Christopher W. McAndrew

The Receptor Tyrosine Kinase FGFR4 Negatively Regulates NF-kappaB Signaling

Background NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis. Methodology/Principal Findings Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit), an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α) to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling. Conclusions/Significance These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFκB signaling will not be limited to FGFR4 alone.

Nordihydroguaiaretic Acid Inhibits an Activated Fibroblast Growth Factor Receptor 3 Mutant and Blocks Downstream Signaling in Multiple Myeloma Cells

Activating mutations within fibroblast growth factor receptor 3 (FGFR3), a receptor tyrosine kinase, are responsible for human skeletal dysplasias including achondroplasia and the neonatal lethal syndromes, Thanatophoric Dysplasia (TD) type I and II. Several of these same FGFR3 mutations have also been identified somatically in human cancers, including multiple myeloma, bladder carcinoma, and cervical cancer. Based on reports that strongly activated mutants of FGFR3 such as the TDII (K650E) mutant signal preferentially from within the secretory pathway, the inhibitory properties of nordihydroguaiartic acid (NDGA), which blocks protein transport through the Golgi, were investigated. NDGA was able to inhibit FGFR3 autophosphorylation both in vitro and in vivo. In addition, signaling molecules downstream of FGFR3 activation such as signal transducers and activators of transcription (STAT)1, STAT3, and mitogen-activated protein kinase (MAPK) were inhibited by NDGA treatment. Using HEK293 cells expressing activated FGFR3-TDII, together with several multiple myeloma cell lines expressing activated forms of FGFR3, NDGA generally resulted in a decrease in MAPK activation by 1 hour, and resulted in increased apoptosis over 24 hours. The effects of NDGA on activated FGFR3 derivatives targeted either to the plasma membrane or the cytoplasm were also examined. These results suggest that inhibitory small molecules such as NDGA that target a specific subcellular compartment may be beneficial in the inhibition of activated receptors such as FGFR3 that signal from the same compartment.

Spy1 expression prevents normal cellular responses to DNA damage : Inhibition of apoptosis and checkpoint activation

Spy1 is the originally identified member of the Speedy/Ringo family of vertebrate cell cycle regulators, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Here we report a role for Spy1 in apoptosis and checkpoint activation in response to UV irradiation. Using an inducible system allowing for regulated expression of Spy1, we show that Spy1 expression prevents activation of caspase-3 and suppresses apoptosis in response to UV irradiation. Spy1 expression also allows for UV irradiation-resistant DNA synthesis and permits cells to progress into mitosis, as demonstrated by phosphorylation on histone H3, indicating that Spy1 expression can inhibit the S-phase/replication and G2/M checkpoints. We demonstrate that Spy1 expression inhibits phosphorylation of Chk1, RPA, and histone H2A.X, which may directly contribute to the decrease in apoptosis and checkpoint bypass. Furthermore, mutation of the conserved Speedy/Ringo box, known to mediate interaction with CDK2, abrogates the ability of Spy1 to inhibit apoptosis and the phosphorylation of Chk1 and RPA. The data presented indicate that Spy1 expression allows cells to evade checkpoints and apoptosis and suggest that Spy1 regulation of CDK2 is important for the response to DNA damage.

Spy1 enhances phosphorylation and degradation of the cell cycle inhibitor p27.

The cyclin dependent kinase inhibitor (CKI) p27Kip1 binds to cyclin E/CDK2 complexes and prevents premature S-phase entry. During late G1 and throughout S phase, p27 phosphorylation at T187 leads to its subsequent degradation, which relieves CDK2 inhibition to promote cell cycle progression. However, critical events that trigger CDK2 complexes to phosphorylate p27 remain unclear. Utilizing recombinant proteins, we demonstrate that human Speedy (Spy1) activates CDK2 to phosphorylate p27 at T187 in vitro. Addition of Spy1 or Spy1/CDK2 to a preformed, inhibited cyclin E/CDK2/p27 complex also promoted this phosphorylation. Furthermore, Spy1 protected cyclin E/CDK2 from p27 inhibition toward histone H1, in vitro. Inducible Spy1 expression in U2OS cells reduced levels of endogenous p27 and exogenous p27WT, but not a p27T187A mutant. Additionally, Spy1 expression in synchronized HeLa cells enhanced T187 phosphorylation and degradation of endogenous p27 in late G1 and throughout S phase. Our studies provide evidence that Spy1 expression enhances CDK2-dependent p27 degradation during late G1 and throughout S phase.

