FGF Stimulation Research Articles

Chemical modification of NSC12 leads to a specific FGF-trap with antitumor activity in multiple myeloma.

Inhibition of FGF/FGFR signaling is a promising strategy for the treatment of malignances dependent from FGF stimulation, including multiple myeloma (MM). The steroidal derivative NSC12 (compound 1) is a pan-FGF trap endowed with antitumor activity invivo. Chemical modifications of compound 1 were explored to investigate structure-activity relationships, focusing on the role of the bis(trifluoromethyl)1,3-propanediol chain, the stereochemistry at C20 and functionalization of C3 position. Our studies unveiled compound 25b, the pregnane 3-keto 20R derivative of compound 1 as an effective agent, blocking the proliferation of MM cells invitro by inhibiting FGF-dependent receptor activation and slowing MM growth invivo. Importantly, the absence of the hydroxyl group at C3 prevents binding to estrogen receptors, which might concur to the antitumor activity observed for compound 1, leading to a specific FGF/FGFR system inhibitor, and further supporting the role of FGFR in anticancer therapy in MM.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

SummaryDUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

Constitutive Dicer1 phosphorylation accelerates metabolism and aging in vivo

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.

Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries

The use of stem or progenitor cells from bone marrow, or peripheral or umbilical cord blood is becoming more common for treatment of diabetic foot problems. These cells promote neovascularization by angiogenic factors and they promote epithelium formation by stimulating cell replication and migration under certain pathological conditions. We investigated the role of CD34 + stem cells from human umbilical cord blood in wound healing using a rat model. Rats were randomly divided into a control group and two groups with diabetes induced by a single dose of 55 mg/kg intraperitoneal streptozocin. Scarred areas 5 mm in diameter were created on the feet of all rats. The diabetic rats constituted the diabetes control group and a diabetes + stem cell group with local injection into the wound site of 0.5 × 106 CD34 + stem cells from human umbilical cord blood. The newly formed skin in the foot wounds following CD34 + stem cell treatment showed significantly improvement by immunohistochemistry and TUNEL staining, and were closer to the wound healing of the control group than the untreated diabetic animals. The increase in FGF expression that accompanied the local injection of CD34 + stem cells indicates that FGF stimulation helped prevent apoptosis. Our findings suggest a promising new treatment approach to diabetic wound healing.

The receptor protein tyrosine phosphatase PTPRB negatively regulates FGF2-dependent branching morphogenesis.

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 SY30-01: Discovery of the highly selective covalent FGFR1-4 inhibitor PRN1371, currently in development for the treatment of solid tumors

