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1.
Cell Mol Life Sci ; 80(10): 311, 2023 Oct 02.
Article in English | MEDLINE | ID: mdl-37783936

ABSTRACT

Fibroblast growth factor 1 (FGF1) acts by activating specific tyrosine kinase receptors on the cell surface. In addition to this classical mode of action, FGF1 also exhibits intracellular activity. Recently, we found that FGF1 translocated into the cell interior exhibits anti-apoptotic activity independent of receptor activation and downstream signaling. Here, we show that expression of FGF1 increases the survival of cells treated with various apoptosis inducers, but only when wild-type p53 is present. The p53-negative cells were not protected by either ectopically expressed or translocated FGF1. We also confirmed the requirement of p53 for the anti-apoptotic intracellular activity of FGF1 by silencing p53, resulting in loss of the protective effect of FGF1. In contrast, in p53-negative cells, intracellular FGF1 regained its anti-apoptotic properties after transfection with wild-type p53. We also found that FGF1 directly interacts with p53 in cells and that the binding region is located in the DBD domain of p53. We therefore postulate that intracellular FGF1 protects cells from apoptosis by directly interacting with p53.


Subject(s)
Fibroblast Growth Factor 1 , Tumor Suppressor Protein p53 , Fibroblast Growth Factor 1/genetics , Fibroblast Growth Factor 1/metabolism , Fibroblast Growth Factor 1/pharmacology , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Apoptosis
2.
Br J Cancer ; 127(11): 1939-1953, 2022 11.
Article in English | MEDLINE | ID: mdl-36097178

ABSTRACT

BACKGROUND: Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic. METHODS: We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model. RESULTS: Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90-dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth. CONCLUSIONS: Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS.


Subject(s)
Rhabdomyosarcoma, Embryonal , Rhabdomyosarcoma , Humans , Mice , Animals , Phosphatidylinositol 3-Kinases , Receptor, Fibroblast Growth Factor, Type 4/genetics , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/metabolism , Protein Kinase Inhibitors/pharmacology , Cell Proliferation , Cell Line, Tumor
3.
FEBS J ; 289(12): 3419-3421, 2022 06.
Article in English | MEDLINE | ID: mdl-35146913

ABSTRACT

Cervical cancer is one of the most frequently diagnosed gynecological malignancies among women worldwide. The main cause of cervical cancer is the human papilloma virus (HPV). Mahmood et al. investigated in detail the role of the fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling axis in the development of the invasive form of the disease. The presented work clearly indicates the FGFR signaling as an additional but important factor for the development of a highly malignant tumor. Comment on https://doi.org/10.1111/febs.16331.


Subject(s)
Receptors, Fibroblast Growth Factor , Uterine Cervical Neoplasms , Female , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Humans , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptors, Fibroblast Growth Factor/genetics , Signal Transduction , Uterine Cervical Neoplasms/genetics
4.
Cells ; 11(3)2022 02 02.
Article in English | MEDLINE | ID: mdl-35159330