Speedy/RINGO regulation of CDKs in cell cycle, checkpoint activation and apoptosis

Speedy/RINGO family members bind and activate cyclin dependent kinases (CDKs), although these proteins have no homology to known cyclin proteins. Members of this family are required for and enhance meiotic maturation, in addition to having novel roles in regulating the mitotic mammalian cell cycle and the DNA damage response. Here we discuss how the specialized functions of these proteins differ from classical cyclin-mediated activation of CDKs. Through atypical activation of CDKs, bypass of conventional inhibitory mechanisms, and unique substrate selection, Speedy/RINGO proteins contribute to cell cycle, checkpoint, and apoptotic regulation. Furthermore, we address the recently established correlation between Spy1 and cancer in terms of the specialized functions of the Speedy/RINGO family.

The atypical CDK activator Spy1 regulates the intrinsic DNA damage response and is dependent upon p53 to inhibit apoptosis

The intrinsic damage response is activated by DNA damage that arises during the cell division process. The ability of the cell to repair this damage during proliferation is important for normal cell growth and, when disrupted, may lead to increased mutatagenesis and tumorigenesis. The atypical CDK activator, Spy1, was previously shown to promote cell survival, prevent apoptosis, and inhibit checkpoint activation in response to DNA damage. Prior studies have shown that Spy1 is up-regulated in breast carcinomas and accelerates mammary tumorigenesis in vivo. In this report, first, we demonstrate that the ability of Spy1 to inhibit apoptosis and bypass UV-induced checkpoint activation is dependent on the presence of the gene regulatory protein p53 and the CKI p21. Second, we demonstrate that Spy1 expression has the following effects: prevents repair of cyclobutane pyrimidine dimers through bypass of nucleotide excision repair; increases the cellular mutation frequency; and reduces the formation of cyclin E induced γH2A.X foci. Lastly, we show that knockdown of endogenous Spy1 leads to γH2A.X foci formation, Chk1 phosphorylation, and proliferation defects, demonstrating a functional role for Spy1 in the intrinsic DNA damage response. These results also demonstrate that Spy1 fulfills a novel regulatory role in the intrinsic DNA damage response and maintains the balance between checkpoint activation, apoptosis, repair, and cell cycle progression in response to exogenous or intrinsic damage. Furthermore, the overexpression of Spy1 as a contributing factor in cancer progression will most likely be confined to p53-positive cells.

Tyrosine Phosphorylation Allows Integration of Multiple Signaling Inputs by IKKβ

Signaling regulated by NFκB and related transcription factors is centrally important to many inflammatory and autoimmune diseases, cancer, and stress responses. The kinase that directly regulates the canonical NFκB transcriptional pathway, Inhibitor of κB kinase β (IKKβ), undergoes activation by Ser phosphorylation mediated by NIK or TAK1 in response to inflammatory signals. Using titanium dioxide-based phosphopeptide enrichment (TiO2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), we analyzed IKKβ phosphorylation in human HEK293 cells expressing IKKβ and FGFR2, a Receptor tyrosine kinase (RTK) essential for embryonic differentiation and dysregulated in several cancers. We attained unusually high coverage of IKKβ, identifying an abundant site of Tyr phosphorylation at Tyr169 within the Activation Loop. The phosphomimic at this site confers a level of kinase activation and NFκB nuclear localization exceeding the iconic mutant S177E/S181E, demonstrating that RTK-mediated Tyr phosphorylation of IKKβ has the potential to directly regulate NFκB transcriptional activation.