Abstract Discovering drug candidates that exhibit durable on-target pharmacology upon oral dosing has been extremely challenging for the pharmaceutical industry. The introduction of a covalent bond between a small molecule drug and its pharmacological target has the potential to achieve prolonged target inhibition with minimal systemic drug exposure. Several irreversible covalent inhibitors employing this strategy have been approved or are in late stage clinical development for the treatment of various cancer indications such as multiple myeloma (carfilzomib), lymphoma (ibrutinib), lung cancer (afatinib) and breast cancer (neratinib). However, the discovery process for covalent drugs presents unique challenges. Identification and optimization of lead molecules require screening assays that measure the degree of covalent engagement with the target, both in vitro and in vivo. In addition, the reactive chemistries often employed for covalent bonding can impart poor ADME properties, requiring IV administration or high oral doses. Principia Biopharma, a pioneer in covalent drug discovery and development, has overcome many of these challenges in the discovery of PRN1371, a covalent irreversible inhibitor of fibroblast growth factor receptor (FGFR) 1-4 with long target residence time. Aberrant signaling of the FGF/FGFR pathway is an oncogenic driver in many solid tumors. Early reports from Phase 1/2 studies with reversible ATP competitive inhibitors have demonstrated encouraging efficacy from FGFR inhibition in the treatment of bladder, lung, and breast cancers. However, some results are difficult to interpret due to the poor selectivity of the drugs used. Aberrant pathway activation may be a consequence of FGF ligand overexpression or changes to the FGF receptor including mutations, translocations/fusions, and protein overexpression. Such genetic alterations in one or more isoforms of FGFR have been reported in many solid tumors including bladder, breast, cervical, endometrial, gastric, hepatocellular, lung, and ovarian cancers. There remains a compelling medical need to identify a highly selective inhibitor of all four FGFR family members, FGFR1-4, to improve clinical responses in patients while minimizing off-target toxicity. PRN1371 is the culmination of a drug discovery program focused upon the creation of an oral, low dose, highly selective, potent, and irreversible inhibitor of FGFR1-4. Starting from a 7-oxopyridopyrimidine core, a series of covalent inhibitors were designed to incorporate a Michael acceptor appropriately positioned to engage Cys 488 in a covalent bond. Extensive SAR was generated using a FRET-based biochemical occupancy assay to measure the durability of drug binding over time. Molecules were optimized for prolonged FGFR residence time and potent inhibition of pERK in HUVEC cells upon FGF stimulation. Advanced leads with suitable cellular potency were studied in vivo, using an FGF-induced CCL2 readout in mice. The relationship between plasma drug concentration (PK) and pharmacological inhibition of CCL2 production (PD) in mice provided essential guidance in selecting optimal drug candidates. To assess the ADME liabilities of the Michael acceptor, in vitro microsomal stability and thiol reactivity assays were used. The final selection of PRN1371 as a clinical candidate was based upon the ideal combination of exquisite selectivity, potent and prolonged pharmacodynamic inhibition of FGFR1-4 in vivo and high predicted human oral bioavailability. PRN1371 is now evaluated in a Phase 1/2 open-label, multicenter clinical trial for solid tumors (ClinicalTrials.gov: NCT02608125). Citation Format: Ken A. Brameld. Discovery of the highly selective covalent FGFR1-4 inhibitor PRN1371, currently in development for the treatment of solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY30-01.

Sustained Inhibition of Proliferative Response After Transient FGF Stimulation Is Mediated by Interleukin 1 Signaling.

Transient FGF stimulation of various cell types results in FGF memory--a sustained blockage of efficient proliferative response to FGF and other growth factors. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates proinflammatory NFκB signaling, which is also critical for FGF memory formation. The search for FGF-induced mediators of FGF memory revealed that FGF stimulates HDAC-dependent expression of the inflammatory cytokine IL1α. Similarly to FGF, transient cell treatment with recombinant IL1α inhibits the proliferative response to further FGF and EGF stimulation, but does not prevent FGF receptor-mediated signaling. Interestingly, like cells pretreated with FGF1, cells pretreated with IL1α exhibit enhanced restructuring of actin cytoskeleton and increased migration in response to FGF stimulation. IRAP, a specific inhibitor of IL 1 receptor, and a neutralizing anti-IL1α antibody prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. A similar effect results following treatment with the anti-inflammatory agents aspirin and dexamethasone. Thus, FGF memory is mediated by proinflammatory IL1 signaling. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound-induced hyperplasia.

Induction of endogenous interleukin 1 alpha limits to one cycle the proliferative response to FGF

We found that transient FGF stimulation of various cell types results in the novel phenomenon termed “FGF memory” – a sustained blockage of efficient proliferative response to FGF, PDGF or EGF. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates the proinflammatory NFkappaB signaling, which is also critical for FGF memory formation. The search of potential FGF‐induced mediators of FGF memory revealed that FGF induces a sustained expression of the inflammatory cytokine IL1alpha, and this induction is abolished by HDAC inhibitors. Similarly to FGF pretreatment, transient cell treatment with recombinant IL1alphainhibits the proliferative response to further FGF stimulation. However, cells pretreated with FGF or IL1alpha exhibit an enhanced restructuring of actin cytoskeleton and migration in response to FGF. IL1alpha‐prestimulated cells respond to FGF treatment by ERK1/2 phosphorylation and expression of cyclin D1, but not of cyclin A. Both IRAP, a specific inhibitor of IL1 receptor, and neutralizing anti‐IL1alphaantibodies prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. Thus, FGF memory is mediated by the endogenous IL1alpha. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound‐induced hyperplasia.

