi Chiyomaru, Shinichiro Fukuhara, Inik Chang, Guoren Deng, Yuichiro Tanaka, Rajvir Dahiya. MicroRNA 4723-5p is novel tumor suppressor microRNA in prostate cancer that directly regulates Abelson family of nonreceptor protein tyrosine kinases. [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 3068. doi:10.1158/1538-7445.AM2013-3068

MicroRNA-497 increases apoptosis in MYCN amplified neuroblastoma cells by targeting the key cell cycle regulator WEE1

BackgroundNeuroblastoma is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor in high-risk disease (25-40%). MiR-497 was previously identified by our laboratory as a member of a miRNA expression signature, predictive of neuroblastoma patient survival and has been reported as a tumor suppressor in a variety of other cancers. WEE1, a tyrosine kinase regulator of the cell cycle and predicted target of miR-497, has emerged as an oncogene in several cancer types and therefore represents an attractive potential target for novel therapy approaches in high-risk neuroblastoma. Our aim was to investigate the potential tumor suppressive role of miR-497 in high-risk neuroblastoma.MethodsExpression levels of miR-497 and WEE1 in tissues and cells were determined using RT-PCR. The effect of miR-497 and siWEE1 on cell viability was evaluated using MTS assays, apoptosis levels were determined using FACS analysis of Annexin V/PI stained cells, and target protein expression was determined using western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. Results were reported as mean±S.E.M and differences were tested for significance using 2-tailed Students t-test.ResultsWe determined that miR-497 expression was significantly lower in high-risk MYCN amplified (MNA) tumors and that low miR-497 expression was associated with worse EFS and OS in our cohort. Over-expression of miR-497 reduced cell viability and increased apoptosis in MNA cells. We identified WEE1 as a novel target for miR-497 in neuroblastoma. Furthermore, our analysis showed that high WEE1 levels are significantly associated with poor EFS and OS in neuroblastoma and that siRNA knockdown of WEE1 in MNA cell lines results in significant levels of apoptosis, supporting an oncogenic role of WEE1 in neuroblastoma. Cisplatin (CDDP) treatment of both miR-497 over-expressing cells and WEE1 inhibited cells, resulted in a significant increase in apoptosis in MNA cells, describing a synergistic effect and therefore a potential therapeutic for high-risk neuroblastoma.ConclusionOur study’s results are consistent with miR-497 being a candidate tumor suppressor in neuroblastoma, through the direct targeting of WEE1. These findings re-enforce the proposal of WEE1 as a therapeutic target in neuroblastoma.

Redox regulation of protein kinases

Reactive oxygen species (ROS) have been long regarded as by-products of a cascade of reactions stemming from cellular oxygen metabolism, which, if they accumulate to toxic levels, can have detrimental effects on cellular biomolecules. However, more recently, the recognition of ROS as mediators of cellular communications has led to their classification as signalling mediators in their own right. The prototypic redox-regulated targets downstream of ROS are the protein tyrosine phosphatases, and the wealth of research that has focused on this area has come to shape our understanding of how redox-signalling contributes to and facilitates protein tyrosine phosphorylation signalling cascades. However, it is becoming increasingly apparent that there is more to this system than simply the negative regulation of protein tyrosine phosphatases. Identification of redox-sensitive kinases such as Src led to the slow emergence of a role for redox regulation of tyrosine kinases. A flow of evidence, which has increased exponentially in recent times as a result of the development of new methods for the detection of oxidative modifications, demonstrates that, by concurrent oxidative activation of tyrosine kinases, ROS fine tune the duration and amplification of the phosphorylation signal. A more thorough understanding of the complex regulatory mechanism of redox-modification will allow targeting of both the production of ROS and their downstream effectors for therapeutic purposes. The present review assesses the most relevant recent literature that demonstrates a role for kinase regulation by oxidation, highlights the most significant findings and proposes future directions for this crucial area of redox biology.

Oncogenic Signaling by Leukemia-Associated Mutant Cbl Proteins

Members of the Cbl protein family (Cbl, Cbl-b, and Cbl-c) are E3 ubiquitin ligases that have emerged as critical negative regulators of protein tyrosine kinase (PTK) signaling. This function reflects their ability to directly interact with activated PTKs and to target them as well as their associated signaling components for ubiquitination. Given the critical roles of PTK signaling in driving oncogenesis, recent studies in animal models and genetic analyses in human cancer have firmly established that Cbl proteins function as tumor suppressors. Missense mutations or small in-frame deletions within the regions of Cbl protein that are essential for its E3 activity have been identified in nearly 5% of leukemia patients with myelodysplastic/myeloproliferative disorders. Based on evidence from cell culture studies, in vivo models and clinical data, we discuss the potential signaling mechanisms of mutant Cbl-driven oncogenesis. Mechanistic insights into oncogenic Cbl mutants and associated animal models are likely to enhance our understanding of normal hematopoietic stem cell homeostasis and provide avenues for targeted therapy of mutant Cbl-driven cancers.