ABSTRACT

Fibroblast growth factor 1 (FGF1) is considered primarily as a ligand for FGF surface receptors (FGFRs) through which it activates a number of cellular responses. In addition to its canonical mode of action, FGF1 can act intracellularly, before secretion or after internalization and translocation from the cell exterior. The role of FGF1 inside the cell is to provide additional protection against apoptosis and promote cell survival. The FGF1 protein contains a specific N-terminal nuclear localization sequence (NLS) that is essential for its efficient transport to the nucleus. Here, we investigated the role of this sequence in the anti-apoptotic response of FGF1. To this end, we produced recombinant FGF1 variants with mutated or deleted NLS and added them to apoptosis-induced cells in which FGFR1 was inactive, either as a result of chemical inhibition or kinase-dead mutation. After internalization, all FGF1 variants were able to protect the differentiated cells from serum starvation-induced apoptosis. To verify the results obtained for NLS mutants, we knocked down LRRC59, a protein that mediates the nuclear transport of FGF1. Upon LRRC59 silencing, we still observed a decrease in caspase 3/7 activity in cells treated exogenously with wild-type FGF1. In the next step, FGF1 variants with mutated or deleted NLS were expressed in U2OS cells, in which apoptosis was then induced by various factors (e.g., starvation, etoposide, staurosporine, anisomycin and actinomycin D). Experiments were performed in the presence of specific FGFR inhibitors to eliminate FGFR-induced signaling, potentially activated by FGF1 proteins released from damaged cells. Again, we found that the presence of NLS in FGF1 is not required for its anti-apoptotic activity. All NLS variants tested were able to act as wild type FGF1, increasing the cell viability and mitochondrial membrane potential and reducing the caspase 3/7 activity and PARP cleavage in cells undergoing apoptosis, both transiently and stably transfected. Our results indicate that the nuclear localization of FGF1 is not required for its intracellular anti-apoptotic activity in differentiated cells and suggest that the mechanism of the stress response differs according to the level of cell differentiation.


Subject(s)
Apoptosis , Cell Nucleus , Fibroblast Growth Factor 1 , Active Transport, Cell Nucleus , Caspase 3/metabolism , Cell Differentiation/physiology , Cell Line, Tumor , Cell Nucleus/metabolism , Fibroblast Growth Factor 1/genetics , Humans
5.
Biomacromolecules ; 22(10): 4169-4180, 2021 10 11.
Article in English | MEDLINE | ID: mdl-34542998

ABSTRACT

Worldwide, cancer is the second leading cause of death. Regardless of the continuous progress in medicine, we still do not have a fully effective anti-cancer therapy. Therefore, the search for new targeted anti-cancer drugs is still an unmet need. Here, we present novel protein-drug conjugates that inhibit tumor growth in a mouse model of human breast cancer. We developed conjugates based on fibroblast growth factor (FGF2) with improved biophysical and biological properties for the efficient killing of cancer cells overproducing fibroblast growth factor receptor 1 (FGFR1). We used hydrophilic and biocompatible PEG4 or PEG27 molecules as a spacer between FGF2 and the toxic agent monomethyl auristatin E. All conjugates exhibited a cytotoxic effect on FGFR1-positive cancer cell lines. The conjugate with the highest hydrodynamic size (42 kDa) and cytotoxicity was found to efficiently inhibit tumor growth in a mouse model of human breast cancer.


Subject(s)
Antineoplastic Agents , Fibroblast Growth Factor 2 , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Humans , Mice , Oligopeptides
7.
Int J Mol Sci ; 22(12)2021 Jun 11.
Article in English | MEDLINE | ID: mdl-34208396

ABSTRACT

Non-small cell lung cancer (NSCLC) continues to be the leading cause of cancer death worldwide. Recently, targeting molecules whose functions are associated with tumorigenesis has become a game changing adjunct to standard anti-cancer therapy. As evidenced by the results of preclinical and clinical investigations, whole-body irradiations (WBI) with X-rays at less than 0.1-0.2 Gy per fraction can induce remissions of various neoplasms without inciting adverse side effects of conventional chemo- and radiotherapy. In the present study, a murine model of human NSCLC was employed to evaluate for the first time the anti-neoplastic efficacy of WBI combined with inactivation of CTLA-4, PD-1, and/or HSP90. The results indicate that WBI alone and in conjunction with the inhibition of the function of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the programmed death-1 (PD-1) receptor immune checkpoints (ICs) and/or heat shock protein 90 (HSP90) markedly reduced tumorigenesis in mice implanted by three different routes with the syngeneic Lewis lung cancer cells and suppressed clonogenic potential of Lewis lung carcinoma (LLC1) cells in vitro. These results were associated with the relevant changes in the profile of pro- and anti-neoplastic immune cells recruited to the growing tumors and the circulating anti- and pro-inflammatory cytokines. In contrast, inhibition of the tested molecular targets used either separately or in combination with each other did not exert notable anti-neoplastic effects. Moreover, no significant synergistic effects were detected when the inhibitors were applied concurrently with WBI. The obtained results supplemented with further mechanistic explanations provided by future investigations will help design the effective strategies of treatment of lung and other cancers based on inactivation of the immune checkpoint and/or heat shock molecules combined with low-dose radiotherapy.