P53 Suppression Mediates Blasts Survival, Proliferation and CD34 Maintenance in Pediatric Acute Myeloid Leukemia

Aberrations in the DNA damage responses contributes to the uncontrolled proliferation and therapy resistance in many cancers. Acute myeloid leukemia (AML) presents enhanced DNA damage as compared to MDS characterized by induced levels γ-2HAX and phosphorylated ATM that correlated with AML blast percentages (Boehrer et al. Oncogene 2009). The low incidence of p53 mutations (7% in adult AML, The Cancer Genome Atlas Research Network, N Engl J Med 2013) in AML indicates downstream suppression of DNA damage control system proteins, as was proposed previously by overexpression of MDM2 and BCL-2 (Wojcik et al. Neoplasma 2005). However, the role of p53 and downstream effectors regulating the DNA damage pathway in CD34+ AML blasts is still relatively unclear.Surprisingly, using RPPA analysis, we found a common significant increase in p53 phosphorylation at serine 15 in pediatric AML as compared to CD34+ NBM (AML n=31 and NBM n=10, Mann-Whitney U, P = 0.003). Therefore, we challenged to target p53 using a shRNA approach. In the sh-p53 model, primary pediatric AML samples transduced with sh-p53 showed a significant increase in AML proliferation over time as compared to scrambled control (paired-samples t-test, P = 0.005). The overall AML cell viability was significantly improved in sh-p53 AML cultures (paired-samples t-test P = 0.001). DNA cell cycle analysis revealed that sh-p53 AML cultures contained significant increased percentages of cells in S and G2/M phases of the cell cycle as compared to their scrambled controls (paired samples t-test, P = 0.015). The CD34+ leukemic population was maintained in p53 knockdown cultures and thereby outcompeted scrambled control cultures. Knockdown of p53 in primary pediatric AML samples resulted in a higher proliferation rate, repressed differentiation and prolonged survival of AML blasts, especially in MLL-rearranged AML. Interestingly, we previously showed that MLL-rearranged AML samples express significantly higher BCL-2 peptide phosphorylation as compared to NBM (Kampen et al. Leukemia 2014). Growth factors are extrinsic cues that can regulate BCL-2 expression (Pidgeon et al. Br J Cancer 2001). Growth factors like VEGF, have been shown to be involved in leukemia pathogenesis in the bone marrow where leukemic stem cells are preserved in tightly controlled niches (Kampen et al. Cell Mol Life Sci 2013). Accordingly, we found that FGF and VEGFC stimulation of CD34+ AML blasts both significantly accelerated BCL-2 mRNA expression by 28-fold and 2-fold, indicating that indeed the niche is important for inhibiting pro-apoptotic p53-dependent signals further downstream the pathway, which protects AML CD34+ blasts (Student’s t-test, both P < 0.001), where p53 and p21 mRNA expression remained unchanged. The availability of VEGFR-2 and FGFR1 previously highlighted their therapeutic targeting potential in AML (Kampen et al. Leukemia 2006, Kentsis et al, Nature Med 2012). This study presents the effect of p53 suppression on AML blast proliferation, survival and maintenance in pediatric AML and indicates the importance of exploring extrinsic factors that contribute to DNA damage control system suppression in vivo for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Regulation of metastasis of pediatric multiple myeloma by MMP13.

The molecular mechanism underlying metastasis of pediatric multiple myeloma (MM) remains elusive. Here, we showed that the levels of MMP13 are significantly higher in MM from young patients than those from adult patients. Moreover, a strong correlation of the MMP13 and phosphorylated fibroblast growth factor receptor 4 (FGFR4) levels was detected in MM from young patients. To prove a causal link between activation of fibroblast growth factor receptors (FGFR) signaling pathway and MMP13 expression, we used a human MM line, RPMI-8226 (8226), to study the underlying molecular basis. We found that FGF1-induced FGFR4 phosphorylation in 8,226 cells resulted in significant activation of MMP13, and consequently, an increase in cancer invasiveness. FGFR4 inhibition in 8,226 cells abolished FGF1-stimulated MMP13 expression, suggesting that activation of FGFR signaling pathway in MM may promote cancer metastasis by inducing MMP13 expression. To define the signaling cascades downstream of FGFR4 activation for MMP13 activation, we applied specific inhibitors for PI3K, Jun N-terminal kinase (JNK), and ERK/MAPK, respectively, to the FGF1-stimulated 8,226 cells. We found that only inhibition of ERK1/2 significantly decreased the activation of MMP13 in response to FGF stimulation, suggesting that activation of FGFR signaling may activate ERK/MAPK, rather than JNK or PI3K pathway to activate MMP13 expression in 8,226 cells. Our study thus highlights FGFR4 signaling pathway and MMP13 as novel therapeutic targets for MM.