Abstract 3208: Development of applications for acoustic membrane microparticle assay technology for translational medicine.

The detection and study of proteins and their interactions utilizing small samples of cells or tissues is an ongoing problem in cancer research. Recent advances in the early detection of many cancers have resulted in smaller sized tumors for biopsy and evaluation. This has created a need for sensitive and robust assays for pathway related proteins. One mission for the Antibody and Protein Purification Unit (APPU) of the National Cancer Institute has been to evaluate emerging technologies and create new applications for these technologies. Using Acoustic Membrane MicroParticle (AMMP) assays on the ViBE Workstation (Bioscale, Lexington, MA) for solution phase immunoassays, the APPU has developed several applications which support the great utility and breadth of use for this technology in translational research. AMMP assays employ a homogenous, sandwich assay format with paired antibodies, one coupled to a magnetic bead and the second tagged for capture on the sensor, and the analyte forming a complex which interacts with a vibrating membrane sensor. The assay measures the concentration of the analyte by a resonant frequency shift of the membrane caused by the protein interaction – without matrix interference or signal scatter seen in optical techniques. A highly sensitive assay for Prolactin showed no serum interference, which is a common problem in optical immunoassays for circulating peptide hormones. An assay for the important marker C-Met required less than 125 nanograms of cell lysate protein for detection and quantitation. An AMMP assay was further able to quantitate C-Met on the surface of Hep2 cells using an antibody capture method. In an application for detection and quantitation of a member of the MAPK kinase family, an ultrasensitive assay for the detection of MEK1 was developed as well as a highly specific solution phase assay for pMEK1 (S218/S222) as shown by cognate phosphopeptide inhibition. And finally in an application which we believe to be truly unique, a solution heterodimer assay for the ERK1/MEK1 interaction was shown to be susceptible to inhibitor treatment. In conclusion, the AMMP technology was able to perform a wide range of applications required by translational research studies. The assays showed superior lower limit of detection and were reproducible, only requiring nanogram quantities of lysate proteins. The ability to measure protein/protein interactions in solution phase is especially useful for drug development and evaluation of drug effects in targeted therapies. Citation Format: Christopher McAndrew, Christopher Heger, Ashley Saab, W Matthew Dickerson, Paul K. Goldsmith. Development of applications for acoustic membrane microparticle assay technology for translational medicine. [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 3208. doi:10.1158/1538-7445.AM2013-3208

Chapter 237 – Signaling from Fibroblast Growth Factor Receptors in Development and Disease

Publisher Summary Fibroblast growth factor receptors (FGFRs) constitute a family of four (FGFR1–4) structurally related, cell surface receptor tyrosine kinases (RTKs), with 55–72% homology. FGFRs are involved in a variety of biological processes, including cell growth, migration, differentiation, survival, and apoptosis, and are essential for embryonic and neural development, skeletal and organ formation, and adult tissue homoeostasis. The three main signaling pathways associated with FGFR activation include the Ras/MAPK, PI 3-kinase, and PLCg pathways. All but one of the mutations known for the Fgfr genes are gain-of-function mutations, and activation of these receptors is associated with many developmental and skeletal disorders. Additionally, FGFR and FGF overexpression has been observed in many tumor samples, and mutations are also likely to be involved in carcinogenesis. Also, during embryonic development, FGFR signaling is essential for organ growth and patterning of the embryo. Activation of FGFRs can result in a variety of outcomes by initiating various intracellular signaling pathways. In many cases, the pathways activated depend on the cell type or stage of differentiation, leading to specific activation of downstream targets. Specific mutations in the Fgfr1–3 genes lead to congenital bone diseases classified as chondrodysplasia and craniosynostosis syndromes, which cause dwarfism, deafness, and abnormalities of the skeleton, skin, and eye. Finally, FGFRs and many of their ligands play roles in cancer progression by angiogenesis, changes in cell morphology, increased motility, and tumor cell proliferation.