Subject(s)
Heat-Shock Proteins/metabolism , Immune Checkpoint Proteins/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/radiotherapy , Neoplasm Transplantation , Radiotherapy Dosage , Whole-Body Irradiation , Animals , Clone Cells , Lung/pathology , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating , Macrophages/pathology , Mice, Inbred C57BL , Tumor Stem Cell Assay
8.
Cells ; 10(6)2021 05 28.
Article in English | MEDLINE | ID: mdl-34071546

ABSTRACT

FGFR (fibroblast growth factor receptor) signaling controls fundamental processes in embryonic, fetal and adult human life. The magnitude, duration, and location of FGFR signaling must be strictly controlled in order to induce the correct biological response. Uncontrolled receptor signaling has been shown to lead to a variety of diseases, such as skeletal disorders and cancer. Here we review the numerous cellular mechanisms that regulate and turn off FGFR signaling, once the receptor is activated. These mechanisms include endocytosis and endocytic sorting, phosphatase activity, negative regulatory proteins and negative feedback phosphorylation events. The mechanisms act together simultaneously or sequentially, controlling the same or different steps in FGFR signaling. Although more work is needed to fully understand the regulation of FGFR signaling, it is clear that the cells in our body have evolved an extensive repertoire of mechanisms that together keep FGFR signaling tightly controlled and prevent excess FGFR signaling.


Subject(s)
Fibroblast Growth Factors/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Animals , Cell Line , Humans
9.
Cancers (Basel) ; 12(10)2020 Oct 15.
Article in English | MEDLINE | ID: mdl-33076489

ABSTRACT

Fibroblast growth factor receptors (FGFRs) are emerging targets for directed cancer therapy. Presented here is a new FGFR1-targeting conjugate, the peptibodyF2, which employs peptibody, a fusion of peptide and the Fc fragment of human IgG as a selective targeting agent and drug carrier. Short peptide based on FGF2 sequence was used to construct a FGFR1-targeting peptibody. We have shown that this peptide ensures specific delivery of peptibodyF2 into FGFR1-expressing cells. In order to use peptibodyF2 as a delivery vehicle for cytotoxic drugs, we have conjugated it with MMAE, a drug widely used in antibody-drug conjugates for targeted therapy. Resulting conjugate shows high and specific cytotoxicity towards FGFR1-positive cells, i.e., squamous cell lung carcinoma NCI-H520, while remaining non-toxic for FGFR1-negative cells. Such peptibody-drug conjugate can serve as a basis for development of therapy for tumors with overexpressed or malfunctioning FGFRs.

10.
Cells ; 8(6)2019 05 29.
Article in English | MEDLINE | ID: mdl-31146385

ABSTRACT

Tight regulation of signaling from receptor tyrosine kinases is required for normal cellular functions and uncontrolled signaling can lead to cancer. Fibroblast growth factor receptor 2 (FGFR2) is a receptor tyrosine kinase that induces proliferation and migration. Deregulation of FGFR2 contributes to tumor progression and activating mutations in FGFR2 are found in several types of cancer. Here, we identified a negative feedback loop regulating FGFR2 signaling. FGFR2 stimulates the Ras/MAPK signaling pathway consisting of Ras-Raf-MEK1/2-ERK1/2. Inhibition of this pathway using a MEK1/2 inhibitor increased FGFR2 signaling. The putative ERK1/2 phosphorylation site at serine 780 (S780) in FGFR2 corresponds to serine 777 in FGFR1 which is directly phosphorylated by ERK1/2. Substitution of S780 in FGFR2 to an alanine also increased signaling. Truncated forms of FGFR2 lacking the C-terminal tail, including S780, have been identified in cancer and S780 has been found mutated to leucine in bladder cancer. Substituting S780 in FGFR2 with leucine increased FGFR2 signaling. Importantly, cells expressing these mutated versions of S780 migrated faster than cells expressing wild-type FGFR2. Thus, ERK1/2-mediated phosphorylation of S780 in FGFR2 constitutes a negative feedback loop and inactivation of this feedback loop in cancer cells causes hyperactivation of FGFR2 signaling, which may result in increased invasive properties.