Transitory FGF Treatment Results in the Long‐Lasting Suppression of the Proliferative Response to Repeated FGF Stimulation

FGF applied as a single growth factor to quiescent mouse fibroblasts induces a round of DNA replication, however continuous stimulation results in arrest in the G1 phase of the next cell cycle. We hypothesized that FGF stimulation induces the establishment of cell memory, which prevents the proliferative response to repeated or continuous FGF application. When a 2-5 days quiescence period was introduced between primary and repeated FGF treatments, fibroblasts failed to efficiently replicate in response to secondary FGF application. The establishment of "FGF memory" during the first FGF stimulation did not require DNA synthesis, but was dependent on the activity of FGF receptors, MEK, p38 MAPK and NFκB signaling, and protein synthesis. While secondary stimulation resulted in strongly decreased replication rate, we did not observe any attenuation of morphological changes, Erk1/2 phosphorylation and cyclin D1 induction. However, secondary FGF stimulation failed to induce the expression of cyclin A, which is critical for the progression from G1 to S phase. Treatment of cells with a broad range histone deacetylase inhibitor during the primary FGF stimulation rescued the proliferative response to the secondary FGF treatment suggesting that the establishment of "FGF memory" may be based on epigenetic changes. We suggest that "FGF memory" can prevent the hyperplastic response to cell damage and inflammation, which are associated with an enhanced FGF production and secretion. "FGF memory" may present a natural obstacle to the efficient application of recombinant FGFs for the treatment of ulcers, ischemias, and wounds.

Abstract P4-06-08: Clinical utility of functional analysis of FGFR kinase family for selecting patients who may benefit from FGFR inhibitors

Abstract Background: Fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) are currently under investigation as therapeutic targets for the treatment of breast cancer. Members of this kinase family have been associated with breast cancer progression and resistance to current therapeutics. FGFR1 amplifications range from 7.5–17% in breast cancers and are associated with a shorter overall survival. FGFR1 amplified cell lines drive activation of AKT, ERK, and RSK and are highly sensitive to FGFR1 inhibition, suggesting an oncogenic addiction to FGFR1. FGFR2 is activated in lapatinib-resistant, HER2-positive cells. Likewise, FGFR3 expression levels are elevated in tamoxifen-resistant, ER positive patients. FGFR 4 overexpression correlated with poor response to chemotherapy due to activation of MAPK and increase in B-cell lymphoma extra large (BCL-XL) levels. Using an immuno-microarray system, this study seeks to determine FGFR signal transduction pathway modulation post FGFR inhibitor treatment in breast cancer cell lines expressing varying levels of FGFR1, 2, 3 or 4. Method: FGFR 1, 2, 3, and 4 amplified cell lines (MDA-MB-134VI (KRAS), SNU16, RT112, and MDA-MB-453) were treated with varying dosages (1, 10, 100, and 1000nM) of several FGFR inhibitors (AZD4547 (pan FGFR), PD173074 (FGFR1,3) and Ponatinib-AP24534 (FGFR1)) either in presence or absence of corresponding FGF (FGF1, FGF7, FGF9, and FGF19) stimulation. Expression and activation of FGFR 1, 2, 3, and 4 and downstream signaling proteins FRS2, AKT, ERK, MEK, and RSK were measured utilizing a proximity based immuno-microarray. Results: FGFR1, 2, 3, 4 protein expression was detected in each of the corresponding four FGFR1, 2, 3, 4 amplified cell lines. Total protein levels remained unchanged with or without FGFs stimulation. Levels of FGFR1 phosphorylation increased 5 folds in MDA-MB-134VI stimulated with FGF1. FGFR2 and FGFR3 activation increased by 2–3 folds in SNU16 and RT112 stimulated with FGF7, and FGF9 respectively. FGFR4 activation levels in MDA-MB-453 remained unchanged due to Y367C mutation. ERK was activated in both FGF1 stimulated and unstimulated group in MDA-MB134 due to the KRAS mutation. RSK activation increased in both SNU16 and RT112 when stimulated with FGF7, and FGF9 respectively. FGFR inhibitors treated data analysis is currently in progress and will be presented. Conclusion: We have demonstrated utility of specific FGFR analysis using well defined model systems. FGFR- profiling should be considered during the clinical work-up in order to select patients who may benefit from regimen containing specific FGFR inhibitors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-08.

