Intracellular FGF1 protects cells from apoptosis through direct interaction with p53.

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.

Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma.

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.

Fibroblast Growth Factor 2 Conjugated with Monomethyl Auristatin E Inhibits Tumor Growth in a Mouse Model.

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.

Effects of a Unique Combination of the Whole-Body Low Dose Radiotherapy with Inactivation of Two Immune Checkpoints and/or a Heat Shock Protein on the Transplantable Lung Cancer in Mice.

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.

Protein Tyrosine Phosphatase Receptor Type G (PTPRG) Controls Fibroblast Growth Factor Receptor (FGFR) 1 Activity and Influences Sensitivity to FGFR Kinase Inhibitors.

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.

Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling.

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.

The novel HSP90 inhibitor NVP-AUY922 shows synergistic anti-leukemic activity with cytarabine in vivo.

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.

Proximity Labeling Reveals Molecular Determinants of FGFR4 Endosomal Transport.

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.

Instability restricts signaling of multiple fibroblast growth factors.

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.

Nuclear import of exogenous FGF1 requires the ER-protein LRRC59 and the importins Kpnα1 and Kpnß1.

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnß1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnß1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnß1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.

Translocation of exogenous FGF1 into cytosol and nucleus is a periodic event independent of receptor kinase activity.

Fibroblast growth factor 1 (FGF1) has the property to become translocated from the extracellular space into the cell cytosol and nucleus. Membrane translocation of FGF1 occurs subsequent to endocytic uptake and is strictly FGF-receptor (FGFR) dependent. Here we have investigated the timing of FGF1 translocation in relation to FGFR1 signalling. We found that the translocation of FGF1 is a periodic event that occurs with 24h intervals. Serum-starved cells translocated the growth factor with peak occurrences ~6 h, ~30 h, and ~54 h after the addition of FGF1. The periodic FGF1 translocation was totally independent of the FGFR1 tyrosine kinase activity as it proceeded unchanged when the kinase activity was chemically inhibited or the kinase domain was deleted. Furthermore, FGF1 translocation was not restricted to a particular phase of the cell cycle or dependent on cell cycle progression. The results demonstrate that the FGF1/FGFR1 complex constitutes a signalling module that independently of the receptor tyrosine kinase can convey a signal that initiates a strictly timed and periodic release of endocytosed FGF1 into the cytosol/nucleus.

FGF-FGFR signaling promotes the development of invasive cervical cancer.

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.

Nuclear Localization Sequence of FGF1 Is Not Required for Its Intracellular Anti-Apoptotic Activity in Differentiated Cells.

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.

Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells.

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.

Roles of the FGF-FGFR Signaling System in Cancer Development and Inflammation.

For multi-cellular organisms to organize tissues, their cells must communicate with each other [...].

Negative Regulation of FGFR (Fibroblast Growth Factor Receptor) Signaling.

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.

Increased protein stability of FGF1 can compensate for its reduced affinity for heparin.

Human FGF1 (fibroblast growth factor 1) is a powerful signaling molecule with a short half-life in vivo and a denaturation temperature close to physiological. Binding to heparin increases the stability of FGF1 and is believed to be important in the formation of FGF1.fibroblast growth factor receptor (FGFR) active complex. In order to reveal the function of heparin in FGF1.FGFR complex formation and signaling, we constructed several FGF1 variants with reduced affinity for heparin and with diverse stability. We determined their biophysical properties and biological activities as well as their ability to translocate across cellular membranes. Our study showed that increased thermodynamic stability of FGF1 nicely compensates for decreased binding of heparin in FGFR activation, induction of DNA synthesis, and cell proliferation. By stepwise introduction of stabilizing mutations into the K118E (K132E) FGF1 variant that shows reduced affinity for heparin and is inactive in stimulation of DNA synthesis, we were able to restore the full mitogenic activity of this mutant. Our results indicate that the main role of heparin in FGF-induced signaling is to protect this naturally unstable protein against heat and/or proteolytic degradation and that heparin is not essential for a direct FGF1-FGFR interaction and receptor activation.

FGF2-Derived PeptibodyF2-MMAE Conjugate for Targeted Delivery of Cytotoxic Drugs into Cancer Cells Overexpressing FGFR1.

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.

Size limitation in translocation of fibroblast growth factor 1 fusion proteins across the endosomal membrane.

After binding to its receptor on the surface of mammalian cells and subsequent endocytosis, FGF1 is translocated across the membrane into the cytosol. The growth factor is then further transported into the nucleus. In order to characterize more closely the translocation mechanism utilized by FGF1, we introduced additional amino acids into FGF1 to test the size dependence of the translocated substrate. We constructed mutants containing an increasing number of copies of the myc tag (1-13 copies) in a surface loop of the FGF1 molecule. All of the constructs bound to specific FGF receptors and to heparin and were taken up by endocytosis. However, only FGF1 mutants harboring up to three myc tags (53 amino acids) were translocated while mutants with five myc tags (77 amino acids) or more were not translocated through the membrane. We further showed that insertion of other, unrelated polypeptides into FGF1, i.e., 3xFLAG tag (22 amino acids) and streptavidin binding peptide (50 amino acids), was also translocated. Larger insertions into FGF1, like the CBP-SBP tag (82 amino acids) or ricin A-chain (272 amino acids), resulted in fusion proteins that failed to translocate. The presented data imply that it is possible to employ FGF1 to import various polypeptides into the cytosol and nucleus of cells. Furthermore, the strict size dependence of FGF1 fusion proteins in membrane translocation argues against simple leakage of FGF1 from ruptured endosomal membranes but rather points to a specific translocation apparatus involving a proteinaceous pore.

Cancer Mutations in FGFR2 Prevent a Negative Feedback Loop Mediated by the ERK1/2 Pathway.

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.