Subject(s)
Feedback, Physiological , MAP Kinase Signaling System , Mutation/genetics , Neoplasms/genetics , Receptor, Fibroblast Growth Factor, Type 2/genetics , Amino Acid Sequence , Cell Line, Tumor , Disease Progression , Epidermal Growth Factor/pharmacology , Feedback, Physiological/drug effects , Fibroblast Growth Factor 1/pharmacology , Humans , MAP Kinase Signaling System/drug effects , Phosphorylation/drug effects , Receptor, Fibroblast Growth Factor, Type 2/chemistry , Serine/genetics , Signal Transduction
11.
Int J Mol Sci ; 20(8)2019 Apr 12.
Article in English | MEDLINE | ID: mdl-31013829

ABSTRACT

Mitogen-activated protein kinases (MAPK): Erk1 and Erk2 are key players in negative-feedback regulation of fibroblast growth factor (FGF) signaling. Upon activation, Erk1 and Erk2 directly phosphorylate FGF receptor 1 (FGFR1) at a specific serine residue in the C-terminal part of the receptor, substantially reducing the tyrosine phosphorylation in the receptor kinase domain and its signaling. Similarly, active Erks can also phosphorylate multiple threonine residues in the docking protein FGF receptor substrate 2 (FRS2), a major mediator of FGFR signaling. Here, we demonstrate that in NIH3T3 mouse fibroblasts and human osteosarcoma U2OS cells stably expressing FGFR1, in addition to Erk1 and Erk2, p38 kinase is able to phosphorylate FRS2. Simultaneous inhibition of Erk1/2 and p38 kinase led to a significant change in the phosphorylation pattern of FRS2 that in turn resulted in prolonged tyrosine phosphorylation of FGFR1 and FRS2 and in sustained signaling, as compared to the selective inhibition of Erks. Furthermore, excessive activation of p38 with anisomycin partially compensated the lack of Erks activity. These experiments reveal a novel crosstalk between p38 and Erk1/2 in downregulation of FGF-induced signaling.


Subject(s)
Fibroblast Growth Factor 1/metabolism , MAP Kinase Signaling System , p38 Mitogen-Activated Protein Kinases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/pharmacology , Animals , MAP Kinase Signaling System/drug effects , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Mice , NIH 3T3 Cells , Phosphorylation
12.
J Mol Biol ; 430(21): 4087-4101, 2018 10 19.
Article in English | MEDLINE | ID: mdl-30099027

ABSTRACT

FGF1 and FGF2 bind to specific cell-surface tyrosine kinase receptors (FGFRs) and activate intracellular signaling that leads to proliferation, migration or differentiation of many cell types. Besides this classical mode of action, under stress conditions, FGF1 and FGF2 are translocated in a receptor-dependent manner via the endosomal membrane into the cytosol and nucleus of the cell. However, despite many years of research, the role of translocated FGF1 and FGF2 inside the cell remains unclear. Here, we reveal an anti-apoptotic activity of intracellular FGF1 and FGF2, which is independent of FGFR activation and downstream signaling. We observed an inhibition of cell apoptosis induced by serum starvation or staurosporine upon treatment with exogenous FGF1 or FGF2, despite the presence of highly potent FGFR inhibitors. Similar results were found when the tyrosine kinase of FGFR1 was completely blocked by a specific mutation. Moreover, the anti-apoptotic effect of the growth factors was abolished by known inhibitors of the translocation of FGF1 and FGF2 from the endosomes to the interior of the cell. Interestingly, FGF2 showed higher anti-apoptotic activity than FGF1. Since FGF2 is not phosphorylated by PKCδ and is present inside the nucleus longer than is FGF1, we speculated that the different activities could reflect their diverse nuclear export kinetics. Indeed, we observed that FGF1 mutations preventing binding to nucleolin and therefore phosphorylation in the nucleus affect the anti-apoptotic activity of FGF1. Taken together, our data indicate that the translocation of FGF1 and FGF2 protects cells against apoptosis and promotes cell survival.