Epidermal Growth Factor Receptor (EGFR)-mediated Positive Feedback of Protein-tyrosine Phosphatase ϵ (PTPϵ) on ERK1/2 and AKT Protein Pathways Is Required for Survival of Human Breast Cancer Cells

Increased tyrosine phosphorylation has been correlated with human cancer, including breast cancer. In general, the activation of tyrosine kinases (TKs) can be antagonized by the action of protein-tyrosine phosphatases (PTPs). However, in some cases PTPs can potentiate the activation of TKs. In this study, we have investigated the functional role of PTPε in human breast cancer cell lines. We found the up-regulation and activation of receptor PTPε (RPTPε) in MCF-7 cells and MDA-MB-231 upon PMA, FGF, and serum stimulation, which depended on EGFR and ERK1/2 activity. Diminishing the expression of PTPε in human breast cancer cells abolished ERK1/2 and AKT activation, and decreased the viability and anchorage-independent growth of the cells. Conversely, stable MCF-7 cell lines expressing inducible high levels of ectopic PTPε displayed higher activation of ERK1/2 and anchorage-independent growth. Our results demonstrate that expression of PTPε is up-regulated and activated in breast cancer cell lines, through EGFR, by sustained activation of the ERK1/2 pathway, generating a positive feedback regulatory loop required for survival of human breast cancer cells.

Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.

FGF-dependent regulation of VEGF receptor 2 expression in mice

Numerous studies have suggested a link between the angiogenic FGF and VEGF signaling pathways; however, the nature of this link has not been established. To evaluate this relationship, we investigated VEGF signaling in ECs with disrupted FGF signaling in vitro and in vivo. ECs lacking FGF signaling became unresponsive to VEGF, caused by downregulation of VEGF receptor 2 (VEGFR2) expression after reduced Vegfr2 enhancer activation. FGF mediated VEGFR2 expression via activation of Erk1/2. Transcriptional analysis revealed that Ets transcription factors controlled VEGFR2 expression in an FGF- and Erk1/2-dependent manner. Mice with defective FGF signaling exhibited loss of vascular integrity and reduced vascular morphogenesis. Thus, basal FGF stimulation of the endothelium is required for maintenance of VEGFR2 expression and the ability to respond to VEGF stimulation and accounts for the hierarchic control of vascular formation by FGFs and VEGF.

Regulation of the growth factor signaling modulator, sprouty 2, by PHDs and pVHL/E3 ligase

Sprouty (Spry) proteins modulate receptor tyrosine kinase signaling and inhibit cellular processes. While levels of Spry isoforms, especially Spry2, have been shown to influence the growth of tumors from the lung, liver, breast, and prostate, little is known about the interaction of Spry with proteins involved in the hypoxic response. Here, we demonstrate that HA‐Spry 2 levels are elevated after exposure to hypoxia (3% O2) and DMOG (200μM), conditions that are known to inhibit the activity of prolyl hydroxylases (PHDs). Spry2 interacts with PHDs 1–3 as well as von Hippel‐Lindau tumor suppressor protein (pVHL)—two key players in the activation of the hypoxic response. Overexpression of all PHDs under normoxic conditions (21%O2) resulted in decreased HA‐Spry2 levels in HeLa cells. Similar results were obtained when pVHL was overexpressed in HEK293T cells. Moreover, expression of shRNA against PHD1 and PHD3 or treatment with siRNA against pVHL resulted in increased levels of overexpressed HA‐Spry2. Knockdown of endogenous PHD1 in HEK293T cells caused a further reduction in the phosphorylation of ERK upon FGF stimulation when HA‐Spry 2 was present, confirming that Spry2/PHD interactions represent an alternate mechanism by which Spry2 function can be regulated.Supported by NIH grant GM073181