Subject(s)
Apoptosis , Fibroblast Growth Factor 1/metabolism , Fibroblast Growth Factor 2/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Line , Ectopic Gene Expression , Fibroblast Growth Factor 1/genetics , Fibroblast Growth Factor 1/pharmacology , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 2/pharmacology , Gene Expression , Humans , Mice , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Transport , Receptors, Fibroblast Growth Factor/antagonists & inhibitors , Receptors, Fibroblast Growth Factor/genetics , Receptors, Fibroblast Growth Factor/metabolism
13.
Mol Cell Proteomics ; 17(5): 850-870, 2018 05.
Article in English | MEDLINE | ID: mdl-29371290

ABSTRACT

Recently, FGFR1 was found to be overexpressed in osteosarcoma and represents an important target for precision medicine. However, because targeted cancer therapy based on FGFR inhibitors has so far been less efficient than expected, a detailed understanding of the target is important. We have here applied proximity-dependent biotin labeling combined with label-free quantitative mass spectrometry to identify determinants of FGFR1 activity in an osteosarcoma cell line. Many known FGFR interactors were identified (e.g. FRS2, PLCG1, RSK2, SRC), but the data also suggested novel determinants. A strong hit in our screen was the tyrosine phosphatase PTPRG. We show that PTPRG and FGFR1 interact and colocalize at the plasma membrane where PTPRG directly dephosphorylates activated FGFR1. We further show that osteosarcoma cell lines depleted for PTPRG display increased FGFR activity and are hypersensitive to stimulation by FGF1. In addition, PTPRG depletion elevated cell growth and negatively affected the efficacy of FGFR kinase inhibitors. Thus, PTPRG may have future clinical relevance by being a predictor of outcome after FGFR inhibitor treatment.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 5/metabolism , Cell Line, Tumor , Down-Regulation , Fibroblast Growth Factors/pharmacology , Gene Knockdown Techniques , Humans , Osteosarcoma/metabolism , Phosphorylation/drug effects , Protein Binding/drug effects , Proteomics , Reproducibility of Results
14.
ACS Omega ; 2(7): 3792-3805, 2017 Jul 31.
Article in English | MEDLINE | ID: mdl-30023704

ABSTRACT

Antibody-drug conjugates (ADCs) are a new class of anticancer therapeutics that combine the selectivity of targeted treatment, ensured by monoclonal antibodies, with the potency of the cytotoxic agent. Here, we applied an analogous approach, but instead of an antibody, we used fibroblast growth factor 2 (FGF2). FGF2 is a natural ligand of fibroblast growth factor receptor 1 (FGFR1), a cell-surface receptor reported to be overexpressed in several types of tumors. We developed and characterized FGF2 conjugates containing a defined number of molecules of highly cytotoxic drug monomethyl auristatin E (MMAE). These conjugates effectively targeted FGFR1-expressing cells, were internalized upon FGFR1-mediated endocytosis, and, in consequence, revealed high cytotoxicity, which was clearly related to the FGFR1 expression level. Among the conjugates tested, the most potent was that bearing three MMAE molecules, showing that the cytotoxicity of protein-drug conjugates in vitro is directly dependent on drug loading.

15.
J Proteome Res ; 15(10): 3841-3855, 2016 10 07.
Article in English | MEDLINE | ID: mdl-27615514

ABSTRACT

The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.


Subject(s)
Endosomes/metabolism , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Biotinylation , Cell Line, Tumor , Clathrin/metabolism , Endocytosis , Humans , Microscopy/methods , Protein Transport , Signal Transduction , Staining and Labeling , trans-Golgi Network/metabolism
16.
Exp Cell Res ; 340(2): 220-6, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26748184

ABSTRACT

HSP90 is a molecular chaperone essential for stability, activity and intracellular sorting of many proteins, including oncoproteins, such as tyrosine kinases, transcription factors and cell cycle regulatory proteins. Therefore, inhibitors of HSP90 are being investigated for their potential as anti-cancer drugs. Here we show that the HSP90 inhibitor NVP-AUY922 induced degradation of the fusion oncoprotein FOP2-FGFR1 in a human acute myeloid leukemia (AML) cell line, KG-1a. Concordantly, downstream signaling cascades, such as STAT1, STAT3 and PLCγ were abrogated. At concentrations that caused FOP2-FGFR1 degradation and signaling abrogation, NVP-AUY922 treatment caused significant cell death and inhibition of proliferation of KG-1a cells in vitro. In an animal model for AML, NVP-AUY922 administrated alone showed no anti-leukemic activity. However, when NVP-AUY922 was administered in combination with cytarabine, the two compounds showed significant synergistic anti-leukemic activity in vivo. Thus NVP-AUY922 and cytarabine combination therapy might be a prospective strategy for AML treatment.


Subject(s)
Antineoplastic Agents/pharmacology , Cytarabine/pharmacology , HSP90 Heat-Shock Proteins/metabolism , Isoxazoles/pharmacology , Leukemia/metabolism , Resorcinols/pharmacology , Signal Transduction/drug effects , Animals , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Leukemia/drug therapy , Male , Neoplasm Proteins/metabolism
17.
Cell Mol Life Sci ; 72(12): 2445-59, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25854632

ABSTRACT

Fibroblast growth factors (FGFs) deliver extracellular signals that govern many developmental and regenerative processes, but the mechanisms regulating FGF signaling remain incompletely understood. Here, we explored the relationship between intrinsic stability of FGF proteins and their biological activity for all 18 members of the FGF family. We report that FGF1, FGF3, FGF4, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF20, and FGF22 exist as unstable proteins, which are rapidly degraded in cell cultivation media. Biological activity of FGF1, FGF3, FGF4, FGF6, FGF8, FGF10, FGF16, FGF17, and FGF20 is limited by their instability, manifesting as failure to activate FGF receptor signal transduction over long periods of time, and influence specific cell behavior in vitro and in vivo. Stabilization via exogenous heparin binding, introduction of stabilizing mutations or lowering the cell cultivation temperature rescues signaling of unstable FGFs. Thus, the intrinsic ligand instability is an important elementary level of regulation in the FGF signaling system.


Subject(s)
Bone Neoplasms/metabolism , Breast Neoplasms/metabolism , Cell Proliferation , Chondrosarcoma/metabolism , Fibroblast Growth Factors/chemistry , Fibroblast Growth Factors/metabolism , Signal Transduction , Animals , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Chondrosarcoma/genetics , Chondrosarcoma/pathology , Circular Dichroism , Female , Fibroblast Growth Factors/classification , Fibroblast Growth Factors/genetics , Humans , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation/genetics , Protein Stability , Rats , Temperature , Tumor Cells, Cultured
18.
PLoS One ; 9(3): e90687, 2014.
Article in English | MEDLINE | ID: mdl-24595027