A Novel Fibroblast Growth Factor-1 (FGF1) Mutant that Acts as an FGF Antagonist

BackgroundCrosstalk between integrins and FGF receptors has been implicated in FGF signaling, but the specifics of the crosstalk are unclear. We recently discovered that 1) FGF1 directly binds to integrin αvβ3, 2) the integrin-binding site and FGF receptor (FGFR) binding site are distinct, and 3) the integrin-binding-defective FGF1 mutant (R50E) is defective in inducing FGF signaling although R50E still binds to FGFR and heparin and induces transient ERK1/2 activation.Principal FindingsWe tested if excess R50E affect DNA synthesis and cell survival induced by WT FGF1 in BaF3 mouse pro-B cells expressing human FGFR1. R50E suppressed DNA synthesis and cell proliferation induced by WT FGF1. We tested if WT FGF1 and R50E generate integrin-FGF1-FGFR ternary complex. WT FGF1 induced ternary complex formation (integrin-FGF-FGFR1) and recruitment of SHP-2 to the complex in NIH 3T3 cells and human umbilical endothelial cells, but R50E was defective in these functions. It has been reported that sustained ERK1/2 activation is integrin-dependent and crucial to cell cycle entry upon FGF stimulation. We thus determined the time-course of ERK1/2 activation induced by WT FGF1 and R50E. We found that WT FGF1 induced sustained activation of ERK1/2, but R50E was defective in this function.Conclusions/SignificanceOur results suggest that 1) R50E is a dominant-negative mutant, 2) Ternary complex formation is involved in FGF signaling, 3) The defect of R50E to bind to integrin may be directly related to the antagonistic action of R50E. Taken together, these results suggest that R50E has potential as a therapeutic in cancer.

The identification of FGF-dependent phosphorylation events in embryonic stem cells using mass spectrometry

Fibroblast growth factor-four (FGF4) signalling is essential for embryonic stem (ES) cells to become competent to undergo differentiation and enter lineage commitment pathways. The signalling pathway whereby this occurs is as yet not fully characterized. In this study, high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis was used in conjunction with phosphopeptide enrichment using TiO2 affinity chromatography to detect peptides phosphorylated in response to FGF stimulation of mouse ES cells. Many phosphorylation events were observed in FGF-treated ES cells and some phosphopeptides were unambiguously identified that associate with four different proteins: Lig1, Eif3b, 6430527G18Rik and Sin3a. We propose a novel pathway in which FGF signalling enables differentiation of ES cells via activation of Sin3a, a transcription repressor, which regulates expression of pluripotency genes.

Sprouty2 Interacts with Protein Kinase Cδ and Disrupts Phosphorylation of Protein Kinase D1

The Sprouty (Spry) proteins act as inhibitors of the Ras/ERK pathway downstream of receptor tyrosine kinases. In this study, we report a novel interaction between protein kinase C delta (PKCdelta) and Spry2. Endogenous PKCdelta and Spry2 interact in cells upon basic fibroblast growth factor stimulation, indicating a physiological relevance for the interaction. This interaction appeared to require the full-length Spry2 protein and was conformation-dependent. Conformational constraints were released upon FGFR1 activation, allowing the interaction to occur. Although this interaction did not affect the phosphorylation of PKCdelta by another kinase, it reduced the phosphorylation of a PKCdelta substrate, protein kinase D1 (PKD1). Spry2 was found to interact more strongly with PKCdelta with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKCdelta, it was more likely to form a trimeric complex with both PKCdelta and PKD1. Formation of the complex was found to be dependent on an existing PKCdelta-PKD1 interaction. By disrupting the interaction between PKCdelta and PKD1, Spry2 was unable to associate with PKCdelta to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKCdelta and PKD1 was increased, and the transfer of phosphate groups from PKCdelta to PKD1 was at least partly blocked by Spry2. The action of Spry2 on PKCdelta resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKCdelta signaling. By disrupting the capacity of PKCdelta to phosphorylate its cognate substrates, Spry2 may serve to modulate PKCdelta signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome.

FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for naïve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.