ABSTRACT

Extracellular fibroblast growth factor 1 (FGF1) acts through cell surface tyrosine kinase receptors, but FGF1 can also act directly in the cell nucleus, as a result of nuclear import of endogenously produced, non-secreted FGF1 or by transport of extracellular FGF1 via endosomes and cytosol into the nucleus. In the nucleus, FGF1 can be phosphorylated by protein kinase C δ (PKCδ), and this event induces nuclear export of FGF1. To identify intracellular targets of FGF1 we performed affinity pull-down assays and identified nucleolin, a nuclear multifunctional protein, as an interaction partner of FGF1. We confirmed a direct nucleolin-FGF1 interaction by surface plasmon resonance and identified residues of FGF1 involved in the binding to be located within the heparin binding site. To assess the biological role of the nucleolin-FGF1 interaction, we studied the intracellular trafficking of FGF1. In nucleolin depleted cells, exogenous FGF1 was endocytosed and translocated to the cytosol and nucleus, but FGF1 was not phosphorylated by PKCδ or exported from the nucleus. Using FGF1 mutants with reduced binding to nucleolin and a FGF1-phosphomimetic mutant, we showed that the nucleolin-FGF1 interaction is critical for the intranuclear phosphorylation of FGF1 by PKCδ and thereby the regulation of nuclear export of FGF1.


Subject(s)
Cell Nucleus/metabolism , Fibroblast Growth Factor 1/metabolism , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Active Transport, Cell Nucleus , Animals , Cell Line , Fibroblast Growth Factor 1/analysis , Fibroblast Growth Factor 2/metabolism , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , Phosphorylation , Nucleolin
19.
Sci Signal ; 6(262): ra11, 2013 Feb 12.
Article in English | MEDLINE | ID: mdl-23405013

ABSTRACT

Fibroblast growth factor 1 (FGF1) controls cellular activities through the activation of specific cell-surface FGF receptors (FGFRs). Transphosphorylation of tyrosine residues in the kinase domain of FGFRs leads to activation of intracellular signaling cascades, including those mediated by mitogen-activated protein kinases (MAPKs). FGFRs also contain a serine-rich C-terminal tail. We identified a regulatory mechanism of FGFR signaling involving phosphorylation of Ser(777) in the C-terminal region of FGFR1 by the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2. Prevention of the phosphorylation of Ser(777) in FGFR1 or mutation of Ser(777) to alanine enhanced FGF-stimulated receptor tyrosine phosphorylation and increased cell proliferation, cell migration, and axonal growth. A form of FGFR1 with a phosphomimetic mutation at Ser(777) exhibited reduced signaling. Activation of MAPKs by other receptor tyrosine kinases also resulted in phosphorylation of Ser(777) in FGFR1, thereby enabling crosstalk regulation of FGFR activity by other signaling pathways. Our data reveal a negative feedback mechanism that controls FGF signaling and thereby protects the cell from excessive activation of FGFR.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Serine/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Enzyme Activation , Humans , Molecular Sequence Data , Mutation , Phosphorylation , Receptor, Fibroblast Growth Factor, Type 1/chemistry , Receptor, Fibroblast Growth Factor, Type 1/genetics , Sequence Homology, Amino Acid
20.
Int J Nanomedicine ; 7: 5915-27, 2012.
Article in English | MEDLINE | ID: mdl-23226697

ABSTRACT

Fibroblast growth factor receptors (FGFRs) are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we designed, constructed, and characterized FGFR-targeted gold nanoconjugates suitable for infrared-induced thermal ablation (localized heating leading to cancer cell death) based on gold nanoparticles (AuNPs). We showed that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs. To assure thermal stability, protease resistance, and prolonged half-life of the targeting protein, we employed highly stable FGF1 variant that retains the biological activities of the wild type FGF1. Novel FGF1 variant, AuNP conjugates are specifically internalized only by the cells expressing FGFRs, and they significantly reduce their viability after irradiation with near-infrared light (down to 40% of control cell viability), whereas the proliferation potential of cells lacking FGFRs is not affected. These results demonstrate the feasibility of FGF1-coated AuNPs for targeted cancer therapy.


Subject(s)
Fibroblast Growth Factor 1/pharmacokinetics , Gold/therapeutic use , Hyperthermia, Induced/methods , Metal Nanoparticles/therapeutic use , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/therapy , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Cell Line, Tumor , Cell Survival/radiation effects , Gold/chemistry , Humans , Infrared Rays/therapeutic use , Metal Nanoparticles/chemistry , Treatment Outcome
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